Backert - Helicobacter Pylori Research 2016

Steffen
Backert · Yoshio Yamaoka
Editors
Helicobacter
pylori
Research
From Bench to Bedside
Helicobacter pylori Research
ThiS is a FM Blank Page
Steffen Backert • Yoshio Yamaoka
Editors
Helicobacter pylori Research

From Bench to Bedside
Editors
Steffen Backert Yoshio Yamaoka
Division of Microbiology Faculty of Medicine
Department of Biology Department of Environmental and
Friedrich Alexander University Preventive Medicine
Erlangen-Nuremberg Oita University
Erlangen, Germany Yufu, Japan
Department of Medicine-Gastroenteology
Baylor College of Medicine
Houston, USA
ISBN 978-4-431-55934-4 ISBN 978-4-431-55936-8 (eBook)

DOI 10.1007/978-4-431-55936-8
Library of Congress Control Number: 2016935851
© Springer Japan 2016

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Foreword
Helicobacter pylori, the Gastric Bacterium Which Still

Infects Half the World’s Population, Is an Important
Part of Gastroenterology and Infectious Disease
It is exciting to participate in this new “state-of-the-art” book about Helicobacter

pylori, the gastric bacterium which still infects half of the human race and which
promises to challenge clinicians and scientists for decades to come. The book is
structured so as to place H. pylori in perspective as the gastric bacterium attached
itself to prehistoric humans and followed their migrations, through the stone-age
into the twenty-first century. Thus the first chapter, by Yoshan Moodley, sets us up
to ask the questions which can then be answered in depth by the other experts.
Where did H. pylori come from? How does it survive in the stomach? What unique
adaptations have taken place, which allow it to persist throughout life? If it
colonised mankind for so long, was there once a useful purpose in having
H. pylori? Is it still useful in some way? As complete genome sequencing technol-
ogy becomes widespread, many will use this in-depth initial chapter as a hypothesis
generating knowledge-base for our own molecular epidemiology studies. Examples
are even given of newer technologies such as RNAseq, for examining gene expres-
sion and intergenic control regions. Later, Ichizo Kobayashi predicts that further
breakthroughs via OMICS analyses will also provide more insight into the evolu-
tion of both the genome and methylome of this highly diverse model organism.
Once the big picture has been described, we want to know how H. pylori
accomplishes this and “what makes it tick?” So we delve into H. pylori biochem-
istry realising that the metabolism of the bug mirrors the microenvironment of the
gastric mucosa (Fischer and deReuse, Praszkier, Sutton and Ferrero; Backert,
Zanotti, Lind, Asche and Tegtmeyer; Cover, Holland and Blanke; Arnqvist;
Wessler; Pernitzsch, Darfeuille and Sharma). In a hostile acidic milieu, but under
the mucus layer, H. pylori solve the problems of immediate survival but then must
deal with the host’s attempts to remove the invader. Certainly, its very plastic
v
vi Foreword
genome is an advantage coupled with the massive numbers of organisms competing

for life in that niche. But other mechanisms exist whereby H. pylori regulate the
immune response, optimising its nutrition without allowing the mucosa to be totally
disrupted by too vigorous tissue reaction. Subtle degrees of positive and negative
feedback must be at work. A clear explanation of the interrelated virulence factors,
primarily CagA and VacA, and then the several newer surface antigens is given in
subsequent chapters, perhaps explaining how H. pylori disguises itself from the
immune response.
As we move from basic to clinical research in H. pylori, animal models of the
infection become even more important. Chapters on this theme describe the
100-year-long history of various related Helicobacters in animals and more recent
insights into their genomics and taxonomy (Flahon, Haesebrouck and Smet;
Solnick, Eaton and Peek; Müller and Hartung; Oshima, Nakayama and Oshima;
McLean; Nicoll, Saw, Hold and El-Omar). How the animal infections provide
guidance into the realm of animal models remains important. Chapters on the trials
and tribulations of current animal models serve to emphasise that the perfect animal
model is yet to be discovered, unless it is still the human. Nevertheless, selection of
or direct manipulation of small animal genetics serves to tease out important
predictors of epidemiology, colonisation, inflammation and carcinogenesis.
In this context, the clinician will be delighted to study the detailed clinical
chapters on human diseases related to H. pylori infection (Boltin and Niv; Genta
and Lash; Wroblewski and Peek; Sagaert; Koletzko and Megraud). The reasons to
treat H. pylori and features of the host and the bacterium which drive the various
phenotypes of asymptomatic, ulcerated and malignant gastric disorders will give
confidence to the clinician seeing patients with gastric disorders. In addition, the
spectre of rarer non-gastric disorders is exposed, as these will be seen from time to
time and the gastroenterologist or infectious disease specialist will be expected to
assist decisions into their management too. Very relevant here is the controversial
area of immune modulation from H. pylori. It is relevant to many areas, but the
tantalising possibility exists that H. pylori could still play a useful role, as an
immune modulator, like “oil on troubled waters” to decrease the apparent overshoot
which commonly occurs in allergy susceptible individuals. Certainly the negative
association with asthma in New York children and in adults with various inflam-
matory gastrointestinal diseases is worthy of study and is authoritatively reviewed
in immunology-related and paediatric chapters.
Finally, it is great to see that the common, real-world issues of H. pylori
treatment are dealt with by authors who have many years of experience successfully
curing difficult to treat patients (Molina-Infante and Graham; Malfertheiner and
Selgrad; Torres, Correa, Herrero, Piazuelo and Ferrecio; Miftahussurur and
Yamaoka; Vale, Vitor and Oleastro; Raghavan and Quiding-Järbrink). While the
level of antibiotic resistance has been creeping up, most notably to macrolides
and quinolones, logical planning of treatment, with sophisticated microbiological
testing, can give us confidence. Several new antibiotics and/or combinations of
well-known drugs mean there is a bright, H. pylori-free future for patients with the
bacterium.
Foreword vii
Finally, we are shown glimpses of the twenty-first century future where H. pylori
infection might be prevented, or eradicated rather simply, in each continent. This is
already starting and will become easier as the knowledge in this book is dissemi-
nated. Strategies to improve hygiene, vaccinate, treat and even suppress H. pylori
with foods and probiotic supplements are all relevant here and are now being tested.
In summary then, every science graduate will enjoy the intelligent structure of
this book which uses in-depth explanation of the current knowledge to suggest how
gastric colonisation with H. pylori might be a tool to unlock secrets related to the
physiology of the gastrointestinal tract and the gastrointestinal immune system. The
clinician also will be delighted with the extensive discussions of important disease
associations and then, most importantly, how to render the patient H. pylori
negative.
The Helicobacter Research Laboratory Barry Marshall

The Marshall Centre for Infectious Diseases
Research and Training, PaLM M504
Perth WA 6009, Australia
2015
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Contents
Part I Bacteriology and Molecular Biology

1 Helicobacter pylori: Genetics, Recombination, Population
Structure, and Human Migrations . . . . . . . . . . . . . . . . . . . . . . . . . 3
Yoshan Moodley
2 Adaptation of Helicobacter pylori Metabolism to Persistent
Gastric Colonization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Frédéric Fischer and Hilde De Reuse
3 Virulence Mechanisms of Helicobacter pylori: An Overview . . . . . . 57
Judyta Praszkier, Philip Sutton, and Richard L. Ferrero
4 Roles of the cagPAI and CagA on Gastroduodenal Diseases . . . . . . 89
Steffen Backert, Giuseppe Zanotti, Judith Lind, Carmen Isabell Asche,
and Nicole Tegtmeyer
5 Helicobacter pylori Vacuolating Toxin . . . . . . . . . . . . . . . . . . . . . . . 113
Timothy L. Cover, Robin L. Holland, and Steven R. Blanke
6 Roles of the BabA and the SabA Adhesins in Gastroduodenal
Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Anna Arnqvist
7 Emerging Novel Virulence Factors of Helicobacter pylori . . . . . . . . 165
Silja Wessler
8 The Primary Transcriptome and Noncoding RNA Repertoire
of Helicobacter pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Sandy R. Pernitzsch, Fabien Darfeuille, and Cynthia M. Sharma
9 Genome Evolution: Helicobacter pylori as an Extreme Model . . . . . 217
Ichizo Kobayashi
ix
x Contents
10 Non-Helicobacter pylori Helicobacter Infections in Humans

and Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Bram Flahou, Freddy Haesebrouck, and Annemieke Smet
Part II Immune Responses and Host Factors

11 Animal Models of Helicobacter pylori Infection . . . . . . . . . . . . . . . . 273
Jay V. Solnick, Kathryn A. Eaton, and Richard M. Peek Jr.
12 Helicobacter pylori and the Host Immune Response . . . . . . . . . . . . 299
Anne Müller and Mara L. Hartung
13 The Role of Inflammatory Responses in Mouse Gastric
Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Hiroko Oshima, Mizuho Nakayama, and Masanobu Oshima
14 Influence of Host Gene Polymorphisms on Development
of Gastroduodenal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Mairi H. McLean, Ruairidh Nicoll, Cheryl Saw, Georgina L. Hold,
and Emad M. El-Omar
Part III Helicobacter pylori-Associated Diseases

15 Helicobacter pylori and Nonmalignant Diseases . . . . . . . . . . . . . . . . 365
Doron Boltin and Yaron Niv
16 Helicobacter pylori and Gastrointestinal Polyps . . . . . . . . . . . . . . . 387
Robert M. Genta and Richard H. Lash
17 Helicobacter pylori Infection and Gastric Cancer . . . . . . . . . . . . . . 403
Richard M. Peek Jr. and Lydia E. Wroblewski
18 Helicobacter pylori Infection and MALT Lymphoma . . . . . . . . . . . 423
Xavier Sagaert
19 Helicobacter pylori Infection in Children . . . . . . . . . . . . . . . . . . . . . 443
Sibylle Koletzko and Francis Mégraud
Part IV Treatment of Helicobacter pylori

20 Helicobacter pylori Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
Javier Molina-Infante and David Y. Graham
21 Management of H. pylori Infection in Europe . . . . . . . . . . . . . . . . . 491
Peter Malfertheiner and Michael Selgrad
22 Population-Based Strategies for Helicobacter pylori-Associated
Disease Management: Latin American Perspective . . . . . . . . . . . . . 503
Javier Torres, Pelayo Correa, Rolando Herrero, M. Blanca Piazuelo,
and Catterina Ferreccio
Contents xi
23 Population-Based Strategies for Helicobacter pylori-Associated

Disease Management: Asian Perspective . . . . . . . . . . . . . . . . . . . . . 519
Muhammad Miftahussurur and Yoshio Yamaoka
24 Probiotics as an Alternative Therapy for Helicobacter
pylori-Associated Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543
Filipa F. Vale, Jorge M.B. Vı́tor, and Monica Oleastro
25 Vaccination Against Helicobacter pylori Infection . . . . . . . . . . . . . . 575
Sukanya Raghavan and Marianne Quiding-Järbrink
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
Part I
Bacteriology and Molecular Biology
Chapter 1
Helicobacter pylori: Genetics, Recombination,
Population Structure, and Human
Migrations
Yoshan Moodley
Abstract Humans and their stomach bacterium Helicobacter pylori share a coevo-
lutionary relationship that spans at least the last 100,000 years and quite possibly
even longer. Population and evolutionary genetic research has demonstrated a
species-wide phylogeographic structure that faithfully mirrors that of its human
host. However, because of its very high genetic diversity and fast generation time,
H. pylori DNA sequences are often better able to resolve prehistoric human
migrations than human DNA markers. The worldwide genetic diversity of
H. pylori has, thus far, been divided into 7 populations and 14 subpopulations,
most of which are now established markers for recent and prehistoric human
migrations. Key developments such as the inference of hypothetical ancestral
populations and the implementation of coalescent models have provided a clearer
understanding of the population historical role of admixture and recombination and
helped superimpose a chronology onto the human H. pylori association, from which
the timing, direction, and magnitude of migration events can be accurately inferred.
However, there remain large parts of the world from which H. pylori has never been
cultured, and this, along with a move to sequence whole genomes rather than just
housekeeping genes, will form the basis of future evolutionary genetic research.
Keywords Helicobacter pylori • Coevolution • Population • Recombination •

Phylogeography • Coalescence • Migrations
1.1 Introduction
In 1984, Barry Marshall and Robin Warren discovered the spiral-shaped stomach
bacterium Helicobacter pylori, and for demonstrating its role in gastritis and
peptide ulcer formation (Marshall and Warren 1984; Marshall et al. 1985), they
Y. Moodley (*)
Department of Zoology, University of Venda, Private Bag X5050, Thohoyandou 0950,
South Africa
e-mail: [email protected]
© Springer Japan 2016 3

S. Backert, Y. Yamaoka (eds.), Helicobacter pylori Research,
DOI 10.1007/978-4-431-55936-8_1
4 Y. Moodley
were awarded the Nobel Prize for medicine in 2005. Since its discovery, ongoing
research has shown that this bacterium is present in 50 % of human stomachs
worldwide, the vast majority of whom are asymptomatic. H. pylori is usually
contracted early in childhood, and once acquired, bacterial colonization is generally
lifelong. The bacterium can be transmitted within families (e.g., from parents to
children, Kivi et al. 2003; Tindberg et al. 2001) but also between unrelated people
living in close proximity (Delport et al. 2006; Schwarz et al. 2008). Unusually high
mutation (Bj€ orkholm et al. 2001; Morelli et al. 2010) and homologous recombina-
tion rates (Falush et al. 2001; Suerbaum et al. 1998) have resulted in very high DNA
sequence diversity that is much greater than that of other bacteria (Achtman
et al. 1999) and 50-fold greater than its human host (Li and Sadler 1991). Unlike
chromosomal mutations, which can occur at random in any part of the species
distribution, recombination necessarily requires mixed colonization (Falush
et al. 2001; Raymond et al. 2004; Kersulyte et al. 1999; Taylor et al. 1995) and,
by extrapolation, very close human proximity. The end result is a human bacterial
species whose range-wide genetic structure mirrors that of its host. In the following
review, I will outline the stages of development of evolutionary genetic research
that have helped transform our understanding of H. pylori – from a newly discov-
ered human pathogen to our species’ oldest-known and most faithful commensal.
1.2 The Coevolution of Helicobacter pylori and Humans
1.2.1 H. pylori’s Housekeeping Genes
Meaningful population and evolutionary analyses of DNA data must dissect out
demographic processes (genetic drift and gene flow) from selection processes, since
both have the ability to alter allele frequencies. Typically, the selection issue is
circumvented in eukaryotic species by only analyzing genes or regions that are, or
are at least thought to be, selectively neutral – such as mitochondrial DNA (Cann
et al. 1987), nuclear intronic sequences (Matthee et al. 2001), and repeat elements
(Bruford and Wayne 1993). The smaller, more conserved genomes of bacteria,
however, make such approaches untenable. Instead, the genes chosen for analyses
are necessary housekeeping genes, encoding cytoplasmic enzymes. They are dis-
tributed across the H. pylori genome (Achtman et al. 1999) and are unlinked to
genes encoding putative outer membrane or secreted or hypothetical proteins that
might be under selection. They are more likely therefore to represent genome-wide
selectively neutral variation.
The most incisive evolutionary or population genetic knowledge about H. pylori
has, until very recently, been inferred from the DNA sequences of a set of seven
housekeeping gene fragments. These genes, atpA, efp, mutY, ppa, trpC, ureA, and
yphC, were originally the basis of an attempt at multilocus sequence typing (MLST)
of H. pylori (Achtman et al. 1999) along the lines of what was then recently and
1 Helicobacter pylori: Genetics, Recombination, Population Structure. . . 5
successfully carried out in Neisseria meningitidis (Maiden et al. 1998) and Strep-
tococcus pneumoniae (Enright and Spratt 1998). The aim of MLST was to stan-
dardize DNA sequence variability in pathogenic microorganisms for efficient
comparison of data between different laboratories. Typically, fragments of six to
eight housekeeping genes are sequenced, and each unique allele (or haplotype) was
given an arbitrary numerical identifier – a sequence type (ST). The resulting allelic
profiles then allowed researchers to trace the clonal evolutionary relationships of
pathogens, assuming that identical STs were identical by descent – that is, they
evolved only once. While this scheme was successful in identifying clonal com-
plexes in several bacterial species, H. pylori’s high mutation and recombination
rates meant that strains possessing the same ST were the exception rather than the
rule (Falush et al. 2003b; Linz et al. 2007). Contrary to its wide utility in other
bacterial species, the MLST scheme simply did not provide the necessary variation
to adequately quantify population structure in H. pylori (Achtman et al. 1999).
Instead multilocus nucleotide variation of housekeeping gene fragments was used
to deduce evolutionary relationships. This approach proved more fruitful, as
researchers were able to make use of a wealth of existing and constantly developing
analyses of DNA sequence evolution such as Bayesian inference, phylogenetic
reconstruction, and coalescent modeling. However, despite the failure of the MLST
approach to bring order to H. pylori genetic variation, the seven housekeeping gene
sequences are still commonly referred to as the MLST genes.
1.2.2 Geographic Clustering of Housekeeping Gene

Sequences
H. pylori’s unusually high genetic diversity led to initial suggestions that high
recombination and random mating rendered its population structure panmictic
(Go et al. 1996; Salaün et al. 1998; Achtman et al. 1999), which means that it
had no population or clonal structure. However, with increased sample sizes,
geographic patterns of strain relatedness based on sequence similarity slowly
became apparent (Kersulyte et al. 2000; Mukhopadhyay et al. 2000) with some
early studies suggesting co-migration and association of H. pylori and humans for at
least 11,000 years ago (Ghose et al. 2002; Yamaoka et al. 2002). However, it was
not until model-based Bayesian cluster and assignment analyses were applied to
global multilocus DNA sequence data sets of hundreds of strains (Falush
et al. 2003b; Linz et al. 2007) could the extent of worldwide population structure
in this species start being appreciated. Populations appeared to correlate with their
continent of origin which argued strongly against a worldwide panmictic popula-
tion. Originally, Falush and coworkers (2003b) defined four main modern
populations – hpAfrica1, hpAfrica2, hpEastAsia, and hpEurope. Additional
regional substructuring within some of these populations led to the designation of
subpopulations – hpAfrica1 could be broken down into hspWAfrica and hspSAfrica
6 Y. Moodley
Fig. 1.1 Global overview of genetic structure in H. pylori. (a) The global population structure of
H. pylori as determined through Bayesian cluster analysis of the housekeeping gene sequences of
1716 individual strains. Seven populations have been discovered thus far. Locations indicate
geographic substructure within each population. (b) Worldwide geographic structure of seven
H. pylori populations. Obvious geographic discontinuities in the distribution of hpEurope and
hpAfrica1 reflect recent human movements associated with European colonial expansion and the
slave trade, respectively. The structured natural distributions of populations allow them to be used
as markers for human migrations
and hpEastAsia into hspEAsia, hspAmerind, and hspMaori. While numbers of

populations and subpopulations continue to be discovered or refined, the original
method of population definition (e.g., no admixture, using STRUCTURE (Pritchard
et al. 2000) software) is now standard. To date, seven modern H. pylori populations
have been defined globally (Fig. 1.1a). These include the original four plus
hpAsia2, hpNEAfrica (Linz et al. 2007), and hpSahul (Moodley et al. 2009). An
increase in the number of available strains for analyses has also resulted in an
increase in the resolution of subpopulations distributed within almost every modern
population (see Table 1.1 for a summary).
1.2.2.1 Recent Human Population Movements
This strong geographic structuring in the global data reflected major recent and
long-term events in human settlement history, many of which were then intractable
to human DNA. This arose most likely because of the much slower mutation rates
of markers typically used to infer human demographic movements such as mito-
chondrial DNA and Y chromosome (Hudjashov et al. 2007). Therefore, human
Table 1.1 Summary of the modern populations and subpopulations of Helicobacter pylori,
including their geographic range as far as it is known
Population Subpopulation Geographic range
hpAfrica2 hspNorthSan Namibia, Angola
hspSouthSan South Africa
hpAfrica1 hspWAfrica Senegal, the Gambia, Burkina Faso, Morocco, Algeria,
Nigeria, Cameroon, South Africa
hspCAfrica Cameroon, Namibia
hspSAfrica Namibia, Angola, South Africa
hpNEAfrica hspENEAfrica Sudan, Ethiopia, Somalia, Algeria
hspCNEAfrica Sudan, Cameroon, Nigeria, Algeria
hpSahul hspAustralia Australia
hspNGuinea New Guinea
hpAsia2 hspLadakh India (Himalayas)
hspIndia India, Bangladesh, Malaysia, Thailand, the Philippines
hpEurope Recombinant Europe as far east as Southeast Asia
population
hpEastAsia hspEAsia China, India, Malaysia, Singapore, Taiwan, Cambodia, Viet-
nam, Japan, Korea
hspAmerind Canada, the USA, Venezuela, Colombia, Peru
hspMaori Taiwan, the Philippines, Japan, Samoa, New Caledonia,
Wallis and Futuna, New Zealand
DNA lineages tend to sort more slowly, leading to slower population differentiation
and decreased population resolution.
Obvious inconsistencies in the geographic structuring (Fig. 1.1b) were immedi-
ate and strong evidence of recent human population movements, many of which are
difficult to deduce from human DNA. The presence of hpEurope outside its natural
range in the Americas, South Africa, the Philippines, Australia, and the South
Pacific Islands reflects the expansion of Europeans during the colonial era begin-
ning about 500 years ago (Falush et al. 2003b; Linz et al. 2007). It shows clearly
that colonizing or migrating humans carried with them more than just their own
genes and suggests that, in many cases, H. pylori lived in intimate association with
the people they were colonizing. The particularly high frequency of hpEurope in
South America relative to hspAmerind is sometimes taken as evidence of compe-
tition and replacement of less diverse indigenous bacterial populations
(Domı́nguez-Bello et al. 2008; Kersulyte et al. 2010). However, this hypothesis
does not explain why, even in open modern-day communities, hspAmerind has not
been completely replaced by hpEurope. Alternatively, the low frequency of
hspAmerind in South America may have resulted in the near extinction of their
hosts with the introduction of diseases such as smallpox by old-world colonizers.
Other apparent geographic inconsistencies were the presence of the hpAfrica1 in
North and South America, where West African people were forcibly brought to the
Americas during the colonial-era slave trade, and the distribution of hpEastAsia in
8 Y. Moodley
Thailand and Malaysia, carried there by Chinese traders in the last 200 years (Linz
et al. 2007; Breurec et al. 2011).
1.2.2.2 Prehistoric Human Migrations
When subpopulation-level structure was analyzed, geographic patterns also

suggested large-scale human movements since the end of the Last Glacial Maxi-
mum about 18,000 years ago.
Bantu speakers: hpAfrica1
The distribution of hpAfrica1 spans the length of Africa from Morocco and Algeria
in the north to South Africa in the south (Fig. 1.2). Geographic substructuring was
detected across this considerable distribution, and cluster analyses defined three
closely related subpopulations in West and North Africa (hspWAfrica, Falush
et al. 2003b), southern Africa (hspSAfrica) (Falush et al. 2003b), and most recently
in Central Africa (hspCAfrica, Nell et al. 2013). This differentiation was interpreted
as a marker for the expansion of Bantu, a group of languages within the Niger-
Congo language family that was spread across sub-Saharan Africa within the last
5000 years from an original homeland in Nigeria/Cameroon. The expansion was
enabled through Neolithic technological developments that saw the migration of
Iron Age agriculturalists into the summer-rainfall regions of subequatorial Africa
that were climatically suitable for their crops, displacing or absorbing resident
pastoralists and Stone Age hunter-gatherer communities (Diamond and Bellwood
2003). Debate still continues about the possibility of a Western alternative to the
classically accepted eastern migration route into southern Africa via East Africa
(Newman 1995; Pakendorf et al. 2011). The discovery of the subpopulation
hspCAfrica in Cameroon and Angola but not in South Africa and Namibia
(hspSAfrica) supports the existence of a migration route traveling along the west
coast of Africa (Fig. 1.2) but which was halted by the Namib Desert prior to
reaching South Africa. hspSAfrica is presumed to have evolved during Bantu
migrations along the east coast that brought the Nguni speakers to southern Africa.
Additionally, hpAfrica1 is also a marker for migrations elsewhere in Africa. The
presence of hspWAfrica in North Africa may be taken as evidence of gene flow
across the Sahara (Linz et al. 2007), and hspSAfrica in Madagascar suggests
migration across the Mozambique Channel by Bantu speakers during or after
their migration along the east coast of Africa (Linz et al. 2014).
Nilo-Saharan speakers: hpNEAfrica
In the central Sahel and North Africa, hpAfrica1 shares its distribution with the
population hpNEAfrica (Linz et al. 2007), and its frequency decreases eastward to
Fig. 1.2 Subpopulation structure among H. pylori populations in Africa. The discovery of
hspCAfrica in Cameroon and Namibia supports a second wave of Bantu migrations along the
west coast of Africa. Among Nilo-Saharan speakers, the evolution of hspENEAfrica coincides
with the spread of fishing communities across the Sahara during the Holocene humid period
the Nile and the Horn of Africa while that of hpNEAfrica increases. Therefore the
spread of Bantu farmers across the more arid regions of the eastern Sahel appears to
have been limited, possibly due to the incompatibility of tropical crops in more arid
climates and perhaps the presence of another, older society of Nilo-Saharan speak-
ing pastoralists. Thus, H. pylori also provides a convenient marker for the spread of
Nilo-Saharan languages in the form of hpNEAfrica (Fig. 1.2). Here again, geo-
graphic substructure provides clues as to postglacial movements of among Nilo-
Saharans. Nell and coworkers (2013) split hpNEAfrica into hspEastNEA and
hspCentralNEA, roughly along the Nile Valley – Sudan, which straddles the Nile,
contains both subpopulations at high frequency. The presence of hspCentralNEA in
10 Y. Moodley
Cameroon, Nigeria, and Algeria suggests this subpopulation as a marker for human
expansions during the Holocene humid period (6000–9000 years ago) that carried
Nilo-Saharan languages westward from its home in northeast Africa into the
waterlogged Sahara and beyond (Fig. 1.2).
Australians and New Guineans: hpSahul
The peopling of Sahul (the former continent comprising Australia and New Guinea)
is thought to have occurred relatively soon after humans left Africa, following the
southern coastal route passing southern India, the Andaman Archipelago (then
joined to the Asian mainland), and Sundaland (Fig. 1.3). Although human unipa-
rental markers show that indigenous Australians are closely related to New
Guineans, they were also indistinguishable from East and southern Asian lineages
because of incomplete sorting (Hudjashov et al. 2007). On the other hand, H. pylori
from these two populations clustered into a single population (named hpSahul,
Moodley et al. 2009) and could be further separated into discrete subpopulations,
hspAustralia and hspNGuinea (Fig. 1.3), which suggests that they have been
isolated from each other since sea levels rose after the Last Glacial Maximum.
Fig. 1.3 Geographic structuring of subpopulations in the Indo-Pacific is consistent with several
human migration events. hpSahul is a marker for post out-of-Africa migration along the southern
coastal route, hspAmerind evolved prior to the peopling of the Americas, and hspMaori was
carried from Taiwan into the Pacific by Austronesian seafarers
Central and Southeast Asians: hpAsia2
Of all the H. pylori populations to have evolved outside Africa, hpAsia2 is perhaps
the most intriguing. It evolved among the people who either did not follow a
southern coastal migration route, or perhaps those who settled during the early
phases of this migration and later began expanding north into the Western and
Central Asian hinterland. Its distribution was almost certainly more widespread in
the past, prior to the evolution of hpEurope, and hpAsia2 may even have accom-
panied the first modern humans into Europe 40,000 years ago. The present-day
hpAsia2 has largely been replaced by hpEurope in western Eurasia, but it still can
be differentiated into subpopulations hspLadakh from the isolated Himalayan
region of northern India and hspIndia, which is found among Indians, Malays,
and Thais (Tay et al. 2009; Breurec et al. 2011). Its presence in northern Europe
among Finnish and Estonians suggests that it may also be a marker for the spread of
Uralic languages out of Siberia. Whether other relict hpAsia2 bacteria persist in the
mountainous regions of Afghanistan, Pakistan, Tajikistan, Xinjiang, and remote
northern Asia/Siberia is yet to be discovered.
Native Americans, Han Chinese, and the Austronesians: hpEastAsia
Among subpopulations of hpEastAsia, hspAmerind was isolated among diverse

indigenous American populations in North and South America, and its uniqueness
argues against the introduction of the East Asian bacteria into the Americas from
recent Chinese or Japanese immigrants. Instead, these differentiated East Asian
strains provided the first evidence that H. pylori accompanied humans on their
migrations across the Bering Strait and into the Americas (Falush et al. 2003b)
(Fig. 1.3). Similarly, the homogeneous distribution of the subpopulation hspEAsia
across East Asia to Korea and Japan and the fragmentation of the three major
language families (Hmong-Mien, Tai-Kadai, and Austroasiatic) originally spoken
in South China suggested the expansion of the Chinese language (family, Sino-
Tibetan) during the last 3000 years but mainly during the expansion of Zhou
Dynasty (1100–221 BC, Diamond 1998). The detection of the subpopulation
hspMaori, first isolated in the stomachs of New Zealand Maori but then observed
in the Philippines and Samoa (Linz et al. 2007) and again among indigenous in New
Caledonians, Wallis and Futuna Islanders, and Taiwanese (Moodley et al. 2009),
suggested it as a marker for the expansion of Austronesian language and culture
into the Pacific.
1.2.3 Formal Comparisons with Human DNA Data
The strong global and regional geographic structure of population clusters, tanta-
lizingly similar to genetic structure observed in human DNA, suggested an ancient
12 Y. Moodley
association between H. pylori and its host. Startling formal comparisons between
human and H. pylori DNA provided the first quantitative evidence for a long-term
coevolutionary relationship. In a matrix comparison of pairs of equivalent H. pylori
and human populations, up to 73 % (Linz et al. 2007) and 62 % (Moodley
et al. 2012) of the global diversity in housekeeping gene sequences could be
explained by human microsatellite and mitochondrial genomic diversity,
respectively.
The genetic structure of a global human sample emphasizes the overarching
influence of genetic drift on neutral human DNA markers (Prugnolle et al. 2005;
Ramachandran et al. 2005). Under an isolation-by-distance (IBD) model, adjacent
populations would be expected to exchange genes relatively regularly compared to
populations that are situated further apart from each other. Thus, the geographic
distance between a pair of populations is correlated with the neutral genetic
distance (or divergence) between populations. IBD was also found to hold true
for H. pylori populations, further supporting an ancient coevolutionary relationship
(Linz et al. 2007).
Perhaps the most compelling evidence for long-term coevolution was provided
by the first successful human exodus from Africa, around 60,000 years ago. Upon
leaving Africa, migrating human populations were subjected to a series of founder
effects in each of which only a proportion of people left an area that was already
settled. Combined with the added erosive effect of genetic drift in small
populations, these serial founder effects have left an indelible signature of our
evolutionary origin in our DNA, as human genetic diversity decreases significantly
with distance away from East Africa, the likely cradle of modern humans
(Prugnolle et al. 2005; Ramachandran et al. 2005). The same significant relation-
ship between genetic diversity and geographic distance was also demonstrated in
H. pylori, indicating an African origin for both host and bacterium (Linz
et al. 2007). Simulations using a one-dimensional “stepping-stone model” of
migration indicated that anatomically modern humans migrated from Africa around
56,000 years ago (Liu et al. 2006) and H. pylori likewise approximately
58,000 years ago (Linz et al. 2007). Taken together, these data strongly imply
that H. pylori arose in Africa and our forefathers carried this pathogen in their
stomachs on their migrations out of Africa and to the furthest inhabitable corners of
the world.
1.2.4 Recombination and Its Effect on Evolutionary

Inference
Strong geographic structuring of population assignment clusters and clear parallels

with human DNA seemed counterintuitive in a bacterium with particularly high
rates of recombination or admixture (Falush et al. 2001; Suerbaum et al. 1998).
However, unlike mutation, recombination requires the physical exchange of
bacteria between hosts, and this can only happen when host individuals live in close
proximity to each other. Recombination, therefore, is geographically restricted, and
its effect is less pronounced between populations than within populations.
Nevertheless, the intriguing geographic patterns elucidated by Bayesian cluster-
ing precluded any formal evolutionary genetic analyses of phylogenetic relatedness
between and within populations, which populations were basal or derived, the
direction of migrations, population demography, and interpopulation gene flow.
That is because of the fundamental assumption of most analytical software that the
accumulation of genetic diversity via mutation is lineage specific, that is, it passed
on vertically from parent to offspring – identical by descent. Recombination on the
other hand allows horizontal gene transfer between unrelated individuals. The
effect of this is that mutations that appear identical (identical in state) are not
identical by descent. Mutation may also generate homoplasies, but it is generally
not of high enough frequency in eukaryotes to seriously bias evolutionary infer-
ence. However, in highly recombining species like H. pylori, homoplasies gener-
ated by horizontal gene transfer could seriously bias standard evolutionary analyses
such as reconstruction of clonal phylogenies, dating population splits, and estimat-
ing demographic parameters. A standard neighbor-joining phylogenetic analysis of
a global data set (Fig. 1.4a) using the Kimura two-parameter model of nucleotide
substitution highlights consequences of not explicitly accounting for recombination
Fig. 1.4 Determining interpopulation relatedness in a highly recombining genome. (a)

Individual-based neighbor-joining tree showing the influence of homologous recombination.
Branches separating individuals are much longer than those separating populations, resulting in
a genetic continuum in which only the distantly related hpAfrica is distinguishable. (b) Averaging
sequence variation within each population reduced the effect of local recombination making
interpopulation relationships clearer
14 Y. Moodley
in the substitution model. Due to the excess of homoplasies introduced by recom-

bination, branches that lead to the tips of the tree (individual branches) are much
longer than those separating populations from each other.
1.2.4.1 Population Trees
Given that recombination is usually geographically restricted, one way of mini-

mizing its effect is to average out individual genetic diversity by a priori defining
populations (of e.g., Bayesian clusters, ethnicities, countries, etc.) within each of
which recombination may be common but likely to be much less frequent between
populations.
Figure 1.4b shows the global population tree for H. pylori. Here, relationships
between populations are depicted more accurately than in Fig. 1.4a, as individual
variation is denoted in circle diameters and not branch lengths. Of the modern
populations described, six populations were found to be very closely related to each
other, and these included hpAfrica1, hpNEAfrica, hpAsia2, hpEastAsia, hpEurope,
and hpSahul. The population hpAfrica2 is not only very divergent to all other
H. pylori populations but was only isolated in South Africa, among people with
both African and European ancestry.
Population trees reconstructed using the fixation index FST, calculated between
pairs of populations, have been useful in retrieving subtle changes in DNA
sequence variation, which would otherwise appear to be homogeneously distributed
due to admixture. Analyzing a data set comprising ethnicities carrying the highly
diverse hpEurope, Latifi-Navid and coworkers (2010) resolved an FST population
tree that largely corroborated known geopolitical events in history, such as the Arab
conquest of Iran in the seventh century AD and the Ottoman conquest and the
Uzbek-Iranian conflict of the sixteenth century AD. Breurec and colleagues (2011)
used the same method to disentangle several human demographic movements in
Southeast Asia, where a diversity of three H. pylori populations (hpEurope,
hpAsia2, and hpEastAsia) are hosted. They extended the prehistoric range of
hpEurope into Thailand, Malaysia, and Cambodia, which appears to have been
first introduced to Southeast Asia via ancient migration, most likely from the Indian
subcontinent, and not exclusively from historically documented recent (nineteenth-
century) Indian migration as previously thought (Tay et al. 2009). hpAsia2 (sub-
population hspIndia) was also found to have entered Southeast Asia twice – once
most likely with the arrival of Tai-Kadai speakers into what is now Thailand in the
early second millennium AD and again with the previously mentioned nineteenth-
century Indian migrants (Breurec et al. 2011). Similarly, the subpopulation of
hspEAsia was introduced to present-day Vietnam and Cambodia during the Neo-
lithic diaspora out of the Yangtze and Yellow River Valleys around 2000 BC but
appears to have arrived in Malaysia, Singapore, and Thailand during recent Chinese
migration sometime during the last 200 years. Pairwise FST, therefore, has been
successfully implemented in the inference of past human migrations; however, its
disadvantage is that it depends heavily upon the reliability of the assumption of
relatively low recombination rates between, and only minor substructure within,
predefined populations or ethnicities.
1.2.4.2 The Linkage Model: The Concept of Ancestral Populations
Falush and coworkers (2003a) attempted to deal with the admixture that results
from high recombination by using the Bayesian clustering framework of STRUC-
TURE to infer a finite number of “ancestral” or precursor populations, to which all
nucleotides in a strain’s multilocus DNA sequence can then be assigned, thus
inferring the level and putative source of admixture at the level of the individual
strain. This method, known as the linkage model, relies on the admixture linkage
disequilibrium that results when gene flow (migration) occurs between genetically
distinct populations. Assignment of each nucleotide to an ancestral population,
therefore, is based on linkage to neighboring nucleotides. Although the model
depends on the correct inference of the number of ancestral populations, it was
nevertheless a seminal leap forward in our understanding of admixture, population
history, and evolution in this highly recombining bacterial species and resulted in
several high-profile population genetic studies.
Since recombination historically occurred between strains within populations,
signal for more ancient between-population events still persists in the H. pylori
genome, despite very high within-population genetic diversity. The linkage model
has thus far identified six ancestral populations (Breurec et al. 2011; Falush
et al. 2003b), and one of its major contributions to our present-day understanding
of admixture in H. pylori is the discovery that hpEurope is a recombinant hybrid
population made up of roughly equal African and Central Asian ancestry. The six
populations identified were thus ancestral Europe1 (AE1), ancestral Europe2
(AE2), ancestral EastAsia, ancestral Africa1, and ancestral Africa2 (Fig. 1.5).
Given the geographic location of populations that contained “pure” AE1 and AE2
isolates, neither population appears to have originated in Europe. Instead, the
Fig. 1.5 Ancestral populations of H. pylori inferred by the linkage model. The multilocus
genotype of each strain is divided into “chunks” based on the level of admixture linkage
disequilibrium between nucleotides. Modern populations are a produce of admixture between
ancient populations. hpEurope is clearly a recombinant population made up of mainly Ancestral
Europe1 and Ancestral Europe2 nucleotides
16 Y. Moodley
isolates belonging to the modern hpAsia2 population were found to contain the
highest levels of AE1 ancestry, locating the source of this ancestral population to
somewhere in Central or southern Asia; likewise, the highest AE2 proportions were
found among hpNEAfrica isolates, locating its source somewhere in Northeast
Africa.
Ancestral EastAsia originated in East Asia, ancestral Africa1 in West Africa, and
ancestral Africa2 in South Africa. The proportion of ancestry from each of the five
ancestral sources varied among individual isolates and, when grouped by modern
population, formed a continuum, suggesting extensive admixture between ancestral
populations (Fig. 1.5). This allowed for the detection of more subtle population
events occurring in hybrid zones between adjacent ancestral populations. Ladakh in
northern India is one such zone inhabited by people of two major human religious
groups, Muslims and Buddhists, who have coexisted for almost 1000 years but
remained largely isolated due to cultural and religious differences. Human
microsatellites and mtDNA were only marginally informative in detecting differ-
ences between the two groups. However, a linkage model analysis of H. pylori
housekeeping gene sequences found that isolates from Buddhists showed a cline of
admixture from almost pure ancestral EastAsia to almost pure AE1, clear evidence
for the simultaneous introduction of Buddhism and hpEastAsia by human migration
from Tibet into a preexisting Ladakhi (hpAsia2) population. Conversely, isolates
from Muslims were uniform in AE1 ancestry, indicating that Islam was introduced
to Ladakh by a few missionaries rather than by population migration (Wirth
et al. 2004).
The pattern of ancestry in modern European H. pylori is more complex. Modern
humans inhabited Europe between 40,000 and 46,000 years ago (Mellars 2006;
Higham et al. 2011; Benazzi et al. 2011) but retreated southward into refugia in the
Iberian Peninsula (Torroni et al. 1998, 2001) and Ukraine (Malyarchuk et al. 2008).
It is not known whether these original Europeans were infected with H. pylori or
whether it was first introduced to Europe by the expansion of Neolithic farmers
from the Middle East. Although hpEurope comprises roughly 50:50 AE1/AE2
ancestry, the proportion of AE1 is higher in northern Europe, and the proportion
of AE2 ancestry is higher in southern Europe (Falush et al. 2003b), suggesting more
than one introduction of H. pylori. Using principal component analysis (PCA), Linz
and colleagues (2007) demonstrated multivariate clines of potential migrations into
Europe. These included evidence for the westward spread of domesticated crops
from the Fertile Crescent into Europe by Neolithic farmers (AE2), migration of
Uralic language-speaking peoples from Siberia into Scandinavia (AE1), and a
population expansion from the steppes between the Volga and Don Rivers, associ-
ated with the domestication of the horse.
It is extremely unlikely that the hybridization event that resulted in hpEurope
actually occurred in Europe. This is because almost all Europeans screened to date
contain hpEurope, with very few exceptions. Humans migrating out of Northeast
Africa carrying hpNEAfrica would also have to have passed through regions
inhabited by hpAsia2 carriers prior to their arrival in Europe. Iran, situated at a
crossroad between Europe, Asia, and Africa and containing a large proportion of
the Fertile Crescent in which Neolithic agriculture was developed, was hypothe-
sized as a potential source for one of Europe’s ancestral components (Latifi-Navid
et al. 2010), but the hpEurope strains carried by Iranians were comprised of equal
proportions of AE1 and AE2, similar to other countries in the Middle East (Linz
et al. 2007).
hpWAfrica is indigenous to West Africa. However, in an analysis of strains from
the Gambia, Senegal, and Burkina Faso, Secka and coworkers (2014) found little to
no evidence of AE1 nucleotides in their sample, suggesting that the Nilo-Saharan
expansion across the Sahara (6000–9000 years ago), rather than from repeated
recent contact with Europeans and North Africans.
1.2.5 Enter Coalescence
Inference of population demographic parameters such as migration rates and

effective population sizes and how these change over evolutionary time is made
possible by coalescent theory (Kingman 1982). While coalescent models have
undergone valuable development (Kuhner et al. 1998; Beerli and Felsenstein
1999; Drummond et al. 2005), their usefulness in analyzing highly recombining
H. pylori sequences could be severely hampered by high homoplasy. One solution
is to implement Hudson’s (1985) four-gamete test, to detect recombining regions
between pairs of segregating sites, which can then be removed prior to coalescent
modeling. The four-gamete test relies on a simple assumption that homoplasies are
generated by recombination and not by repeat mutation. Even though H. pylori is
highly recombining, a small proportion of the recombining regions identified by the
four-gamete test would likely be due to repeat mutation. The test is therefore
conservative, discarding as much as half the DNA sequence data. Nevertheless,
housekeeping gene sequences thus stripped of recombining regions and subjected
to coalescent analyses using a two-population “isolation-with-migration” model
(Hey and Nielsen 2004) have allowed estimates of bidirectional migration rates
between population pairs that are consistent with known human demographic
events (Moodley et al. 2009, 2012). It was thus established that gene flow (migra-
tion) occurred from East Asia into the Americas, but not the other way around, and
notable back migration was detected between Africa and the Middle East and
between Central and East Asia, highlighting the interface between these regions
as “highways” for human and bacterial gene flow. Newer composite likelihood
approaches (e.g., Jaatha software, Naduvilezhath et al. 2011) now provide a more
flexible user-defined “isolation-with-migration” coalescent framework in which
homologous recombination and changes in effective population size can be directly
modeled. This method uses the joint allele frequency spectrum between two
populations as a summary statistic to compute the composite likelihood of posterior
demographic parameters.
The coalescent has also been used to model recombination directly from DNA
sequence data to produce a genealogy that describes the clonal relationships among
18 Y. Moodley
individual lineages. The ClonalFrame software (Didelot and Falush 2007) uses a
probabilistic Bayesian framework where the number, size, and location of recom-
bination events are model parameters whose posterior distributions are estimated by
Markov chain Monte Carlo (MCMC) simulation. The only drawback of this method
is that it does not explicitly model the source of imported stretches of DNA
sequences but instead assumes that novel mutations are introduced at a uniform
rate. This would tend to underestimate the level of recombination relative to
mutation. Nevertheless, the method has been used to successfully infer robust
near-clonal genealogies at both inter- and intrapopulation scales in a number of
studies (Moodley et al. 2009, 2012; Nell et al. 2013), adding greatly to our current
knowledge of the evolutionary history of H. pylori.
Coalescent models work backward in time, estimating pairwise divergence in
population-level models and the height of nodes at which individual lineages
coalesce in a genealogy. While these two measures are not strictly the same, the
first estimates the divergence between two populations, which would normally
predate the time calculated from tree or node coalescence, since any given popu-
lation may contain lineages that have not fully sorted. This restriction can be
circumvented by defining biologically meaningful populations (e.g., those defined
by STRUCTURE analysis), rather than more arbitrarily sorting strains into country
or ethnic populations. The implementation of coalescent models in analyzing
housekeeping gene sequences in H. pylori has greatly improved our understanding
of the coevolution of this stomach bacterium and its hosts, shedding light on
significant human migrations.
1.2.5.1 The Global H. pylori Phylogeny
Until the implementation of coalescent models, the phylogenetic relationships

among H. pylori populations were only vaguely understood, the most striking and
obvious observation from both population and individual neighbor-joining phylog-
enies being the distinctiveness of hpAfrica2 from all other populations (Falush
et al. 2003b; Linz et al. 2007). However, genealogical analyses using ClonalFrame
produced the first global clonal phylogeny for H. pylori, resolving the interrelation-
ships between all known populations (Moodley et al. 2009). This phylogeny was
calibrated by attributing certain branches on the tree to known human population
splitting events and then smoothing the rates of evolution so that times could be
readily compared across branches and the timing of debated human migrations
could be estimated. The global phylogeny was refined to include a greater diversity
of African strains and an out-group taxon, H. cetorum, isolated from cetaceans and
H. pylori’s closest known relative (Moodley et al. 2012).
The global phylogeny (Fig. 1.6) showed clearly that H. pylori is structured into
two superlineages: one containing hpAfrica2 and its close relative H. acinonychis
and the other containing all other populations in a single monophyletic clade. This
second superlineage is further split into a clade containing the two sister African
populations hpAfrica1 and hpNEAfrica and a clade containing all non-African
Fig. 1.6 The global H. pylori phylogeny obtained through simultaneous coalescent modeling of
mutation and recombination. Housekeeping gene sequence diversity is structured into two clonal
superlineages that coalesce to approximately 100,000 years. hpSahul is phylogenetically interme-
diate between African and Asian bacteria, and the San hunter-gatherers of southern Africa are the
original hosts of hpAfrica2. Lineages are color-coded according to subpopulations in Figs. 1.3 and
1.4
H. pylori. Within this second clade, hpSahul is basal-most and hpAsia2 and
hpEastAsia are sister populations. Subpopulation structure was also resolved within
hpEastAsia, showing that hspAmerind is more distantly related to hspEAsia and
hspMaori, which form a sister subclade.
1.2.5.2 Age of the H. pylori Human Association
If H. pylori accompanied humans out of Africa, then the association between the
two species must date back prior to that event, seeing as the bacterium would first
have to have been acquired by humans and then proliferated to infect the majority
of the population by the time the event took place 60,000 years ago. A rooted, fully
resolved, and calibrated global clonal phylogeny allowed the estimation of a lower
limit for this association. This was the time at which the two H. pylori superlineages
coalesced to a single common ancestor, and it was found to have occurred approx-
imately 100,000 years ago (range: 88,000–116,000 years, Fig. 1.6, Moodley
et al. 2012). However, because of lineage sorting and population bottlenecks,
older lineages that might extend this time frame may have gone extinct. It is
therefore possible that the human H. pylori association could be considerably
older than this minimum estimate. That Helicobacter species were not successfully
identified in gastric biopsies from chimpanzees does not fully reject the hypothesis
20 Y. Moodley
of a continuous hominoid-Helicobacter association spanning several million years,

because of potential technical difficulties in sampling and culturing bacteria and
host population structuring. More research in this area, sampling from different
populations of bonobos and gorillas, may help push the length of our association
further back in time.
1.2.5.3 Sahul Was Colonized Only Once
The dates and numbers of migrations to Sahul are controversial. Using a unique
collection of strains from the stomachs of aboriginal Australians and New Guinean
Highlanders, Moodley and coworkers (2009) showed that each ethnic group formed
a unique subclade (or subpopulation) of its own, with no strain sharing. These
subclades formed a single monophyletic group, whose coalescence dated back to
31,000–37,000 years ago (Fig. 1.6). This timing is consistent with the majority of
the Pleistocene human archaeological sites but predates the earliest (Pope and
Terrell 2007). More importantly, the monophyly of hpSahul evidences a single
introduction of H. pylori into Sahul, and once it had split into hspAustralia and
hspNGuinea, demographic analyses showed little to no migration between the
subpopulations. The phylogenetic position of hpSahul, directly intermediate
between African and Asian populations, firmly identified it as the marker for the
first successful human population migrations outside Africa, along the southern
coastal into Southeast Asia and eventually to Sahul. The H. pylori in other human
population relicts of this migration, such as the Andaman Islanders, the Semang of
Malaysia, and the Mani of Thailand, may therefore provide closer genetic links
between hpSahul and African populations.
1.2.5.4 The Austronesian Expansion
The expansion of the Austronesian language family was the greatest prehistoric
language expansion the world has ever seen, spreading the Malayo-Polynesian
subfamily from Madagascar in the Indian Ocean to the Easter Islands in the Pacific.
With the exception of Madagascar (Linz et al. 2014), the H. pylori subpopulation
hspMaori has been observed in the stomachs of Polynesians, Filipinos (Linz
et al. 2007), Melanesians, and indigenous Taiwanese (Moodley et al. 2009). A
clonal phylogeny for this subpopulation placed Taiwanese strains basal to all
others, with sequentially derived branches marking migrations to the Philippines,
Melanesia, and Polynesia. Coalescent estimates of bidirectional migration rates
between the East Asian mainland and Taiwan, and between Taiwan and the Pacific,
show that the overwhelming majority of migration took place in the outward
direction. Together with significantly higher genetic diversity in Taiwanese strains,
these results established Taiwan as the source for the co-expansion of Austronesian
and hspMaori.
1.2.5.5 San Hunter-Gatherers Are the Original Hosts of hpAfrica2
hpAfrica2 is the most distant and curiously distributed of all H. pylori populations.
Its location at the southern end of Africa led to speculation that it might be
associated with the ancient San hunter-gatherers of southern Africa. Its high
frequency among existing San ethnicities in southern Africa strengthened this
hypothesis. Furthermore, highly similar topologies between rooted global
H. pylori human mtDNA phylogenies show that hpAfrica2 and the San are basal
in the evolutionary histories of their respective species. However, hpAfrica2 is also
frequently isolated among Bantu speakers in southern Africa (Falush et al. 2003b;
Linz et al. 2007; Moodley et al. 2009; Duncan et al. 2013), and it was not obvious
which of the two groups was hpAfrica2’s natural host. However, a clonal phylogeny
of only hpAfrica2 strains revealed that San isolates were both more diverse and
basal to strains isolated from Bantus (Fig. 1.6). Reciprocally, hpAfrica1 strains
isolated in San were more derived in an hpAfrica1 phylogeny than Bantu-isolated
strains. This placed the San as the unequivocal original hosts of the divergent
hpAfrica2 (Moodley et al. 2012). The inclusion of strains from three San ethnicities
also resolved the two hitherto unknown subpopulations hspNorthSan and
hspSouthSan. The northern ethnicities Khwe and !Xun, speaking Central and
Northern Khoisan languages, respectively, were basal to the southern Khomani
who spoke southern Khoisan, and migration between the two groups since their
split 32,000–47,000 years ago has been predominantly from north to south.
Moodley and colleagues (2012) also refined the timing of the split between
hpAfrica2 and H. acinonychis to 43,000–56,000 years ago (Fig. 1.6), which sug-
gests that the latter species was contracted by large felines from a San host.
1.2.5.6 A Second More Recent Out-of-Africa Migration
hpEurope is a hybrid population comprising nucleotides from two ancestral

populations, AE1 and AE2, that evolved in Central or southern Asia and Northeast
Africa, respectively. Consequently, and unlike its human hosts, hpEurope is highly
diverse, more diverse than most populations in Africa, except southern Africa
where the presence of the hpAfrica2 superlineages markedly inflates levels of
genetic variation. This high diversity is inconsistent with a single human
H. pylori migration out of Africa 60,000 years ago. This is because one of
hpEurope’s ancestral components AE2, exemplified in the modern-day
hpNEAfrica, had not yet evolved by then. Coalescent simulations indicate that
hpNEAfrica splits from hpAfrica1 sometime between 36,000 and 52,000 years ago,
predating the 60,000-year event (Fig. 1.6). AE2 must therefore have left Africa
sometime after it evolved in order to come into contact and hybridize with AE2 in
western Asia, therefore invoking a second migration of humans out of Africa.
Archaeological evidence that Middle Paleolithic technology was predominant in
southern Asia, but more recently developed Upper Paleolithic or “Mode 4” stone
22 Y. Moodley
tools occurred exclusively in North Africa, the Levant, and Europe (Mellars 2006;
Foley and Lahr 2003), corroborates the idea of a second African exodus. However,
evidence from uniparentally inherited human DNA markers is still lacking (Macau-
lay et al. 2005; Oppenheimer 2012).
1.2.5.7 Pygmy Hunter-Gatherers Contracted H. pylori Recently from

Neolithic Bantus
Nell and coworkers investigated the population demographics of H. pylori trans-

mission between hunter-gatherer Baka pygmies and their neighboring Neolithic
communities in Cameroon (Nell et al. 2013). They used a composite likelihood
approach that included population expansion and Aboriginal populations
andrecombination; they found high gene flow between H. pylori populations in
Baka and agriculturalists and that the Baka bacterial population was constant rather
than expanding. Baka and agriculturalist strains coalesced to a population split
between 856 and 3981 years ago, consistent with anthropological evidence that
Baka came into recent secondary contact with Neolithic communities
3000–5000 years ago (Schoenbrun 2001). Furthermore, the high number of
unrelated strains among the Baka community and their derived positions on a
ClonalFrame phylogeny indicated that Baka contracted H. pylori from their agri-
culturalist neighbors. A susceptible-infectious (SI) epidemiological model showed
that population-wide infection decreased with population census size, thus
explaining the particularly low rate of infection (20.8 %) in the small (~40,000
individuals) Baka population. Given these analyses, it is most likely that Baka were
H. pylori free prior to contact with Neolithic Bantu agriculturalists, from whom
they contracted the bacterium during the last 4000 years.
1.2.6 Outlook: Genomics, Aboriginal Populations,

and Ancient DNA
While the population, phylogeographic and evolutionary, research outlined in this

review has helped revolutionized our understanding of the long and intricate
coevolutionary relationship between humans and H. pylori, there are many ques-
tions still open to future research.
Thus far, almost all evolutionary and population genetic information on
H. pylori has been inferred from the original MLST set of seven housekeeping
gene sequences. With the costs of genome sequencing technologies decreasing
yearly and more H. pylori genomes becoming available, whole genome analysis
is likely to lead to greater resolution of bacterial and host demography. Already,
over 72 H. pylori genomes, mostly sequenced with 454 technology, are available on
public databases, although some populations such as hpNEAfrica and hpSahul are
not yet represented at the genome level. Nevertheless, future evolutionary analyses
could potentially make use of a wealth of genetic information from 1.6 to 1.7
megabase genomes containing approximately 1500 open reading frames. However,
genome analysis has its own drawback. The assumption of selective neutrality, vital
for evolutionary analyses of housekeeping genes, would no longer apply to whole
genomes where several hundred loci are likely to be under varying degrees of
selection. Loci under selection or selective sweeps would have to be identified
beforehand and excluded from analyses. Alternatively, population demography
could be modeled via coalescent simulations that compute a large number of
possible scenarios, to which the observed genomic data is compared, identifying
regions that do not fit the best neutral model. A benefit of methods is the quanti-
fication of regions of the H. pylori genome that are under selection. An understand-
ing of these would not only help resolve the history of co-migration but could also
identify the loci responsible for medical symptoms and why these have become
prevalent in some populations and not others.
Greater sampling coverage among the world’s aboriginal people may help
elucidate human demographic events in hitherto unstudied ethnicities such as
among click-speaking East African Pygmies, the Hadza and Sandawe, among
Nilo-Saharans and Bantus along the east coast of Africa, and among the myriad
ethnicities in Siberia, islands of Southeast Asia, Oceania, and the Americas.
Ancient DNA may be another avenue of research worth investigating. Increas-
ingly reliable capture methods have already made it possible to retrieve whole
human genomes from mummified ancient human remains (Keller et al. 2012;
Skoglund et al. 2012; Sánchez-Ouinto et al. 2012). If remains include the gastro-
intestinal tract, it may also be possible to capture ancient H. pylori genomes. Since
most recent research in this field has been conducted on remains discovered in
Europe, obtaining H. pylori from these remains may provide crucial insights as to
whether the first hunter-gatherer humans in Europe were infected. Solving this
puzzle may also help localize the hybridization event that resulted in the recombi-
nant hpEurope population.
References
Achtman M, Azuma T, Berg DE et al (1999) Recombination and clonal groupings within

Helicobacter pylori from different geographical regions. Mol Microbiol 32(3):459–470.
doi:10.1046/j.1365-2958.1999.01382.x
Beerli P, Felsenstein J (1999) Maximum-likelihood estimation of migration rates and effective
population numbers in two populations using a coalescent approach. Genetics 152(2):763–773
Benazzi S, Douka K, Fornai C et al (2011) Early dispersal of modern humans in Europe and
implications for Neanderthal behaviour. Nature 479(7374):525–529. doi:10.1038/
Nature10617
Bj€
orkholm B, Sj€olund M, Falk PG et al (2001) Mutation frequency and biological cost of antibiotic
resistance in Helicobacter pylori. Proc Natl Acad Sci U S A 98(25):14607–14612. doi:10.
1073/pnas.241517298
24 Y. Moodley
Breurec S, Guillard B, Hem S et al (2011) Evolutionary history of Helicobacter pylori sequences

reflect past human migrations in southeast Asia. PLoS One 6(7):e22058. doi:10.1371/journal.
pone.0022058
Bruford MW, Wayne RK (1993) Microsatellites and their application to population genetic
studies. Curr Opin Genet Dev 3:939–943. doi:10.1016/0959-437X(93)90017-J
Cann R, Stoneking M, Wilson A (1987) Mitochondrial DNA and human evolution. Nature 325
(6099):31–36. doi:10.1038/325031a0
Delport W, Cunningham M, Olivier B et al (2006) A population genetics pedigree perspective on
the transmission of Helicobacter pylori. Genetics 174(4):2107–2118. doi:10.1534/genetics.
106.057703
Diamond J (1998) Guns, germs, and steel: the fates of human societies. W.W. Norton and
Company, New York
Diamond J, Bellwood P (2003) Farmers and their languages: the first expansions. Science 300
(5619):597–603. doi:10.1126/science.1078208
Didelot X, Falush D (2007) Inference of bacterial microevolution using multilocus sequence data.
Genetics 175(3):1251–1266. doi:10.1534/genetics.106.063305
Domı́nguez-Bello MG, Pérez ME, Bortolini MC et al (2008) Amerindian Helicobacter pylori
strains go extinct, as European strains expand their host range. PLoS One 3(10):E3307. doi:10.
1371/Journal.Pone.0003307
Drummond AJ, Rambaut A, Shapiro B et al (2005) Bayesian coalescent inference of past
population dynamics from molecular sequences. Mol Biol Evol 22(5):1185–1192. doi:10.
1093/molbev/msi103
Duncan SS, Bertoli MT, Kersulyte D et al (2013) Genome sequences of three hpAfrica2 strains of
Helicobacter pylori. Genome Ann 1(5):e00729-13. doi:10.1128/genomeA.00729-13
Enright MC, Spratt BG (1998) A multilocus sequence typing scheme for Streptococcus
pneumoniae: identification of clones associated with serious invasive disease. Microbiology
144:3049–3060. doi:10.1099/00221287-144-11-3049
Falush D, Kraft C, Taylor NS et al (2001) Recombination and mutation during long-term gastric
colonization by Helicobacter pylori: estimates of clock rates, recombination size, and minimal
age. Proc Natl Acad Sci USA 98(26):15056–15061. doi:10.1073ypnas.251396098
Falush D, Stephens M, Pritchard JK (2003a) Inference of population structure using multilocus
genotype data: linked loci and correlated allele frequencies. Genetics 164(4):1567–1587
Falush D, Wirth T, Linz B et al (2003b) Traces of human migrations in Helicobacter pylori
populations. Science 299(5612):1582–1585. doi:10.1126/science.1080857
Foley R, Lahr MM (2003) On stony ground: lithic technology, human evolution, and the emer-
gence of culture. Evol Anthropol 12(3):109–122. doi:10.1002/evan.10108
Ghose C, Perez-Perez GI, Dominguez-Bello M-G et al (2002) East asian genotypes of
Helicobacter pylori strains in Amerindians provide evidence for its ancient human carriage.
Proc Natl Acad Sci U S A 99(23):15107–15111. doi:10.1073/pnas.242574599
Go MF, Kapur V, Graham DY et al (1996) Population genetic analysis of Helicobacter pylori by
multilocus enzyme electrophoresis: extensive allelic diversity and recombinational population
structure. J Bacteriol 178(13):3934–3938
Hey J, Nielsen R (2004) Multilocus methods for estimating population sizes, migration rates and
divergence time, with applications to the divergence of Drosophila pseudoobscura and
D. persimilis. Genetics 167(2):747–760. doi:10.1534/genetics.103.024182
Higham T, Compton T, Stringer C et al (2011) The earliest evidence for anatomically modern
humans in northwestern Europe. Nature 479(7374):521–524. doi:10.1038/Nature10484
Hudjashov G, Kivisild T, Underhill PA et al (2007) Revealing the prehistoric settlement of
Australia by Y chromosome and mtDNA analysis. Proc Natl Acad Sci U S A 104
(21):8726–8730. doi:10.1073/pnas.0702928104
Hudson RR (1985) The sampling distribution of linkage disequilibrium under an infinite allele
model without selection. Genetics 109(3):611–631
Keller A, Graefen A, Ball M et al (2012) New insights into the Tyrolean Iceman’s origin and
phenotype as inferred by whole-genome sequencing. Nat Commun 3:698. doi:10.1038/
Ncomms1701
Kersulyte D, Chalkauskas H, Berg DE (1999) Emergence of recombinant strains of Helicobacter
pylori during human infection. Mol Microbiol 31(1):31–43. doi:10.1046/j.1365-2958.1999.
01140.x
Kersulyte D, Mukhopadhyay AK, Velapatino B et al (2000) Differences in genotypes of
Helicobacter pylori from different human populations. J Bacteriol 182(11):3210–3218.
doi:10.1128/Jb.182.11.3210-3218.2000
Kersulyte D, Kalia A, Gilman RH et al (2010) Helicobacter pylori from Peruvian Amerindians:
traces of human migrations in strains from remote Amazon, and genome sequence of an
Amerind Strain. PLoS One 5(11):e15076. doi:10.1371/journal.pone.0015076
Kingman JFC (1982) The coalescent. Stoch Proc Appl 13(3):235–248. doi:10.1016/0304-4149
(82)90011-4
Kivi M, Tindberg Y, S€orberg M et al (2003) Concordance of Helicobacter pylori strains within
families. J Clin Microbiol 41(12):5604–5608. doi:10.1128/jcm.41.12.5604-5608.2003
Kuhner MK, Yamato J, Felsenstein J (1998) Maximum likelihood estimation of population growth
rates based on the coalescent. Genetics 149(1):429–434
Latifi-Navid S, Ghorashi SA, Siavoshi F et al (2010) Ethnic and geographic differentiation of
Helicobacter pylori within Iran. PLoS One 5(3):E9645. doi:10.1371/Journal.Pone.0009645
Li WH, Sadler LA (1991) Low nucleotide diversity in man. Genetics 129(2):513–523
Linz B, Balloux F, Moodley Y et al (2007) An African origin for the intimate association between
humans and Helicobacter pylori. Nature 445(7130):915–918. doi:10.1038/nature05562
Linz B, Vololonantenainab CRR, Seck A et al (2014) Population genetic structure and isolation by
distance of Helicobacter pylori in Senegal and Madagascar. PLoS One 9(1):e87355. doi:10.
1371/journal.pone.0087355
Liu H, Prugnolle F, Manica A et al (2006) A geographically explicit genetic model of worldwide
human-settlement history. Am Hum Genet 79(2):230–237. doi:10.1086/505436
Macaulay V, Hill C, Achilli A et al (2005) Single, rapid coastal settlement of Asia revealed by
analysis of complete mitochondrial genomes. Science 308(5724):1034–1036. doi:10.1126/
science.1109792
Maiden MCJ, Bygraves JA, Feil E et al (1998) Multilocus sequence typing: a portable approach to
the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad
Sci U S A 95(6):3140–3145. doi:10.1073/pnas.95.6.3140
Malyarchuk B, Grzybowski T, Derenko M et al (2008) Mitochondrial DNA phylogeny in eastern
and western Slavs. Mol Biol Evol 25(8):1651–1658. doi:10.1093/molbev/msn114
Marshall BJ, Warren JR (1984) Unidentified curved bacilli in the stomach of patients with gastritis
and peptic ulceration. Lancet 1(8390):1311–1315. doi:10.1016/S0140-6736(84)91816-6
Marshall BJ, Armstrong JA, McGechie DB et al (1985) Attempt to fulfil Koch’s postulates for
pyloric Campylobacter. Med J Aust 142(8):436–439
Matthee CA, Burzlaff JD, Taylor JF et al (2001) Mining the mammalian genome for artiodactyl
systematics. Syst Biol 50(3):367–390. doi:10.1080/106351501300317987
Mellars P (2006) Going east: new genetic and archaeological perspectives on the modern human
colonization of Eurasia. Science 313(5788):796–800. doi:10.1126/science.1128402
Moodley Y, Linz B, Yamaoka Y et al (2009) The peopling of the Pacific from a bacterial
perspective. Science 323(5913):527–530. doi:10.1126/science.1166083
Moodley Y, Linz B, Bond RP et al (2012) Age of the association between Helicobacter pylori and
man. PLoS Pathog 8(5):e1002693. doi:10.1371/journal.ppat.1002693
Morelli G, Didelot X, Kusecek B et al (2010) Microevolution of Helicobacter pylori during
prolonged infection of single hosts and within families. PLoS Genet 6(7):e1001036. doi:10.
1371/journal.pgen.1001036
26 Y. Moodley
Mukhopadhyay AK, Kersulyte D, Jeong JY et al (2000) Distinctiveness of genotypes of

Helicobacter pylori in Calcutta, India. J Bacteriol 182(11):3219–3227. doi:10.1128/Jb.182.
11.3219-3227.2000
Naduvilezhath L, Rose LE, Metzler D (2011) Jaatha: a fast composite-likelihood approach to
estimate demographic parameters. Mol Ecol 20(13):2709–2723. doi:10.1111/j.1365-294X.
2011.05131.x
Nell S, Eibach D, Montano V et al (2013) Recent acquisition of Helicobacter pylori by Baka
Pygmies. PLoS Genet 9(9):e1003775. doi:10.1371/journal.pgen.1003775
Newman JL (1995) The peopling of Africa: a geographic interpretation. Yale University Press,
New Haven
Oppenheimer S (2012) Out-of-Africa, the peopling of continents and islands: tracing uniparental
gene trees across the map. Phil Trans R Soc B 367(1590):770–784. doi:10.1098/rstb.2011.0306
Pakendorf B, Bostoen K, de Filippo C (2011) Molecular perspectives on the Bantu expansion: a
synthesis. Lang Dyn Chang 1:50–88. doi:10.1163/221058211X570349
Pope KO, Terrell JE (2007) Environmental setting of human migrations in the circum-Pacific
region. J Biogeogr 35:1–21. doi:10.1111/j.1365-2699.2007.01797.x
Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus
genotype data. Genetics 155(2):945–959
Prugnolle F, Manica A, Balloux F (2005) Geography predicts neutral genetic diversity of human
populations. Curr Biol 15(5):R159–R160. doi:10.1016/j.cub.2005.02.038
Ramachandran S, Deshpande O, Roseman CC et al (2005) Support from the relationship of genetic
and geographic distance in human populations for a serial founder effect originating in Africa.
Proc Natl Acad Sci U S A 102(44):15942–15947. doi:10.1073/pnas.0507611102
Raymond J, Thiberge JM, Chevalier C et al (2004) Genetic and transmission Analysis of
Helicobacter pylori strains within a family. Emerg Infect Dis 10(10):1816–1821. doi:10.
1128/JB.01696-06
Salaün L, Audibert C, Le Lay G et al (1998) Panmictic structure of Helicobacter pylori demon-
strated by the comparative study of six genetic markers. FEMS Microbiol Lett 161
(2):231–239. doi:10.1016/S0378-1097(98)00080-9
Sánchez-Ouinto F, Schroeder H, Ramirez O et al (2012) Genomic affinities of two 7,000-year-old
Iberian hunter-gatherers. Curr Biol 22(16):1494–1499. doi:10.1016/j.cub.2012.06.005
Schoenbrun D (2001) Representing the Bantu expansions: what’s at stake? Int J Afr Hist Stud 34
(1):1–4. doi:10.2307/3097284
Schwarz S, Morelli G, Kusecek B et al (2008) Horizontal versus familial transmission of
Helicobacter pylori. PLoS Pathog 4(10):e1000180. doi:10.1371/journal.ppat.1000180
Secka O, Moodley Y, Antonio M et al (2014) Population genetic analyses of Helicobacter pylori
isolates from Gambian adults and children. PLoS ONE 9:e109466
Skoglund P, Malmstr€om H, Raghavan M et al (2012) Origins and genetic legacy of neolithic
farmers and hunter-gatherers in Europe. Science 336(6080):466–469. doi:10.1126/science.
1216304
Suerbaum S, Smith JM, Bapumia K et al (1998) Free recombination within Helicobacter pylori.
Proc Natl Acad Sci U S A 95(21):12619–12624. doi:10.1073/pnas.95.21.12619
Tay CY, Mitchell H, Dong QJ et al (2009) Population structure of Helicobacter pylori among
ethnic groups in Malaysia: recent acquisition of the bacterium by the Malay population. BMC
Microbiol 9:126. doi:10.1186/1471-2180-9-126
Taylor NS, Fox JG, Akopyants NS et al (1995) Long-term colonization with single and multiple
strains of Helicobacter pylori assessed by DNA fingerprinting. J Clin Microbiol 33(4):918–923
Tindberg Y, Bengtsson C, Granath F et al (2001) Helicobacter pylori infection in Swedish school
children: lack of evidence of child-to-child transmission outside the family. Gastroenterology
121(2):310–316. doi:10.1053/gast.2001.26282
Torroni A, Bandelt HJ, D’Urbano L et al (1998) mtDNA analysis reveals a major late paleolithic
population expansion from southwestern to northeastern Europe. Am Hum Genet 62
(5):1137–1152. doi:10.1086/301822
Torroni A, Bandelt HJ, Macaulay V et al (2001) A signal, from human mtDNA, of postglacial
recolonization in Europe. Am Hum Genet 69(4):844–852. doi:10.1086/323485
Wirth T, Wang X, Linz B et al (2004) Distinguishing human ethnic groups by means of sequences
from Helicobacter pylori: lessons from Ladakh. Proc Natl Acad Sci USA 101(14):4746–4751.
doi:10.1073/pnas.0306629101
Yamaoka Y, Orito E, Mizokami M et al (2002) Helicobacter pylori in North and South America
before Columbus. FEBS Lett 517(1–3):180–184. doi:10.1016/S0014-5793(02)02617-0
Chapter 2
Adaptation of Helicobacter pylori Metabolism
to Persistent Gastric Colonization
Frédéric Fischer and Hilde De Reuse
Abstract Helicobacter pylori is an amazingly successful pathogen that persis-

tently colonizes the hostile gastric niche. Persistence of H. pylori colonization is
an important parameter in the clinical outcome of the infection. H. pylori has
evolved original mechanisms to colonize and persist within the stomach in spite
of the harsh acidic conditions encountered in this environment. Genetic, physio-
logical, and biochemical analyses of H. pylori have revealed peculiar properties of
its metabolism, some of which are central in the adaptation to the gastric environ-
ment. The abundant enzyme urease is essential for H. pylori resistance to acidity
through urea breakdown and production of buffering ammonia. Acid adaptation and
management of the potentially toxic amounts of ammonia are closely associated
through original transport and metabolic pathways, some of which involve protein
complexes. Several metabolic enzymes have been shown to act in addition as
genuine virulence factors in particular as immune modulators. This is illustrated
by gamma-glutamyl transpeptidase, asparaginase, and arginase. Finally, given the
central role of the nickel-dependent enzyme urease, the uptake and intracellular
trafficking pathways of this metal essential for H. pylori colonization will be
presented. In conclusion, we propose that the constrains of the small H. pylori
genome and a very specialized niche has resulted in a close association and in
overlapping networks between mechanisms of persistence, acid adaptation and
metabolic pathways.
Keywords Helicobacter pylori • Acid adaptation • Ammonia metabolism •

Urease • Hydrogenase • Nickel • Metallochaperone • Gamma-glutamyl
transpeptidase • Asparaginase • Arginase • Gastric colonization
F. Fischer • H. De Reuse (*)

Department of Microbiology, Unité Pathogenèse de Helicobacter, ERL CNRS 3526, Institut
Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France

DOI 10.1007/978-4-431-55936-8_2
30 F. Fischer and H. De Reuse
2.1 Introduction
The stomach has long been regarded as a sterile environment because of its extreme
acidity. It is now known that the stomach of about half of the human population is
infected by Helicobacter pylori.
After acquisition during childhood, the infection by H. pylori becomes chronic if
not treated. Severe pathologies like gastric cancer can occur after decades of
H. pylori colonization and intense inflammation supporting the view that persis-
tence of the infection is one important parameter in its clinical outcome. It is thus
important to define the mechanisms that make this bacterium such a successful and
persistent pathogen in its unique and hostile acidic gastric niche. H. pylori is a
neutrophilic microorganism which means that it grows at pH values between 6 and
8. However, H. pylori has evolved original mechanisms to colonize and persist
within the stomach in spite of the harsh acidic conditions encountered in this
environment. Genetic, physiological, and biochemical analyses of H. pylori have
revealed peculiar properties of its metabolism, some of which are central in the
adaptation to the gastric environment. The nickel-dependent urease enzyme plays a
major role in this process through urea breakdown and production of buffering
ammonia. The present chapter is focused on the physiological and metabolic
adaptations adopted by H. pylori to successfully colonize the stomach and to persist
in its host. The first part of this chapter will explain how survival and pathogenesis
rely on urease enzymatic activity and ammonia metabolism, the second part will
concern metabolic enzymes that are in addition virulence factors, while the third
part will shed light on the importance of nickel availability to enable successful
colonization and persistence.
2.2 H. pylori Facing Acidity
Apart from the acidophiles that exclusively grow at low external pH, numerous
neutrophilic bacteria are able to survive low-pH exposure. This is, for instance, the
case for bacteria such as Escherichia coli, Vibrio cholerae, and Salmonella spp. that
need to survive the passage through the stomach but are unable to sustain growth
under these conditions. In order to overcome the adverse effects of acidity, most
bacteria use acid resistance systems that either involve a reversed F1F0-ATPase
activity for proton extrusion in response to the membrane potential change or amino
acid decarboxylation/protonation pathways coupled to proton efflux (Foster 2004)
to maintain cytoplasmic pH homeostasis.
Contrary to the other bacteria passing through the gastric lumen and that cannot
divide, H. pylori thrives very well within the acidic gastric environment and can
even reach densities as high as 106 cfu/g in the human stomach (Fig. 2.1). However,
H. pylori does not possess the “classical” resistance mechanisms described above.
Only when urea is provided, H. pylori resists low pH exposure and can multiply.
2 Adaptation of Helicobacter pylori Metabolism to Persistent Gastric Colonization 31
Fig. 2.1 Life cycle of

H. pylori in the human
stomach. (A) when
H. pylori enters the human
body through the oral route
(1), it is exposed to the
extremely acidic pH of the
stomach lumen (2).
(B) However, its
colonization site is the
moderately acidic gastric
mucus (3) where about 80 %
of H. pylori cells are found
(3). Around 20 % of the
bacteria actually adhere to
the gastric epithelial cells
where they cause
inflammation and tissue
injury (4). (C) to face
acidity, H. pylori
reprograms transcription of
numerous genes. The ArsR-
S two-component system is
central in the acid response
of H. pylori regulating
either positively or
negatively the expression of
several genes
H. pylori is thus able to survive and multiply under very acidic conditions, but relies
on a completely different system that is unique to the gastric Helicobacter species
and requires massive ammonia production. As we will discuss, acid adaptation
involves the nickel-dependent urease enzyme, capable of hydrolyzing urea to
produce ammonia and bicarbonate, as well as different enzymes of the ammonia
metabolism.
2.2.1 The Dangers of Low pH
Exposure to low pH has dramatic effects on the cells: it triggers protein denatur-
ation, alters enzymatic activity, enhances DNA depurination, and generates mem-
brane damage (Foster 2004). In Gram-negative bacteria, the periplasmic space is
the first compartment to experience pH decrease. This drastically perturbs the
inward proton gradient and directly decreases the membrane potential, which, in
turn, hinders the proton influx toward the periplasm. This affects all the membrane
potential-dependent processes, such as ATP synthesis or respiration. Then, accu-
mulation of protons can cause the cytoplasmic pH to decrease, disrupting metabolic
pathways, leading to growth arrest, and finally to cell death.
2.2.2 Acid Adaptation and Acclimation of H. pylori

to the Gastric Niche
Upon entry through the oral route, H. pylori reaches the highly acidic lumen
(median pH 1.5) (Fig. 2.1). The bacterium only survives for minutes in this
challenging environment and has to migrate rapidly toward the epithelium surface
(Schreiber et al. 2005). About 80 % of the H. pylori cells are detected in the
moderately acidic gastric mucus (pH 5) (Schreiber et al. 2004). Once there,
H. pylori may also experience large pH variations under fasting (pH 1) and
digestive (pH 4.5–6) conditions. The capacity to deal with this low pH was referred
to as “acid acclimation” or “acid adaptation” (Fig. 2.1). In addition, acidity also
varies depending on the infection location within the stomach, from the mildly
acidic antrum to the highly acidic corpus zones. Many parameters can vary either
spatially (lumen, mucus, surface of the epithelial cells, corpus, antrum) and/or
overtime within the gastric environment. These parameters not only include the
pH value but also the availability (stability, solubility) of nutrients such as amino
acids, peptides, and metal ions, etc. It is therefore not surprising that the mecha-
nisms involved in H. pylori acid acclimation are not only related to pH managing
processes but also to nutrient uptake (Stingl and De Reuse 2005), to chemotaxis,
and to motility.
2.2.3 Establishing the Response to Acidity
Once it faces a pH decrease, H. pylori triggers a transcriptional reprogramming to

express genes involved in acid adaptation and mechanisms of protection against
protons.
The global transcriptional response of H. pylori to acidity has been analyzed
either in vitro using bacteria exposed to acidity (Allan et al. 2001; Ang et al. 2001;
Wen et al. 2003; Merrell et al. 2003) or grown under moderately acidic conditions
(as in our previous study (Bury-Moné et al. 2004)) or in vivo during Mongolian
gerbil colonization (Scott et al. 2007). Gene expression patterns poorly matched
when comparing these analyses, both in the nature and the number of regulated
genes. This can be explained by the use of different H. pylori strains but also
because cells were exposed to different culture media and/or acid conditions.
Despite those differences, clear common trends could be extracted from these
results, some of which were confirmed subsequently.
A major strategy for acid resistance in H. pylori is ammonia production. The
transcriptomic studies unambiguously showed that the operon containing the genes
coding for the urease structural subunits (ureAB operon) and the operon encoding
the urea channel and urease maturation accessory proteins (ureIEFGH operon)
were acid-induced in H. pylori. In addition, the expression of two other ammo-
nia-producing enzymes, the AmiE amidase and AmiF formamidase, was found to
be acid-induced and will be discussed below (Skouloubris et al. 1997, 2001). Acid
protection strategy of H. pylori is also suggested by the repeated observation of
downregulation in the expression of membrane proteins including transporters,
permeases, and outer membrane proteins.
Another common observation was the enhanced expression of genes related to
motility including structural proteins of the flagellar apparatus and motor-related
proteins. Stimulation of H. pylori motility by acid was experimentally demonstrated
(Merrell et al. 2003) and may be indicative of a strategy to escape acidity or, at
least, might suggest a pH-driven response directing the bacteria through the mucus
pH gradient toward a suitable site for multiplication and/or adhesion, as suggested
by Schreiber et al. (2004). In parallel, major modifications in the expression of
genes coding for proteins related to, or regulated by, metal ion availability was
evidenced (Bury-Moné et al. 2004). Solubility of nickel and other metals is known
to be strongly enhanced at low pH. Urease maturation and activity strictly depend
on two nickel ion cofactors within the active site. Therefore, acid resistance is
tightly linked to nickel metabolism in H. pylori. This aspect will be discussed
below.
Most of the studies on transcriptional regulation in H. pylori showed the
involvement of three major regulators of the acid response network: (i) the two
metal regulators Fur and NikR that respond to iron and to nickel, respectively
(Van Vliet et al. 2003; Contreras et al. 2003; Bury-Moné et al. 2004), and (ii) the
ArsRS (HP0165-HP0166 in strain 26695 (Tomb et al. 1997)) two-component
regulatory system (TCS) that seems to act as a master regulator (Bury-Moné

et al. 2004; Pflock et al. 2005; Muller et al. 2009).
When H. pylori is exposed to acidic pH, the ArsS sensor histidine kinase, located
in the inner membrane, becomes phosphorylated on its His94 residue (Fig. 2.1).
This residue of the output domain is crucial for pH sensing. ArsS P then transfers
its phosphate to its cognate regulator ArsR that acts as an activator or a repressor on
a variety of pH-responsive genes. ArsR is an essential protein for H. pylori growth
and its phosphorylation is essential for acid acclimation (Muller et al. 2009). The
ArsRS system regulates many genes encoding central proteins in the acid response
that will be discussed in this chapter (Pflock et al. 2006; Wen et al. 2007). These
include genes encoding urease, the AmiE and AmiF aliphatic amidases, the
α-carbonic anhydrase, arginase, nickel storage proteins, and several others. The
cytoplasmic FlgS histidine kinase which governs the expression of some flagellar
genes seems to play an additional minor role in the H. pylori transcriptional
response to acidity (Marcus et al. 2012).
2.3 Urease, the Major Player in H. pylori Resistance

to Acidity
H. pylori urease is composed of two subunits, UreA (HP0073) and UreB (HP0072).
UreA is a 26.5 kDa protein and the 61.6 kDa UreB subunit carries the active site
containing the bi-nickel metallic complex essential for activity. Assembly of this
metallocenter is performed by accessory proteins (see below) (Farrugia et al. 2013;
De Reuse et al. 2013; Stingl and De Reuse 2005). Each UreA/B dimer associates
with two other dimers, forming a (UreA/B)3 complex, which can then cluster with
other trimers, to create a giant dodecameric ((UreA/B)3)4 complex whose structure
has been solved (Ha et al. 2001). In vivo, urease is present in huge amounts within
H. pylori cells and can represent up to 10 % of all proteins.
2.3.1 Urease Enzymatic Activity
Urease catalyzes the hydrolysis of urea (CO(NH2)2) into ammonia (NH3) and
carbamate, the latter being spontaneously degraded into another molecule of
ammonia and carbon dioxide. CO2 mostly exists as a dissolved gas form, and the
H2CO3 carbonic acid form only represents 1 % of the species present in solution.
At pH 7, it rapidly turns to bicarbonate (HCO3). The H. pylori cytoplasmic
carbonic anhydrase can further catalyze the interconversion between CO2 and
HCO3. Consequently, the overall reactions lead to molecules capable of proton
buffering. The NH4+/NH3 couple represents a strong proton buffering system, since
its pKA is 9.2, meaning that at neutral or acidic pH, ammonia is almost
Fig. 2.2 Acid resistance in H. pylori and revised model for the role of the α-carbonic anhydrase.
Panel (A) when pH decreases and falls below 6.0, urea (1) enters the cell through the UreI channel
(2). Once in the cytoplasm, it is degraded by urease (3) into ammonia and CO2. Upon protonation,
ammonia is transformed into NH4+ ions (4), thereby buffering the cytoplasm. In solution, CO2
rapidly turns to bicarbonate HCO3, buffering another H+ (5). HCO3 can be exported into the
periplasm where the membrane-bound α-carbonic anhydrase catalyzes a dehydration reaction that
consumes protons to produce H2O and CO2 (6). Panel (B) carbonic anhydrases catalyze either the
hydration of CO2 or the dehydration of HCO3 in a pH-dependent manner. The catalytic efficiency
of the hydration reaction increases with pH, with a pKA value of ~7.0. The opposite is observed for
the dehydration reaction. Thus, at the periplasmic pH of 6.0, the dehydration reaction (producing
water and CO2) is strongly favored, while the hydration reaction is not. In case of low-pH
exposure, this will avoid periplasmic over-acidification. Panel (C) if NH3 production leads to
periplasmic alkalinization, the α-CA will rather catalyze the hydration reaction (producing HCO3
and H+). This balancing model should allow H. pylori to maintain a periplasmic pH value around
6.0
exclusively converted into its ammonium form NH4+. The massive ammonia
production by urease activity thus represents the first countermeasure against
proton overload under acidic conditions (Mobley and Hausinger 1989; Labigne
et al. 1991) (Fig. 2.2).
The urease catalytic constant measured at steady-state is high, ranging from
1,400 to 2,700 s1 (Hu and Mobley 1990), implying a very rapid turnover for urea
degradation on one side and nitrogen mineralization on the other side. Based on its
kinetic properties, the urease complex should function at a maximal rate at the
concentration range of urea found within the human stomach (1–3 mM) (Mobley
and Hausinger 1989; Hu and Mobley 1990). Consequently, urease produces mas-
sive amounts of ammonia upon urea entry within the cells, which allow H. pylori to
rapidly resist pH decrease and proton overload.
2.3.2 Acid-Gated Transport of Urea by the UreI Channel
Urease is a cytoplasmic enzyme that is not required for growth at neutral pH

in vitro, but becomes essential under acidic conditions (Fig. 2.2). The H. pylori
acid acclimation is lost when the urease genes are deleted or when the UreI urea
channel gene is disrupted (Skouloubris et al. 1998; Scott et al. 2000; Bury-Moné
et al. 2001). Thus, acid acclimation not only depends on urea degradation and
ammonia production but also on urea uptake across the inner membrane. Urease
and UreI are indeed essential for H. pylori to colonize several animal models (Eaton
et al. 1991; Skouloubris et al. 1998; Mollenhauer-Rektorschek et al. 2002).
UreI, whose gene lies between the ureAB and ureEFGH gene clusters, has been
identified as a transmembrane channel transporting urea from the periplasmic space
into the cytoplasm where it is hydrolyzed by urease to produce ammonia
(Skouloubris et al. 1998; Weeks et al. 2000; Weeks and Sachs 2001). UreI is a
proton-gated channel responding to the decrease of periplasmic pH, the channel
starts to open up when the pH falls below 6.0, and selectively transports urea (Bury-
Moné et al. 2001; Gray et al. 2011; Weeks et al. 2000). The UreI structure has been
recently solved (Strugatsky et al. 2013): it has a novel channel architecture com-
posed of six protomers assembled in a hexameric ring surrounding a central bilayer
plug of ordered lipids. Residues important for the selectivity of the channel have
been identified, and the dynamics of urea conduction was characterized providing
hints on its capacity to transport urea even at low concentration gradients (McNulty
et al. 2013). It has been proposed that UreA-B and the UreE accessory proteins are
recruited to UreI at the inner membrane (Voland et al. 2003) and that UreI is in
addition permeable to NH3/NH4+ and CO2 (Scott et al. 2010). Additional data will
be necessary to reinforce these views.
When exposed to a low external pH value of 2.5, the H. pylori membrane
potential, which is 100 mV at neutral pH, starts to considerably fall. When urea
is provided, this value stabilizes around 100 mV, indicating that urease is at least
partly involved in pH homeostasis of the periplasm that is maintained at about 6.1
(Scott et al. 1998). The control of the periplasm pH upon acid exposure is till now
unique to H. pylori and implies a complex response dependent on the α-carbonic
anhydrase (see below).
2.3.3 Regulation of the Expression of the Urease Genes
Urease genes are clustered into two operons. The ureA-ureB dicistronic part is
followed by an operon composed of ureI and the ureEFGH genes required for
urease maturation and metalation. The urease operon is controlled by the acid-
responsive ArsRS two-component system (Marcus et al. 2012), as well as by NikR
(Van Vliet et al. 2001, 2002; Muller et al. 2011), the latter responding to intracel-
lular nickel concentration. Urease transcriptional balance thus depends on both
acidity and metal content (van Vliet et al. 2004b; Muller et al. 2011). This
regulation reflects the need for maximal urease activity under acidic conditions,
taking into account its maturation by nickel insertion (De Reuse et al. 2013) (see
paragraph 2.6).
2.3.4 Role of the Carbonic Anhydrases in H. pylori
Carbonic anhydrases (CA) catalyze the interconversion of carbon dioxide and

bicarbonate and are involved in functions such as CO2 transport or trapping and
pH homeostasis. Two genes encoding carbonic anhydrases are found in the
H. pylori genome (hp1186 and hp0004 in strain 26695). The first one encodes a
periplasmic α-type CA bound to the inner membrane (Marcus et al. 2005) and
homologous to those found in vertebrates. The second one codes for a cytoplasmic
ß-CA of a different structural type that is mostly found in prokaryotes and plants
(Fig. 2.2). Both enzymes are metalloproteins that require one Zn2+ ion to catalyze
the reversible hydration of carbon dioxide (CO2) and dehydration of bicarbonate
(HCO3) (reviewed in Frost and McKenna (2014)). CO2 occupies a central position
in the physiology of H. pylori owing the large amounts of carbon dioxide produced
by urease-mediated urea hydrolysis, the constant bicarbonate supply in the stom-
ach, and its capnophilic nature (strict dependence on CO2 for growth). In addition, it
has been shown that H. pylori displays a chemotactic response to bicarbonate
through a methyl-accepting chemotaxis receptor protein system, which seems to
be coupled with urea and arginine sensing (Cerda et al. 2011).
Both α-CA and β-CA have been shown to be dispensable for in vitro growth
under neutral conditions in several H. pylori strains (Marcus et al. 2005; Bury-
Mone et al. 2008). We only observed a growth defect phenotype with the single and
double CA deletion mutants of strain SS1 (Bury-Mone et al. 2008). These mutants
were in addition strongly affected in their capacity to colonize the mouse model
(Bury-Mone et al. 2008). 13C-NMR measurements demonstrated that most CA
activity can be attributed to the α-CA, with the β-CA enzyme playing a minor
role (Bury-Mone et al. 2008).
Interestingly, the α-CA plays a role in the urea-dependent response of H. pylori
to acidic pH (Fig. 2.2b). Indeed, when the α-CA was absent, a delayed production of
ammonia from urea was observed (Bury-Mone et al. 2008). The enzyme does not
participate in urease regulation or maturation, indicating that α-CA truly partici-

pates in the pH-dependent response. Another study unraveled the involvement of
the α-CA in periplasmic pH buffering (Marcus et al. 2005). The authors observed
that an α-CA knockout strain (H. pylori 26695) could not survive in acidic medium
compared to the wild-type strain, a phenotype that was not observed in our study
(Bury-Mone et al. 2008). This could be explained by different genetic backgrounds
and/or the use of poorly defined complex media for growth (Bury-Mone
et al. 2008). Using fluorescent dyes to monitor intracellular and periplasmic pH
and measurements of the membrane potential in response to urea uptake by whole
H. pylori cells, Marcus and coworkers (Marcus et al. 2005) concluded that the α-CA
is involved in the buffering of the periplasm and, consequently, in the membrane
potential rescue upon acid exposure. The HCO3/CO2 system displays a pKA 6.1.
Thus, a model was proposed in which the CO2 gas molecule produced by urease
passively diffuses across the inner membrane toward the periplasm, where it is
hydrated by the α-CA into HCO3 and H+. This proton would then immediately be
buffered by NH3 to form NH4+. This would ensure the maintenance of a periplasmic
pH 6.1 keeping an –100 mV inner membrane potential (Marcus et al. 2005). The
cytoplasmic β-CA would be involved in the system as well, converting the HCO3
produced by urea degradation into CO2. In support of this model is the observation
by the same group that UreI is not only involved in urea uptake but also capable of
extruding CO2 and NH4+ from the cytoplasm to the periplasm (Scott et al. 2010).
However, our close analysis of the enzymatic properties of the CA enzymes
leads us to conclude that this model needs to be reexamined for the following
reasons (Fig. 2.2b). Because the activity of these enzymes relies on a zinc ion, their
catalytic activity strongly depends on pH. The hydration reaction (CO2 + H2O !
HCO3 + H+) efficiency increases as a sigmoid when the pH increases, fitting an
acid/base titration curve with a pKA value of 7.0 (Fig. 2.2b). This property
originates from the Zn-hydroxylated center found within the active site (Frost and
McKenna 2014). On the contrary, the catalytic efficiency of the dehydration
reaction (HCO3 + H+ ! CO2 + H2O) decreases as a sigmoid curve when pH
increases (Fig. 2.2b). This was clearly demonstrated by Chirica et al. (2001) with
purified H. pylori α-CA. Consequently, the CA hydration/dehydration reaction ratio
directly depends on pH, thereby determining the role of CA as a function of pH
variations (Fig. 2.2b).
According to these enzymatic properties, it is unlikely that at the pH 6 found
within the periplasm, the α-CA hydrates CO2 to produce HCO3 and H+, as
proposed in the model of Marcus et al. (2005). Under such conditions, one would
rather expect the dehydration activity to be increased over hydration.
We propose a more complete model that would work as follows (Fig. 2.2c). At
periplasmic pH of approx. 6, the α-CA mostly catalyzes the dehydration reaction,
thereby consuming protons and producing CO2. The same reaction would even be
strongly favored at acidic pH (<6.0), meaning that under neutral or acidic condi-
tions, α-CA is involved in proton consumption and buffering, increasing the pH in
the periplasm. If NH4+ can be exported to the periplasm as stated by Scott
et al. (2010), then it would be able to release protons in the alkalinizing periplasm
and counterbalance this pH variation, to bring it back to a more acidic range. When
urease catalyzes urea breakdown, however, it triggers a rapid and massive NH3
accumulation, which increases the cytoplasmic and the periplasmic pH value.
Indeed, at pH 6, with a pKA value of 9.2, ammonia efficiently pumps in protons
and turns to its NH4+ ionic form, favoring a rapid and strong alkalinization. If the
periplasmic pH starts to increase as well, the α-CA would start to catalyze the
hydration (HCO3 and H+-forming) reaction more efficiently, thereby optimally
producing buffering HCO3 and H+ to counterbalance ammonia-driven alkaliniza-
tion (Fig. 2.2c). In conclusion, we propose that urease and α-CA should rather be
regarded as a tangled-up balancing system avoiding over-acidification and over-
alkalinization of both the periplasm and the cytoplasm (Fig. 2.2).
2.4 Ammonia Metabolism in H. pylori
Considering the large amounts of ammonia that are produced by urease in response
to the acid pH exposure, H. pylori is expected to have counterbalancing systems to
avoid toxic massive ammonia accumulation (De Reuse and Skouloubris 2001). In
bacteria, ammonia is assimilated (“stored”) into amino acids through amination of
α-ketoglutarate into glutamate (Glu) and then by amidation into glutamine (Gln).
Not surprisingly, in H. pylori, nitrogen from urea has been demonstrated to be
incorporated into amino acids (Williams et al. 1996). In addition, urease is required
for colonization of gnotobiotic piglets with an artificially neutralized stomach
suggesting a metabolic function of urea degradation in addition to its role in acid
resistance (Eaton and Krakowka 1994). H. pylori was found to have minimalist
pathways for nitrogen/ammonia assimilation that are illustrated in Fig. 2.3.
2.4.1 Minimalist Pathways for Nitrogen Assimilation
In H. pylori Glu is synthesized from ammonia and α-ketoglutarate by a single

glutamate dehydrogenase (GdhA). Indeed, this organism does not possess the
classical glutamine oxoglutarate aminotransferase (GOGAT) system found in
other bacteria (for a review, see Leigh and Dodsworth (2007)). Gln is then synthe-
sized from Glu and ammonia by the essential glutamine synthetase (GlnA,
HP0512). This enzyme cannot be inactivated in H. pylori even when Gln is supplied
in the medium (Garner et al. 1998; Stingl et al. 2008). In bacteria, Gln plays a
central role, since it is involved in almost all the amidation reactions that rely on an
amido group donor in the purine/pyrimidine biosynthetic pathway, amino acid
biosynthesis, as well as in the bacterial cell wall synthesis (Leigh and Dodsworth
2007). Another way to store ammonia is to perform aspartate (Asp) synthesis from
fumarate and ammonia using aspartate ammonia lyase (AspA) and then to amidate
it with an asparagine synthetase to produce asparagine (Asn). However, in
Fig. 2.3 Asparagine and glutamine metabolism in H. pylori. Asn (1, green label) and Gln (1,
orange label) are both imported in the periplasm, where they are deamidated by the AnsB or GGT
enzymes, respectively, providing Asp and Glu and ammonium. Asp and Glu are then transported
across the inner membrane through the DcuA and GltS transporters, respectively, toward the
cytoplasm. Glu can also be synthesized by the glutamate dehydrogenase (GdhA) (2) and Asp by
the aspartate ammonia lyase (AspA) (2, III). Glu serves as a substrate for the Gln synthetase
(GlnA) (3) that provides Gln (I ). In H. pylori Asn is not synthesized in a free form. Gln can also be
synthesized in a tRNA-dependent manner in H. pylori (steps 4–6, II). In this pathway, Glu is first
misacylated by a glutamyl-tRNA synthetase (GluRS2) onto tRNAGln (4, orange); then this
intermediate gets amidated by the GatCAB complex (5, orange), to provide the final Gln-tRNA
Gln
molecule suitable for translation (5, orange). These reactions are performed by the
Gln-transamidosome complex (II). Asn is specifically synthesized for translation in an analogous
tRNA-dependent manner (steps 3–5, green), starting with misacylation of Asp onto tRNAAsn by
the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) (3, green), followed by amidation
by the GatCAB enzyme (4, green) to provide Asn-tRNAAsn (5, green). All reactions occur within
the Asn-transamidosome complex (AspRS-ND/tRNAAsn/GatCAB) (IV)
H. pylori, whole-genome analysis revealed the absence of an Asn biosynthetic

pathway (Marais et al. 1999).
2.4.2 Central Role of Glutamine Synthetase in Ammonia

Metabolism and Possible Coupling with Urease
Glutamine synthetase (GlnA) that catalyzes the incorporation of ammonia into Glu
to form Gln thus plays a central role in the ammonia metabolism of H. pylori. This
pathway is usually tightly controlled through several levels of regulation including
GlnA adenylation. H. pylori GlnA does not seem to be subject to this classical retro-
inhibition pathway; therefore, Gln synthesis is probably constitutive (Garner
et al. 1998).
Some data are supporting the view that there is a coupling between the activities
of glutamine synthetase (GlnA) and urease in H. pylori. During our analysis of the
urease protein interactome by tandem affinity purification (TAP), we found that
UreA specifically interacts with GlnA (Stingl et al. 2008). We proposed that this
physical interaction of GlnA with UreA allows optimization of the incorporation of
urease-derived ammonium into glutamate to produce glutamine through metabolic
channeling, a mechanism that has been reported for other protein complexes (see
below). In support of this view, a recent study showed that in an H. pylori strain
artificially overexpressing GlnA, urea-derived ammonia production is enhanced
about sixfold (Miller and Maier 2014). Thus, the metabolism of Glu/Gln and
probably of amidated amino acids is closely connected to the ammonia production
in H. pylori. The next paragraphs will describe ammonia-producing and ammonia-
assimilating enzymes found in H. pylori.
2.4.3 Transport and Metabolism of Amidated Amino Acids
In bacteria, amino acids are mostly used for protein synthesis, but are also taken up
as energy, nitrogen, and carbon sources. H. pylori preferentially uses amino acids as
an energy source and interestingly consumes very large amounts of Asp and Glu as
well as their amide counterparts, Asn and Gln (Mendz and Hazell 1995; Stark
et al. 1997). In this paragraph, we will present the particularities of the metabolism
of these two amidated amino acids Gln and Asn in H. pylori.
2.4.3.1 Asp/Asn and Glu/Gln Uptake in H. pylori
In H. pylori, the important asparaginase and glutaminase activities are catalyzed by

two highly active periplasmic enzymes, gamma-glutamyl transpeptidase (GGT,
HP1118) and L-asparaginase (AnsB HP0723) that produce ammonia and Asp or
Glu from Asn and Gln, respectively (Cappelletti et al. 2008; Shibayama et al. 2007;
Leduc et al. 2010). We tested the role of these enzymes in the uptake of Asp/Asn
and Glu/Gln in intact H. pylori cells (Leduc et al. 2010). While Asp and Glu uptake
were not dependent on these activities, Gln and Asn strictly required GGT and
AnsB activities, respectively, to be taken up (Shibayama et al. 2007; Leduc
et al. 2010). We identified two inner membrane transporters for Asp (DcuA,
HP0724) and for Glu (GltS, HP1506) and demonstrated that they are the sole
uptake systems for these two amino acids in H. pylori and are unable to take up
Gln and Asn (Leduc et al. 2010). We further demonstrated that Asn deamidation by
AnsB is a prerequisite to its import by the DcuA transporter. The same is true in the
case of Gln, where its deamidation by GGT is required for the GltS transporter to
import Glu. This explains why an H. pylori GlnA mutant cannot be obtained even
when Gln is provided in the growth medium. Consequently, H. pylori does not
import Asn and Gln under their amidated forms, but uses a coupled deamidation/
transport system that provides Glu and Asp to the cytoplasm, thereby fueling the
periplasm with ammonia. Most interestingly, each of the four components of this
coupled deamidation/transport systems, namely, GGT/GltS and AnsB/DcuA, is
essential for colonization of the animal model (Leduc et al. (2010) and references
therein). Their in vivo essentiality can be at least partly attributed to the requirement
of amino acids in the gastric environments. However, for GGT and AsnB, important
roles linking virulence and amino acid metabolism were demonstrated and will be
discussed in paragraph 2.5.
2.4.3.2 Asparagine and Glutamine in Translation
The entire pool of proteinogenic amino acids has to be available in the cells to fuel
protein translation. In H. pylori, Gln is imported under the form of Glu upon
deamidation, and glutamine synthetase (GlnA) enables intracellular Gln biosynthe-
sis. A major issue arises when considering Asn since no predicted Asn synthetase
gene was identified in the genome (Marais et al. 1999; Fischer et al. 2012). More-
over, H. pylori is also particular in that it is completely deprived of the enzymes
ubiquitously implicated in Asn and Gln binding to their respective transfer RNA
(tRNA) (Fischer et al. 2012; Chuawong and Hendrickson 2006; Huot et al. 2011;
Skouloubris et al. 2003), namely, the asparaginyl-tRNA synthetase (AsnRS) and
the glutaminyl-tRNA synthetase (GlnRS). Several studies showed that the metab-
olism of amidated amino acids (Asn and Gln) is particular in H. pylori and involves
a specific tRNA-dependent biosynthetic pathway. The system involves an essential
enzyme, GatCAB that is one of the H. pylori ammonia-assimilating enzymes.
2.4.3.3 How Are Asn-tRNA and Gln-tRNA Generated in H. pylori?
In order to be incorporated into proteins, amino acids first have to be aminoacylated

onto their cognate tRNA in the form of an aminoacyl-tRNA (aa-tRNA) by
aminoacyl-tRNA synthetases (aaRSs). However, both AsnRS and GlnRS are absent
in H. pylori, making Asn-tRNAAsn and Gln-tRNAGln synthesis impossible. Like in
a large majority of prokaryotes and in organelles, H. pylori uses ancestral tRNA-
dependent amino acid biosynthesis routes to fuel translation with Asn-tRNAAsn and
Gln-tRNAGln (Sheppard et al. 2008).
These pathways involve the formation of ribonucleoprotein complexes that
require the presence of an aaRS, a tRNA, and a tRNA-dependent ammonia-depen-
dent amidotransferase (AdT) composed of three subunits, called GatCAB. The first
complex, named the Gln-transamidosome, leads to the formation of Gln-tRNAGln
and involves in H. pylori an unconventional glutamyl-tRNA synthetase (GluRS2,
HP0643) (Skouloubris et al. 2003), the orphan tRNAGln, and the GatCAB AdT.
GluRS2 is conserved among epsilonproteobacteria. Similarly, the
Asn-transamidosome, containing the aspartyl-tRNA synthetase (AspRS,
HP0617), the orphan tRNAAsn, and GatCAB, provides Asn-tRNAAsn in a second
biosynthetic pathway (Fischer et al. 2012; Silva et al. 2013).
In the Gln-transamidosome (GluRS2/tRNAGln/GatCAB), the GluRS2 enzyme
loads Glu onto the tRNAGln moiety, thereby producing a misacylated (“wrong”)
Glu-tRNAGln species. In H. pylori, GluRS2 is specifically devoted to the
misacylation of tRNAGln with Glu for the tRNA-dependent Gln biosynthesis
(Skouloubris et al. 2003). This intermediate is then immediately processed by the
GatCAB AdT, which amidates the Glu carboxylate into an amide, in an
ATP-dependent reaction (Sheppard et al. 2007; Huot et al. 2011). The
Asn-transamidosome functions in a similar way, but first involves a nondiscri-
minating AspRS (ND-AspRS, HP0617) that charges tRNAAsn with Asp, providing
the Asp-tRNAAsn intermediate necessary for the next step. The ND-AspRS is also
required for normal Asp-tRNAAsp synthesis (Chuawong and Hendrickson 2006).
Then, the GatCAB AdT amidates the Asp moiety onto the tRNAAsn molecule and
provides Asn-tRNAAsn for protein translation.
GatCAB is an essential enzyme composed of three subunits, with GatA being the
amidase, capable of hydrolyzing a free Gln donor, providing ammonium that is then
channeled along a molecular tunnel toward the GatB-active site, where the
misacylated aa-tRNA lies and where amidation occurs (Nakamura et al. 2006).
This enzyme constituted a “classical” example illustrating the existence of ammo-
nia channeling. Gln is by far the best substrate for GatA (Sheppard et al. 2007), but
in H. pylori GatCAB is also capable of using NH4+ directly, presumably because
massive ammonia concentration may fill up the molecular channel between GatA
and GatB (Sheppard et al. 2007). Therefore, it can be considered that in H. pylori,
GatCAB constitutes an additional system for ammonia assimilation into amino
acids.
2.4.3.4 The AmiE and AmiF Aliphatic Amidases
Aliphatic amidases are enzymes catalyzing the hydrolysis of short-chain amides to

produce ammonia and the corresponding organic acid. H. pylori possesses two such
enzymes AmiE (HP0294) (Skouloubris et al. 1997) and AmiF (HP1238)
(Skouloubris et al. 2001; Bury-Moné et al. 2003). The expression of both
corresponding genes is strongly upregulated in response to acidity and to iron or
nickel (Bury-Moné et al. 2004; van Vliet et al. 2004a). AmiE is a “classical”
aliphatic amidase; in vitro it is active on propionamide, acrylamide, or acetamide
(Skouloubris et al. 1997). We found that the AmiF protein is a novel type of
formamidase hydrolyzing formamide to form formic acid and ammonia. AmiF is
an AmiE paralogue with a restricted substrate specificity that possibly has evolved
to achieve enzymatic specialization after ancestral gene duplication (Skouloubris
et al. 2001). The three-dimensional structure of the AmiF enzyme has been solved
and indeed defines a new group of formamidases (Hung et al. 2007). These
H. pylori enzymes are unable to catalyze urea breakdown. The in vivo function
and substrates of both enzymes are still unknown, but their catalytic activities
liberate ammonia, which clusters them into the ammonia pool-forming proteins
of H. pylori. Neither of the amidases are important for colonization in the mouse
model (Bury-Moné et al. 2003). Interestingly, the amidases are found only in
Helicobacter species able to colonize the stomach, suggesting that their acquisition
might be related to selective pressure in this particular gastric environment (Bury-
Moné et al. 2003).
2.5 Metabolic Enzymes Involved in Virulence
A close relation between metabolism and virulence has been established in many
pathogens. In H. pylori, this is perfectly illustrated by two periplasmic deamidases
required for in vivo colonization, gamma-glutamyl transpeptidase (GGT),
asparaginase, and also by arginase. The role of GGT will not be discussed here
since it is extensively presented in Chap. 7.
2.5.1 Asparaginase
Compared to GGT, there is much less information available on the role of the
second periplasmic deamidase of H. pylori, the asparaginase AnsB that catalyzes
the conversion of Asn to Asp and ammonia (Leduc et al. 2010; Shibayama
et al. 2011). Purified AnsB protein was shown to be cytotoxic when applied to
cultured AGS and MKN28 gastric epithelial cells (Cappelletti et al. 2008). As for
GGT, AnsB was identified as a factor responsible for cell-cycle inhibition of
fibroblasts and gastric cell lines (Scotti et al. 2010). However, in a more recent
study, it was shown that the depletion of Asn by asparaginase contributes poorly to
induction of the inflammatory response during H. pylori infection (Rimbara
et al. 2013). Additional studies are needed to understand the precise role of the
H. pylori asparaginase that might be less determinant for the outcome of the
pathologies than GGT. Indeed, no correlation between the severity of the
H. pylori-associated disease and the level of asparaginase activity was observed
(Rimbara et al. 2013).
2.5.2 Arginase
Arginases are enzymes that convert arginine to urea and ornithine. H. pylori was
found to present a strong membrane-bound arginase activity, and it was proposed
that its endogenously produced urea might be used by urease. The corresponding
RocF arginase protein is constitutively expressed and was extensively characterized
biochemically (Mendz et al. 1998; McGee et al. 2004). It uses cobalt as cofactor, as
opposed to mammalian arginases which use manganese and has an acidic pH
optimum. Interestingly, arginase activity increases the resistance of H. pylori to
acid in an arginine-dependent fashion (McGee et al. 1999). However, H. pylori
arginase is not required for colonization of wild-type mice or of arginase II
knockout mice indicating that the major in vivo source for urea is neither bacterial
arginase nor host arginase II (Kim et al. 2011). Most interestingly, in H. pylori as in
some other pathogens, arginase acts as an immune modulator (Das et al. 2010).
Indeed, at physiologically relevant arginine concentrations, an H. pylori arginase
mutant elicits higher amounts of nitric oxide (NO) production in RAW macro-
phages and accordingly is more affected in its survival. In contrast, peritoneal
macrophages from iNOS/ mice failed to affect the survival of the rocF mutant.
It was concluded that arginase allows H. pylori to evade the immune response by
quenching arginine from the inducible nitric oxide synthase (iNOS) thereby
downregulating eukaryotic NO production and protecting itself from killing
(Gobert et al. 2001). In addition, the H. pylori arginase impairs host T-cell function
by reducing CD3ζ chain expression (Zabaleta et al. 2004). Thus, arginase might
also play a very important role during H. pylori infection.
2.6 Metabolism of Nickel, an Essential Metal

for the Virulence of Helicobacter pylori
Metal acquisition is a critical process for all organisms, because metal ions are often
found at very low concentrations in their environments. In spite of their low
quantities within the cells, they are essential since many enzymes depend on
metal cofactors for their catalytic activity. To face this scarcity, bacteria have
evolved very efficient uptake systems to acquire metals from the environment, as
well as systems allowing their intracellular storage, distribution, and maintenance
of homeostasis. Indeed, at nonphysiological high intracellular concentrations,
metals are toxic. The uptake, storage, distribution, incorporation, and efflux mech-
anisms are together referred to as “trafficking systems.”
2.6.1 Nickel Is a Virulence Determinant for H. pylori
Nickel (Ni2+) is a central metal for the acid adaptation and survival of H. pylori
within the stomach (for reviews, see Stingl and De Reuse (2005), De Reuse
et al. (2013), Rowinska-Zyrek et al. (2014)). Indeed, for the urease enzyme to be
active, nickel has to be present in sufficient amounts to enable its maturation.
Accordingly, the total intracellular nickel concentration is about 50 times that of
E. coli (Schauer 2007). Since urease is strongly overexpressed and present in very
high amounts when the bacterium faces acidic conditions, H. pylori must import
large amounts of nickel to ensure its optimal activity in a challenging environment.
If urease is absent or not maturated, H. pylori is unable to colonize the stomach of
animal models. In H. pylori, [NiFe] hydrogenase is a second enzyme that requires
Ni2+ as a cofactor (Maier and Bock 1996). Its activity enables H. pylori to use H2 as
an energy source, and this enzyme is required for optimal colonization of the mouse
model (Olson and Maier 2002). Thus, since Ni2+ is critical for both urease and
hydrogenase catalytic activities and for proper colonization and survival in the
stomach, this metal can be considered as a virulence determinant.
2.6.2 Nickel Transport and Efflux
In Gram-negative bacteria, energized transport of metabolites such as iron

siderophore complexes through the outer membrane (OM) relies on the TonB
machinery and on TonB-dependent transporters (TBDTs). Since nickel is found
at very low concentrations within the human stomach, it was expected that H. pylori
uses efficient uptake systems to obtain appropriate amounts of this metal from its
environment. Indeed, we discovered in H. pylori the first nickel transport system
across a bacterial OM (Schauer et al. 2007, 2008). This uptake machinery requires
the FrpB4 TBDT and is energized by the TonB machinery comprising ExbB-ExbD-
TonB (Schauer et al. 2007, 2008). The role of ExbD in the control of the nickel
activation of urease and thus in pH homeostasis was subsequently confirmed
(Marcus et al. 2013). Expression of the frpB4 gene is repressed by NikR in the
presence of nickel, which suggests that this transport system is fully active under
nickel-limiting conditions. In addition, FrpB4 is acid-activated allowing H. pylori
to optimize urease maturation by nickel incorporation under conditions where its
activity needs to be maximal for acid adaptation. Moreover, nickel is more soluble
at acidic pH, which makes an acid-induced uptake system appropriate in the gastric
environment. Additional mechanisms certainly exist that could allow, for example,
nickel entry at neutral pH. FecA3, another H. pylori TBDT whose synthesis is under
the control of nickel, might be an alternative nickel uptake system (Ernst
et al. 2006; Romagnoli et al. 2011).
In H. pylori, once nickel has crossed the OM, it can be taken up through the
cytoplasmic membrane (CM) by NixA, a high-affinity and low-capacity nickel
transporter (Fulkerson and Mobley 2000) of the NiCoT family, whose expression
is also repressed by nickel (Wolfram et al. 2006; Muller et al. 2011). Because NixA
mutant retains half of urease activity and its capacity to colonize the mouse model is
not totally abolished (Nolan et al. 2002), alternative ways of nickel entry across the
CM must exist.
Once it has crossed the CM, nickel has to be directed to its proper targets while
avoiding potential damages caused by free metal ions. If nickel is in excess with
respect to H. pylori cellular needs, it has either to be stored or to be exported out of
the cell. One nickel export system was described in H. pylori. It is a proton-driven
RND-type metal efflux-pump encoded by the cznABC genes (Stahler et al. 2006).
Inactivation of this pump increases H. pylori sensitivity to nickel, cadmium, and
zinc and impairs colonization of the gerbil stomach (Stahler et al. 2006),
underlining the importance of metal homeostasis for H. pylori virulence.
2.6.3 Nickel Chaperones and Storage Proteins
Metal overload is toxic to the cells. In H. pylori, several storage proteins, also called
metallochaperones, have evolved to ensure that nickel remains correctly balanced
and gets optimally incorporated into urease and hydrogenase.
2.6.3.1 Role of HspA, the Helicobacter-Specific GroES Homolog
In H. pylori, the sole member of the highly conserved and essential GroES
co-chaperonin family is HspA (Suerbaum et al. 1994). This protein is original in
that it possesses a unique His- and Cys-rich C-terminal extension specifically found
within the Helicobacter genus. This unusual extension was shown to bind nickel
ions (Kansau et al. 1996), and the hspA gene is upregulated by NikR in response to
nickel (Muller et al. 2011).
HspA is an abundant protein in vivo, and one of its roles in H. pylori is to bind
free nickel ions to store them in the cytoplasm and prevent the cell from metal
overload. Indeed, using an H. pylori mutant expressing a C-terminal truncated form
of HspA, we observed that this extension is involved in nickel sequestration
(Schauer et al. 2010). This mutant strain expressed wild-type urease activity and
was strongly affected in its hydrogenase activity. We concluded that HspA
constitutes a nickel storage protein pool specifically devoted to hydrogenase mat-

uration. How nickel is mobilized from HspA and whether HspA provides nickel to
other proteins remain to be determined (Schauer et al. 2010).
2.6.3.2 Hpn and Hpn-2: Two Remarkable Histidine-Rich Proteins
H. pylori also possesses two proteins of unusual amino-acid composition that have
no orthologs outside the Helicobacter species. Hpn and Hpn-2 (also called Hpn-
like) are two small proteins (7 and 8 kDa) that are extremely rich in His-residues: 47
and 25 % of the total residues, respectively. Hpn-2 contains additional poly-
Glutamine stretches representing 40 % of the total residues. These two proteins
form multimers and bind nickel in vitro, as expected for His-rich peptides (Gilbert
et al. 1995; Ge et al. 2011; Zeng et al. 2008 and 2011; Rowinska-Zyrek et al. 2011).
Like hspA, transcription of both genes is upregulated by NikR in response to
nickel (Muller et al. 2011). Because hpn and hpn-2 mutants were reported to be
more sensitive to high exogenous nickel concentrations than a wild type strain,
these proteins were suggested to be involved in nickel storage and detoxification via
sequestration of excess nickel (Mobley et al. 1999; Seshadri et al. 2007). We
recently demonstrated (Vinella et al. 2015) that in H. pylori only Hpn is involved
in nickel sequestration and that Hpn and Hpn-2 interact with each other. In addition,
their combined activities were found to participate in a nickel transfer pathway to
urease. Using bioinformatics and top-down proteomics to identify the predicted
proteins, we established that Hpn-2 is only expressed by H. pylori and its closely
related species Helicobacter acinonychis. Hpn was detected in every gastric
Helicobacter species tested and is absent from the enterohepatic Helicobacter
species. Phylogenomic analysis revealed that Hpn acquisition was concomitant
with the specialization of Helicobacter to colonization of the gastric environment.
Finally, both proteins are essential for colonization of a mouse model by H. pylori.
Taken together, these results strongly suggest that during evolution, the acquisition
of Hpn by gastric Helicobacter species was decisive for their capacity to colonize
the stomach.
2.6.4 Urease and Hydrogenase Maturation
Urease and hydrogenase are two nickel enzymes necessary for the virulence of
H. pylori. Complex mechanisms are required for their maturation by nickel incor-
poration. H. pylori is till now the only organism in which a molecular cross talk
between the maturation machineries of these two enzymes has been observed. This
again underlines the utmost importance of the control of nickel distribution and
trafficking in H. pylori.
2.6.4.1 Urease Maturation
Urease maturation requires four accessory proteins, UreE, UreF, UreG, and UreH,
whose gene cluster lies downstream the ureAB operon. The whole process takes
place within a multi-protein maturation complex (for a review, see Farrugia
et al. (2013)). Interaction between the urease structural and accessory proteins
was observed by yeast two-hybrid analysis (Rain et al. 2001; Voland et al. 2003).
Using tandem affinity purification, we isolated from H. pylori cells a complex
composed of the UreA/B structural units, together with the complete UreE/F/G/H
activation complex (Stingl et al. 2008). The ((UreA/B)3)4 apo-urease complex is
thought to recruit a UreF/G/H complex and the UreE·Ni2+ chaperone to catalyze the
insertion of two nickel ions. UreE is a metallochaperone that binds one nickel ion
with a high affinity (KD 150 nM) and interacts with UreG (Bellucci et al. 2009).
UreH is a scaffold protein necessary for the recruitment of UreF; this heterodimer
then binds UreG. This latter protein is an intrinsically unstructured GTPase of weak
catalytic activity that dimerizes upon binding of a metal ion (Zambelli et al. 2009).
This protein is also capable of binding nickel (KD 5 μM), although less efficiently
than UreE. It has been suggested that the binding of the UreF/H complex onto
urease induces conformational changes, allowing nickel ion and carbon dioxide to
access its active site. UreF was shown to gate the GTPase activity of UreG, which
would enhance its GTPase activity and as a result the fidelity of urease
metallocenter assembly (Boer and Hausinger 2012). Recently, it was reported that
UreF binds two nickel ions per dimer, with a micromolar KD and site-directed
mutagenesis suggested an additional role for a Ni2+-UreF complex in urease
maturation (Zambelli et al. 2014).
A recent crystal structure of the H. pylori UreF/G/H complex reveals how UreF
and UreH facilitate UreG dimerization and how this leads to the correct assembly of
its metal-binding site (Fong et al. 2013). The addition of nickel and GTP to the
UreF/G/H complex causes the release of the UreG dimer that binds nickel at the
dimeric interface. In vitro, the nickel-loaded UreG dimer was shown to activate
urease in the presence of UreF/H and in the absence of the UreE metallochaperone.
Thus, in the fully assembled UreE/F/G/H, nickel could be channeled from UreE to
UreG, before its insertion in the urease-active site. How nickel is transferred from
UreE to the binding site of UreG and how the complete activation complex interacts
with urease have still to be determined.
2.6.4.2 Hydrogenase Maturation
[NiFe] hydrogenases catalyze the reversible heterolytic cleavage of dihydrogen.

The active site of these enzymes is buried within the large subunit (HydB) and
contains an Fe(CO)(CN)2 unit and a nickel ion. The small subunit (HydA) carries
the Fe-S clusters necessary for dihydrogen activation and electron transfer
(Fontecilla-Camps et al. 2007). H. pylori possesses a respiratory-type [NiFe]

hydrogenase that catalyzes the oxidation of dihydrogen (Maier et al. 1996).
Hydrogenase maturation requires specialized metallochaperones for nickel
delivery and accessory proteins that perform the posttranslational modifications
required for the enzyme to be active. HydB contains a complex NiFe(CN)2CO
center, and its maturation requires six accessory proteins for nickel delivery and
insertion (HypA and HypB), for Fe delivery (HypC, HypD), and for cyanide
(CN) ligand biosynthesis and insertion (HypE, HypF). The whole process has
mostly been characterized in E. coli. HypA and HypB act together to insert the
nickel ion necessary to complete the metallic center within the active site. Upon
nickel delivery, a C-terminal peptide gets cleaved by an endopeptidase to obtain the
matured large subunit containing the NiFe(CN)2CO metallic complex. The αβ
heterodimer is then translocated to the periplasmic space through the Tat secretion
pathway (for a review, see Watanabe et al. (2012)).
2.6.4.3 A Molecular Cross Talk Between Urease and Hydrogenase

Maturation Machineries
An interesting particularity of nickel trafficking in H. pylori is the interconnectivity

between the urease and hydrogenase maturation pathways. This was discovered
using H. pylori mutants; the data showed that HypA and HypB are both required not
only for hydrogenase maturation but also for full urease activation (Olson
et al. 2001). In agreement with this, our H. pylori interactome analysis evidenced
that HypB is actually physically associated to the urease maturation complex
(Stingl et al. 2008). Using optical biosensing methods, the H. pylori HypA and
UreG proteins were shown to compete with each other for UreE recognition (Benoit
et al. 2012). Importantly, in vitro translocation of nickel between the two
metallochaperones HypA and UreE was demonstrated (Yang et al. 2014). In
addition, experiments performed with purified recombinant HypA indicated that
this protein is sufficient for the recovery of full urease activity in cell lysates from a
hypA deletion mutant (Herbst et al. 2010). These results suggest that the function of
the hydrogenase accessory proteins HypA in urease activation relies on nickel
delivery or exchange rather than on catalytic activity regulation.
2.7 Conclusions and Outlook
In conclusion, this chapter presents how unusual metabolic properties of H. pylori

contribute to its spectacular capacity to persistently colonize a hostile niche. Its
ability to multiply at low pH relies on massive ammonia production whose potential
toxicity is prevented by coupling to protein complexes. In H. pylori as in other
pathogens, some metabolic enzymes are in addition directly involved in virulence
acting in particular as immune modulators. Finally, the dependence on the nickel
enzyme urease for acid resistance renders nickel a virulence determinant whose
transport and trafficking is tightly controlled.
We propose that the constrains of the small H. pylori genome and a very
specialized niche have resulted in a close association and in overlapping networks
between mechanisms of persistence/acid adaptation and metabolic pathways.
References
Allan E, Clayton CL, McLaren A, Wallace DM, Wren BW (2001) Characterization of low-pH
responses of Helicobacter pylori using genomic DNA arrays. Microbiology 147:2285–2292
Ang S, Lee C, Peck K, Sindici M, Matrubutham U, Gleeson MA, Wang J (2001) Acid-induced
expression in Helicobacter pylori: study in genomic scale by microarray. Infect Immun
69:1679–1686
Bellucci M, Zambelli B, Musiani F, Turano P, Ciurli S (2009) Helicobacter pylori UreE, a urease
accessory protein: specific Ni2 +- and Zn2 +-binding properties and interaction with its cognate
UreG. Biochem J 422:91–100
Benoit SL, Mj L, Hill SA, Maier RJ (2012) Helicobacter pylori hydrogenase accessory protein
HypA and urease accessory protein UreG compete with each other for UreE recognition.
Biochim Biophys Acta 1820:1519–1525
Benoit SL, Miller EF, Maier RJ (2013) Helicobacter pylori stores nickel to aid its host coloniza-
tion. Infect Immun 81:580–584
Boer JL, Hausinger RP (2012) Klebsiella aerogenes UreF: identification of the UreG binding site
and role in enhancing the fidelity of urease activation. Biochemistry 51:2298–2308
Bury-Moné S, Skouloubris S, Labigne A, De Reuse H (2001) The Helicobacter pylori UreI
protein: role in adaptation to acidity and identification of residues essential for its activity
and for acid activation. Mol Microbiol 42:1021–1034
Bury-Moné S, Skouloubris S, Dauga C, Thiberge J-M, Dailidiene D, Berg DE, Labigne A, De
Reuse H (2003) Presence of active aliphatic amidases in Helicobacter species able to colonize
the stomach. Infect Immun 71:5613–5622
Bury-Moné S, Thiberge J-M, Contreras M, Maitournam A, Labigne A, De Reuse H (2004)
Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen
Helicobacter pylori. Mol Microbiol 53:623–638
Bury-Mone S, Mendz GL, Ball GE, Thibonnier M, Stingl K, Ecobichon C, Ave P, Huerre M,
Labigne A, Thiberge J-M, De Reuse H (2008) Roles of α and β carbonic anhydrases of
Helicobacter pylori in the urease-dependent response to acidity and in colonization of the
murine gastric mucosa. Infect Immun 76(2):497–509
Cappelletti D, Chiarelli LR, Pasquetto MV, Stivala S, Valentini G, Scotti C (2008) Helicobacter
pylori L-asparaginase: a promising chemotherapeutic agent. Biochem Biophys Res Commun
377:1222–1226
Cerda O, Nú~ nez-Villena F, Soto S, Ugalde J, Lopez-Solı́s R, Toledo H (2011) tlpA gene
expression is required for arginine and bicarbonate chemotaxis in Helicobacter pylori. Biol
Res 44:277–282
Chirica L, Elleby B, Lindskog S (2001) Cloning, expression and some properties of alpha-carbonic
anhydrase from Helicobacter pylori. Biochim Biophys Acta 1544:55–63
Chuawong P, Hendrickson T (2006) The nondiscriminating aspartyl-tRNA synthetase from
Helicobacter pylori: anticodon-binding domain mutations that impact tRNA specificity and
heterologous toxicity. Biochemistry 45:8079–8087
Contreras M, Thiberge JM, Mandrand-Berthelot MA, Labigne A (2003) Characterization of the
roles of NikR, a nickel-responsive pleiotropic autoregulator of Helicobacter pylori. Mol
Microbiol 49(4):947–963
Das P, Lahiri A, Lahiri A, Chakravortty D (2010) Modulation of the arginase pathway in the
context of microbial pathogenesis: a metabolic enzyme moonlighting as an immune modulator.
PLoS Pathog 6:e1000899
De Reuse H, Skouloubris S (2001) Metabolism of nitrogenous compounds. In: Mobley H,
Mendz G, Hazell S (eds) Helicobacter pylori: physiology and genetics. ASM Press,
Washington, DC
De Reuse H, Vinella D, Cavazza C (2013) Common themes and unique proteins for the uptake and
trafficking of nickel, a metal essential for the virulence of Helicobacter pylori. Front Cell Infect
Microbiol 3:94. doi:10.3389/fcimb.2013.00094
Eaton KA, Krakowka S (1994) Effect of gastric pH on urease-dependent colonization of gnoto-
biotic piglets by Helicobacter pylori. Infect Immun 62(9):3604–3607
Eaton KA, Brooks CL, Morgan DR, Krakowka S (1991) Essential role of urease in pathogenesis of
gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun 59:2470–2475
Ernst FD, Stoof J, Horrevoets WM, Kuipers EJ, Kusters JG, van Vliet AHM (2006) NikR mediates
nickel-responsive transcriptional repression of the Helicobacter pylori outer membrane pro-
teins FecA3 (HP1400) and FrpB4 (HP1512). Infect Immun 74(12):6821–6828
Farrugia M, Macomber L, Hausinger R (2013) Biosynthesis of the urease metallocenter. J Biol
Chem 288:13178–13185
Fischer F, Huot J, Lorber B, Diss G, Hendrickson T, Becker H, Lapointe J, Kern D (2012) The
asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in
non-discriminating aspartyl-tRNA synthetase safeguards the genetic code. Nucleic Acids
Res 40:4965–4976
Fong YH, Wong HC, Yuen MH, Lau PH, Chen YW, Wong KB (2013) Structure of UreG/UreF/
UreH complex reveals how urease accessory proteins facilitate maturation of Helicobacter
pylori urease. PLoS Biol 11:e1001678
Fontecilla-Camps JC, Volbeda A, Cavazza C, Nicolet Y (2007) Structure/function relationships of
[NiFe]- and [FeFe]-hydrogenases. Chem Rev 107:4273–4303
Foster JW (2004) Escherichia coli acid resistance: tales of an amateur acidophile. Nat Rev
Microbiol 2:898–907
Frost SC, McKenna R (2014) Carbonic anhydrase: mechanism, regulation, links to disease, and
industrial applications, vol 75, Subcellular biochemistry. Springer, Dordrecht
Fulkerson JF, Mobley HLT (2000) Membrane topology of the NixA nickel transporter of
Helicobacter pylori: two nickel transport-specific motifs within transmembrane helices II
and III. J Bacteriol 182(6):1722–1730
Garner RM, Fulkerson J Jr, Mobley HL (1998) Helicobacter pylori glutamine synthetase lacks
features associated with transcriptional and posttranslational regulation. Infect Immun 66
(5):1839–1847
Ge R, Sun X, Wang D, Zhou Q, Sun H (2011) Histidine-rich protein Hpn from Helicobacter pylori
forms amyloid-like fibrils in vitro and inhibits the proliferation of gastric epithelial AGS cells.
Biochim Biophys Acta 1813:1422–1427
Gilbert J, Ramakrishna J, Sunderman F Jr, Wright A, Plaut A (1995) Protein Hpn: cloning and
characterization of a histidine-rich metal-binding polypeptide in Helicobacter pylori and
Helicobacter mustelae. Infect Immun 63(7):2682–2688
Gobert A, McGee D, Akhtar M, Mendz G, Newton J, Cheng Y, Mobley H, Wilson K (2001)
Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: a strategy for
bacterial survival. Proc Natl Acad Sci U S A 98:13844–13849
Gray L, Gu S, Quick M, Khademi S (2011) Transport kinetics and selectivity of HpUreI, the urea
channel from Helicobacter pylori. Biochemistry 50:8656–8663
Ha NC, Oh ST, Sung JY, Cha KA, Lee MH, Oh BH (2001) Supramolecular assembly and acid
resistance of Helicobacter pylori urease. Nat Struct Biol 8(6):505–509
Herbst RW, Perovic I, Martin-Diaconescu V, O’Brien K, Chivers PT, Pochapsky SS, Pochapsky
TC, Maroney MJ (2010) Communication between the zinc and nickel sites in dimeric HypA:
metal recognition and pH sensing. J Am Chem Soc 132:10338–10351
Hu LT, Mobley HL (1990) Purification and N-terminal analysis of urease from Helicobacter
pylori. Infect Immun 58:992–998
Hung C, Liu J, Chiu W, Huang S, Hwang J, Wang W (2007) Crystal structure of Helicobacter
pylori formamidase AmiF reveals a cysteine-glutamate-lysine catalytic triad. J Biol Chem
282:12220–12229
Huot J, Fischer F, Corbeil J, Madore E, Lorber B, Diss G, Hendrickson T, Kern D, Lapointe J
(2011) Gln-tRNAGln synthesis in a dynamic transamidosome from Helicobacter pylori, where
GluRS2 hydrolyzes excess Glu-tRNAGln. Nucleic Acids Res 39:9306–9315
Kansau I, Guillain F, Thiberge J-M, Labigne A (1996) Nickel binding and immunological
properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA).
Mol Microbiol 22:1013–1023
Kim SH, Langford M, Boucher J, Testerman T, McGee D (2011) Helicobacter pylori arginase
mutant colonizes arginase II knockout mice. World J Gastroenterol 17:3300–3309
Labigne A, Cussac V, Courcoux P (1991) Shuttle cloning and nucleotide sequence of Helicobacter
pylori genes responsible for urease activity. J Bacteriol 173:1920–1931
Leduc D, Gallaud J, Stingl K, De Reuse H (2010) Coupled amino acid deamidase-transport
systems essential for Helicobacter pylori colonization. Infect Immunol 78:2782–2792
Leigh J, Dodsworth J (2007) Nitrogen regulation in bacteria and archaea. Annu Rev Microbiol
61:349–377
Maier T, Bock A (1996) Generation of active NiFe hydrogenase in vitro from a nickel-free
precursor form. Biochemistry 35(31):10089–10093
Maier RJ, Fu C, Gilbert J, Moshiri F, Olson J, Plaut AG (1996) Hydrogen uptake hydrogenase in
Helicobacter pylori. FEMS Microbiol Lett 141(1):71–76
Marais A, Mendz GL, Hazell SL, Megraud F (1999) Metabolism and genetics of Helicobacter
pylori: the genome era. Microbiol Mol Biol Rev 63(3):642–674
Marcus EA, Moshfegh AP, Sachs G, Scott D (2005) The periplasmic alpha-carbonic anhydrase
activity of Helicobacter pylori is essential for acid acclimation. J Bacteriol 187:729–738
Marcus E, Sachs G, Wen Y, Feng J, Scott DR (2012) Role of the Helicobacter pylori sensor kinase
ArsS in protein trafficking and acid acclimation. J Bacteriol 194:5545–5551
Marcus E, Sachs G, Scott D (2013) The role of ExbD in periplasmic pH homeostasis in
Helicobacter pylori. Helicobacter 18:363–372
McGee DJ, Radcliff FJ, Mendz GL, Ferrero RL, Mobley HL (1999) Helicobacter pylori rocF is
required for arginase activity and acid protection in vitro but is not essential for colonization of
mice or for urease activity. J Bacteriol 181(23):7314–7322
McGee DJ, Zabaleta J, Viator RJ, Testerman TL, Ochoa AC, Mendz GL (2004) Purification and
characterization of Helicobacter pylori arginase, RocF: unique features among the arginase
superfamily. Eur J Biochem 271:1952–1962
McNulty R, Ulmschneider J, Luecke H, Ulmschneider M (2013) Mechanisms of molecular
transport through the urea channel of Helicobacter pylori. Nat Commun 4:2900
Mendz GL, Hazell SL (1995) Aminoacid utilization by Helicobacter pylori. Int J Biochem Cell
Biol 27(10):1085–1093
Mendz GL, Holmes EM, Ferrero RL (1998) In situ characterization of Helicobacter pylori
arginase. Biochim Biophys Acta 1388(2):465–477
Merrell DS, Goodrich ML, Otto G, Tompkins LS, Falkow S (2003) pH-regulated gene expression
of the gastric pathogen Helicobacter pylori. Infect Immun 71:3529–3539
Miller E, Maier R (2014) Ammonium metabolism enzymes aid Helicobacter pylori acid resis-
tance. J Bacteriol 196:3074–3081
Mobley HLT, Hausinger RP (1989) Microbial ureases: significance, regulation, and molecular
characterization. Microbiol Rev 53:85–108
Mobley HLT, Garner RM, Chippendale GR, Gilbert JV, Kane AV, Plaut AG (1999) Role of Hpn
and NixA of Helicobacter pylori in susceptibility and resistance to bismuth and other metal
ions. Helicobacter 4(3):162–169
Mollenhauer-Rektorschek M, Hanauer G, Sachs G, Melchers K (2002) Expression of UreI is
required for intragastric transit and colonization of gerbil gastric mucosa by Helicobacter
pylori. Res Microbiol 153:659–666
Muller S, Gotz M, Beier D (2009) Histidine residue 94 is involved in pH sensing by histidine
kinase ArsS of Helicobacter pylori. PLoS One 4:e6930
Muller C, Bahlawane C, Aubert S, Delay CM, Schauer K, Michaud-Soret I, De Reuse H (2011)

Hierarchical regulation of the NikR-mediated nickel response in Helicobacter pylori. Nucleic
Acids Res 39:7564–7575
Nakamura A, Yao M, Chimnaronk S, Sakai N, Tanaka I (2006) Ammonia channel couples
glutaminase with transamidase reactions in GatCAB. Science 312:1954–1958
Nolan KJ, McGee DJ, Mitchell HM, Kolesnikow T, Harro JM, O’Rourke J, Wilson JE, Danon SJ,
Moss ND, Mobley HLT, Lee A (2002) In vivo behaviour of a Helicobacter pylori SS1 nixA
mutant with reduced urease activity. Infect Immun 70:685–691
Olson JW, Maier RJ (2002) Molecular hydrogen as an energy source for Helicobacter pylori.
Science 298(5599):1788–1790
Olson JW, Mehta NS, Maier RJ (2001) Requirement of nickel metabolism proteins HypA and
HypB for full activity of both hydrogenase and urease in Helicobacter pylori. Mol Microbiol
39:176–182
Pflock M, Kennard S, Delany I, Scarlato V, Beier D (2005) Acid-induced activation of the urease
promoters is mediated directly by the ArsRS two-component system of Helicobacter pylori.
Infect Immunol 73:6437–6445
Pflock M, Kennard S, Finsterer N, Beier D (2006) Acid-responsive gene regulation in the human
pathogen Helicobacter pylori. J Biotechnol 126:52–60
Rain J-C, Selig L, De Reuse H, Battaglia V, Reverdy C, Simon S, Lenzen G, Petel F, Wojcik J,
Schächter V, Chemama Y, Labigne A, Legrain P (2001) The protein-protein interaction map of
the bacterial pathogen Helicobacter pylori. Nature 409:211–215
Rimbara E, Mori S, Kim H, Shibayama K (2013) Role of γ-glutamyltranspeptidase in the
pathogenesis of Helicobacter pylori infection. Microbiol Immunol 57:665–673
Romagnoli S, Agriesti F, Scarlato V (2011) In vivo recognition of the fecA3 target promoter by
Helicobacter pylori NikR. J Bacteriol 193:1131–1141
Rowinska-Zyrek M, Witkowska D, Bielinska S, Kamysz W, Kozlowski H (2011) The -Cys-Cys-
motif in Helicobacter pylori’s Hpn and HspA proteins is an essential anchoring site for metal
ions. Dalton Trans 40:5604–5610
Rowinska-Zyrek M, Zakrzewska-Czerwinska J, Zawilak-Pawlik A, Kozlowski H (2014) Ni2+
chemistry in pathogens – a possible target for eradication. Dalton Trans 43:8976–8989
Schauer K (2007) Étude du Métabolisme du Nickel chez Helicobacter pylori, vol 7, Université
Paris. Denis Diderot, Paris
Schauer K, Gouget B, Carriere M, Labigne A, de Reuse H (2007) Novel nickel transport
mechanism across the bacterial outer membrane energized by the TonB/ExbB/ExbD machin-
ery. Mol Microbiol 63:1054–1068
Schauer K, Rodionov DA, de Reuse H (2008) New substrates for TonB-dependent transport: do
we only see the ‘tip of the iceberg’? Trends Biochem Sci 33(7):330–338
Schauer K, Muller C, Carriere M, Labigne A, Cavazza C, De Reuse H (2010) The Helicobacter
pylori GroES cochaperonin HspA functions as a specialized nickel chaperone and sequestra-
tion protein through its unique C-terminal extension. J Bacteriol 192:1231–1237
Schreiber S, Konradt M, Groll C, Scheid P, Hanauer G, Werling H-O, Josenhans C, Suerbaum S
(2004) The spatial orientation of Helicobacter pylori in the gastric mucus. Proc Natl Acad Sci
U S A 101:5024–5029
Schreiber S, Bücker R, Groll C, Azevedo-Vethacke M, Garten D, Scheid P, Friedrich S,
Gatermann S, Josenhans C, Suerbaum S (2005) Rapid loss of motility of Helicobacter pylori
in the gastric lumen in vivo. Infect Immun 73:1584–1589
Scott DR, Weeks D, Hong C, Postius S, Melchers K, Sachs G (1998) The role of internal urease in
acid resistance of Helicobacter pylori. Gastroenterology 114:58–70
Scott DR, Marcus EA, Weeks DL, Lee A, Melchers K, Sachs G (2000) Expression of the
Helicobacter pylori ureI gene is required for acidic pH activation of cytoplasmic urease. Infect
Immunol 68:470–477
Scott DR, Marcus EA, Wen Y, Oh J, Sachs G (2007) Gene expression in vivo shows that
Helicobacter pylori colonizes an acidic niche on the gastric surface. Proc Natl Acad Sci U S
A 104:7235–7240
Scott D, Marcus E, Wen Y, Singh S, Feng J, Sachs G (2010) Cytoplasmic histidine kinase
(HP0244)-regulated assembly of urease with UreI, a channel for urea and its metabolites,
CO2, NH3, and NH4+, is necessary for acid survival of Helicobacter pylori. J Bacteriol
192:94–103
Scotti C, Sommi P, Pasquetto M, Cappelletti D, Stivala S, Mignosi P, Savio M, Chiarelli L,
Valentini G, Bolanos-Garcia V, Merrell D, Franchini S, Verona M, Bolis C, Solcia E, Manca R,
Franciotta D, Casasc OA, Filipazzi P, Zardini E, Vannini V (2010) Cell-cycle inhibition by
Helicobacter pylori L-asparaginase. PLoS One 5:e13892
Seshadri S, Benoit SL, Maier RJ (2007) Roles of His-rich hpn and hpn-like proteins in
Helicobacter pylori nickel physiology. J Bacteriol 189(11):4120–4126
Sheppard K, Akochy P, Salazar J, S€oll D (2007) The Helicobacter pylori amidotransferase
GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and
Glu-tRNAGln. J Biol Chem 282:11866–11873
Sheppard K, Yuan J, Hohn M, Jester B, Devine K, S€oll D (2008) From one amino acid to another:
tRNA-dependent amino acid biosynthesis. Nucleic Acids Res 36:1813–1825
Shibayama K, Wachino J-I, Arakawa Y, Saidijam M, Rutherford NG, Henderson PJF (2007)
Metabolism of glutamine and glutathione via g-glutamyltranspeptidase and glutamate trans-
port in Helicobacter pylori: possible significance in the pathophysiology of the organism. Mol
Shibayama K, Takeuchi H, Wachino J, Mori S, Arakawa Y (2011) Biochemical and pathophys-
iological characterization of Helicobacter pylori asparaginase. Microbiol Immunol
55:408–417
Silva G, Fatma S, Floyd A, Fischer F, Chuawong P, Cruz A, Simari R, Joshi N, Kern D,
Hendrickson T (2013) A tRNA-independent mechanism for transamidosome assembly pro-
motes aminoacyl-tRNA transamidation. J Biol Chem 288:3816–3822
Skouloubris S, Labigne A, De Reuse H (1997) Identification and characterization of an aliphatic
amidase in Helicobacter pylori. Mol Microbiol 25:989–998
Skouloubris S, Thiberge JM, Labigne A, De Reuse H (1998) The Helicobacter pylori UreI protein
is not involved in urease activity but is essential for bacterial survival in vivo. Infect Immun 66
(9):4517–4521
Skouloubris S, Labigne A, De Reuse H (2001) The AmiE aliphatic amidase and AmiF
formamidase of Helicobacter pylori: natural evolution of two enzyme paralogs. Mol Microbiol
40:596–609
Skouloubris S, de Pouplana LR, de Reuse H, Hendrickson TL (2003) A noncognate aminoacyl-
tRNA synthetase that may resolve a missing link in protein evolution. Proc Natl Acad Sci U S
A 100(20):11297–11302
Stahler FN, Odenbreit S, Haas R, Wilrich J, Van Vliet AH, Kusters JG, Kist M, Bereswill S (2006)
The novel Helicobacter pylori CznABC metal efflux pump is required for cadmium, zinc, and
nickel resistance, urease modulation, and gastric colonization. Infect Immun 74(7):3845–3852
Stark RM, Suleiman MS, Hassan IJ, Greenman J, Millar MR (1997) Amino acid utilisation and
deamination of glutamine and asparagine by Helicobacter pylori. J Med Microbiol 46:793–800
Stingl K, De Reuse H (2005) Staying alive overdosed: how does Helicobacter pylori control
urease activity? Int J Med Microbiol 295(5):307–315
Stingl K, Schauer K, Ecobichon C, Labigne A, Lenormand P, Rousselle JC, Namane A, de Reuse
H (2008) In vivo interactome of Helicobacter pylori urease revealed by tandem affinity
purification. Mol Cell Proteomics 7(12):2429–2441
Strugatsky D, McNulty R, Munson K, Chen C, Soltis S, Sachs G, Luecke H (2013) Structure of the
proton-gated urea channel from the gastric pathogen Helicobacter pylori. Nature 493:255–258
Suerbaum S, Thiberge JM, Kansau I, Ferrero RL, Labigne A (1994) Helicobacter pylori hspA-
hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immu-
nogenicity. Mol Microbiol 14(5):959–974
Tomb J-F, White O, Kerlavage AR, Clayton RA, Sutton GG, Fleischmann RD, Ketchum KA,
Klenk HP, Gill S, Dougherty BA et al (1997) The complete genome sequence of the gastric
pathogen Helicobacter pylori. Nature 388:539–547
Van Vliet AHM, Kuipers E, Waidner B, Davies BJ, De Vries N, Penn CW, Vandenbroucke-Grauls
CMJE, Kist M, Bereswill S, Kusters JG (2001) Nickel-responsive induction of urease expres-
sion in Helicobacter pylori is mediated at the transcriptional level. Infect Immunol
69:4891–4897
van Vliet AHM, Poppelaars SW, Davies BJ, Stoof J, Bereswill S, Kist M, Penn CW, Kuipers EJ,
Kusters JG (2002) NikR mediates nickel-responsive transcriptional induction of urease expres-
sion in Helicobacter pylori. Infect Immun 70(6):2846–2852
Van Vliet AHM, Stoof J, Poppelaars SW, Heijens A, Kuipers EJ, Kusters JG (2003) Acid- and
nickel-responsive transcriptional induction of ammonia-producing enzymes in Helicobacter
pylori. Gastroenterology 124(4):A52–A52
van Vliet AHM, Ernst FD, Kuipers EJ, Heijens A, Stoof J, Kusters JG (2004a) The NikR protein
governs transcriptional regulation of NixA-mediated nickel-uptake in Helicobacter pylori.
Gastroenterology 126(4):A402–A402
van Vliet AHM, Kuipers EJ, Stoof J, Poppelaars SW, Kusters JG (2004b) Acid-responsive gene
induction of ammonia-producing enzymes in Helicobacter pylori is mediated via a metal-
responsive repressor cascade. Infect Immun 72(2):766–773
Vinella D, Fischer F, Vorontsov E, Gallaud J, Malosse C, Michel V, Cavazza C, Robbe-Saule M,
Richaud P, Chamot-Rooke J, Brochier-Armanet C, de Reuse H (2015) Evolution of
Helicobacter: acquisition by gastric species of two Histidine-rich proteins essential for colo-
nization. PLoS Pathog 11(12). doi:10.1371/journal.ppat.1005312
Voland P, Weeks D, Marcus E, Prinz C, Sachs G, Scott D (2003) Interactions among the seven
Helicobacter pylori proteins encoded by the urease gene cluster. Am J Physiol Gastrointest
Liver Physiol 284:G96–G106
Watanabe S, Sasaki D, Tominaga T, Miki K (2012) Structural basis of [NiFe] hydrogenase
maturation by Hyp proteins. Biol Chem 393:1089–1100
Weeks DL, Sachs G (2001) Sites of pH regulation of the urea channel of Helicobacter pylori. Mol
Weeks DL, Eskandari S, Scott DR, Sachs G (2000) A H+-gated urea channel: the link between
Helicobacter pylori urease and gastric colonization. Science 287:482–485
Wen Y, Marcus EA, Matrubutham U, Gleeson MA, Scott DR, Sachs G (2003) Acid-adaptive
genes of Helicobacter pylori. Infect Immun 71:5921–5939
Wen Y, Feng J, Scott DR, Marcus EA, Sachs G (2007) The HP0165-HP0166 two-component
system (ArsRS) regulates acid-induced expression of HP1186 alpha-carbonic anhydrase in
Helicobacter pylori by activating the pH-dependent promoter. J Bacteriol 189:2426–2434
Williams CL, Preston T, Hossack M, Slater C, McColl KEL (1996) Helicobacter pylori utilises
urea for amino acid synthesis. FEMS Immunol Med Microbiol 13:87–94
Wolfram L, Haas E, Bauerfeind P (2006) Nickel represses the synthesis of the nickel permease
NixA of Helicobacter pylori. J Bacteriol 188(4):1245–1250
Yang X, Li H, Cheng T, Xia W, Lai Y, Sun H (2014) Nickel translocation between
metallochaperones HypA and UreE in Helicobacter pylori. Metallomics 6:1731–1736
Zabaleta J, McGee D, Zea A, Hernandez C, Rodriguez P et al (2004) Helicobacter pylori arginase
inhibits T cell proliferation and reduces the expression of the TCR zeta-chain (CD3zeta). J
Immunol 173:586–593
Zambelli B, Turano P, Musiani F, Neyroz P, Ciurli S (2009) Zn2+-linked dimerization of UreG
from Helicobacter pylori, a chaperone involved in nickel trafficking and urease activation.
Proteins Struct Funct Bioinform 74(1):222–239
Zambelli B, Berardi A, Martin-Diaconescu V, Mazzei L, Musiani F, Maroney M, Ciurli S (2014)
Nickel binding properties of Helicobacter pylori UreF, an accessory protein in the nickel-based
activation of urease. J Biol Inorg Chem 19:319–334
Zeng Y, Yang N, Sun H (2011) Metal-binding properties of an Hpn-like histidine-rich protein.
Chem Eur J 17:5852–5860
Zeng YB, Zhang DM, Li H, Sun H (2008) Binding of Ni2+ to a histidine- and glutamine-rich
protein, Hpn-like. J Biol Inorg Chem 13:1121–1131
Chapter 3
Virulence Mechanisms of Helicobacter pylori:
An Overview
Judyta Praszkier, Philip Sutton, and Richard L. Ferrero
Abstract Helicobacter pylori is a highly successful human pathogen, able to

establish a chronic infection in the harsh environment of the stomach. These
bacteria express a variety of virulence factors that promote their survival under
acidic conditions, motility and spatial orientation in gastric mucus and adherence to
epithelial cells. Other pathogenicity-associated mechanisms contribute to chronic
gastritis by inducing pro-inflammatory responses and by manipulating cellular
responses in host cells. Although H. pylori elicits a strong inflammatory response,
the immune system fails to clear the infection. The pathogen employs a range of
evasion strategies to dampen or reduce host immune responses. These strategies
enable H. pylori to establish an equilibrium with its host, so that the vast majority of
the chronically infected individuals do not develop severe disease. However, in a
subset of patients, disturbance of this equilibrium in favour of the pathogen may
lead to the development of gastroduodenal ulceration, mucosa-associated lymphoid
tissue (MALT) lymphoma or adenocarcinoma.
Keywords Virulence factors • Pathogenesis • Urease • Motility • Adhesion •

Immunomodulation • Apoptosis • Autophagy
3.1 Introduction
Helicobacter pylori is one of the most successful human pathogens, colonising

more than 50 % of the world’s population (Suerbaum and Josenhans 2007). The
infection is usually acquired in early childhood (Weyermann et al. 2009) and, in the
absence of aggressive antibiotic therapy, typically persists for life (Suerbaum and
J. Praszkier • R.L. Ferrero (*)

C/- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research,
Monash University, 27-31 Wright Street, Clayton 3168, Victoria, Australia
e-mail: [email protected]; [email protected]
P. Sutton
Mucosal Immunology Research, Murdoch Childrens Research Institute, Flemington Road,
Parkville 3052, Victoria, Australia

DOI 10.1007/978-4-431-55936-8_3
58 J. Praszkier et al.
Josenhans 2007). Most of the colonised individuals develop asymptomatic chronic

gastritis, but in 10–20 % of cases, H. pylori infection is associated with the
development of severe gastroduodenal disease, including peptic ulcers, gastric
MALT lymphoma or adenocarcinoma (Kusters et al. 2006). H. pylori infection is
the strongest known risk factor for gastric adenocarcinoma, and in 1994 the
International Agency for Research on Cancer classified H. pylori as a class I
carcinogen. The clinical outcome of H. pylori infection depends on a complex
interplay of many factors, including the virulence determinants expressed by the
colonising strain(s), the genetic background of the host and environmental factors.
H. pylori strains show extensive genetic diversity, which is a consequence of the
high mutation (Bjorkholm et al. 2001) and recombination frequencies (Suerbaum
et al. 1998) in this bacterium. H. pylori has undergone a long co-evolution with its
human host. Indeed, it is estimated that the association of H. pylori with modern
humans predates by some 40,000 years the human migration from East Africa that
occurred approximately 60,000 years ago (Moodley et al. 2012) (see Chap. 9 for
more details). During this long association with humans, H. pylori has evolved
sophisticated mechanisms to persistently colonise its host and avoid elimination by
the immune system. These mechanisms range from colonisation factors that allow
the bacterium to survive the harsh acidic environment of the stomach and establish
persistent infection in the gastric mucosa, to complex strategies involving virulence
factors that enable H. pylori to evade and manipulate both innate and adaptive
immune responses. The lack of disease progression in the vast majority of persis-
tently colonised individuals points to a delicate balance between the host and the
pathogen. This chapter summarises the bacterial factors and biological processes
that enable H. pylori to establish persistent colonisation and chronic inflammation
of the human gastric mucosa. A more in-depth analysis of several of these products
and processes will be provided in the chapters to follow.
3.2 H. pylori Colonisation and Adherence
3.2.1 Escape from the Stomach Lumen
In order to reach its ideal ecological niche, H. pylori must survive the extremely
acidic environment of the stomach lumen and penetrate the outer mucous gel layer
of the stomach. Once in the mucus, H. pylori resides in a very specific niche with an
external pH of approximately 5–6. The bacterium is, however, able to increase the
pH of its immediate surrounds, as well as of its cytosol and periplasm, by producing
urease, which hydrolyses urea to ammonium ions and carbon dioxide (Marshall
et al. 1990). Urease is composed of UreA and UreB subunits (Labigne et al. 1991)
which are assembled into a catalytically active, nickel-containing dodecamer via
the actions of accessory proteins UreE, UreF, UreG and UreH (Mobley et al. 1995).
Urease activity is up-regulated under acidic conditions, by a proton-gated urea
3 Virulence Mechanisms of Helicobacter pylori: An Overview 59
channel formed by the inner membrane protein, UreI, allowing rapid entry of urea
into the bacteria (Skouloubris et al. 1998; Weeks et al. 2000). H. pylori can further
tightly control urease activity in response to both an acidic pH and increasing
concentrations of nickel ions. This occurs via up-regulation of urease gene expres-
sion by the acid-responsive signalling regulon (ArsRS) (Pflock et al. 2006) and the
nickel response regulator, NikR (van Vliet et al. 2002), respectively. H. pylori
mutants lacking either urease activity (through disruption of ureB) or a functional
UreI were shown to be defective for colonisation in animal models of infection
(Eaton et al. 2002; Skouloubris et al. 1998), thus demonstrating the essential role of
urease in H. pylori pathogenesis (see also Chap. 2).
Having overcome the acidic lumen, H. pylori must next confront the viscous
mucous gel covering the gastric epithelium. Gastric mucus varies in its viscoelastic
properties, from a soft gel at acidic pH to a viscous solution at neutral pH. H. pylori
is well adapted to this environment and is able to move rapidly, in a corkscrew like
manner, through highly viscous solutions that impede the motility of rod-shaped
organisms, such as Escherichia coli (Hazell et al. 1986). The spiral cell shape of
H. pylori is believed to enhance its ability to penetrate the mucus, and mutants
lacking a helical twist show a colonisation defect. Production and maintenance of
the spiral morphology require coordinated activity of multiple enzyme networks
that modify the peptidoglycan composition of the cell wall (Bonis et al. 2010;
Sycuro et al. 2012).
In addition to its spiral shape, H. pylori uses flagella-mediated motility to move
through both the gastric lumen and mucus to reach and maintain itself close to the
epithelial surface. The importance of motility for H. pylori is illustrated by the fact
that mutants lacking flagella were unable to colonise the gnotobiotic piglet model of
infection (Eaton et al. 1996). H. pylori bacteria have two to five sheathed, unipolar
flagella which are composed of the basal body, the hook/universal joint and the
filament. The flagellar filament is composed of repeating units of two flagellins,
FlaA (53 kilodalton, kDa) and FlaB (54 kDa) (Suerbaum et al. 1993). The flaA and
flaB genes are not co-located on the chromosome, nor is their transcription
co-regulated (Suerbaum et al. 1993). The flagellar system comprises a network of
over 40 mostly unclustered and temporally regulated genes, whose transcription is
hierarchical and tightly controlled by the three RNA polymerase sigma factors of
H. pylori: σ28 (FliA), σ54 (RpoN) and σ80 (Douillard et al. 2009; Josenhans
et al. 2002; McGowan et al. 2003; Niehus et al. 2004). H. pylori also has an anti-
sigma factor, FlgM, which acts as an antagonist to FlaA (Colland et al. 2001;
Niehus et al. 2004). Flagellar assembly requires interaction with the peptidoglycan
layer through which the flagella has to be extruded. The peptidoglycan-degrading
enzymes of the lytic transglycosylase family, Slt and MltD, are required for full
motility in H. pylori (Roure et al. 2012). Inactivation of mltD, but not slt, was shown
to have a significant impact on H. pylori colonisation in vivo (Roure et al. 2012).
Motile bacteria use chemotaxis for spatial orientation, coupling control of
flagellar rotation with environmental sensing (Wadhams and Armitage 2004).
H. pylori uses four methyl-accepting chemoreceptor proteins (TlpA, TlpB, TlpC
and TlpD) to sense the external stimuli and repellent ligands. This information is
then relayed via CheW, to a histidine kinase, CheA, which phosphorylates the
response regulator, CheY. The phosphorylated CheY interacts with the flagellar
motor to alter the rotational direction of the flagellum. H. pylori mutants lacking
cheA, cheW or cheY are non-chemotactic and show colonisation defects (Terry
et al. 2005). In addition, H. pylori encodes a novel chemotaxis regulator, ChePep,
which preferentially localises to the flagellar pole. H. pylori chePep mutants cannot
control the rotation of their flagella, but are motile. They are attenuated for
colonisation of the stomach and fail to establish bacterial colonies deep in the
gastric glands of mice. Interestingly, ChePep homologues are present and function-
ally conserved in ε-proteobacteria, but not in other bacterial classes (Howitt
et al. 2011).
Microarray analyses of H. pylori showed that both exposure to low pH in vitro
and infection of the gerbil stomach in vivo resulted in increased expression of many
of the genes involved in motility and chemotaxis (Merrell et al. 2003; Scott
et al. 2007). These findings are consistent with data showing that exposure of
H. pylori to an acidic environment leads to a large increase in both the proportion
and the speed of motile bacteria (Merrell et al. 2003). H. pylori exploits the pH
gradient of the stomach, which ranges from pH 1.8 in the lumen to a near-neutral
pH at the mucus-mucosal interface, to guide it to the epithelial surface. This pH-
tactic behaviour is dependent on the chemotaxis receptor, TlpB (Croxen
et al. 2006). H. pylori tlpB mutants were shown to be motile but could not colonise
interleukin-12 p40 (IL-12 p40)-deficient C57BL/6 mice (Croxen et al. 2006).
Expression of tlpB is regulated at the posttranscriptional level by an abundant
small RNA (sRNA), regulator of polymeric G-repeats (RepG), which targets a
homopolymeric G-repeat in the leader region of the tlpB mRNA. The length of this
G-repeat, which varies from 6 to 16 guanine residues in different H. pylori strains,
influences both the level and type (repression or activation) of regulation. There is
also evidence that the length of the tlpB G-repeat can change during infection,
suggesting that differential expression of tlpB may be involved in host adaptation
(Pernitzsch et al. 2014).
3.2.2 Adhesion of H. pylori to Gastric Epithelial Cells
Although most of the H. pylori in the mucosa are free-swimming, some 20 % of the
bacteria adhere to the surface of the epithelial cells (Hessey et al. 1990). Binding of
H. pylori to gastric epithelial cells involves the interactions between specific
bacterial adhesins and their cognate receptors on the surfaces of host cells. The
known H. pylori adhesins all belong to the major outer membrane protein (OMP)
family 1 (Alm et al. 2000). This family of proteins is further divided into the
Helicobacter outer membrane porins (Hop) and Hop-related (Hor) subgroups
(Alm et al. 2000; Odenbreit et al. 2009). All but one of the adhesins identified to
date are members of the Hop family. The best characterised of these adhesins are
the blood group antigen-binding (BabA) and sialic acid-binding (SabA) proteins,
the outer inflammatory protein A (OipA) and the adherence-associated lipoproteins,

AlpA and AlpB.
BabA binds to the human fucosylated Lewisb antigen (Leb) and related terminal
fucose residues on blood group O (H antigen), A and B antigens present on the
surface of gastric epithelial cells (Aspholm-Hurtig et al. 2004; Gerhard et al. 1999;
Ilver et al. 1998). BabA binding affinity for O, A and B antigens correlates with the
blood group expressed by the human host, supporting the notion of adaptation to the
host during persistent infection and transmission between hosts (Aspholm-Hurtig
et al. 2004). In contrast, SabA binds to sialyl-Lewis x (sLex) and Lewis a (sLea)
antigens whose synthesis is up-regulated during H. pylori-induced inflammation
(Mahdavi et al. 2002). SabA also binds to erythrocytes (Aspholm et al. 2006) and
may play a role in inflammation, by binding and activating neutrophils (Unemo
et al. 2005). More details on the effects of BabA and SabA on gastroduodenal
disease can be found in Chap. 6.
OipA (formerly called HopH) is involved in the adherence of H. pylori to gastric
cell lines (Dossumbekova et al. 2006; Yamaoka et al. 2004), but its host receptor
has yet to be identified. Expression of functional OipA, which is regulated by
slipped-strand mispairing within a CT-rich region present at the 50 terminus of
oipA, promoted IL-8 induction in vitro. Conversely, oipA inactivation in cagPAI+
clinical isolates resulted in approximately 50 % lower IL-8 responses in epithelial
cells (Yamaoka et al. 2004). The notion that OipA has a role in IL-8 production
in vivo is supported by the observation that OipA expression was significantly
associated with high levels of IL-8 in the gastric mucosa of infected patients
(Yamaoka et al. 2002). The molecular basis for the effect of OipA on IL-8
production was investigated in a study of the IL-8 promoter in gastric cell cultures
(Yamaoka et al. 2004). This study showed that maximal induction of IL-8 tran-
scription required activation of an interferon-stimulated responsive element
(ISRE)-like element by the interferon regulatory factor (IRF)-1 (Yamaoka
et al. 2004). Moreover, OipA was reported to selectively induce phosphorylation
of signal transducer and activator of transcription 1 (STAT1), an upstream mediator
of IRF-1 signalling. These results were recapitulated in vivo, where it was found
that STAT1 phosphorylation in human gastric biopsy specimens correlated with the
presence of functional OipA in the infecting H. pylori strain (Yamaoka et al. 2004).
It has also been proposed that OipA plays a role in IL-1β, IL-17 and TNF expression
and inflammation in the stomach (Sugimoto et al. 2009). Analysis of clinical
isolates showed that the presence of functional OipA is associated with high
H. pylori density, severe neutrophil infiltration, duodenal ulcer disease and gastric
cancer (Yamaoka et al. 2002, 2006; Franco et al. 2008). The role of OipA in gastric
disease is supported by data showing that inactivation of oipA reduces the incidence
of cancer in Mongolian gerbils and decreases nuclear translocation of β-catenin
(Franco et al. 2008), a cellular protein important for cell adhesion and regulation of
genes implicated in carcinogenesis. In vitro studies with murine dendritic cells
(DCs) showed that OipA suppresses DC maturation and decreases production of
IL-10 (Teymournejad et al. 2014); however, the biological significance of this
finding requires further investigation.
AlpA and AlpB, which are encoded by the alpA/B operon, are also required for
specific adhesion of H. pylori to human gastric epithelial cells (Odenbreit
et al. 1999). Furthermore, these proteins were shown to be important for colonisa-
tion in guinea pig (de Jonge et al. 2004) and murine (Lu et al. 2007) models of
infection. Analysis of 200 clinical strains showed that AlpA and AlpB were
expressed in all strains, suggesting that these adhesins are likely to have important
functions (Odenbreit et al. 2009). The target of both AlpA and AlpB is laminin, a
component of the host extracellular matrix (Senkovich et al. 2011).
3.3 Major H. pylori Virulence Factors Involved

in Pathogenesis
3.3.1 cag Pathogenicity Island (cagPAI)
The cagPAI is a horizontally acquired insertion element of 40-kilobases (kb),

consisting of approximately 31 genes, whose presence in a functional form is
associated with an increased risk of severe gastroduodenal disease (Covacci
et al. 1999; see also Chap. 4). cagPAI encodes a bacterial type IV secretion system
(T4SS) and its only known effector protein, CagA, which translocates into gastric
epithelial cells (Censini et al. 1996; Fischer et al. 2001; Odenbreit et al. 2000). The
presence of a cagPAI appears to influence the topography of colonisation within the
stomach, as cagPAI- H. pylori strains were mostly present in the mucous gel layer
or near the apical surface of epithelial cells, whereas the cagPAI+ strains were
found closely adjacent to gastric epithelial cells or in the intercellular epithelial
spaces (Camorlinga-Ponce et al. 2004).
The H. pylori T4SS is induced by contact with the host cell and forms a large
complex spanning the inner and outer membranes of the bacterium, with a pilus-
like structure that protrudes from the bacterial surface (Rohde et al. 2003). It is
currently unclear how the H. pylori T4SS is able to deliver not only CagA but also
H. pylori cell wall peptidoglycan, into the host cell. The internalised peptidoglycan
is recognised by the cytoplasmic pathogen-recognition molecule, nucleotide-
binding oligomerisation domain-containing protein 1 (NOD1) (Viala et al. 2004).
NOD1 sensing of H. pylori peptidoglycan triggers in epithelial cells a pro-inflam-
matory signalling cascade, characterised by the translocation of nuclear factor-κB
(NF-κB) to the nucleus (Viala et al. 2004) and activation of the mitogen-activated
protein kinases (MAPKs), p38 and extracellular signal-regulated kinase (ERK),
leading to induction of CXC chemokine responses (Allison et al. 2009). H. pylori
can also activate this pro-inflammatory signalling cascade via the actions of outer
membrane vesicles (OMVs) which deliver peptidoglycan to cytosolic NOD1
(Hutton et al. 2010; Kaparakis et al. 2010). Both T4SS- and OMV-dependent
activations of the NOD1 signalling pathway involve cholesterol-rich
microdomains, or lipid rafts, in host-cell membranes (Hutton et al. 2010; Kaparakis
et al. 2010). Interestingly, CagA translocation into host cells also requires the
presence of functional lipid raft domains (Jimenez-Soto et al. 2009; Lai
et al. 2008). H. pylori T4SS delivery of CagA (Jimenez-Soto et al. 2009; Kwok
et al. 2007) and peptidoglycan (Hutton et al. 2010; Kaparakis et al. 2010) into cells
was shown to be dependent upon binding of the cagPAI-encoded protein, CagL, to
its cognate host-cell receptor, α5β1 integrin. CagA itself was shown to interact with
the host factors, β1 integrin (Jimenez-Soto et al. 2009; Kwok et al. 2007) and
phosphatidylserine (Murata-Kamiya et al. 2010).
Once CagA has translocated into epithelial cells, it localises to the plasma
membrane and undergoes tyrosine phosphorylation within the EPIYA motif that
is found in tandemly arranged segments located in the C-terminal half of the
protein. The number and organisation of these segments differ between H. pylori
strains and are thought to contribute to differences in strain pathogenicity (Argent
et al. 2004; Higashi et al. 2002). There are four distinct EPIYA segments (A to D),
each of which contains a single EPIYA motif, with the EPIYA A, B and C segments
predominating in H. pylori isolates from Western countries and EPIYA A, B and D
segments predominating in the generally more virulent East Asian isolates (Higashi
et al. 2002). The cellular kinases responsible for phosphorylating the EPIYA motifs
within CagA are oncoproteins belonging to the Src and Abl family kinases (Poppe
et al. 2007; Selbach et al. 2002; Tammer et al. 2007).
CagA translocation and tyrosine phosphorylation lead to a perturbation of
mammalian signal transduction cascades, morphological effects such as cell cyto-
skeletal rearrangement, elongation and scattering that has been designated the
“hummingbird” phenotype, as well as modification of cellular functions (Selbach
et al. 2003; Tammer et al. 2007; Tsutsumi et al. 2003). These in vitro observations
are recapitulated in vivo, with the finding that CagA is actively translocated to
gastric epithelial cells and tyrosine-phosphorylated and binds Src homology region
2 (SH2) domain-containing phosphatase-2 (SHP-2) in inflamed human gastric
mucosa (Tsutsumi et al. 2003). The ability of CagA to perturb host-cell functions
is dependent on its SHP-2 binding activity, which is determined by the number and
sequences of tyrosine phosphorylation sites (Higashi et al. 2002). It should be noted
that non-phosphorylated CagA also contributes to pathogenesis, through interac-
tions that lead to induction of pro-inflammatory and mitogenic responses, suppres-
sion of apoptosis, loss of cell polarity and disruption of gastric barrier function (see
Chap. 4 for a detailed discussion).
3.3.2 Vacuolating Cytotoxin VacA
VacA is a pore-forming toxin secreted by a classical autotransporter pathway (see

Chap. 5 for more details). The protein is synthesised as a 140 kDa precursor, which
is processed to produce a 33-amino acid signal peptide, the mature 88 kDa secreted
toxin, an approximately 12 kDa secreted peptide and a C-terminal domain that
remains associated with the bacteria (Cover and Blaser 1992; Schmitt and Haas
1994; Telford et al. 1994). The secreted VacA can undergo spontaneous proteolytic
cleavage into the N-terminal p33 and C-terminal p55 fragments that are thought to
represent the functional domains of VacA (Torres et al. 2005) and that remain
non-covalently associated (Cover et al. 1997; Lupetti et al. 1996; Telford
et al. 1994). The p33 domain is important for membrane channel formation
(McClain et al. 2001; Ye et al. 1999), whereas the p55 domain is required for
binding to host cells (Garner and Cover 1996). Both domains are required for toxin
oligomerisation (Gangwer et al. 2007; Genisset et al. 2006). Active VacA induces
structural and functional changes in epithelial cells in vitro, the most noticeable
being formation of large intracellular vacuoles, the phenotype that gave the toxin its
name (Leunk et al. 1988).
Most of the secreted VacA was shown to bind to cultured epithelial cells and to
use lipid rafts as entry sites so as to be internalised by clathrin-independent
endocytosis (Gauthier et al. 2005). A number of studies have indicated that VacA
may also exert an antagonistic effect on CagA functions (see Chap. 5 for a detailed
discussion). Once intracellular, VacA causes a wide range of alterations to the host
cell. The large membrane-bound vacuoles induced by VacA in the cytoplasm of
gastric cells originate from the late endosomal pathway and are a consequence of
disruption of the late endosomal/lysosomal compartments (Papini et al. 1994).
However, the role of these cytoplasmic vacuoles in H. pylori pathogenesis is
unclear. As discussed in Sect. 6 below, VacA induces apoptosis of gastric epithelial
cells, independently of its vacuolating activity and also promotes autophagy in
these cells.
All H. pylori strains encode vacA, yet display considerable heterogeneity in their
production of the vacuolating cell phenotype (Atherton et al. 1995). This diversity
is largely due to polymorphisms in the vacA gene. The highest level of sequence
diversity is found in the signal (s), intermediate (i) and middle (m) regions of vacA
and forms the basis of a classification system. The signal sequences of the s1 and s2
alleles of VacA are processed at different sites, such that the mature s2 toxin
contains a 12-amino acid hydrophilic extension at its N-terminus, which abolishes
its cytotoxic activity and reduces its ability to form membrane channels, without
abrogating toxin secretion (Letley et al. 2003; McClain et al. 2001). The i region,
defined as either i1 or i2, is important for toxicity (Rhead et al. 2007; Winter
et al. 2014); however, its role in VacA functions is not yet known. The m region
of VacA contains the cell-binding site, with m1-type toxins having higher binding
affinities for host cells than m2-type toxins and also showing different cell-type
specificities (Pagliaccia et al. 1998; Wang et al. 2001).
H. pylori strains with the s1/m1 vacA alleles have higher levels of vacuolating
activity in vitro than those carrying s1/m2 alleles (Atherton et al. 1995). Epidemi-
ological studies are consistent with these in vitro observations, as H. pylori strains
that encode s1 and m1 vacA alleles are associated with a higher risk of gastric
carcinoma than strains with s2 and m2 alleles. Furthermore, s1/m1 vacA genotypes
are strongly associated with peptic ulcers (Atherton et al. 1995, 1997; Strobel
et al. 1998). The i1 allele of vacA shows a strong association with gastric adeno-
carcinoma (Rhead et al. 2007; Winter et al. 2014). Interestingly, in the murine
model of infection, H. pylori bacteria producing the s2/i2 form of VacA colonised
mice more efficiently than those producing the s1/i1 form of VacA or those lacking
VacA, potentially suggesting a different biological role for the weakly active s2/i2
toxin. Strains producing more active VacA induced more severe and extensive
metaplasia and inflammation in the mouse stomach than strains producing s2/i2
toxin (Winter et al. 2014). Thus, specific vacA alleles may contribute to the
pathogenicity and clinical outcomes of H. pylori infection.
3.3.3 Other Putative Autotransporter Proteins of H. pylori
H. pylori genomes contain three vacA-like genes encoding proteins of 260–348 kDa
(Tomb et al. 1997). The C-terminal regions of these proteins show homology to the
C-terminal region of VacA, which is a β-barrel domain that is required for secretion
of VacA through an autotransporter (type V) pathway. On the basis of this simi-
larity, three proteins are predicted to be autotransporters: immunomodulating
autotransporter (ImaA), flagella-associated autotransporter A (FaaA) and VacA-
like protein C (VlpC) (Radin et al. 2013; Sause et al. 2012). These proteins are all
present on the surface of H. pylori (Radin et al. 2013; Sause et al. 2012). However,
whereas ImaA and VlpC localise to a bacterial pole, FaaA localises to a sheath
overlying the flagellar filament and bulb and is important for flagellar morphology
and function. The faaA mutant strain shows decreased motility, reduced flagellar
stability and an increased proportion of flagella in nonpolar sites (Radin et al. 2013).
Expression levels of imaA, faaA and vlpC were up-regulated during colonisation
of the mouse stomach (Radin et al. 2013; Sause et al. 2012). imaA was identified as
belonging to the ArsRS regulon and thus its increased expression in vivo is likely a
response to gastric acid (Sause et al. 2012). The mechanism(s) by which faaA and
vlpC expression levels are regulated remain(s) unknown. Consistent with the idea
that ImaA, FaaA and VlpC may have important roles in colonisation, competition
experiments in mice showed that mutants for each of these autotransporters were
outcompeted by wild-type bacteria in vivo (Radin et al. 2013; Sause et al. 2012).
Indeed, a single challenge study confirmed that an H. pylori faaA mutant was
attenuated in its ability to colonise, when compared with the wild-type strain;
however, this was apparent during the early (4 days post-infection) but not late
(1 month post-infection) stages of infection (Radin et al. 2013; Sause et al. 2012). It
was suggested that FaaA may be important early in the infection process, when
fully formed and functional flagella are required for H. pylori entry into the mucous
layer (Radin et al. 2013; Sause et al. 2012). Similar to the findings above, mice
challenged with H. pylori imaA mutant bacteria alone or in competition with wild-
type bacteria demonstrated that this mutant also had a colonisation defect in vivo
(Sause et al. 2012). In this case, however, ImaA reduced expression of inflamma-
tory chemokines and cytokines in infected stomachs and cultured epithelial cells,
suggesting that this autrotransporter may be important for dampening host immune
responses (Sause et al. 2012). The immunomodulatory activity of ImaA was
observed in H. pylori strains that harbour a cagPAI, suggesting that ImaA down-
regulates the inflammatory responses triggered by the T4SS (Sause et al. 2012).
Interestingly, ImaA exhibits some similarity to the bacterial integrin-binding pro-
tein, invasin (Sause et al. 2012). As the T4SS pilus is known to mediate its
pro-inflammatory effects through binding to α5β1 integrin (Hutton et al. 2010;
Kwok et al. 2007), it was suggested that ImaA and the T4SS may compete for
integrin binding (Sause et al. 2012). Thus, in the absence of ImaA, the T4SS is
better able to deliver the pro-inflammatory effectors, CagA and peptidoglycan
(Sause et al. 2012).
3.3.4 γ-Glutamyl Transpeptidase
γ-Glutamyl transpeptidase (GGT) is produced by all strains of H. pylori (Chevalier

et al. 1999). It plays an important role in the amino acid metabolism of H. pylori, by
synthesising glutamate from both glutamine and glutathione, neither of which can
be assimilated from the environment by this bacterium (Shibayama et al. 2007).
H. pylori GGT hydrolyses glutamine and glutathione outside the cell, with the
resulting products of this reaction being glutamate and ammonia and glutamate and
cysteinylglycine, respectively. The glutamate produced is transported by a Na+-
dependent reaction into H. pylori cells (Shibayama et al. 2007). The enzyme is
catalytically active even at the low pH of the gastric mucosa and its expression
appears to be constitutive (Chevalier et al. 1999). GGT is synthesised as a 60 kDa
inactive proenzyme that undergoes autocatalytic processing to form an enzymati-
cally active heterodimer of 40 and 20 kDa subunits (Chevalier et al. 1999;
Shibayama et al. 2003).
Several lines of evidence indicate that GGT is a virulence factor of H. pylori.
Although deletion of ggt did not impair the in vitro growth of H. pylori (Chevalier
et al. 1999), ggt mutants were attenuated for colonisation of mice and gnotobiotic
piglets (Chevalier et al. 1999; McGovern et al. 2001; Oertli et al. 2013). The degree
of attenuation appears to depend on the H. pylori strain and/or the experimental
animals used. It has been suggested that GGT may be associated with H. pylori-
induced peptic ulcer disease (PUD) in that H. pylori isolates from patients with
PUD showed significantly higher levels of GGT activity than those from patients
with non-ulcer dyspepsia (Gong et al. 2010). Definitive evidence for this suggestion
is, however, currently lacking.
GGT is thought to contribute to the H. pylori-induced damage to the gastric
epithelial cells by promoting apoptosis and by modulating the immune response.
Purified, enzymatically active GGT induced apoptosis and reduced viability in AGS
gastric epithelial cells (Shibayama et al. 2003). Furthermore, GGT was shown to
induce production of H2O2, leading to DNA damage, apoptosis and activation of
inflammatory pathways (Gong et al. 2010). It has been suggested that GGT might
contribute to the damaging effect of H. pylori on gastric cells by depleting gluta-
mine and glutathione, which are important nutrients for maintenance of healthy
gastrointestinal tissue (Shibayama et al. 2007). As discussed in Sect. 4.3 and in

more detail in Chap. 5, GGT also exerts immunomodulatory effects on T-cells.
3.3.5 High Temperature Requirement A (HtrA) Serine

Protease
H. pylori HtrA belongs to a family of serine proteases that is widely conserved in

both single and multicellular organisms. This family of proteins can be distin-
guished from other serine proteases by their sequence homology and oligomeric
structure, as well as by the presence of a protease domain and one or two carboxy
terminal PDZ (post synaptic density protein, Drosophila disc large tumour suppres-
sor, Dlg1, and zonula occludens-1 protein) domains (Clausen et al. 2011). HtrA
serine proteases are involved in important cellular processes, including bacterial
virulence (Clausen et al. 2011). The HtrA secreted by H. pylori (Bumann
et al. 2002) cleaves the extracellular domain of the cell adhesion protein
E-cadherin, present on the surface of host cells, resulting in the loss of cell-cell
contact and enabling the bacterial entry into the intracellular space of epithelial
cells (Hoy et al. 2010). H. pylori HtrA cleavage of E-cadherin was highly efficient
at physiological and high temperatures and at pH 5–8, with highest activity
observed at pH 6–7 (Hoy et al. 2013). Expression of HtrA was up-regulated by
oxidative stress (Huang and Chiou 2011) and environmental acidification (Merrell
et al. 2003). These characteristics of HtrA might aid H. pylori in colonising the
gastric environment. Interestingly, HtrA also appears to be essential for H. pylori
survival in vitro (Hoy et al. 2010; Salama et al. 2004), suggesting that the protein
may have functions other than the cleavage of E-cadherin. Indeed, many HtrAs play
an important role in protein quality control, with some also acting as chaperones to
stabilise specific proteins (Clausen et al. 2011).
3.3.6 Other Pro-inflammatory Virulence Factors of H. pylori
Various new bacterial virulence factors are emerging as putative contributors to

H. pylori pathogenesis in human gastric mucosa. For example, it has been suggested
that the TNFα-inducing protein (Tipα, HP0596) contributes to H. pylori oncoge-
nicity. Tipα is a homodimeric protein that has been shown to be important for
colonisation of mouse gastric mucosa (Godlewska et al. 2008) and is secreted
independently of the T4SS (Suganuma et al. 2005). This protein binds specifically
to nucleolin, a cell surface receptor on gastric epithelial cells (Watanabe
et al. 2010), whereupon it is internalised into the cytosol and then nucleus
(Suganuma et al. 2008). In a mouse gastric epithelial cell line (MGT-40), Tipα
was shown to induce expression of chemokine genes, such as Ccl2, Ccl7, Ccl20,
Cxcl1, Cxcl2, Cxcl5 and Cxcl10 (Kuzuhara et al. 2007). Tipα was also shown to
induce epithelial-mesenchymal transition in human gastric cancer cell lines
(Watanabe et al. 2014).
Duodenal ulcer-promoting gene A (dupA) was originally found to be associated
with an increased risk for duodenal ulcers and a reduced risk for gastric atrophy and
cancer (Lu et al. 2005). Subsequent studies, however, indicated that this association
held in some geographical regions, but not in others (Abadi et al. 2012; Alam
et al. 2012; Arachchi et al. 2007; Argent et al. 2007; Gomes et al. 2008; Imagawa
et al. 2010; Nguyen et al. 2010; Shiota et al. 2010). dupA is associated with
increased IL-8 production from gastric mucosa in vivo (Hussein et al. 2010; Lu
et al. 2005). This gene is located in a plasticity region and encodes a protein that is
functionally homologous to the T4SS ATPase protein, VirB4. In some H. pylori
genomes, dupA is located adjacent to homologues of other vir genes, and a
complete dupA cluster was predicted to form a third T4SS, tfs3a (Kersulyte
et al. 2009). The presence of a complete dupA cluster was found to significantly
increase the risk of duodenal ulcer compared to H. pylori infection with an
incomplete dupA cluster or without the dupA gene (Jung et al. 2012). These data
suggest that the epidemiological studies into the role of dupA in pathogenicity
should be revisited, with the focus on the presence of an intact dupA cluster. Indeed,
the extensive genetic diversity of H. pylori, which contributes to its success as a
pathogen, also increases the difficulty of delineating the molecular basis of the
pathogenesis of H. pylori-induced diseases.
3.4 Avoidance and Modulation of the Host Immune

Response
Although H. pylori is an extracellular pathogen, this bacterium is able to disrupt

epithelial integrity (Amieva et al. 2003). Thus, H. pylori products are likely to enter
the lamina propria and come into contact with immune cells (Necchi et al. 2007).
Although controversial, there is also some evidence to suggest that the bacterium
can invade and replicate within intracellular compartments of epithelial cells,
macrophages and DCs (Ricci et al. 2011). H. pylori induces vigorous inflammatory
host responses, with a large influx of neutrophils, macrophages, DCs and lympho-
cytes, but the immune system fails to clear the infection, attesting to the success of
the various sophisticated strategies used by the pathogen to evade and subvert this
system. These strategies include: evasion of the innate immune system, modulation
of phagocytosis and neutrophil functions, inhibition of lymphocyte proliferation,
and skewing of T-cell-mediated adaptive immune responses toward tolerogenicity
(Baldari et al. 2005).
3.4.1 Evasion of Detection by the Innate Immune System
Host cells are able to detect conserved components of microorganisms, known as

microbe-associated molecular patterns (MAMPs), via pattern recognition receptors
(PRRs). The best characterised PRRs are the Toll-like receptors (TLRs), which
recognise specific classes of MAMPs and respond by activating intracellular sig-
nalling pathways that lead to activation of the master transcriptional regulator,
NF-κB, and pro-inflammatory gene expression. H. pylori has developed several
strategies that largely allow it to avoid detection by TLRs (Takeda and Akira 2005).
The best understood of these strategies involve lipopolysaccharide (LPS) and
flagellin.
LPS consists of an O side chain, a core oligosaccharide and lipid A, which is
anchored to the bacterial membrane. The lipid A of H. pylori LPS is predominantly
tetraacylated, whereas the LPS of E. coli, which is 1,000-fold more biologically
active than that of H. pylori, is hexaacylated (Moran et al. 1997). Pathogenic
bacteria can evade the host innate immune system by concealing or removing the
negatively charged phosphate groups present on the lipid A disaccharide backbone.
The resultant net loss of negative surface charges makes the bacterial membrane
more resistant to cathelicidin antimicrobial peptides (CAMPs). CAMPs, which are
found in macrophages and neutrophils and at the mucosal surface, are an important
component of the host innate immune response and a link between the innate and
adaptive immune systems (Diamond et al. 2009). The low biological activity of
H. pylori LPS has been shown to be due to the removal of phosphate groups from
the 10 - and 40 -positions of lipid A by two lipid phosphatases, LpxE and LpxF,
respectively (Cullen et al. 2011). Dephosphorylated LPS is attenuated for TLR4
activation and highly resistant to CAMPs. Importantly, dephosphorylation of lipid
A by LpxE and LpxF is required for effective colonisation and survival of H. pylori
in mice (Cullen et al. 2011). Studies indicated that H. pylori LPS initiates inflam-
matory signalling in human epithelial cells via TLR2, rather than the more classical
sensor of Gram-negative LPS, TLR4 (Smith et al. 2011; Yokota et al. 2007). TLR2
recognises a variety of microbial components, including lipoproteins, lipoteichoic
acid and atypical LPS molecules whose structures differ from those recognised by
TLR4 in the number of acyl chains in the lipid A moiety (Takeda and Akira 2005).
Recognition of many but not all bacterial flagellins by TLR5, which is present on
the membrane of various cell types, including epithelial cells, leads to activation of
the innate immune system (Takeda and Akira 2005). However, the major flagellin
of H. pylori, FlaA, is much less well recognised by TLR5 than the flagellins of other
enteric mucosal pathogens, such as Salmonella typhimurium (Gewirtz et al. 2004).
Moreover, unlike the flagellins of Escherichia and Salmonella, FlaA is not released
from the bacteria. The evolutionarily conserved recognition sequence for TLR5 is
located in the N-terminal D1 domain of bacterial flagellin, within a region that is
required for flagellar filament assembly and motility. Substitution of amino acids
89–96 of the flagellin (FliC) from S. typhimurium, with the corresponding amino
acids from H. pylori FlaA, abolishes its recognition by TLR5 but also renders the
bacteria non-motile (Andersen-Nissen et al. 2005). H. pylori has preserved its
motility by selecting for compensatory changes in other regions of FlaA, suggesting

that avoidance of detection by TLR5 is important for the persistence of H. pylori at
mucosal sites (Andersen-Nissen et al. 2005).
3.4.2 Modulation of Phagocytosis and Neutrophil Function
The engulfment and killing of microorganisms by the process known as phagocy-

tosis are an important part of host innate defence against many pathogens. The role
of macrophages in H. pylori pathogenesis, however, remains very controversial.
Indeed, professional phagocytes appear to be ineffective in killing H. pylori.
Reduced levels of H. pylori opsonisation by phagocytes have been attributed to
its urease activity (Rokita et al. 1998) and the environmental conditions in the
stomach (Berstad et al. 1997). Furthermore, cagPAI+ H. pylori strains are able to
retard phagocytosis in a cagPAI-dependent but CagA-, VacA- and urease-
independent manner (Allen et al. 2000; Ramarao et al. 2000). Following their
engulfment, these more virulent H. pylori strains stimulate rapid and extensive
homotypic phagosome fusion, leading to formation of megasomes containing large
numbers of viable H. pylori. Formation of these megasomes, which were shown to
be stable for 24 h, requires live, metabolically active H. pylori (Allen et al. 2000).
cagPAI+ H. pylori strains induce the recruitment and retention of coronin 1 protein
on phagosomes and prevent phagosome fusion with lysosomes (Zheng and Jones
2003). This inhibition of phagosome maturation is dependent on VacA and urease
(Schwartz and Allen 2006; Zheng and Jones 2003). Although H. pylori strains that
do not encode cagPAI, and which express a non-toxigenic form of VacA, are
capable of subverting bacterial killing by macrophages for up to 24 h, their survival
is inferior to that shown by cagPAI+ bacteria (Zheng and Jones 2003). The ability of
toxigenic alleles of VacA to modulate autophagy may also contribute to the
survival of H. pylori in macrophages, by allowing the surviving phagocytosed
bacteria to escape killing (Raju et al. 2012). Despite the in vitro evidence for
H. pylori survival in macrophages, further investigations are required in in vivo
models to confirm the biological relevance of these observations.
One mechanism by which H. pylori has been shown to be capable of interfering
with its phagocytosis by antigen-presenting cells is via the actions of a cholesterol-
α-glucosyltransferase (HP0421). This enzyme, also known as type 1 capsular
polysaccharide biosynthesis protein J (CapJ), catalyses the conversion of choles-
terol to cholesteryl α-glucosides (Lebrun et al. 2006; Wunder et al. 2006). Although
H. pylori is auxotrophic for cholesterol, its envelope contains high concentrations
of cholesteryl glucosides (Tannaes and Bukholm 2005). The pathogen extracts
cholesterol from the plasma membranes of epithelial cells, but excessive choles-
terol promotes phagocytosis of the bacteria by antigen-presenting cells, thereby
enhancing T-cell activation. Conversely, α-glucosylation of cholesterol by
cholesterol-α-glucosyltransferase abrogates phagocytosis of H. pylori and T-cell
activation (Wunder et al. 2006). In addition to these effects, cholesterol
glucosylation by CapJ is important for tight binding of H pylori to gastric epithelial

cells and for the assembly of a functional T4SS, as a capJ mutant was impaired in
its ability to translocate CagA into the cytosol of host cells (Wang et al. 2012).
3.4.3 Inhibition of Lymphocyte Proliferation
H pylori uses the secreted proteins VacA (Gebert et al. 2003) and GGT (Shibayama
et al. 2007), to inhibit lymphocyte activation and proliferation. VacA is able to
inhibit proliferation of primary human B lymphocytes, as well as CD4+ and CD8+
T-cells (Torres et al. 2007). The VacA receptor on human immune cells is the β2
(CD18) integrin subunit (Sewald et al. 2008). In transformed Jurkat T-cells, VacA
was shown to down-regulate transcription of IL-2, required for efficient lymphocyte
activation and proliferation (Gebert et al. 2003). VacA does this by blocking
nuclear translocation of the global regulator of immune response genes, nuclear
factor of activated T-cells (NFAT). In activated primary human T-cells, VacA has
also been shown to inhibit IL-2-driven cell-cycle progression independently of IL-2
secretion, by blocking the activation of regulatory proteins important for G1 cell-
cycle transition (Oswald-Richter et al. 2006; Sundrud et al. 2004). Interestingly,
murine T-cells, splenocytes and CD4+ T-cells do not significantly respond to VacA
and this resistance is, at least in part, due to the impaired binding of VacA to murine
cells (Algood et al. 2007; Sewald et al. 2008).
In common with H. pylori VacA, GGT inhibits proliferation of stimulated
primary T-cells and peripheral blood mononuclear cells (PBMCs), but without
affecting secretion of IL-2 (or IFN-γ) and without induction of apoptosis (Oertli
et al. 2013; Schmees et al. 2007). The inhibition of lymphocyte proliferation
involves induction of cell-cycle arrest in G1 phase through disruption of
Ras-dependent signalling and requires the structural integrity of the catalytic
domain of GGT and the presence of glutamine (Oertli et al. 2013; Schmees
et al. 2007). It has been suggested that the inhibitory effect of GGT on T-cells is
mediated indirectly by the formation of metabolites during transpeptidation (Oertli
et al. 2013; Schmees et al. 2007).
3.4.4 Skewing of Adaptive Immune Responses Toward

Tolerogenicity
H. pylori bacteria can manipulate adaptive immune responses to promote their

persistence. One mechanism by which this may occur is via the preferential
induction of regulatory T-cell (Treg) responses. This is evident in heavily colonised
but asymptomatic carriers, who show Treg-predominant responses (Robinson
et al. 2008). Similar findings were observed in a mouse model, in which depletion
of Tregs led to spontaneous clearance of the infection (Arnold et al. 2011).

H. pylori-induced disease in humans was associated with low Treg responses and
significantly higher levels of gastric T-helper 1 (Th1) and Th2 cells, whereas in
disease-free infected subjects the balance was shifted toward elevated Tregs and a
reduced T-helper response (Robinson et al. 2008). Thus, it was suggested that
disease is a consequence of an inadequate regulatory response to H. pylori infection
(Robinson et al. 2008).
DCs play a crucial role as a link between innate and adaptive immunity, being
exquisitely adept at acquiring, processing and presenting antigens to T-cells. DCs
present antigens in a way that promotes tolerance, at least in part, via regulation of
Treg responses (Maldonado and von Andrian 2010). Priming by tolerogenic DCs
converts naive T-cells into FoxP3+ Tregs through antigen presentation in the
absence of co-stimulatory signals or cytokines (Maldonado and von Andrian
2010). H. pylori is able to reprogram DCs toward a tolerogenic phenotype
in vitro and in vivo. Indeed, DCs that have been exposed to H. pylori appear to
preferentially prime Tregs over pro-inflammatory Th1 and Th17 responses and also
fail to produce pro-inflammatory cytokines (Kao et al. 2010; Oertli et al. 2012;
Wang et al. 2010). The importance of DCs in the development of H. pylori-specific
immune tolerance is highlighted by the finding that systemic depletion of DCs
breaks tolerance and facilitates clearance of the bacteria (Oertli et al. 2012).
H. pylori VacA and GGT proteins play critical, non-redundant and
non-synergistic roles in the tolerising effects of this pathogen on murine DCs
in vitro and in vivo, by mechanisms that are independent of their suppressive
activities on T-cells. Isogenic H. pylori mutants lacking either GGT or VacA are
incapable of preventing LPS-induced DC maturation, fail to drive DC tolerisation
and are attenuated for mouse colonisation (Oertli et al. 2013). Furthermore, vacA
mutants induce stronger Th1 and Th17 responses and more severe gastric pathology
(Oertli et al. 2013). VacA and GGT were reported to induce the expression of
miR-155 and Foxp3 in human lymphocytes via a cAMP-dependent pathway (Fassi
Fehri et al. 2010). Both VacA and GGT promote efficient induction of Tregs
in vivo, while VacA is required to prevent allergen-induced asthma. The immuno-
modulatory effects of GGT are dependent on its enzymatic activity, whereas those
of VacA are not linked to its vacuolating cytotoxicity, as strains expressing the
toxigenic (s1/m1) or non-toxigenic (s2/m2) forms of VacA are equally tolerogenic
in vitro (Oertli et al. 2013).
3.5 Mitigation of Inflammatory Responses
H. pylori infection leads to chronic gastritis, which generates reactive oxygen

species (ROS) and nitric oxide (NO). The pathogen limits the bactericidal effects
of these pro-inflammatory mediators, enabling it to chronically colonise its host.
The inflammatory response induced by H. pylori generates large amounts of ROS,
which encompass superoxide anions, hydroxyl radicals and hydrogen peroxide.
H. pylori survive these oxidative stress conditions using a variety of stress resis-
tance proteins. These include catalase (KatA), superoxide dismutase (SodB) and
three peroxidases, an alkylhydroperoxide reductase (AhpC) and two thiolper-
oxidases (Tpx and bacterioferritin comigratory protein, Bcp), which catalyse the
breakdown of hydrogen peroxide, superoxide and organic peroxides, respectively.
H. pylori also encodes the neutrophil activating protein (NapA), which sequesters
toxic levels of iron, and NADPH quinone reductase, MdaB (Stent et al. 2012).
Furthermore, H. pylori bacteria respond to inactivation of important oxidative stress
resistance proteins by increasing the expression of their oxidative stress resistance
proteins, including KatA, SodB and NapA (Olczak et al. 2005). Disruption of katA,
sodB, ahpC, tpx, bcp or mdaB in H. pylori results in an oxidative stress-sensitive
phenotype that severely affects the ability of mutants to colonise the stomach
(Harris et al. 2003; Olczak et al. 2002, 2003; Seyler et al. 2001; Wang and Maier
2004; Wang et al. 2005).
Infection by H. pylori leads to up-regulation of inducible nitric oxide synthase
(iNOS) in the gastric mucosa, leading to mucosal damage. Data obtained using
cultured macrophages indicate that this induction of iNOS is dependent on the
urease released from H. pylori (Gobert et al. 2002). H. pylori bacteria mitigate the
bactericidal effects of NO, which is generated during the conversion of L-arginine
to L-citrulline by iNOS, via the actions of an arginase, RocF (Gobert et al. 2001).
The constitutively produced RocF inhibits production of NO by competing with the
host for L-arginine, which is hydrolysed to L-ornithine and urea; the latter is then
used as a substrate by urease. Loss of RocF activity leads to significant
NO-dependent killing of H. pylori in vitro (Gobert et al. 2001). However, rocF is
not essential for H. pylori colonisation of wild-type (McGee et al. 1999) or arginase
II knockout mice (Kim et al. 2011). RocF activity is stimulated by Trx1 (HP0824),
one of the two thioredoxins of H. pylori. Trx1 acts as a chaperone, converting
denatured or suboptimally folded RocF into its catalytically active structure and
reversing the damage caused by reactive oxygen and nitrogen intermediates
(McGee et al. 2006).
3.6 Modulation of Apoptosis and Autophagy by H. pylori
Apoptosis and autophagy are intricately connected but opposing processes that can
be induced in response to cellular stress and must be finely balanced to regulate cell
death and survival. Perturbation of this balance can lead to pathologies such as
cancer. H. pylori is capable of inducing and inhibiting both apoptosis and
autophagy. The major known H. pylori virulence factors involved in these pro-
cesses are VacA, CagA and GGT.
3.6.1 Apoptosis
VacA induces apoptosis of gastric epithelial cells by targeting the mitochondria,

where it accumulates in the inner membrane and causes depolarisation, outer
membrane permeability, cytochrome C release and mitochondrial fragmentation
(Cover et al. 2003; Galmiche et al. 2000; Willhite et al. 2003; Yamasaki
et al. 2006). The ability of VacA to form anion-selective membrane channels is
required for cytochrome C release, mitochondrial outer membrane permeabilisation
(MOMP) and cell death (Willhite and Blanke 2004). VacA induces the activation of
the pro-apoptotic proteins, Bax and Bcl-2 homologous antagonist/killer (Bak), thus
leading to apoptosis (Yamasaki et al. 2006). VacA-mediated MOMP and activation
of Bak require the mitochondrial recruitment and hyperactivation of dynamin-
related protein 1 (Drp1), a critical regulator of mitochondrial fission within cells
(Jain et al. 2011). GGT induces apoptosis in gastric epithelial cells via a
mitochondria-mediated pathway (Kim et al. 2007) and by inducing the loss of
survivin, an inhibitor of apoptosis (Valenzuela et al. 2013). Recently, another
putative H. pylori virulence factor, HP0986 (TNFR1 interacting endonuclease A,
TieA), was reported to actively induce apoptosis in cultured human and murine
macrophages via a Fas-mediated pathway (Alvi et al. 2011).
There is also evidence that H. pylori can prevent or block apoptosis. In a
transgenic mouse model, H. pylori CagA was shown to interact with the
apoptosis-stimulating protein of p53 (ASPP2) and thereby inhibit apoptosis by
promoting proteasomal degradation of the p53 tumour suppressor (Buti
et al. 2011). Host cells that had been co-cultured with H. pylori and then treated
with the p53-activating drug doxorubicin were more resistant to apoptosis than cells
not exposed to the bacterium (Buti et al. 2011). In a separate study, it was reported
that CagA is also able to mediate activation of the pro-survival factor ERK and the
anti-apoptotic protein, myeloid leukaemia cell differentiation protein 1 (Mcl-1)
(Mimuro et al. 2007). Using the Mongolian gerbil model, the authors showed that
H. pylori is able to activate the ERK-Mcl-1 pathway in vivo so as to suppress
apoptosis in gastric pit cells, thereby leading to gland hyperplasia and persistent
bacterial colonisation (Mimuro et al. 2007). In agreement with these findings,
another group demonstrated H. pylori CagA-dependent induction of Mcl-1 expres-
sion via up-regulation of a known negative regulator of Mcl-1, the tumour suppres-
sor microRNA (miRNA) miR-320 (Noto et al. 2013). Consistent with the
Mongolian gerbil data, H. pylori was shown to induce Mcl-1 expression in a
CagA-dependent manner in the murine gastric mucosa, as well as in tissues from
a human population at high risk for gastric cancer (Noto et al. 2013). Moreover,
Mcl-1 epithelial expression levels increased at each stage of neoplastic progression
in gastric tissues from human subjects infected with cagA+ strains of H. pylori
(Noto et al. 2013). Thus, down-regulation of miR-320 and subsequent induction of
Mcl-1 by cagA+ H. pylori strains suppresses apoptosis, potentially promoting
H. pylori persistence within the gastric mucosa but also possibly gastric
carcinogenesis.
3.6.2 Autophagy
Autophagy is a tightly controlled major catabolic pathway in eukaryotes, which is

required for the lysosomal/vacuolar degradation of cytoplasmic proteins and organ-
elles. H. pylori induces autophagy in gastric epithelial cells (Tang et al. 2012;
Terebiznik et al. 2009), as well as in professional phagocytes (Wang et al. 2010).
VacA is necessary and sufficient to induce autophagy in gastric epithelial cells, with
the induction of autophagy responsible for a decrease in the levels of intracellular
VacA and vacuole biogenesis within intoxicated cells. Thus, autophagy may
represent a mechanism by which host cells limit VacA-mediated damage
(Terebiznik et al. 2009). In the AZ-521 human gastric epithelial cell line, VacA-
induced autophagy was shown to be mediated by VacA binding to and
internalisation via the low-density-lipoprotein receptor-related protein 1 (LRP1)
(Yahiro et al. 2012). Knockdown of LRP1 abrogated VacA internalisation and
significantly down-regulated autophagy in vitro (Yahiro et al. 2012). LRP1 is also
required for the induction of autophagy-mediated degradation of CagA in response
to m1 forms of VacA in AGS gastric epithelial cells. Signalling through p53
degradation is involved in m1VacA-induced autophagy in these cells (Tsugawa
et al. 2012).
In addition to the effects of VacA in promoting autophagy, this cytotoxin can
also block autophagy. Indeed, prolonged exposure of human gastric epithelial cells
to VacA was found to disrupt toxin-induced autophagy, by blocking the maturation
of autolysosomes (Raju et al. 2012). VacA alters the degradative properties of the
endocytic pathway by subverting the sorting and activation of cathepsin enzymes
(Satin et al. 1997). VacA-induced autolysosomes lack the key lysosomal hydrolase,
cathepsin D, and so cannot complete the process of degrading their cargo, leading to
accumulation of ROS and the signalling adaptor, p62 (Raju et al. 2012). Interest-
ingly, impairment of autophagy and the accumulation of p62 lead to enhanced
tumourigenicity (Moscat and Diaz-Meco 2009). Further work is required to deter-
mine whether VacA subversion of autophagy may promote gastric cancer
development.
Finally, H. pylori can modulate autophagy through a mechanism involving the
microRNA, MIR30B (Tang et al. 2012). Expression of MIR30B was elevated in
gastric mucosal tissues from infected patients, as well as during infection of gastric
epithelial cell lines, and this effect was a specific response to H. pylori. Moreover,
elevated MIR30B expression in human gastric tissues was inversely correlated with
the mRNA levels of the genes encoding two of the proteins that are important in
regulating autophagy, autophagy-related protein 12 (ATG12) and BCL2-
interacting coiled-coil protein (BECN1). Inhibition of autophagy by MIR30B was
demonstrated to increase the number of VacA-dependent large vacuoles and
enhanced the intracellular survival of the pathogen, demonstrating that autophagy
is involved in regulating the levels of intracellular VacA (Tang et al. 2012). Taken
together, these data show that H. pylori VacA is able to use different strategies to
interfere with autophagy in gastric epithelial cells.
H. pylori is arguably one of the most successful human pathogens, persistently

colonising the gastric mucosa of more than 50 % of the world’s population. This
pathogen induces vigorous inflammatory host responses. However, due to the many
effective strategies employed by the bacterium to subvert host immune responses,
the infection cannot be easily cleared. H. pylori uses its numerous virulence factors
to establish chronic infection, alter cellular signalling cascades, cause damage to the
mucosa and modulate host immune responses. The multitude of virulence factors,
together with their complex interactions and allelic variations, render it difficult to
dissect the individual contributions of these factors to the chronicity of H. pylori
infection and its long-term consequences. Moreover H. pylori is not only highly
heterogeneous but also genetically unstable, adding to the difficulty in studying the
virulence mechanisms of this human pathogen. As discussed here, H. pylori can
alter cell proliferation, apoptosis and autophagy processes, as well as down-regulate
cellular tumour suppressor genes. All together, these changes contribute to onco-
genesis and the development of more severe gastric disease. Although the last
decade has seen great advances in our understanding of the virulence mechanisms
of H. pylori, much of this knowledge has been gained from experiments conducted
in vitro or in animal models and awaits confirmation from clinical and epidemio-
logical studies. Such studies must encompass populations from diverse geographic
locations, as both bacterial and host polymorphisms are likely to contribute to the
pathogenesis of H. pylori infection in humans.
References
Abadi AT, Taghvaei T, Wolfram L, Kusters JG (2012) Infection with Helicobacter pylori strains
lacking dupA is associated with an increased risk of gastric ulcer and gastric cancer develop-
ment. J Med Microbiol 61:23–30
Alam J, Maiti S, Ghosh P, De R, Chowdhury A, Das S, Macaden R, Devarbhavi H, Ramamurthy T,
Mukhopadhyay AK (2012) Significant association of the dupA gene of Helicobacter pylori
with duodenal ulcer development in a South-east Indian population. J Med Microbiol
61:1295–1302
Algood HM, Torres VJ, Unutmaz D, Cover TL (2007) Resistance of primary murine CD4+ T cells
to Helicobacter pylori vacuolating cytotoxin. Infect Immun 75:334–341
Allen LA, Schlesinger LS, Kang B (2000) Virulent strains of Helicobacter pylori demonstrate
delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages. J Exp Med
191:115–128
Allison CC, Kufer TA, Kremmer E, Kaparakis M, Ferrero RL (2009) Helicobacter pylori induces
MAPK phosphorylation and AP-1 activation via a NOD1-dependent mechanism. J Immunol
183:8099–8109
Alm RA, Bina J, Andrews BM, Doig P, Hancock RE, Trust TJ (2000) Comparative genomics of
Helicobacter pylori: analysis of the outer membrane protein families. Infect Immun
68:4155–4168
Alvi A, Ansari SA, Ehtesham NZ, Rizwan M, Devi S, Sechi LA, Qureshi IA, Hasnain SE, Ahmed
N (2011) Concurrent proinflammatory and apoptotic activity of a Helicobacter pylori protein
(HP986) points to its role in chronic persistence. PLoS ONE 6:e22530
Amieva MR, Vogelmann R, Covacci A, Tompkins LS, Nelson WJ, Falkow S (2003) Disruption of
the epithelial apical-junctional complex by Helicobacter pylori CagA. Science 300:1430–1434
Andersen-Nissen E, Smith KD, Strobe KL, Barrett SL, Cookson BT, Logan SM, Aderem A (2005)
Evasion of Toll-like receptor 5 by flagellated bacteria. Proc Natl Acad Sci U S A
102:9247–9252
Arachchi HS, Kalra V, Lal B, Bhatia V, Baba CS, Chakravarthy S, Rohatgi S, Sarma PM,
Mishra V, Das B, Ahuja V (2007) Prevalence of duodenal ulcer-promoting gene (dupA) of
Helicobacter pylori in patients with duodenal ulcer in North Indian population. Helicobacter
12:591–597
Argent RH, Kidd M, Owen RJ, Thomas RJ, Limb MC, Atherton JC (2004) Determinants and
consequences of different levels of CagA phosphorylation for clinical isolates of Helicobacter
pylori. Gastroenterology 127:514–523
Argent RH, Burette A, Miendje Deyi VY, Atherton JC (2007) The presence of dupA in
Helicobacter pylori is not significantly associated with duodenal ulceration in Belgium,
South Africa, China, or North America. Clin Infect Dis 45:1204–1206
Arnold IC, Lee JY, Amieva MR, Roers A, Flavell RA, Sparwasser T, Muller A (2011) Tolerance
rather than immunity protects from Helicobacter pylori-induced gastric preneoplasia. Gastro-
enterology 140:199–209
Aspholm M, Olfat FO, Norden J, Sonden B, Lundberg C, Sjostrom R, Altraja S, Odenbreit S,
Haas R, Wadstrom T, Engstrand L, Semino-Mora C, Liu H, Dubois A, Teneberg S, Arnqvist A,
Boren T (2006) SabA is the H. pylori hemagglutinin and is polymorphic in binding to sialylated
glycans. PLoS Pathog 2:e110
Aspholm-Hurtig M, Dailide G, Lahmann M, Kalia A, Ilver D, Roche N, Vikstrom S, Sjostrom R,
Linden S, Backstrom A, Lundberg C, Arnqvist A, Mahdavi J, Nilsson UJ, Velapatino B,
Gilman RH, Gerhard M, Alarcon T, Lopez-Brea M, Nakazawa T, Fox JG, Correa P,
Dominguez-Bello MG, Perez-Perez GI, Blaser MJ, Normark S, Carlstedt I, Oscarson S,
Teneberg S, Berg DE, Boren T (2004) Functional adaptation of BabA, the H. pylori ABO
blood group antigen binding adhesin. Science 305:519–522
Atherton JC, Cao P, Peek RM Jr, Tummuru MK, Blaser MJ, Cover TL (1995) Mosaicism in
vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with
cytotoxin production and peptic ulceration. J Biol Chem 270:17771–17777
Atherton JC, Peek RM Jr, Tham KT, Cover TL, Blaser MJ (1997) Clinical and pathological
importance of heterogeneity in vacA, the vacuolating cytotoxin gene of Helicobacter pylori.
Gastroenterology 112:92–99
Baldari CT, Lanzavecchia A, Telford JL (2005) Immune subversion by Helicobacter pylori.
Trends Immunol 26:199–207
Berstad AE, Brandtzaeg P, Stave R, Halstensen TS (1997) Epithelium related deposition of
activated complement in Helicobacter pylori associated gastritis. Gut 40:196–203
Bjorkholm B, Sjolund M, Falk PG, Berg OG, Engstrand L, Andersson DI (2001) Mutation
frequency and biological cost of antibiotic resistance in Helicobacter pylori. Proc Natl Acad
Sci U S A 98:14607–14612
Bonis M, Ecobichon C, Guadagnini S, Prevost MC, Boneca IG (2010) A M23B family
metallopeptidase of Helicobacter pylori required for cell shape, pole formation and virulence.
Bumann D, Aksu S, Wendland M, Janek K, Zimny-Arndt U, Sabarth N, Meyer TF, Jungblut PR
(2002) Proteome analysis of secreted proteins of the gastric pathogen Helicobacter pylori.
Infect Immun 70:3396–3403
Buti L, Spooner E, Van der Veen AG, Rappuoli R, Covacci A, Ploegh HL (2011) Helicobacter
pylori cytotoxin-associated gene A (CagA) subverts the apoptosis-stimulating protein of p53
(ASPP2) tumor suppressor pathway of the host. Proc Natl Acad Sci U S A 108:9238–9243
Camorlinga-Ponce M, Romo C, Gonzalez-Valencia G, Munoz O, Torres J (2004) Topographical

localisation of cagA positive and cagA negative Helicobacter pylori strains in the gastric
mucosa; an in situ hybridisation study. J Clin Pathol 57:822–828
Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, Borodovsky M, Rappuoli R, Covacci A
(1996) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-
associated virulence factors. Proc Natl Acad Sci U S A 93:14648–14653
Chevalier C, Thiberge JM, Ferrero RL, Labigne A (1999) Essential role of Helicobacter pylori
γ-glutamyltranspeptidase for the colonization of the gastric mucosa of mice. Mol Microbiol
31:1359–1372
Clausen T, Kaiser M, Huber R, Ehrmann M (2011) HTRA proteases: regulated proteolysis in
protein quality control. Nat Rev Mol Cell Biol 12:152–162
Colland F, Rain JC, Gounon P, Labigne A, Legrain P, De Reuse H (2001) Identification of the
Helicobacter pylori anti-sigma 28 factor. Mol Microbiol 41:477–487
Covacci A, Telford JL, Del Giudice G, Parsonnet J, Rappuoli R (1999) Helicobacter pylori
virulence and genetic geography. Science 284:1328–1333
Cover TL, Blaser MJ (1992) Purification and characterization of the vacuolating toxin from
Helicobacter pylori. J Biol Chem 267:10570–10575
Cover TL, Hanson PI, Heuser JE (1997) Acid-induced dissociation of VacA, the Helicobacter
pylori vacuolating cytotoxin, reveals its pattern of assembly. J Cell Biol 138:759–769
Cover TL, Krishna US, Israel DA, Peek RM Jr (2003) Induction of gastric epithelial cell apoptosis
by Helicobacter pylori vacuolating cytotoxin. Cancer Res 63:951–957
Croxen MA, Sisson G, Melano R, Hoffman PS (2006) The Helicobacter pylori chemotaxis
receptor TlpB (HP0103) is required for pH taxis and for colonization of the gastric mucosa.
J Bacteriol 188:2656–2665
Cullen TW, Giles DK, Wolf LN, Ecobichon C, Boneca IG, Trent MS (2011) Helicobacter pylori
versus the host: remodeling of the bacterial outer membrane is required for survival in the
gastric mucosa. PLoS Pathog 7:e1002454
de Jonge R, Durrani Z, Rijpkema SG, Kuipers EJ, van Vliet AH, Kusters JG (2004) Role of the
Helicobacter pylori outer-membrane proteins AlpA and AlpB in colonization of the guinea pig
stomach. J Med Microbiol 53:375–379
Diamond G, Beckloff N, Weinberg A, Kisich KO (2009) The roles of antimicrobial peptides in
innate host defense. Curr Pharm Des 15:2377–2392
Dossumbekova A, Prinz C, Mages J, Lang R, Kusters JG, Van Vliet AH, Reindl W, Backert S,
Saur D, Schmid RM, Rad R (2006) Helicobacter pylori HopH (OipA) and bacterial pathoge-
nicity: genetic and functional genomic analysis of hopH gene polymorphisms. J Infect Dis
194:1346–1355
Douillard FP, Ryan KA, Hinds J, O’Toole PW (2009) Effect of FliK mutation on the transcrip-
tional activity of the {sigma}54 sigma factor RpoN in Helicobacter pylori. Microbiology
155:1901–1911
Eaton KA, Suerbaum S, Josenhans C, Krakowka S (1996) Colonization of gnotobiotic piglets by
Helicobacter pylori deficient in two flagellin genes. Infect Immun 64:2445–2448
Eaton KA, Gilbert JV, Joyce EA, Wanken AE, Thevenot T, Baker P, Plaut A, Wright A (2002) In
vivo complementation of ureB restores the ability of Helicobacter pylori to colonize. Infect
Immun 70:771–778
Fassi Fehri L, Koch M, Belogolova E, Khalil H, Bolz C, Kalali B, Mollenkopf HJ, Beigier-
Bompadre M, Karlas A, Schneider T, Churin Y, Gerhard M, Meyer TF (2010) Helicobacter
pylori induces miR-155 in T cells in a cAMP-Foxp3-dependent manner. PLoS One 5:e9500
Fischer W, Puls J, Buhrdorf R, Gebert B, Odenbreit S, Haas R (2001) Systematic mutagenesis of
the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host
cells and induction of interleukin-8. Mol Microbiol 42:1337–1348
Franco AT, Johnston E, Krishna U, Yamaoka Y, Israel DA, Nagy TA, Wroblewski LE, Piazuelo
MB, Correa P, Peek RM Jr (2008) Regulation of gastric carcinogenesis by Helicobacter pylori
virulence factors. Cancer Res 68:379–387
Galmiche A, Rassow J, Doye A, Cagnol S, Chambard JC, Contamin S, de Thillot V, Just I,

Ricci V, Solcia E, Van Obberghen E, Boquet P (2000) The N-terminal 34 kDa fragment of
Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c
release. EMBO J 19:6361–6370
Gangwer KA, Mushrush DJ, Stauff DL, Spiller B, McClain MS, Cover TL, Lacy DB (2007)
Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain. Proc Natl Acad Sci
U S A 104:16293–16298
Garner JA, Cover TL (1996) Binding and internalization of the Helicobacter pylori vacuolating
cytotoxin by epithelial cells. Infect Immun 64:4197–4203
Gauthier NC, Monzo P, Kaddai V, Doye A, Ricci V, Boquet P (2005) Helicobacter pylori VacA
cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed
to late endosomes. Mol Biol Cell 16:4852–4866
Gebert B, Fischer W, Weiss E, Hoffmann R, Haas R (2003) Helicobacter pylori vacuolating
cytotoxin inhibits T lymphocyte activation. Science 301:1099–1102
Genisset C, Galeotti CL, Lupetti P, Mercati D, Skibinski DA, Barone S, Battistutta R, de
Bernard M, Telford JL (2006) A Helicobacter pylori vacuolating toxin mutant that fails to
oligomerize has a dominant negative phenotype. Infect Immun 74:1786–1794
Gerhard M, Lehn N, Neumayer N, Boren T, Rad R, Schepp W, Miehlke S, Classen M, Prinz C
(1999) Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding
adhesin. Proc Natl Acad Sci U S A 96:12778–12783
Gewirtz AT, Yu Y, Krishna US, Israel DA, Lyons SL, Peek RM Jr (2004) Helicobacter pylori
flagellin evades toll-like receptor 5-mediated innate immunity. J Infect Dis 189:1914–1920
Gobert AP, McGee DJ, Akhtar M, Mendz GL, Newton JC, Cheng Y, Mobley HL, Wilson KT
(2001) Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: a
strategy for bacterial survival. Proc Natl Acad Sci U S A 98:13844–13849
Gobert AP, Mersey BD, Cheng Y, Blumberg DR, Newton JC, Wilson KT (2002) Cutting edge:
urease release by Helicobacter pylori stimulates macrophage inducible nitric oxide synthase.
J Immunol 168:6002–6006
Godlewska R, Pawlowski M, Dzwonek A, Mikula M, Ostrowski J, Drela N, Jagusztyn-Krynicka
EK (2008) Tip-α (hp0596 gene product) is a highly immunogenic Helicobacter pylori protein
involved in colonization of mouse gastric mucosa. Curr Microbiol 56:279–286
Gomes LI, Rocha GA, Rocha AM, Soares TF, Oliveira CA, Bittencourt PF, Queiroz DM (2008)
Lack of association between Helicobacter pylori infection with dupA-positive strains and
gastroduodenal diseases in Brazilian patients. Int J Med Microbiol 298:223–230
Gong M, Ling SS, Lui SY, Yeoh KG, Ho B (2010) Helicobacter pylori γ-glutamyl transpeptidase
is a pathogenic factor in the development of peptic ulcer disease. Gastroenterology
139:564–573
Harris AG, Wilson JE, Danon SJ, Dixon MF, Donegan K, Hazell SL (2003) Catalase (KatA) and
KatA-associated protein (KapA) are essential to persistent colonization in the Helicobacter
pylori SS1 mouse model. Microbiology 149:665–672
Hazell SL, Lee A, Brady L, Hennessy W (1986) Campylobacter pyloridis and gastritis: association
with intercellular spaces and adaptation to an environment of mucus as important factors in
colonization of the gastric epithelium. J Infect Dis 153:658–663
Hessey SJ, Spencer J, Wyatt JI, Sobala G, Rathbone BJ, Axon AT, Dixon MF (1990) Bacterial
adhesion and disease activity in Helicobacter associated chronic gastritis. Gut 31:134–138
Higashi H, Tsutsumi R, Fujita A, Yamazaki S, Asaka M, Azuma T, Hatakeyama M (2002)
Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation
in the tyrosine phosphorylation sites. Proc Natl Acad Sci U S A 99:14428–14433
Howitt MR, Lee JY, Lertsethtakarn P, Vogelmann R, Joubert LM, Ottemann KM, Amieva MR
(2011) ChePep controls Helicobacter pylori Infection of the gastric glands and chemotaxis in
the Epsilonproteobacteria. MBio 2:e00098-11
Hoy B, Lower M, Weydig C, Carra G, Tegtmeyer N, Geppert T, Schroder P, Sewald N, Backert S,

Schneider G, Wessler S (2010) Helicobacter pylori HtrA is a new secreted virulence factor that
cleaves E-cadherin to disrupt intercellular adhesion. EMBO Rep 11:798–804
Hoy B, Brandstetter H, Wessler S (2013) The stability and activity of recombinant Helicobacter
pylori HtrA under stress conditions. J Basic Microbiol 53:402–409
Huang CH, Chiou SH (2011) Proteomic analysis of upregulated proteins in Helicobacter pylori
under oxidative stress induced by hydrogen peroxide. Kaohsiung J Med Sci 27:544–553
Hussein NR, Argent RH, Marx CK, Patel SR, Robinson K, Atherton JC (2010) Helicobacter pylori
dupA is polymorphic, and its active form induces proinflammatory cytokine secretion by
mononuclear cells. J Infect Dis 202:261–269
Hutton ML, Kaparakis-Liaskos M, Turner L, Cardona A, Kwok T, Ferrero RL (2010)
Helicobacter pylori exploits cholesterol-rich microdomains for induction of NF-κB-dependent
responses and peptidoglycan delivery in epithelial cells. Infect Immun 78:4523–4531
Ilver D, Arnqvist A, Ogren J, Frick IM, Kersulyte D, Incecik ET, Berg DE, Covacci A,
Engstrand L, Boren T (1998) Helicobacter pylori adhesin binding fucosylated histo-blood
group antigens revealed by retagging. Science 279:373–377
Imagawa S, Ito M, Yoshihara M, Eguchi H, Tanaka S, Chayama K (2010) Helicobacter pylori
dupA and gastric acid secretion are negatively associated with gastric cancer development.
J Med Microbiol 59:1484–1489
Jain P, Luo ZQ, Blanke SR (2011) Helicobacter pylori vacuolating cytotoxin A (VacA) engages
the mitochondrial fission machinery to induce host cell death. Proc Natl Acad Sci U S A
108:16032–16037
Jimenez-Soto LF, Kutter S, Sewald X, Ertl C, Weiss E, Kapp U, Rohde M, Pirch T, Jung K, Retta
SF, Terradot L, Fischer W, Haas R (2009) Helicobacter pylori type IV secretion apparatus
exploits beta1 integrin in a novel RGD-independent manner. PLoS Pathog 5:e1000684
Josenhans C, Niehus E, Amersbach S, Horster A, Betz C, Drescher B, Hughes KT, Suerbaum S
(2002) Functional characterization of the antagonistic flagellar late regulators FliA and FlgM
of Helicobacter pylori and their effects on the H. pylori transcriptome. Mol Microbiol
43:307–322
Jung SW, Sugimoto M, Shiota S, Graham DY, Yamaoka Y (2012) The intact dupA cluster is a
more reliable Helicobacter pylori virulence marker than dupA alone. Infect Immun
80:381–387
Kao JY, Zhang M, Miller MJ, Mills JC, Wang B, Liu M, Eaton KA, Zou W, Berndt BE, Cole TS,
Takeuchi T, Owyang SY, Luther J (2010) Helicobacter pylori immune escape is mediated by
dendritic cell-induced Treg skewing and Th17 suppression in mice. Gastroenterology
138:1046–1054
Kaparakis M, Turnbull L, Carneiro L, Firth S, Coleman HA, Parkington HC, Le Bourhis L,
Karrar A, Viala J, Mak J, Hutton ML, Davies JK, Crack PJ, Hertzog PJ, Philpott DJ, Girardin
SE, Whitchurch CB, Ferrero RL (2010) Bacterial membrane vesicles deliver peptidoglycan to
NOD1 in epithelial cells. Cell Microbiol 12:372–385
Kersulyte D, Lee W, Subramaniam D, Anant S, Herrera P, Cabrera L, Balqui J, Barabas O,
Kalia A, Gilman RH, Berg DE (2009) Helicobacter pylori’s plasticity zones are novel
transposable elements. PLoS One 4:e6859
Kim KM, Lee SG, Park MG, Song JY, Kang HL, Lee WK, Cho MJ, Rhee KH, Youn HS, Baik SC
(2007) γ-glutamyltranspeptidase of Helicobacter pylori induces mitochondria-mediated apo-
ptosis in AGS cells. Biochem Biophys Res Commun 355:562–567
Kim SH, Langford ML, Boucher JL, Testerman TL, McGee DJ (2011) Helicobacter pylori
arginase mutant colonizes arginase II knockout mice. World J Gastroenterol 17:3300–3309
Kusters JG, van Vliet AH, Kuipers EJ (2006) Pathogenesis of Helicobacter pylori infection. Clin
Microbiol Rev 19:449–490
Kuzuhara T, Suganuma M, Kurusu M, Fujiki H (2007) Helicobacter pylori-secreting protein Tipα
is a potent inducer of chemokine gene expressions in stomach cancer cells. J Cancer Res Clin
Oncol 133:287–296
Kwok T, Zabler D, Urman S, Rohde M, Hartig R, Wessler S, Misselwitz R, Berger J, Sewald N,

Konig W, Backert S (2007) Helicobacter exploits integrin for type IV secretion and kinase
activation. Nature 449:862–866
Labigne A, Cussac V, Courcoux P (1991) Shuttle cloning and nucleotide sequences of
Helicobacter pylori genes responsible for urease activity. J Bacteriol 173:1920–1931
Lai CH, Chang YC, Du SY, Wang HJ, Kuo CH, Fang SH, Fu HW, Lin HH, Chiang AS, Wang WC
(2008) Cholesterol depletion reduces Helicobacter pylori CagA translocation and CagA-
induced responses in AGS cells. Infect Immun 76:3293–3303
Lebrun AH, Wunder C, Hildebrand J, Churin Y, Zahringer U, Lindner B, Meyer TF, Heinz E,
Warnecke D (2006) Cloning of a cholesterol-α-glucosyltransferase from Helicobacter pylori.
J Biol Chem 281:27765–27772
Letley DP, Rhead JL, Twells RJ, Dove B, Atherton JC (2003) Determinants of non-toxicity in the
gastric pathogen Helicobacter pylori. J Biol Chem 278:26734–26741
Leunk RD, Johnson PT, David BC, Kraft WG, Morgan DR (1988) Cytotoxic activity in broth-
culture filtrates of Campylobacter pylori. J Med Microbiol 26:93–99
Lu H, Hsu PI, Graham DY, Yamaoka Y (2005) Duodenal ulcer promoting gene of Helicobacter
Lu H, Wu JY, Beswick EJ, Ohno T, Odenbreit S, Haas R, Reyes VE, Kita M, Graham DY,
Yamaoka Y (2007) Functional and intracellular signaling differences associated with the
Helicobacter pylori AlpAB adhesin from Western and East Asian strains. J Biol Chem
282:6242–6254
Lupetti P, Heuser JE, Manetti R, Massari P, Lanzavecchia S, Bellon PL, Dallai R, Rappuoli R,
Telford JL (1996) Oligomeric and subunit structure of the Helicobacter pylori vacuolating
cytotoxin. J Cell Biol 133:801–807
Mahdavi J, Sonden B, Hurtig M, Olfat FO, Forsberg L, Roche N, Angstrom J, Larsson T,
Teneberg S, Karlsson KA, Altraja S, Wadstrom T, Kersulyte D, Berg DE, Dubois A,
Petersson C, Magnusson KE, Norberg T, Lindh F, Lundskog BB, Arnqvist A,
Hammarstrom L, Boren T (2002) Helicobacter pylori SabA adhesin in persistent infection
and chronic inflammation. Science 297:573–578
Maldonado RA, von Andrian UH (2010) How tolerogenic dendritic cells induce regulatory T cells.
Adv Immunol 108:111–165
Marshall BJ, Barrett LJ, Prakash C, McCallum RW, Guerrant RL (1990) Urea protects
Helicobacter (Campylobacter) pylori from the bactericidal effect of acid. Gastroenterology
99:697–702
McClain MS, Cao P, Iwamoto H, Vinion-Dubiel AD, Szabo G, Shao Z, Cover TL (2001) A
12-amino-acid segment, present in type s2 but not type s1 Helicobacter pylori VacA proteins,
abolishes cytotoxin activity and alters membrane channel formation. J Bacteriol
183:6499–6508
McGee DJ, Radcliff FJ, Mendz GL, Ferrero RL, Mobley HL (1999) Helicobacter pylori rocF is
required for arginase activity and acid protection in vitro but is not essential for colonization of
mice or for urease activity. J Bacteriol 181:7314–7322
McGee DJ, Kumar S, Viator RJ, Bolland JR, Ruiz J, Spadafora D, Testerman TL, Kelly DJ,
Pannell LK, Windle HJ (2006) Helicobacter pylori thioredoxin is an arginase chaperone and
guardian against oxidative and nitrosative stresses. J Biol Chem 281:3290–3296
McGovern KJ, Blanchard TG, Gutierrez JA, Czinn SJ, Krakowka S, Youngman P (2001)
γ-Glutamyltransferase is a Helicobacter pylori virulence factor but is not essential for coloni-
zation. Infect Immun 69:4168–4173
McGowan CC, Necheva AS, Forsyth MH, Cover TL, Blaser MJ (2003) Promoter analysis of
Helicobacter pylori genes with enhanced expression at low pH. Mol Microbiol 48:1225–1239
Mimuro H, Suzuki T, Nagai S, Rieder G, Suzuki M, Nagai T, Fujita Y, Nagamatsu K, Ishijima N,
Koyasu S, Haas R, Sasakawa C (2007) Helicobacter pylori dampens gut epithelial self-renewal
by inhibiting apoptosis, a bacterial strategy to enhance colonization of the stomach. Cell Host
Microbe 2:250–263
Mobley HL, Garner RM, Bauerfeind P (1995) Helicobacter pylori nickel-transport gene nixA:
synthesis of catalytically active urease in Escherichia coli independent of growth conditions.
Moodley Y, Linz B, Bond RP, Nieuwoudt M, Soodyall H, Schlebusch CM, Bernhoft S, Hale J,
Suerbaum S, Mugisha L, van der Merwe SW, Achtman M (2012) Age of the association
between Helicobacter pylori and man. PLoS Pathog 8:e1002693
Moran AP, Lindner B, Walsh EJ (1997) Structural characterization of the lipid A component of
Helicobacter pylori rough- and smooth-form lipopolysaccharides. J Bacteriol 179:6453–6463
Moscat J, Diaz-Meco MT (2009) p62 at the crossroads of autophagy, apoptosis, and cancer. Cell
137:1001–1004
Murata-Kamiya N, Kikuchi K, Hayashi T, Higashi H, Hatakeyama M (2010) Helicobacter pylori
exploits host membrane phosphatidylserine for delivery, localization, and pathophysiological
action of the CagA oncoprotein. Cell Host Microbe 7:399–411
Necchi V, Candusso ME, Tava F, Luinetti O, Ventura U, Fiocca R, Ricci V, Solcia E (2007)
Intracellular, intercellular, and stromal invasion of gastric mucosa, preneoplastic lesions, and
cancer by Helicobacter pylori. Gastroenterology 132:1009–1023
Nguyen LT, Uchida T, Tsukamoto Y, Kuroda A, Okimoto T, Kodama M, Murakami K, Fujioka T,
Moriyama M (2010) Helicobacter pylori dupA gene is not associated with clinical outcomes in
the Japanese population. Clin Microbiol Infect 16:1264–1269
Niehus E, Gressmann H, Ye F, Schlapbach R, Dehio M, Dehio C, Stack A, Meyer TF, Suerbaum S,
Josenhans C (2004) Genome-wide analysis of transcriptional hierarchy and feedback regula-
tion in the flagellar system of Helicobacter pylori. Mol Microbiol 52:947–961
Noto JM, Piazuelo MB, Chaturvedi R, Bartel CA, Thatcher EJ, Delgado A, Romero-Gallo J,
Wilson KT, Correa P, Patton JG, Peek RM Jr (2013) Strain-specific suppression of microRNA-
320 by carcinogenic Helicobacter pylori promotes expression of the antiapoptotic protein
Mcl-1. Am J Physiol Gastrointest Liver Physiol 305:G786–G796
Odenbreit S, Till M, Hofreuter D, Faller G, Haas R (1999) Genetic and functional characterization
of the alpAB gene locus essential for the adhesion of Helicobacter pylori to human gastric
tissue. Mol Microbiol 31:1537–1548
Odenbreit S, Puls J, Sedlmaier B, Gerland E, Fischer W, Haas R (2000) Translocation of
Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science
287:1497–1500
Odenbreit S, Swoboda K, Barwig I, Ruhl S, Boren T, Koletzko S, Haas R (2009) Outer membrane
protein expression profile in Helicobacter pylori clinical isolates. Infect Immun 77:3782–3790
Oertli M, Sundquist M, Hitzler I, Engler DB, Arnold IC, Reuter S, Maxeiner J, Hansson M,
Taube C, Quiding-Jarbrink M, Muller A (2012) DC-derived IL-18 drives Treg differentiation,
murine Helicobacter pylori-specific immune tolerance, and asthma protection. J Clin Invest
122:1082–1096
Oertli M, Noben M, Engler DB, Semper RP, Reuter S, Maxeiner J, Gerhard M, Taube C, Muller A
(2013) Helicobacter pylori γ-glutamyl transpeptidase and vacuolating cytotoxin promote
gastric persistence and immune tolerance. Proc Natl Acad Sci U S A 110:3047–3052
Olczak AA, Olson JW, Maier RJ (2002) Oxidative-stress resistance mutants of Helicobacter
pylori. J Bacteriol 184:3186–3193
Olczak AA, Seyler RW Jr, Olson JW, Maier RJ (2003) Association of Helicobacter pylori
antioxidant activities with host colonization proficiency. Infect Immun 71:580–583
Olczak AA, Wang G, Maier RJ (2005) Up-expression of NapA and other oxidative stress proteins
is a compensatory response to loss of major Helicobacter pylori stress resistance factors. Free
Radic Res 39:1173–1182
Oswald-Richter K, Torres VJ, Sundrud MS, VanCompernolle SE, Cover TL, Unutmaz D (2006)
Helicobacter pylori VacA toxin inhibits human immunodeficiency virus infection of primary
human T cells. J Virol 80:11767–11775
Pagliaccia C, de Bernard M, Lupetti P, Ji X, Burroni D, Cover TL, Papini E, Rappuoli R, Telford

JL, Reyrat JM (1998) The m2 form of the Helicobacter pylori cytotoxin has cell type-specific
vacuolating activity. Proc Natl Acad Sci U S A 95:10212–10217
Papini E, de Bernard M, Milia E, Bugnoli M, Zerial M, Rappuoli R, Montecucco C (1994) Cellular
vacuoles induced by Helicobacter pylori originate from late endosomal compartments. Proc
Natl Acad Sci U S A 91:9720–9724
Pernitzsch SR, Tirier SM, Beier D, Sharma CM (2014) A variable homopolymeric G-repeat
defines small RNA-mediated posttranscriptional regulation of a chemotaxis receptor in
Helicobacter pylori. Proc Natl Acad Sci U S A 111:E501–E510
Pflock M, Finsterer N, Joseph B, Mollenkopf H, Meyer TF, Beier D (2006) Characterization of the
ArsRS regulon of Helicobacter pylori, involved in acid adaptation. J Bacteriol 188:3449–3462
Poppe M, Feller SM, Romer G, Wessler S (2007) Phosphorylation of Helicobacter pylori CagA by
c-Abl leads to cell motility. Oncogene 26:3462–3472
Radin JN, Gaddy JA, Gonzalez-Rivera C, Loh JT, Algood HM, Cover TL (2013) Flagellar
localization of a Helicobacter pylori autotransporter protein. MBio 4:e00613–00612
Raju D, Hussey S, Ang M, Terebiznik MR, Sibony M, Galindo-Mata E, Gupta V, Blanke SR,
Delgado A, Romero-Gallo J, Ramjeet MS, Mascarenhas H, Peek RM, Correa P, Streutker C,
Hold G, Kunstmann E, Yoshimori T, Silverberg MS, Girardin SE, Philpott DJ, El Omar E,
Jones NL (2012) Vacuolating cytotoxin and variants in Atg16L1 that disrupt autophagy
promote Helicobacter pylori infection in humans. Gastroenterology 142:1160–1171
Ramarao N, Gray-Owen SD, Backert S, Meyer TF (2000) Helicobacter pylori inhibits phagocy-
tosis by professional phagocytes involving type IV secretion components. Mol Microbiol
37:1389–1404
Rhead JL, Letley DP, Mohammadi M, Hussein N, Mohagheghi MA, Eshagh Hosseini M, Atherton
JC (2007) A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate
region, is associated with gastric cancer. Gastroenterology 133:926–936
Ricci V, Romano M, Boquet P (2011) Molecular cross-talk between Helicobacter pylori and
human gastric mucosa. World J Gastroenterol 17:1383–1399
Robinson K, Kenefeck R, Pidgeon EL, Shakib S, Patel S, Polson RJ, Zaitoun AM, Atherton JC
(2008) Helicobacter pylori-induced peptic ulcer disease is associated with inadequate regula-
tory T cell responses. Gut 57:1375–1385
Rohde M, Puls J, Buhrdorf R, Fischer W, Haas R (2003) A novel sheathed surface organelle of the
Helicobacter pylori cag type IV secretion system. Mol Microbiol 49:219–234
Rokita E, Makristathis A, Presterl E, Rotter ML, Hirschl AM (1998) Helicobacter pylori urease
significantly reduces opsonization by human complement. J Infect Dis 178:1521–1525
Roure S, Bonis M, Chaput C, Ecobichon C, Mattox A, Barriere C, Geldmacher N, Guadagnini S,
Schmitt C, Prevost MC, Labigne A, Backert S, Ferrero RL, Boneca IG (2012) Peptidoglycan
maturation enzymes affect flagellar functionality in bacteria. Mol Microbiol 86:845–856
Salama NR, Shepherd B, Falkow S (2004) Global transposon mutagenesis and essential gene
analysis of Helicobacter pylori. J Bacteriol 186:7926–7935
Satin B, Norais N, Telford J, Rappuoli R, Murgia M, Montecucco C, Papini E (1997) Effect of
Helicobacter pylori vacuolating toxin on maturation and extracellular release of procathepsin
D and on epidermal growth factor degradation. J Biol Chem 272:25022–25028
Sause WE, Castillo AR, Ottemann KM (2012) The Helicobacter pylori autotransporter ImaA
(HP0289) modulates the immune response and contributes to host colonization. Infect Immun
80:2286–2296
Schmees C, Prinz C, Treptau T, Rad R, Hengst L, Voland P, Bauer S, Brenner L, Schmid RM,
Gerhard M (2007) Inhibition of T-cell proliferation by Helicobacter pylori γ-glutamyl
transpeptidase. Gastroenterology 132:1820–1833
Schmitt W, Haas R (1994) Genetic analysis of the Helicobacter pylori vacuolating cytotoxin:
structural similarities with the IgA protease type of exported protein. Mol Microbiol
12:307–319
Schwartz JT, Allen LA (2006) Role of urease in megasome formation and Helicobacter pylori
survival in macrophages. J Leukoc Biol 79:1214–1225
Scott DR, Marcus EA, Wen Y, Oh J, Sachs G (2007) Gene expression in vivo shows that
Helicobacter pylori colonizes an acidic niche on the gastric surface. Proc Natl Acad Sci U S
A 104:7235–7240
Selbach M, Moese S, Hauck CR, Meyer TF, Backert S (2002) Src is the kinase of the Helicobacter
pylori CagA protein in vitro and in vivo. J Biol Chem 277:6775–6778
Selbach M, Moese S, Hurwitz R, Hauck CR, Meyer TF, Backert S (2003) The Helicobacter pylori
CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactiva-
tion. EMBO J 22:515–528
Senkovich OA, Yin J, Ekshyyan V, Conant C, Traylor J, Adegboyega P, McGee DJ, Rhoads RE,
Slepenkov S, Testerman TL (2011) Helicobacter pylori AlpA and AlpB bind host laminin and
influence gastric inflammation in gerbils. Infect Immun 79:3106–3116
Sewald X, Gebert-Vogl B, Prassl S, Barwig I, Weiss E, Fabbri M, Osicka R, Schiemann M, Busch
DH, Semmrich M, Holzmann B, Sebo P, Haas R (2008) Integrin subunit CD18 Is the
T-lymphocyte receptor for the Helicobacter pylori vacuolating cytotoxin. Cell Host Microbe
3:20–29
Seyler RW Jr, Olson JW, Maier RJ (2001) Superoxide dismutase-deficient mutants of
Helicobacter pylori are hypersensitive to oxidative stress and defective in host colonization.
Shibayama K, Kamachi K, Nagata N, Yagi T, Nada T, Doi Y, Shibata N, Yokoyama K, Yamane K,
Kato H, Iinuma Y, Arakawa Y (2003) A novel apoptosis-inducing protein from Helicobacter
pylori. Mol Microbiol 47:443–451
Shibayama K, Wachino J, Arakawa Y, Saidijam M, Rutherford NG, Henderson PJ (2007)
Metabolism of glutamine and glutathione via γ-glutamyltranspeptidase and glutamate transport
in Helicobacter pylori: possible significance in the pathophysiology of the organism. Mol
Shiota S, Matsunari O, Watada M, Hanada K, Yamaoka Y (2010) Systematic review and meta-
analysis: the relationship between the Helicobacter pylori dupA gene and clinical outcomes.
Gut Pathog 2:13
Skouloubris S, Thiberge JM, Labigne A, De Reuse H (1998) The Helicobacter pylori UreI protein
is not involved in urease activity but is essential for bacterial survival in vivo. Infect Immun
66:4517–4521
Smith SM, Moran AP, Duggan SP, Ahmed SE, Mohamed AS, Windle HJ, O’Neill LA, Kelleher
DP (2011) Tribbles 3: a novel regulator of TLR2-mediated signaling in response to
Helicobacter pylori lipopolysaccharide. J Immunol 186:2462–2471
Stent A, Every AL, Sutton P (2012) Helicobacter pylori defense against oxidative attack. Am J
Physiol Gastrointest Liver Physiol 302:G579–G587
Strobel S, Bereswill S, Balig P, Allgaier P, Sonntag HG, Kist M (1998) Identification and analysis
of a new vacA genotype variant of Helicobacter pylori in different patient groups in Germany. J
Clin Microbiol 36:1285–1289
Suerbaum S, Josenhans C (2007) Helicobacter pylori evolution and phenotypic diversification in a
changing host. Nat Rev Microbiol 5:441–452
Suerbaum S, Josenhans C, Labigne A (1993) Cloning and genetic characterization of the
Helicobacter pylori and Helicobacter mustelae flaB flagellin genes and construction of
H. pylori flaA- and flaB-negative mutants by electroporation-mediated allelic exchange.
Suerbaum S, Smith JM, Bapumia K, Morelli G, Smith NH, Kunstmann E, Dyrek I, Achtman M
(1998) Free recombination within Helicobacter pylori. Proc Natl Acad Sci U S A
95:12619–12624
Suganuma M, Kurusu M, Suzuki K, Nishizono A, Murakami K, Fujioka T, Fujiki H (2005) New
tumor necrosis factor-α-inducing protein released from Helicobacter pylori for gastric cancer
progression. J Cancer Res Clin Oncol 131:305–313
Suganuma M, Yamaguchi K, Ono Y, Matsumoto H, Hayashi T, Ogawa T, Imai K, Kuzuhara T,

Nishizono A, Fujiki H (2008) TNF-α-inducing protein, a carcinogenic factor secreted from
H. pylori, enters gastric cancer cells. Int J Cancer 123:117–122
Sugimoto M, Ohno T, Graham DY, Yamaoka Y (2009) Gastric mucosal interleukin-17 and -18
mRNA expression in Helicobacter pylori-induced Mongolian gerbils. Cancer Sci
100:2152–2159
Sundrud MS, Torres VJ, Unutmaz D, Cover TL (2004) Inhibition of primary human T cell
proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects
on IL-2 secretion. Proc Natl Acad Sci U S A 101:7727–7732
Sycuro LK, Wyckoff TJ, Biboy J, Born P, Pincus Z, Vollmer W, Salama NR (2012) Multiple
peptidoglycan modification networks modulate Helicobacter pylori’s cell shape, motility, and
colonization potential. PLoS Pathog 8:e1002603
Takeda K, Akira S (2005) Toll-like receptors in innate immunity. Int Immunol 17:1–14
Tammer I, Brandt S, Hartig R, Konig W, Backert S (2007) Activation of Abl by Helicobacter
pylori: a novel kinase for CagA and crucial mediator of host cell scattering. Gastroenterology
132:1309–1319
Tang B, Li N, Gu J, Zhuang Y, Li Q, Wang HG, Fang Y, Yu B, Zhang JY, Xie QH, Chen L, Jiang
XJ, Xiao B, Zou QM, Mao XH (2012) Compromised autophagy by MIR30B benefits the
intracellular survival of Helicobacter pylori. Autophagy 8:1045–1057
Tannaes T, Bukholm G (2005) Cholesteryl-6-O-acyl-α-D-glucopyranoside of Helicobacter pylori
relate to relative lysophospholipid content. FEMS Microbiol Lett 244:117–120
Telford JL, Ghiara P, Dell’Orco M, Comanducci M, Burroni D, Bugnoli M, Tecce MF, Censini S,
Covacci A, Xiang Z et al (1994) Gene structure of the Helicobacter pylori cytotoxin and
evidence of its key role in gastric disease. J Exp Med 179:1653–1658
Terebiznik MR, Raju D, Vazquez CL, Torbricki K, Kulkarni R, Blanke SR, Yoshimori T,
Colombo MI, Jones NL (2009) Effect of Helicobacter pylori’s vacuolating cytotoxin on the
autophagy pathway in gastric epithelial cells. Autophagy 5:370–379
Terry K, Williams SM, Connolly L, Ottemann KM (2005) Chemotaxis plays multiple roles during
Helicobacter pylori animal infection. Infect Immun 73:803–811
Teymournejad O, Mobarez AM, Hassan ZM, Moazzeni SM, Ahmadabad HN (2014) In vitro
suppression of dendritic cells by Helicobacter pylori OipA. Helicobacter 19:136–143
Tomb JF, White O, Kerlavage AR, Clayton RA, Sutton GG, Fleischmann RD, Ketchum KA,
Klenk HP, Gill S, Dougherty BA, Nelson K, Quackenbush J, Zhou L, Kirkness EF, Peterson S,
Loftus B, Richardson D, Dodson R, Khalak HG, Glodek A, McKenney K, Fitzegerald LM,
Lee N, Adams MD, Hickey EK, Berg DE, Gocayne JD, Utterback TR, Peterson JD, Kelley JM,
Cotton MD, Weidman JM, Fujii C, Bowman C, Watthey L, Wallin E, Hayes WS,
Borodovsky M, Karp PD, Smith HO, Fraser CM, Venter JC (1997) The complete genome
sequence of the gastric pathogen Helicobacter pylori. Nature 388:539–547
Torres VJ, Ivie SE, McClain MS, Cover TL (2005) Functional properties of the p33 and p55
domains of the Helicobacter pylori vacuolating cytotoxin. J Biol Chem 280:21107–21114
Torres VJ, VanCompernolle SE, Sundrud MS, Unutmaz D, Cover TL (2007) Helicobacter pylori
vacuolating cytotoxin inhibits activation-induced proliferation of human T and B lymphocyte
subsets. J Immunol 179:5433–5440
Tsugawa H, Suzuki H, Saya H, Hatakeyama M, Hirayama T, Hirata K, Nagano O, Matsuzaki J,
Hibi T (2012) Reactive oxygen species-induced autophagic degradation of Helicobacter pylori
CagA is specifically suppressed in cancer stem-like cells. Cell Host Microbe 12:764–777
Tsutsumi R, Higashi H, Higuchi M, Okada M, Hatakeyama M (2003) Attenuation of Helicobacter
pylori CagA x SHP-2 signaling by interaction between CagA and C-terminal Src kinase. J Biol
Chem 278:3664–3670
Unemo M, Aspholm-Hurtig M, Ilver D, Bergstrom J, Boren T, Danielsson D, Teneberg S (2005)
The sialic acid binding SabA adhesin of Helicobacter pylori is essential for nonopsonic
activation of human neutrophils. J Biol Chem 280:15390–15397
Valenzuela M, Bravo D, Canales J, Sanhueza C, Diaz N, Almarza O, Toledo H, Quest AF (2013)

Helicobacter pylori-induced loss of survivin and gastric cell viability is attributable to secreted
bacterial γ-glutamyl transpeptidase activity. J Infect Dis 208:1131–1141
van Vliet AH, Poppelaars SW, Davies BJ, Stoof J, Bereswill S, Kist M, Penn CW, Kuipers EJ,
Kusters JG (2002) NikR mediates nickel-responsive transcriptional induction of urease expres-
sion in Helicobacter pylori. Infect Immun 70:2846–2852
Viala J, Chaput C, Boneca IG, Cardona A, Girardin SE, Moran AP, Athman R, Memet S, Huerre
MR, Coyle AJ, DiStefano PS, Sansonetti PJ, Labigne A, Bertin J, Philpott DJ, Ferrero RL
(2004) Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity
island. Nat Immunol 5:1166–1174
Wadhams GH, Armitage JP (2004) Making sense of it all: bacterial chemotaxis. Nat Rev Mol Cell
Biol 5:1024–1037
Wang G, Maier RJ (2004) An NADPH quinone reductase of Helicobacter pylori plays an
important role in oxidative stress resistance and host colonization. Infect Immun 72:1391–1396
Wang WC, Wang HJ, Kuo CH (2001) Two distinctive cell binding patterns by vacuolating toxin
fused with glutathione S-transferase: one high-affinity m1-specific binding and the other lower-
affinity binding for variant m forms. Biochemistry 40:11887–11896
Wang G, Olczak AA, Walton JP, Maier RJ (2005) Contribution of the Helicobacter pylori thiol
peroxidase bacterioferritin comigratory protein to oxidative stress resistance and host coloni-
zation. Infect Immun 73:378–384
Wang YH, Gorvel JP, Chu YT, Wu JJ, Lei HY (2010) Helicobacter pylori impairs murine
dendritic cell responses to infection. PLoS One 5:e10844
Wang HJ, Cheng WC, Cheng HH, Lai CH, Wang WC (2012) Helicobacter pylori cholesteryl
glucosides interfere with host membrane phase and affect type IV secretion system function
during infection in AGS cells. Mol Microbiol 83:67–84
Watanabe T, Tsuge H, Imagawa T, Kise D, Hirano K, Beppu M, Takahashi A, Yamaguchi K,
Fujiki H, Suganuma M (2010) Nucleolin as cell surface receptor for tumor necrosis factor-α
inducing protein: a carcinogenic factor of Helicobacter pylori. J Cancer Res Clin Oncol
136:911–921
Watanabe T, Takahashi A, Suzuki K, Kurusu-Kanno M, Yamaguchi K, Fujiki H, Suganuma M
(2014) Epithelial-mesenchymal transition in human gastric cancer cell lines induced by
TNF-α-inducing protein of Helicobacter pylori. Int J Cancer 134:2373–2382
Weeks DL, Eskandari S, Scott DR, Sachs G (2000) A H+-gated urea channel: the link between
Helicobacter pylori urease and gastric colonization. Science 287:482–485
Weyermann M, Rothenbacher D, Brenner H (2009) Acquisition of Helicobacter pylori infection in
early childhood: independent contributions of infected mothers, fathers, and siblings. Am J
Gastroenterol 104:182–189
Willhite DC, Blanke SR (2004) Helicobacter pylori vacuolating cytotoxin enters cells, localizes to
the mitochondria, and induces mitochondrial membrane permeability changes correlated to
toxin channel activity. Cell Microbiol 6:143–154
Willhite DC, Cover TL, Blanke SR (2003) Cellular vacuolation and mitochondrial cytochrome c
release are independent outcomes of Helicobacter pylori vacuolating cytotoxin activity that are
each dependent on membrane channel formation. J Biol Chem 278:48204–48209
Winter JA, Letley DP, Cook KW, Rhead JL, Zaitoun AA, Ingram RJ, Amilon KR, Croxall NJ,
Kaye PV, Robinson K, Atherton JC (2014) A role for the vacuolating cytotoxin, VacA, in
colonization and Helicobacter pylori-induced metaplasia in the stomach. J Infect Dis
210:954–963
Wunder C, Churin Y, Winau F, Warnecke D, Vieth M, Lindner B, Zahringer U, Mollenkopf HJ,
Heinz E, Meyer TF (2006) Cholesterol glucosylation promotes immune evasion by
Helicobacter pylori. Nat Med 12:1030–1038
Yahiro K, Satoh M, Nakano M, Hisatsune J, Isomoto H, Sap J, Suzuki H, Nomura F, Noda M,
Moss J, Hirayama T (2012) Low-density lipoprotein receptor-related protein-1 (LRP1)
mediates autophagy and apoptosis caused by Helicobacter pylori VacA. J Biol Chem
287:31104–31115
Yamaoka Y, Kikuchi S, el-Zimaity HM, Gutierrez O, Osato MS, Graham DY (2002) Importance
of Helicobacter pylori oipA in clinical presentation, gastric inflammation, and mucosal inter-
leukin 8 production. Gastroenterology 123:414–424
Yamaoka Y, Kudo T, Lu H, Casola A, Brasier AR, Graham DY (2004) Role of interferon-
stimulated responsive element-like element in interleukin-8 promoter in Helicobacter pylori
infection. Gastroenterology 126:1030–1043
Yamaoka Y, Ojo O, Fujimoto S, Odenbreit S, Haas R, Gutierrez O, El-Zimaity HM, Reddy R,
Arnqvist A, Graham DY (2006) Helicobacter pylori outer membrane proteins and gastrodu-
odenal disease. Gut 55:775–781
Yamasaki E, Wada A, Kumatori A, Nakagawa I, Funao J, Nakayama M, Hisatsune J, Kimura M,
Moss J, Hirayama T (2006) Helicobacter pylori vacuolating cytotoxin induces activation of the
proapoptotic proteins Bax and Bak, leading to cytochrome c release and cell death, indepen-
dent of vacuolation. J Biol Chem 281:11250–11259
Ye D, Willhite DC, Blanke SR (1999) Identification of the minimal intracellular vacuolating
domain of the Helicobacter pylori vacuolating toxin. J Biol Chem 274:9277–9282
Yokota S, Ohnishi T, Muroi M, Tanamoto K, Fujii N, Amano K (2007) Highly-purified
Helicobacter pylori LPS preparations induce weak inflammatory reactions and utilize Toll-
like receptor 2 complex but not Toll-like receptor 4 complex. FEMS Immunol Med Microbiol
51:140–148
Zheng PY, Jones NL (2003) Helicobacter pylori strains expressing the vacuolating cytotoxin
interrupt phagosome maturation in macrophages by recruiting and retaining TACO (coronin 1)
protein. Cell Microbiol 5:25–40
Chapter 4
Roles of the cagPAI and CagA
on Gastroduodenal Diseases
Steffen Backert, Giuseppe Zanotti, Judith Lind, Carmen Isabell Asche,

and Nicole Tegtmeyer
Abstract Helicobacter pylori is a highly successful human-specific Gram-nega-

tive bacterium. Infections with this pathogen in the stomach can induce pathologies
ranging from chronic gastritis, peptic ulcers, to gastric cancer. Highly virulent
H. pylori isolates harbor the cytotoxin-associated genes (cag) pathogenicity island,
which encodes a typical type IV secretion system (T4SS). This T4SS constitutes a
syringe-like pilus structure for the translocation of virulence factors such as the
CagA effector protein into gastric epithelial and immune cells. This is achieved by a
number of T4SS proteins such as CagL, CagI, CagY, and CagA, which can interact
with the host cell integrin member α5β1 followed by transport of CagA across the
host cell membrane. After delivery, CagA undergoes phosphorylation by oncogenic
tyrosine kinases and mimics a host factor for the activation or inactivation of
multiple intracellular signaling cascades. Here we review the current status in the
characterization of phosphorylation-dependent and phosphorylation-independent
signaling events by CagA and the CagA-independent T4SS activities in vivo and
in vitro, which include the induction of membrane dynamics, actin-cytoskeletal
rearrangements, disruption of cell-to-cell junctions, as well as pro-inflammatory,
proliferative and anti-apoptotic nuclear responses. The contribution of these sig-
naling pathways to pathogenesis during H. pylori infection is discussed.
Keywords Helicobacter pylori • Type IV secretion • VirB1–VirB11 • VirD4 •

Signaling • Gastric cancer • Infection • Tyrosine kinase • SH2 domain •
Inflammasome
S. Backert (*) • J. Lind • C.I. Asche • N. Tegtmeyer

Division of Microbiology, Department of Biology, Friedrich Alexander University
Erlangen-Nuremberg, Staudtstr. 5, D-91058 Erlangen, Germany
G. Zanotti
Department of Biomedical Sciences, University of Padua, Viale G. Colombo 3, 35131 Padova,
Italy

DOI 10.1007/978-4-431-55936-8_4
90 S. Backert et al.
4.1 Introduction
Helicobacter pylori is one of the best adapted human pathogens that colonizes the
surface region in the gastric mucosa of the stomach. Approximately half of the
world’s population carries this microbe, often causing asymptomatic gastritis in
infected people, but also more severe gastric diseases such as mucosa-associated
lymphoid tissue (MALT) lymphoma and gastric cancer may arise. Colonization
commonly occurs early in childhood and H. pylori can persist lifelong, if not treated
by antimicrobial therapy. Although H. pylori infections are commonly associated
with elevated inflammation parameters that are generated by the host innate and
adaptive immune systems, the bacteria are not eliminated. Various mechanisms of
immune evasion were documented and H. pylori became a prime example of
chronic bacterial infections. Host-pathogen interactions are highly complex and
determine the clinical outcome of infections. The development of gastric diseases is
controlled by the bacterial genotype, genetic predisposition of the host, and envi-
ronmental factors. For example, specific polymorphisms in host genes with impor-
tant roles in pro-inflammatory and immune-regulatory signal transduction such as
interleukin-1β (IL-1β), interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α),
or Nod-1 (nucleotide oligomerization domain-1) have been linked to a higher risk
of developing H. pylori-triggered gastric diseases (for more details, see Chaps. 3,
13, and 16 of this book). H. pylori strains are highly heterogeneous both in their
DNA sequences and virulence properties. Dozens of bacterial genes have been
described to control the pathogenesis of H. pylori. There are two classical virulence
factors expressed by H. pylori, the CagA protein encoded in the cag (cytotoxin-
associated genes) pathogenicity island (cagPAI) and the vacuolating cytotoxin A
(VacA). VacA interacts with multiple host molecules and can trigger various
downstream signaling cascades as discussed in Chap. 5. Here we summarize our
current knowledge on the multiple cagPAI and CagA functions as well as the
multitude of affected host signaling cascades with focus on their importance in
H. pylori pathogenesis.
4.2 The cagPAI Encodes a Type IV Secretion System
In the H. pylori research field, worldwide interest is focused on the effector protein
CagA. CagA is encoded by highly virulent type I isolates, but is absent in less
virulent type II strains. Thus, the protein has been recognized as a molecular marker
for the cagPAI locus (Hatakeyama 2003; Backert et al. 2015). The cagPAI encodes
functional components of a type IV secretion system (T4SS). This T4SS represents
a pilus-like structure (called the T4SS pilus), induced upon host cell contact and
protruding from the bacterial membrane (Fig. 4.1a). T4SS machineries are evolu-
tionary related to DNA conjugation systems (Backert and Meyer 2006). The class
4 Roles of the cagPAI and CagA on Gastroduodenal Diseases 91
Fig. 4.1 Model for the assembled type IV secretion (T4SS) complex in Helicobacter pylori (Hp).
(a) The T4SS encoded by the cag pathogenicity island is a multicomponent protein complex
spanning the inner and outer membranes. Typical T4SS pili are shown by scanning electron
microscopy, connecting the bacterium with the membrane of AGS gastric epithelial cells (arrows).
(b) The Hp T4SS exhibits homology to the VirB/VirD4 T4SS machinery of Agrobacterium
tumefaciens. T4SS assembly and subcellular localization of the proteins are shown in a simplified
manner. Pilus components, core complex proteins, energetic components, and others factors are
highlighted with different colors as indicated. The reported substrates for the Hp T4SS are CagA
and peptidoglycan. (For more details, see text. Panel a was from Hauck (2007) with kind
permission from Nature Publishing and panel b was adapted from Tegtmeyer and coworkers
(2011) with kind permission from Wiley.)
of T4SSs is highly diverse both with regard to the delivered factors (DNA, proteins,
DNA-protein complexes, or peptidoglycan) and recipient organisms. The latter can
be either a bacterium of the same or other species or even species from different
kingdoms (e.g., mammalian, fungal, or plant cells). In addition to H. pylori,
pathogenicity-linked T4SSs have also been found in Agrobacterium, Legionella,
Bordetella, Bartonella, and other species. T4SSs commonly comprise 11 VirB
proteins (encoded by the virB1–virB11 genes) and the NTPase coupling protein
(VirD4). The prototypic and by far best characterized T4SS is the T-DNA transfer
apparatus of Agrobacterium tumefaciens (Waksman and Orlova 2014). The
agrobacterial VirB proteins can be classified in three groups: (1) the core or putative
channel components (VirB6–VirB10), (2) the pilus-associated proteins (VirB2, and
possibly VirB3 and VirB5), and (3) the energetic factors (the NTPases: VirB4 and
VirB11). In addition, VirB1 is an enzyme having muraminidase activity, allowing
localized lysis of murein layer in the membrane to achieve T4SS assembly at a

specific site of the cell (Backert et al. 2015). A model for the individual steps in
assembling of the agrobacterial T4SS has been described in numerous review
articles (Backert and Meyer 2006; Waksman and Orlova 2014). When having a
look at the H. pylori T4SS, all 11 VirB and VirD4 orthologs are encoded in the
cagPAI as well as some accessory proteins (Fischer 2011), resulting in a T4SS
model (Fig. 4.1b). Immunogold labeling and electron microscopy (EM) have shown
that the tips of the T4SS pilus are covered with CagL (Kwok et al. 2007). In
addition, EM revealed that CagL, CagI, and CagH proteins were involved in
T4SS pilus formation and deletion of a conserved C-terminal hexapeptide motif,
which is shared among these proteins, abolished CagA delivery (Shaffer
et al. 2011). The T4SS pilus is also decorated locally or completely by CagY
(VirB10) (Rohde et al. 2003). CagY is a very large protein (about 250-kDa) that
contains two transmembrane domains with the mid region (also called the repeat
domain) exposed to the extracellular environment (Rohde et al. 2003). It carries an
unusual sequence structure owing to an extraordinary number of direct DNA
repeats that are predicted to result in in-frame intersections or deletions (Barrozo
et al. 2013). Interestingly, CagY rearrangements are driven by the host and are
sufficient to result in gain or loss of function in the T4SS. This molecular switch
allowed modification of the immune response to benefit persistent infection of
H. pylori (Barrozo et al. 2013). Major other components of the T4SS structure,
also visualized by EM, are the VirB7 and VirB9 proteins (Tanaka et al. 2003).
Interestingly, T4SS pilus formation requires a host cell receptor (Kwok et al. 2007).
This is achieved by a number of T4SS proteins, including CagI, CagL, CagY, and
CagA, interacting with the host cell integrin member α5β1, followed by transloca-
tion of CagA into the host (Kwok et al. 2007; Jiménez-Soto et al. 2009). Binding of
CagA to phosphatidylserine has also been shown to play a role in the translocation
process (Murata-Kamiya et al. 2010).
4.3 Crystal Structures of cagPAI Proteins
In the last few years, several pieces of information have been gathered about the
architecture of bacterial T4SSs, both from EM and single-crystal X-ray diffraction
studies. The most complete picture of the apparatus comes from the 18Å–23Å EM
structure of the T4SS of Escherichia coli conjugative plasmid R388 (Low
et al. 2014). This complex includes 8 proteins (from VirB3 to VirB10) that form
a supramolecular assembly spanning the entire cell envelope. The complex can be
divided in two parts, the core complex, characterized by C14 symmetry, and the
inner membrane complex, with a lower C2 symmetry. The two complexes are
connected by a small stalk. In the overall, the entire membrane-spanning machinery
is about 340Å high and 255Å wide. A more detailed description at the atomic level
comes from the crystal structure of the O-layer (Chandran et al. 2009; Terradot and
Waksman 2011) and from EM studies of the outer membrane T4SS core complex
from plasmid pKM101 (Fronzes et al. 2009). The O-layer includes 14 copies of
VirB10, VirB7, and VirB9, with the VirB10 components forming a tetradecameric
channel and the 14 VirB7/VirB9 complexes surrounding and stabilizing it. The
T4SS pilus is composed of the major subunit VirB2 and VirB5 (CagL) (Backert
et al. 2008). The structure of the latter, which decorates the pilus surface, has been
determined (Barden et al. 2013). CagL consists of a four-helix bundle, containing
the Arg-Gly-Asp (RGD) motif that represents a recognition site for integrin binding
(Kwok et al. 2007). Finally, the structure of VirB11 ATPase revealed a hexameric
ring and functions as a gating molecule at the inner membrane, which is proposed to
cycle through closed and open forms by ATP binding/hydrolysis (Yeo et al. 2000;
Hare et al. 2007).
The crystal structures of some other members of the cagPAI, which are impor-
tant for CagA secretion or IL-8 induction, have also been determined: CagZ, a
23-kDa protein essential for CagA translocation, consists of a single compact
L-shaped domain containing seven α-helices (Cendron et al. 2004); CagS is a
23-kDa single-domain protein characterized by an all-α structure and an unusually
high methionine content (Cendron et al. 2007); CagD is a covalent dimer in which
each monomer folds as a single domain that is composed of five β-strands and three
α-helices (Cendron et al. 2009).
The translocated effector protein CagA in strain 26695 consists of 1,186 amino
acids and is structurally organized in several domains. Various crystal structures of
the large N-terminal portion, residues 1–876 and 261–829 (Hayashi et al. 2012) and
residues 1–884 (Kaplan-Türk€oz et al. 2012), consist of three domains, one of which
is made by two sub-domains (or four domains in total, according to a different
interpretation). Domain I (24–221) is composed of 10 α-helices. It makes very few
contacts with the other domains, such that its orientation is mobile with respect to
the rest of the molecule. Domain II (residues 303–644) constitutes the molecular
core and contains a large antiparallel β-sheet flanked by an all α-helical subdomain
(residues 370–446) and two long α-helices. A long, partially flexible α-helix
connects domain II to domain III (residues 645–824), whose structure is that of a
classical four-helix bundle. The C-terminal region (residues 825–1186, about 30%
of the entire protein) of CagA seems to be intrinsically disordered, a fact that could
possibly facilitate the interaction with other proteins inside the host cell. In fact, the
structure of microtubule affinity-regulating kinase 2 (MARK2) in complex with
120 amino acids of the C-terminal region (residues 885–1005) has been determined,
but only a 14-residue peptide of CagA is ordered in the crystal (Nesić et al. 2010).
Recently, the complex between domain I of CagA (residues 25–220) with the
apoptosis stimulating protein of p53-2 (ASPP2) has been also solved (Nešić
et al. 2014), confirming that different parts of CagA interact with different target
proteins to hijack host cell-signaling cascades associated with disease outcome.
4.4 Pathological Function of the cagPAI Type IV Secretion

System
Clinical, epidemiological, and functional studies have shown that the presence of
cagPAI and CagA is associated with the development of gastric disease. Various
animal infection models have been developed and provided comprehensive evi-
dence for the significance of cagPAI and CagA in H. pylori pathogenesis (Ogura
et al. 2000; Rieder et al. 2005; Franco et al. 2005; Noto et al. 2013). For example,
Mongolian gerbils infected with highly virulent cagPAI-positive H. pylori have
been found to develop similar pathology as compared to infected humans. Gerbils
developed gastric dysplasia in almost all animals by 4-week infection, which was
accompanied by adenocarcinomas in ~25% of gerbils (Franco et al. 2005). After
8 weeks, ~75% of infected animals exhibited gastric adenocarcinomas. Impor-
tantly, infection with isogenic mutants indicated that CagA and the T4SS were
necessary for gastric cancer development in the gerbil model (Franco et al. 2005;
Noto et al. 2013). For more details and other models, please refer to Chap. 10 of this
book. In addition to the H. pylori infection model systems, a first direct causal link
between CagA and oncogenesis in vivo was reported by the production of trans-
genic C57BL/6J mice expressing CagA (Ohnishi et al. 2008). After 72 weeks, these
transgenic mice exhibited gastric epithelial hyperplasia and some mice developed
polyps and adenocarcinomas in the stomach and small intestine. Systemic CagA
expression in these mice further induced leukocytosis with IL-3/GM-CSF hyper-
sensitivity, and some animals exhibited myeloid leukemias and B-cell lymphomas
(Ohnishi et al. 2008). These findings were supported using two other model
organisms, zebrafish and Drosophila (Neal et al. 2013; Reid et al. 2012; Muyskens
and Guillemin 2011). Transgenic expression of CagA in these systems exhibited
significantly increased rates of intestinal epithelial cell proliferation, upregulation
of c-Jun N-terminal kinase (JNK) signaling, and Wnt target genes associated with
small cell carcinoma and adenocarcinoma (Neal et al. 2013; Wandler and Guille-
min 2012). These experiments demonstrate that H. pylori can trigger the develop-
ment of gastric adenocarcinoma in gerbils and other model systems in a manner
dependent on a functional T4SS and that sole expression of CagA is sufficient to
produce severe malignant lesions in transgenic mice. Thus, CagA and the cagPAI
T4SS play central roles during H. pylori pathogenesis in vivo.
4.5 Phosphorylation-Dependent Host Cell Signaling

of Translocated CagA
CagA represents a prime example of tyrosine phosphorylatable effector proteins

(CagAPY) of bacteria. Site-directed mutagenesis and mass spectrometry revealed
numerous phosphorylation sites in CagA known as the Glu-Pro-Ile-Tyr-Ala
(EPIYA) motifs A, B, C, and/or D (Hatakeyama 2003; Yamaoka 2010; Backert
et al. 2010). The host tyrosine kinases active on these EPIYA motifs were identified
as members of the c-Src and c-Abl families (Wessler and Backert 2008). The
resulting phosphotyrosines together with some flanking residues commonly act as
recognition motifs for eukaryotic signaling factors. They recruit in particular
cellular binding partners that contain SH2 (Src homology 2) domains, but not
PTB (phosphotyrosine binding) domains and thereby target and subvert eukaryotic
signal transduction pathways in ways that benefit the pathogen (Selbach
et al. 2009). This indicates that CagA was specifically designed during evolution
to target SH2 domain containing host cell factors (Fig. 4.2a). Altogether, 12
phospho-dependent interaction partners have been identified over the years
(Table 4.1). The first reported interaction partner of CagAPY was the tyrosine
phosphatase SHP-2 (Higashi et al. 2002). Since then, nine other host cell factors
were also found to interact with CagA in a phosphorylation-dependent fashion: the
tyrosine phosphatases SHP-1; phosphoinositide-3-kinase (PI3K); the signaling
adaptor proteins Crk, Grb2, and Grb7; the tyrosine kinases Csk, c-Src, and c-Abl;
as well as the Ras GTPase-activating protein Ras-GAP (Tsutsumi et al. 2003;
Suzuki et al. 2005; Tammer et al. 2007; Selbach et al. 2009; Zhang et al. 2015).
Thus, CagAPY seems to mimic a tyrosine-phosphorylated host cell protein and
Fig. 4.2 Model for the role of H. pylori CagA in host cell-signaling processes which may affect
pathogenesis. CagA phosphorylation-dependent (a) and phosphorylation-independent (b) signal
transduction events are shown. CagA is translocated across the host cell membrane of infected
gastric epithelial cells which requires integrin β1 and phosphatidylserine. The tyrosine kinases
c-Src and c-Abl phosphorylate delivered CagA. CagA can then modulate various signaling
cascades associated with cell polarity, cell proliferation, actin-cytoskeletal rearrangements, cell
elongation, disruption of tight and adherens junctions, pro-inflammatory responses, and suppres-
sion of apoptosis, as depicted. Black arrows indicate activated signaling pathways and red arrows
correspond to inactivated cascades. (For more details, see text. Panels a and b were updated from
Backert and coworkers (2010) with kind permission from Wiley.)
96
Table 4.1 Host cell proteins described to interact with phosphorylated CagA and proposed roles in H. pylori infections
Interaction partner Proposed function Experimental evidence Applied methodsa References
Abl Phosphorylation of CagA and CrkII adapter proteins Infection, transfection coIP, IF Tammer et al. (2007)
of CagA Poppe et al. (2007)
Brandt et al. (2007)
CrkI, CrkII, CrkLb Cell scattering, loss of AJs, MAPK signaling Infection, transfection coIP, IF, PD, immunoblot Suzuki et al. (2005)
of CagA Tammer et al. (2007)
Brandt et al. (2007)
Csk c-Src inactivation, loss of CagA-Shp-2 interaction Transfection of CagA coIP Tsutsumi et al. (2003)
Dephosphorylation of cortactin, ezrin, and vinculin Proteomics SILAC/MS Selbach et al. (2009)
Grb2b Cell scattering, activation of Ras-MAPK signaling Proteomics SILAC/MS Selbach et al. (2009)
Grb7 Yet unknown Proteomics, infection SILAC/MS, coIP Selbach et al. (2009)
PI3-kinaseb ATK signaling Proteomics, infection SILAC/MS, coIP Selbach et al. (2009)
Zhang et al. (2015)
Ras-GAP Yet unknown Proteomics SILAC/MS Selbach et al. (2009)
Shp-1 Yet unknown Proteomics, infection SILAC/MS, coIP Selbach et al. (2009)
Shp-2 Cell scattering, activation of ERK Transfection of CagA coIP Higashi et al. (2002)
Tyrosine dephosphorylation of FAK Transfection of CagA coIP Higashi et al. (2004)
Downregulation of hBD3 Infection IF, RT-PCR, immunoblot Tsutsumi et al. (2006)
c-Srcb Phosphorylation of CagA Infection coIP Selbach et al. (2003)
a
Abbreviations: AJs adherens junctions, IF co-localization by immunofluorescence, coIP co-immunoprecipitation, PD pull-down experiments, SILAC/MS
stable isotope labeling with amino acids in cell culture combined with mass spectrometry of bound proteins
b
Using the coexpression system in COS-7 cells, Tsutsumi et al. (2003) overexpressed CagA together with the p85 subunit of PI3-kinase, c-Src, Grb2, or Crk-II,
but none of the above reported interactions during infection between CagA and these proteins were found during these transfection studies
S. Backert et al.
therefore appears to function as a master key or picklock to hijack specific cascades

of the host. The various CagAPY-SH2 domain interactions appear to have complex
roles in H. pylori-triggered cytoskeletal rearrangements, cell scattering, and elon-
gation (summarized in Fig. 4.2a).
Infection of AGS gastric epithelial cells with H. pylori in vitro results in
migration of the cells and an elongated morphology, known as the “hummingbird
phenotype” (Segal et al. 1999). This phenotype requires host cell motility by a yet
unknown T4SS factor (Churin et al. 2003), while cell elongation is clearly induced
by CagAPY (Backert et al. 2001). Transfection experiments have shown that CagA
PY
-SHP-2 interaction activates the phosphatase activity of SHP-2, which contrib-
utes to AGS cell elongation by stimulating the Rap1 ! B-Raf ! Erk signaling
pathway (Higashi et al. 2004). It was also shown that the CagAPY-SHP-2 complex
suppresses the activation of epidermal growth factor receptor (EGFR) and down-
stream signaling, which is associated with enhanced H. pylori survival (Bauer
et al. 2012). The authors found that an antimicrobial peptide, human β-defensin
3 (hBD3), which is highly active against H. pylori, is downregulated by EGFR
suppression. These findings revealed a novel mechanism how T4SS-positive strains
make use of CagA to evade a key innate mucosal defense pathway to support
persistent H. pylori infection (Bauer et al. 2012).
Additional reports demonstrated that the elongation phenotype also requires
tyrosine dephosphorylation of three well-known actin-binding proteins—vinculin,
ezrin, and cortactin (Selbach et al. 2003, 2004; Moese et al. 2007). The phosphatase
involved in this scenario is not SHP-2 and still remains to be identified. Instead it
was shown that CagAPY can hamper c-Src activity in two ways, by direct binding of
both proteins to each other and by interaction of CagAPY with Csk, a negative
regulator of c-Src (Tsutsumi et al. 2003; Selbach et al. 2003). Since c-Src is
the first kinase phosphorylating CagA, inactivation of c-Src by CagAPY forms a
typical negative feedback-loop mechanism, controlling the amount of intracellular
CagAPY. Interestingly, vinculin, ezrin, and cortactin are also targets of c-Src, and
c-Src inactivation by CagAPY causes the tyrosine dephosphorylation of these
factors (Tegtmeyer and Backert 2011). Moreover, binding of CagAPY to PI3K
and/or CrkII is involved in activating the small Rho family GTPase members
Rac1 and Cdc42 (Suzuki et al. 2005; Selbach et al. 2009), while binding of CagA
PY
to SHP-2 or Grb2 can stimulate pro-inflammatory and proliferative responses
through the mitogen-activated protein (MAP) kinase cascades (Fig. 4.2a). Finally,
CagAPY can also interact with SHP-1, Grb7, and Ras-GAP with yet unknown
consequences for the host cell (Fig. 4.2a). Taken together, CagAPY can bind to a
remarkably high number of host cell factors to activate signaling mediating cell
scattering, elongation, and probably other phenotypes.
Translocation and phosphorylation of CagA appear also important for the
interplay of H. pylori with macrophages. Heme oxygenase (HO-1), an anti-
inflammatory enzyme, is released by macrophages in response to CagA phosphor-
ylation in H. pylori-infected human patients and mice, while blocking of phagocy-
tosis prevented CagAPY and HO-1 induction (Gobert et al. 2014). Genetic ablation
of the hmox-1 gene in mice resulted in increased gastritis, which was associated
with enhanced M1/Th1/Th17 responses, reduced regulatory macrophage response,
as well as lower H. pylori colonization. These findings provide a mechanism by
which H. pylori manipulate immune responses, supporting its own survival by
induction of macrophage HO-1 (Gobert et al. 2014).
4.6 Phosphorylation-Independent Signaling of CagA
Early microarray studies of H. pylori-infected T84 cells showed that expression of

670 host genes changed and 479 of these genes occurred independent of the
phosphorylation state of the CagA protein (El-Etr et al. 2004). Thus, not all the
interactions of intracellular CagA require its tyrosine phosphorylation. Subse-
quently, 12 cellular binding partners of non-phosphorylated CagA have been
reported (Table 4.2). Non-phospho CagA interactions have been found to induce
loss of cell polarity, mitogenic responses, and pro-inflammatory signaling
(Fig. 4.2b). The first interaction partner of non-phosphorylated CagA described
has been the adapter protein Grb2 (Mimuro et al. 2002). Interestingly, Grb2 is the
only binding factor that has been described to interact with both
non-phosphorylated and phosphorylated CagA (Mimuro et al. 2002; Selbach
et al. 2009). In particular, non-phosphorylated CagA was shown to utilize Grb2
for recruiting Grb2-associated Sos (son of sevenless), a guanine-exchange factor
(GEF) of the small GTPase Ras, to the plasma membrane (Fig. 4.2b). This CagA/
Grb2/Sos complex triggers Ras-GTP production, which in turn activates the
Raf ! MEK ! ERK signaling pathway contributing to cell scattering (Mimuro
et al. 2002) and stimulation of nuclear responses mediating cell proliferation and
transcription of the anti-apoptotic myeloid cell leukemia sequence-1 (MCL-1)
protein (Mimuro et al. 2007). CagA was also described to function as a mimetic
of the eukaryotic Grb2-associated binder (Gab) adaptor protein in transgenic
Drosophila which feeds into the same MAP kinase signaling pathway (Hatakeyama
2003; Botham et al. 2008). CagA-triggered MAP kinase activation can stimulate
the transcription factor NF-κB mediating the onset of multiple target genes such as
IL-8 (Brandt et al. 2005). In addition, it was shown that CagA targets various tumor
suppressors such as p53 and RUNX3. CagA-positive H. pylori strains more strongly
suppressed p53 as compared with low-risk strains during infection in vivo and
in vitro. Degradation of p53 protein was shown to be induced by CagA-mediated
signaling via host-specific E3 ubiquitin ligases, thus contributing in tumorigenicity
(Wei et al. 2015). In addition, non-phosphorylated CagA can bind to RUNX3,
which is often inactivated in gastric cancer. Interaction with RUNX3 proceeds by a
WW domain located at the amino-terminus of CagA (Tsang et al. 2010). Impor-
tantly, CagA induces the ubiquitination and subsequent degradation of RUNX3, in
this way turning off the transcriptional activity of RUNX3 (Fig. 4.2b). These data
Table 4.2 Host cell proteins described to interact with non-phosphorylated CagA and proposed
roles in H. pylori infections
Interaction Experimental Applied
partner Proposed function evidence methodsa References
α-Pix b Inflammation Infection coIP, MS Baek
et al. (2007)
ASPP2 Activation of ASPP2 and Proteomics LC-MS/MS, Buti
p53 degradation, apoptosis coIP, IF, Y2H et al. (2011),
inhibition Nešić
et al. (2014)
β1-integrin Internalization of CagA Infection Y2H, coIP, Jiménez-Soto
Biacore studies et al. (2009)
CagA Multimerization Transfection coIP, Ren
of CagA mutagenesis et al. (2006)
Calcineurin Dephosphorylation of Transfection Coexpression, Yokoyama
NFAT, nuclear of CagA IF et al. (2005)
translocation
c-Met c Cell scattering, activation Infection, coIP, IF Churin
of PI3-kinase, Erk, transfection et al. (2003),
β-catenin, and NF-κB d of CagA Suzuki
et al. (2009)
E-cadherin Destabilization of AJs, Transfection coIP Murata-
β-catenin signaling of CagA Kamiya
et al. (2007)
GSK-3 Snail-mediated EMT Infection, IF, coIP, Lee
trough GSK-3 depletion transfection immunoblot et al. (2014)
of cagA
p120- E-cadherin/c-Met/ p120 Infection IF, coIP, cell Oliveira
catenin complex, cell scattering, invasion assay, et al. (2006,
invasion immunoblot 2009)
Grb2 e Cell scattering, activation Transfection PD, coIP, IF Mimuro
of Ras-MAPK signaling of CagA et al. (2002)
Par1b/ Inhibition of Par1 activity, Transfection coIP, IF, MF Saadat
MARK2 disruption of apical of CagA et al. (2007),
junctions Zeaiter
et al. (2008)
Par1b Inhibition of mitosis Transfection IF, FACS Umeda
of CagA et al. (2009)
Par1a, Enhancement of cell Transfection coIP, IF Lu et al. (2009)
Par1c, elongation of CagA
Par1d
Par1b Inhibition of Par1 3D structure Crystallization, Nesić
binding, and et al. (2010)
kinase assays
PLC-γ f Cell scattering Infection coIP Churin
et al. (2003)
(continued)
Table 4.2 (continued)

Interaction Experimental Applied
partner Proposed function evidence methodsa References
RUNX3 Oncogene by blocking Infection, Mutagenesis, Tsang
tumor suppressor RUNX3 transfection coIP et al. (2010),
of CagA Liu
et al. (2012)
TAK1g Activation of NF-κB Transfection EMSA, qPCR, Lamb
of CagA, coIP, PD, IF et al. (2009)
infection
ZO-1, JAM Disruption of lateral junc- Infection, IF, MF Amieva
tions and cell motility transfection et al. (2003),
of CagA Bagnoli
et al. (2005)
a
Abbreviations: AJs adherens junctions, EMSA electrophoretic mobility-shift assay, FACS
fluorescence-activated cell sorting, IF co-localization by immunofluorescence, coIP
co-immunoprecipitation, EMT epithelial-mesenchymal transition, MF membrane fractionation
by Iodixanol Gradients, Opti-PrepTM, MS mass spectrometry of bound proteins, PD pull-down
experiments, qPCR quantitative real-time polymerase chain reaction, Y2H yeast two-hybrid screen
b
Dependency of CagAPY was not investigated but α-PIX does not contain an SH2 domain and is
therefore unlikely to interact with CagA in a phospho-specific manner
c
Activation of c-Met was not observed in another study and attributed to cross-reactivity of the
phospho-c-Met antibody with CagAPY (Snider and Cardelli 2009)
d
Activation of CagA-induced NF-κB activation and IL-8 secretion was not observed when CagA
was co-transfected with dominant-negative c-Met in AGS cells (Brandt et al. 2005)
e
An interaction of Grb2 with non-phosphorylated CagA (Mimuro et al. 2002) or CagAPY (Selbach
et al. 2009) was reported during Hp infection; but none of the latter interactions were found in
studies using transfected CagA (Tsutsumi et al. 2003; Churin et al. 2003)
f
PLC-γ coIP revealed a strong background signal for non-injected CagA in a translocation-
defective Δvirb11 mutant
g
An interaction of CagA with TAK1 was excluded in another study showing that anti-TAK1
antibodies artificially precipitate CagA from a T4SS-defective strain (Sokolova et al. 2014)
together suggest the presence of distinct EPIYA-independent domains within

CagA, which have crucial functions in protein targeting and modification of host
cell transcription.
Another remarkable outcome of phosphorylation-independent CagA activities in
polarized epithelial cells is the disturbance of the cell-to-cell integrity (Fig. 4.2b). In
particular, the proper architecture of the gastric epithelium is controlled by highly
organized tight and adherent junctions (Wessler and Backert 2008). Infection and
transfection experiments showed that CagA interferes with these intercellular
junctions in multiple ways. For example, CagA associates with ZO-1 (zona
occludens-1), a tight-junction scaffolding protein and the transmembrane protein
JAM (junctional adhesion molecule), causing an ectopic assembly of tight-junction
components at sites of bacterial attachment (Amieva et al. 2003). The non-
phosphorylated form of CagA has also been reported to bind to the cell-cell
junctional transmembrane protein E-cadherin (Murata-Kamiya et al. 2007). Later
on, immunoprecipitation studies showed that CagA forms a complex with c-Met
recruiting E-cadherin and the armadillo-domain protein p120 catenin, suggesting
that binding of CagA to E-cadherin is probably not direct (Oliveira et al. 2009).
However, there is much debate going on whether or not the 135-kDa c-Met receptor
is phosphorylated and activated upon infection with H. pylori (Snider and Cardelli
2009). Thus, the function of c-Met signaling during H. pylori infection is not fully
clarified and should be investigated more thoroughly in future. Some controversy
also exists whether CagA can disrupt the E-cadherin complex associated with the
release of β-catenin, which has been proposed for transfected CagA or H. pylori-
infected AGS cells (Franco et al. 2005; Murata-Kamiya et al. 2007). It has been
noted by some authors that AGS cells do not express E-cadherin and exhibit
abnormal β-catenin allocation, making them not suitable for the study of related
signaling (Oliveira et al. 2009). Considering this fact, it was shown, using MDCK
cells expressing wild-type E-cadherin and β-catenin without any mutations, that
H. pylori-induced β-catenin signal transduction proceeds independently of CagA
during infection (Sokolova et al. 2008). Similarly, some controversy also exists
with regard to the proposed interaction of CagA with transforming growth factor
beta-activated kinase 1 (TAK1) (Lamb et al. 2009; Sokolova et al. 2014)
(Table 4.2).
However, the importance of CagA in inducing the loss of cell polarity is much
clearer. The host kinase Par1b (partitioning-defective 1), also called MARK2
(microtubule affinity-regulating kinase), is a central regulator of cell polarity and
was reported to have a crucial impact on H. pylori-induced signal transduction.
Non-phosphorylated CagA can directly bind Par1b that results in the inhibition of
its kinase activity, triggering the loss of cell polarity (Saadat et al. 2007; Nesić
et al. 2010). Furthermore, more recent studies showed that CagA not only binds to
Par1b but also to other members of this kinase family (Par1a, Par1c, and Par1d) and
that these interactions contribute to the H. pylori-triggered AGS cell elongation
phenotype (Lu et al. 2009). Recent data also suggest that CagA can also interact
with glycogen synthase kinase 3 (GSK-3) and acts as a pathogenic scaffold protein
that induces a Snail-mediated epithelial-mesenchymal transition via the deple-
tion of GSK-3 activity (Lee et al. 2014). Taken together, these results suggest
that transfected CagA can interfere with Par1 members, c-Met, GSK-3, and
E-cadherin signaling and may also activate NF-κB, thereby contributing to
H. pylori-induced pro-inflammatory responses. Finally, there are two more reported
binding partners of CagA, α-Pix (Baek et al. 2007), and integrin β1 (Jiménez-Soto
et al. 2009; Kaplan-Türk€oz et al. 2012). While the interaction with α-Pix has a
proposed role in inflammation, CagA-integrin β1 interaction may be involved in
delivery of CagA into the host cell (Fig. 4.2b). Importantly, the downstream
pathways of CagA emerged to be highly diverse and possible cross talk among
them and other bacterial factors need to be dissected in more detail.
4.7 T4SS-Dependent but CagA-Independent Cellular

Signaling Induced by H. pylori
In this section, various T4SS-dependent but CagA-independent signaling events

induced by H. pylori will be discussed (Fig. 4.3). Early studies indicated that
H. pylori can actively inhibit its own uptake and killing by professional phagocytes
(Ramarao et al. 2000). This anti-phagocytic effect was dependent on vital bacteria
expressing the T4SS, because various isogenic virB mutants blocked this phenotype
(Ramarao et al. 2000). Interestingly, the actual factor involved was not CagA,
because isogenic ΔcagA mutants also abrogated phagocytosis. These experiments
indicated that H. pylori express a yet unknown T4SS effector with anti-phagocytic
capability that may play a crucial function in the immune escape of this persistent
pathogen (Fig. 4.3). Infection of bone-marrow-derived macrophages by H. pylori-
induced pathology via microRNAs, such as miR-155, as important regulators of
inflammatory and innate immune responses. Increase of miR155 expression was
T4SS-dependent but CagA-independent and resulted in reduced macrophage apo-
ptosis (Koch et al. 2012). However, most of the studies were performed to
Fig. 4.3 Model for the role of H. pylori T4SS-dependent but CagA-independent host cell-
signaling processes which may affect pathogenesis. The multitude of known T4SS-dependent
but CagA-independent pathways involve in the activation of receptor and non-receptor tyrosine
kinases, pro-inflammatory signaling, Rho GTPase activation, scattering and motility of gastric
epithelial cells, as well as suppression of histone phosphorylation and H. pylori phagocytosis by
immune cells. Two particular T4SS factors have been reported to be involved in some but not all of
these responses. The known signaling functions for injected peptidoglycan as well as pilus-
exposed or recombinant CagL are shown. For numerous other pathways, the actual T4SS factor
is yet unknown as also indicated. (For more details see text. This figure was updated from Backert
and coworkers (2010) with kind permission from Wiley.)
investigate the interaction of H. pylori with gastric epithelial cells.

Phosphoproteomics of epithelial cells infected with H. pylori revealed the induction
of multiple tyrosine-phosphorylated proteins. The majority of enriched phospho-
peptides were from kinases of the MAPK family and the use of isogenic mutants
showed that both CagA and the T4SS are key regulators of tyrosine phosphoryla-
tion events (Glowinski et al. 2014). In addition, histone H3 phosphorylation was
found to be altered by a T4SS-dependent but CagA-independent pathway (Fig. 4.3).
Infection with cagPAI-positive H. pylori strains decreased H3 phosphorylation
levels at two phosphorylation sites, serine residue 10 and threonine residue
3 (Fehri et al. 2009; Ding et al. 2010). It appeared that mitotic histone H3 kinases
such as Aurora B and vaccinia-related kinase 1 (VRK1) were not fully activated in
H. pylori-infected cells, leading to a transient pre-mitotic cell cycle arrest (Fehri
et al. 2009). Together, these data demonstrate that H. pylori subvert cellular key
processes, including cell cycle progression, by a not yet identified T4SS effector.
Furthermore, the results of numerous reports revealed that other components of the
T4SS, but not CagA itself, were necessary for the induction of pro-inflammatory
signaling, including the activation of transcription factors NF-κB and AP-1
(Fig. 4.3). This implicated that the T4SS might deliver effectors in addition to
CagA or that the T4SS itself triggers the effect. Despite systematic mutagenesis of
all cagPAI genes and other efforts, the hypothetical additional effector remained
unknown for many years. One proposed candidate was bacterial peptidoglycan,
because it can be identified by Nod1, an intracellular pathogen-recognition mole-
cule (Viala et al. 2004). These results implicated that T4SS-dependent delivery of
peptidoglycan is responsible for activation of Nod1 ! NF-κB-dependent
pro-inflammatory responses such as secretion of IL-8 (Viala et al. 2004). However,
the actual bacterial T4SS factor(s) and pathways that activate both transcription
factors, NF-κB and AP-1, are highly controversial in the literature and still not fully
defined (Backert and Naumann 2010). Remarkably, T4SS-positive H. pylori can
activate the NF-κB-dependent induction of a DNA-editing enzyme (AID) in gastric
epithelial cells, that leads to the accumulation of mutations in tumor suppressor p53
(Matsumoto et al. 2007). Thus, induction of AID by H. pylori infection might be a
mechanism whereby gastric carcinogenesis-related gene mutations accumulate.
Infection of gastric epithelial cells with H. pylori was also reported to profoundly
activate various receptor tyrosine kinases (RTKs) in a T4SS-dependent manner
including EGFR (Keates et al. 2001; Churin et al. 2003), hepatocyte growth factor
receptor c-Met, and Her2/Neu (Churin et al. 2003). Studies on the downstream
signaling indicated that each of these RTKs can activate the MAP kinase members
MEK and ERK1/2 (Fig. 4.3). However, while induction of EGFR has been shown
to induce pro-inflammatory responses leading to the secretion of IL-8 (Keates
et al. 2001), activation of c-Met (but not EGFR or Her2/Neu) was involved in
cell scattering and motogenic responses of infected gastric epithelial cells (Churin
et al. 2003). At later time points of infection, EGFR can be inactivated by CagA as
mentioned above (Bauer et al. 2012). Interestingly, the small Rho GTPases Rac1
and Cdc42 and the non-receptor tyrosine kinase c-Abl are also activated by a T4SS-
dependent but CagA-independent process and play a role in stimulating scattering
and motility of infected gastric epithelial cells (Fig. 4.3). However, the actual T4SS
factor involved for many of the above events is still unknown.
In vitro studies showed a profound role of recombinant CagL in activating the
host tyrosine kinases EGFR, ErbB3/Her3, FAK, and c-Src (Tegtmeyer et al. 2010).
Investigation on the molecular mechanism of EGFR activation by CagL has
demonstrated the involvement of ADAM17, a metalloprotease implicated in cata-
lyzing ectodomain shedding of receptor tyrosine kinase ligands. In non-stimulated
cells, the inactive form of ADAM17 forms a complex with integrin α5β1 (Fig. 4.3).
During acute H. pylori infection, however, it was demonstrated that CagL binding
to integrin α5β1 activates ADAM17 by dissociating ADAM17 from the complex
(Saha et al. 2010). In addition, CagL immobilized on petri dishes binds host cells
and, thus, mimics human fibronectin (Tegtmeyer et al. 2010). Fibronectin is a
250-kDa protein containing an RGD motif that plays crucial roles in promoting
cell adhesion, migration, and intracellular signaling. It was shown that purified
CagL alone can directly trigger intracellular signaling pathways upon contact with
mammalian cells and can even complement the spreading defect of fibronectin-/-
knockout cells in vitro (Tegtmeyer et al. 2010). Treatment of AGS cells with
purified CagL was also demonstrated to be sufficient to result in IL-8 induction,
which required the RGD motif in CagL and activation of integrin α5β1 (Gorrell
et al. 2012). Using different wild-type or ΔcagL mutant strains showed that IL-8
induction occurred independently of CagA translocation. Further studies revealed
another surface-exposed Phe-Glu-Ala-Asn-Glu (FEANE) interaction motif located
close to the RGD site. This site enhanced the interaction of CagL with integrin α5β1
supporting CagA translocation and was referred to as RGD helper sequence, RHS
(Conradi et al. 2012). CagL was also shown to be related to increased gastrin
expression resulting in hypergastrinemia, a major risk factor for gastric adenocar-
cinoma formation. Gastric epithelial cells stably transfected with a human gastrin
promoter luciferase construct increased the promoter activation of gastrin via
integrin-linked kinase (ILK) and integrin αVβ5 (Wiedemann et al. 2012). Another
role of CagL was seen in bone-marrow derived DCs, where it resulted in induction
of pro-IL-1β and formation of mature IL-1β and NLRP3 (NOD-like receptor pyrin
domain-containing 3) induction in response to H. pylori infection. This activity was
mediated via different host innate immune receptors including Toll-like receptor
2 (TLR2) and nucleotide-binding oligomerization domain 2 (NOD2) (Kim
et al. 2013). CagL and the activity of MAP kinases were also important for
H. pylori-mediated regulation of eosinophil migration (Nagy et al. 2011). The
results indicate that H. pylori increases production of the chemokines CCL2,
CCL5, and granulocyte-macrophage colony-stimulating factor (GMC-SF) by gas-
tric epithelial cells and that these molecules induce eosinophil migration. Interest-
ingly, CagL sequence analyses revealed that isolates from the gastric cancer
patients had a higher rate of amino acid sequence polymorphisms—Y58 and
E59—than those of the non-gastric cancer patients (Yeh et al. 2011). The CagL
Y58E59 polymorphism increased risk of gastric cancer up to 4.6-fold and infected
patients had higher integrin α5β1 expression than noninfected patients. Furthermore,
CagL-Y58E59 H. pylori infection predisposed an upward shift in integrin α5β1 in
the corpus, leading to more severe corpus chronic inflammation (Yeh et al. 2011).
However, expression of isogenic CagL Y58/E59 variants in H. pylori strain 26695
significantly blocked translocation and phosphorylation of CagA as compared to
complemented wild-type CagL (Tegtmeyer et al. 2014). The involved signaling
should be studied in detail in future studies.
4.8 Conclusions
H. pylori represents a highly successful human pathogen, which can trigger severe
clinical symptoms in a small subset of patients. The investigation of bacteria-host
interactions and virulence factors such as CagA and the T4SS has provided us with
crucial insights in mechanisms leading to H. pylori pathogenesis. A list of more
than 20 known cellular interaction partners of CagA is quite amazing for a bacterial
effector protein. The current model suggests that CagA mimics a eukaryotic
signaling factor either located in a large multiprotein complex or simultaneously
in various subcellular areas of infected host cells. The large variety of binding
partners also reflects the integrated network of complex signal transduction path-
ways in target cells, which may have important impact on the multi-step pathogen-
esis of H. pylori. In the future, it will be important to search for additional
translocated effector molecules of the cagPAI T4SS and various other T4SSs
present in the H. pylori chromosome (Backert et al. 2015). Finally, the importance
of CagA for H. pylori itself is also not yet clear. Using a polarized epithelium model
system, ΔcagA mutants were shown to be defective in cell surface colonization, but
exogenous addition of iron to the apical medium partially rescues this defect,
suggesting that one of CagA’s effects on host cells is to facilitate iron acquisition
from the host via a mechanism involving the transferrin receptor (Tan et al. 2011).
To test whether CagA is important in promoting iron acquisition in vivo, the
colonization of H. pylori in iron-replete vs. iron-deficient Mongolian gerbils was
carried out. While wild-type H. pylori and ΔcagA mutants colonized iron-replete
gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-
deficient gerbils (Tan et al. 2011). Iron depletion accelerated the development of
H. pylori-induced premalignant and malignant lesions in a CagA-dependent man-
ner (Noto et al. 2013). H. pylori strains harvested from iron-depleted gerbils or
grown under iron-limiting conditions exhibited enhanced virulence and induction
of inflammatory factors. Future studies should investigate the molecular basis of
this important disease-associated phenomenon.
References
Amieva MR, Vogelmann R, Covacci A, Tompkins LS, Nelson WJ, Falkow S (2003) Disruption of
the epithelial apical-junctional complex by Helicobacter pylori CagA. Science 300:1430–1434
Backert S, Meyer TF (2006) Type IV secretion systems and their effectors in bacterial pathogen-
esis. Curr Opin Microbiol 9:207–217
Backert S, Naumann M (2010) What a disorder: proinflammatory signaling pathways induced by
Helicobacter pylori. Trends Microbiol 18:479–486
Backert S, Moese S, Selbach M, Brinkmann V, Meyer TF (2001) Phosphorylation of tyrosine
972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype
in gastric epithelial cells. Mol Microbiol 42:631–644
Backert S, Fronzes R, Waksman G (2008) VirB2 and VirB5 proteins: specialized adhesins in
bacterial type-IV secretion? Trends Microbiol 16:409–413
Backert S, Tegtmeyer N, Selbach M (2010) The versatility of Helicobacter pylori CagA effector
protein functions: The master key hypothesis. Helicobacter 15:163–176
Backert S, Tegtmeyer N, Fischer W (2015) Composition, structure and function of the
Helicobacter pylori cag pathogenicity island encoded type IV secretion system. Future
Baek HY, Lim JW, Kim H (2007) Interaction between the H. pylori CagA and alpha-Pix in gastric
epithelial AGS cells. Ann N Y Acad Sci 1096:18–23
Bagnoli F, Buti L, Tompkins L, Covacci A, Amieva MR (2005) Helicobacter pylori CagA induces
a transition from polarized to invasive phenotypes in MDCK cells. Proc Natl Acad Sci U S A
102:16339–16344
Barden S, Lange S, Tegtmeyer N, Conradi J, Sewald N, Backert S, Niemann HH (2013) A helical
RGD motif promoting cell adhesion: crystal structures of the Helicobacter pylori type IV
secretion system pilus protein CagL. Structure 21:1931–1941
Barrozo RM, Cooke CL, Hansen LM, Gaddy JA, Johnson EM, Cariaga TA, Suarez G, Peek RM Jr,
Cover TL, Solnick JV (2013) Functional plasticity in the type IV secretion system of H. pylori.
Bauer B, Pang E, Holland C, Kessler M, Bartfeld S, Meyer TF (2012) The H. pylori virulence
effector CagA abrogates human β-defensin 3 expression via inactivation of EGFR signaling.
Cell Host Microbe 11:576–586
Botham CM, Wandler AM, Guillemin K (2008) A transgenic Drosophila model demonstrates that
the Helicobacter pylori CagA protein functions as a eukaryotic Gab adaptor. PLoS Pathog 4:
e1000064
Brandt S, Kwok T, Hartig R, K€onig W, Backert S (2005) NF-kappaB activation and potentiation of
proinflammatory responses by the Helicobacter pylori CagA protein. Proc Natl Acad Sci U S A
102:9300–9305
Brandt S, Shafikhani S, Balachandran P, Jin S, Hartig R, K€ onig W, Engel J, Backert S (2007) Use
of a novel coinfection system reveals a role for Rac1, H-Ras, and CrkII phosphorylation in
H. pylori-induced host cell actin cytoskeletal rearrangements. FEMS Immunol Med Microbiol
50:190–205
Buti L, Spooner E, Van der Veen AG, Rappuoli R, Covacci A, Ploegh HL (2011) Helicobacter
pylori cytotoxin-associated gene A (CagA) subverts the apoptosis-stimulating protein of p53
(ASPP2) tumor suppressor pathway of the host. Proc Natl Acad Sci U S A 108:9238–9243
Cendron L, Seydel A, Angelini A, Battistutta R, Zanotti G (2004) Crystal structure of CagZ, a
protein from the H. pylori pathogenicity island that encodes for a Type IV Secretion System. J
Mol Biol 340:881–889
Cendron L, Tasca E, Seraglio T, Seydel A, Angelini A, Battistutta R, Montecucco C, Zanotti G
(2007) The crystal structure of CagS from the Helicobacter pylori pathogenicity island. Pro-
teins 69:440–443
Cendron L, Couturier M, Angelini A, Barison N, Stein M, Zanotti G (2009) The H. pylori CagD
(HP0545, Cag24) protein is essential for CagA translo-cation and maximal induction of IL-8
secretion. J Mol Biol 386:204–217
Chandran V, Fronzes R, Duquerroy S, Cronin N, Navaza J, Waksman G (2009) Structure of the
outer membrane complex of a type IV secretion system. Nature 462:1011–1016
Churin Y, Al-Ghoul L, Kepp O, Meyer TF, Birchmeier W, Naumann M (2003) Helicobacter
pylori CagA protein targets the c-Met receptor and enhances the motogenic response. J Cell
Biol 161:249–255
Conradi J, Tegtmeyer N, Woźna M, Wissbrock M, Michalek C, Gagell C, Cover TL, Frank R,
Sewald N, Backert S (2012) An RGD helper sequence in CagL of Helicobacter pylori assists in
interactions with integrins and injection of CagA. Front Cell Infect Microbiol 2:70
Ding SZ, Fischer W, Kaparakis-Liaskos M, Liechti G, Merrell DS, Ferrero RL, Crowe SE, Haas R,
Hatakeyama M, Goldberg JB (2010) Helicobacter pylori-induced histone modification, asso-
ciated gene expression in gastric epithelial cells, and its implication in pathogenesis. PLoS One
5:e9875
El-Etr SH, Mueller A, Tompkins LS, Falkow S, Merrell DS (2004) Phosphorylation-independent
effects of CagA during interaction between Helicobacter pylori and T84 polarized monolayers.
J Infect Dis 190:1516–1523
Fehri LF, Rechner C, Janssen S, Mak TN, Holland C, Bartfeld S, Brüggemann H, Meyer TF (2009)
Helicobacter pylori-induced modification of the histone H3 phosphorylation status in gastric
epithelial cells reflects its impact on cell cycle regulation. Epigenetics 4:577–586
Fischer W (2011) Assembly and molecular mode of action of the Helicobacter pylori cag type IV
secretion apparatus. FEBS J 278:1203–1212
Franco AT, Israel DA, Washington MK, Krishna U, Collier-Hyams L, Perez-Perez GI,
Hatakeyama M, Whitehead R, Gaus K, O’Brien DP, Romero-Gallo J, Peek RM Jr (2005)
Activation of beta-catenin by carcinogenic Helicobacter pylori. Proc Natl Acad Sci U S A
102:10646–10651
Fronzes R, Schäfer E, Wang L, Saibil HR, Orlova E, Waksman G (2009) Structure of a type IV
secretion system core complex. Science 323:266–268
Glowinski F, Holland C, Thiede B, Jungblut PR, Meyer TF (2014) Analysis of T4SS-induced
signaling by H. pylori using quantitative phosphoproteomics. Front Microbiol 5:356
Gobert AP, Verriere T, Asim M, Barry DP, Piazuelo MB, de Sablet T, Delgado AG, Bravo LE,
Correa P, Peek RM Jr, Chaturvedi R, Wilson KT (2014) Heme Oxygenase-1 Dysregulates
Macrophage Polarization and the Immune Response to Helicobacter pylori. J Immunol
193:3013–3022
Gorrell RJ, Guan J, Xin Y, Hutton ML, McGuckin MA, Ferrero RL, Kwok T (2012) A novel
NOD1- and CagA-independent pathway of IL-8 induction mediated by the H.pylori type IV
system. Cell Microbiol 15:554–570
Hare S, Fischer W, Williams R, Terradot L, Bayliss R, Haas R, Waksman G (2007) Identification,
structure and mode of action of a new regulator of the Helicobacter pylori HP0525 ATPase.
EMBO J 26:4926–4934
Hatakeyama M (2003) Helicobacter pylori CagA-a potential bacterial oncoprotein that function-
ally mimics the mammalian Gab family of adaptor proteins. Microbes Infect 5:143–150
Hauck CR (2007) Microbiology: preparing the shot. Nature 449:798–799
Hayashi T, Senda M, Morohashi H, Higashi H, Horio M, Kashiba Y, Nagase L, Sasaya D,
Shimizu T, Venugopalan N, Kumeta H, Noda NN, Inagaki F, Senda T, Hatakeyama M
(2012) Tertiary structure-function analysis reveals the pathogenic signaling potentia-
tion mechanism of Helicobacter pylori oncogenic effector CagA. Cell Host Microbe
12:20–33
Higashi H, Tsutsumi R, Muto S, Sugiyama T, Azuma T, Asaka M, Hatakeyama M (2002) SHP-2
tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science
295:683–686
Higashi H, Nakaya A, Tsutsumi R, Yokoyama K, Fujii Y, Ishikawa S, Higuchi M, Takahashi A,

Tanaka S, Azuma T, Hatakeyama M (2004) Helicobacter pylori CagA induces
Ras-independent morphogenetic response through SHP-2 recruitment and activation. J Biol
Chem 279:17205–17216
Jiménez-Soto LF, Kutter S, Sewald X, Ertl C, Weiss E, Kapp U, Rohde M, Pirch T, Jung K, Retta
Kaplan-Türk€oz B, Jiménez-Soto LF, Dian C, Ertl C, Remaut H, Louche A, Tosi T, Haas R,
Terradot L (2012) Structural insights into H. pylori oncoprotein CagA interaction with β1
integrin. Proc Natl Acad Sci 109:14640–14645
Keates S, Sougioultzis S, Keates AC, Zhao D, Peek RM Jr, Shaw LM, Kelly CP (2001) cag+
H. pylori induce transactivation of the epidermal growth factor receptor in AGS gastric
epithelial cells. J Biol Chem 276:48127–48134
Kim DJ, Park JH, Franchi L, Backert S, Nú~nez G (2013) The Cag pathogenicity island and
interaction between TLR2/NOD2 and NLRP3 regulate IL-1β production in Helicobacter
pylori infected dendritic cells. Eur J Immunol 43:2650–2658
Koch M, Mollenkopf HJ, Klemm U, Meyer TF (2012) Induction of microRNA-155 is TLR- and
T4SS-dependent in macrophages and inhibits DNA-damage induced apoptosis. Proc Natl Acad
Sci U S A 109:E1153–E1162
K€onig W, Backert S (2007) Helicobacter exploits integrin for type IV secretion and kinase
Lamb A, Yang XD, Tsang YH, Li JD, Higashi H, Peek RM, Blanke SR, Chen LF (2009)
Helicobacter pylori CagA activates NF-κB by targeting TAK1 for TRAF6-mediated Lys
63 ubiquitination. EMBO Rep 10:1242–1249
Lee DG, Kim HS, Lee YS, Kim S, Cha SY, Ota I, Kim NH, Cha YH, Yang DH, Lee Y, Park GJ,
Yook JI, Lee YC (2014) Helicobacter pylori CagA promotes snail-mediated epithelial-mes-
enchymal transition by reducing GSK-3 activity. Nat Commun 5:4423
Liu Z, Xu X, Chen L, Li W, Sun Y, Zeng J, Yu H, Chen C, Jia J (2012) H. pylori CagA inhibits the
expression of Runx3 via Src/MEK/ERK and p38 MAPK pathways in gastric epithelial cell. J
Cell Biochem 113:1080–1086
Low HH, Gubellini F, Rivera-Calzada A, Braun N, Connery S, Dujeancourt A, Lu F, Redzej A,
Fronzes R, Orlova EV, Waksman G (2014) Structure of a type IV secretion system. Nature
508:550–553
Lu H, Murata-Kamiya N, Saito Y, Hatakeyama M (2009) Role of partitioning-defective 1/micro-
tubule affinity-regulating kinases in the morphogenetic activity of Helicobacter pylori CagA. J
Biol Chem 284:23024–23036
Matsumoto Y, Marusawa H, Kinoshita K, Endo Y, Kou T, Morisawa T, Azuma T, Okazaki IM,
Honjo T, Chiba T (2007) Helicobacter pylori infection triggers aberrant expression of
activation-induced cytidine deaminase in gastric epithelium. Nat Med 13:470–476
Mimuro H, Suzuki T, Tanaka J, Asahi M, Haas R, Sasakawa C (2002) Grb2 is a key mediator of
H. pylori CagA protein activities. Mol Cell 10:745–755
Mimuro H, Suzuki T, Nagai S, Rieder G, Suzuki M, Nagai T, Fujita Y, Koyasu S, Haas R,
Sasakawa C (2007) Helicobacter pylori dampens gut epithelial self-renewal by inhibiting
apoptosis, a bacterial strategy to enhance colonization of the stomach. Cell Host Microbe
2:250–263
Moese S, Selbach M, Brinkmann V, Karlas A, Haimovich B, Backert S, Meyer TF (2007) The
Helicobacter pylori CagA protein disrupts matrix adhesion of gastric epithelial cells by
dephosphorylation of vinculin. Cell Microbiol 9:1148–1161
Murata-Kamiya N, Kurashima Y, Teishikata Y, Yamahashi Y, Saito Y, Higashi H, Aburatani H,
Akiyama T, Peek RM Jr, Azuma T, Hatakeyama M (2007) H.pylori CagA interacts with
E-cadherin and deregulates catenin that promotes intestinal transdifferentiation in cells. Onco-
gene 26:4617–4626
Murata-Kamiya N, Kikuchi K, Hayashi T, Higashi H, Hatakeyama M (2010) H. pylori exploits

host membrane phosphatidylserine for delivery, localization, and pathophysiological action of
CagA. Cell Host Microbe 7:399–411
Muyskens JB, Guillemin K (2011) Helicobacter pylori CagA disrupts epithelial patterning by
activating myosin light chain. PLoS One 6:e17856
Nagy TA, Allen SS, Wroblewski LE, Flaherty DK, Slaughter JC, Perez-Perez G, Israel DA, Peek
RM Jr (2011) Helicobacter pylori induction of eosinophil migration is mediated by the cag
pathogenicity island via microbial-epithelial interactions. Am J Pathol 178:1448–1452
Neal JT, Peterson TS, Kent ML, Guillemin K (2013) H. pylori virulence factor CagA increases
intestinal cell proliferation by Wnt pathway activation in a transgenic zebrafish model. Dis
Model Mech 6:802–810
Nesić D, Miller MC, Quinkert ZT, Stein M, Chait BT, Stebbins CE (2010) Helicobacter pylori
CagA inhibits PAR1-MARK family kinases by mimicking host substrates. Nat Struct Mol Biol
17:130–132
Nešić D, Buti L, Lu X, Stebbins CE (2014) Structure of the Helicobacter pylori CagA oncoprotein
bound to the human tumor suppressor ASPP2. Proc Natl Acad Sci U S A 111:1562–1567
Noto JM, Gaddy JA, Lee JY, Piazuelo MB, Friedman DB, Colvin DC, Romero-Gallo J, Tan S,
Morgan DR, Wilson KT, Bravo LE, Correa P, Cover TL, Amieva MR, Peek RM Jr (2013) Iron
deficiency accelerates H. pylori-induced carcinogenesis in rodents and humans. J Clin Invest
123:479–492
Ogura K, Maeda S, Nakao M, Watanabe T, Tada M, Kyutoku T, Yoshida H, Shiratori Y, Omata M
(2000) Virulence factors of H. pylori responsible for gastric diseases in Mongolian gerbil. J
Exp Med 192:1601–1610
Ohnishi N, Yuasa H, Tanaka S et al (2008) Transgenic expression of Helicobacter pylori CagA
induces gastrointestinal and hematopoietic neoplasms in mouse. Proc Natl Acad Sci U S A
105:1003–1008
Oliveira MJ, Costa AC, Costa AM, Henriques L, Suriano G, Atherton JC, Machado JC, Carneiro F,
Seruca R, Mareel M, Leroy A, Figueiredo C (2006) H. pylori induces gastric epithelial cell
invasion in a c-Met and type IV secretion system-dependent manner. J Biol Chem
281:34888–34896
Oliveira MJ, Costa AM, Costa AC, Ferreira RM, Sampaio P, Machado JC, Seruca R, Mareel M,
Figueiredo C (2009) CagA associates with c-Met, E-cadherin, and p120-catenin in a
multiproteic complex that suppresses H. pylori-induced cell-invasive phenotype. J Infect Dis
200:745–755
Poppe M, Feller SM, R€omer G, Wessler S (2007) Phosphorylation of Helicobacter pylori CagA by
c-Abl leads to cell motility. Oncogene 26:3462–3472
Ramarao N, Gray-Owen SD, Backert S, Meyer TF (2000) Helicobacter pylori inhibits phagocy-
tosis by professional phagocytes involving type IV secretion components. Mol Microbiol
37:1389–1404
Reid DW, Muyskens JB, Neal JT, Gaddini GW, Wandler AM, Botham CM, Guillemin K (2012)
Identification of genetic modifiers of CagA-induced epithelial disruption in Drosophila. Front
Cell Infect Microbiol 2:24
Ren S, Higashi H, Lu H, Azuma T, Hatakeyama M (2006) Structural basis and functional
consequence of Helicobacter pylori CagA multimerization in cells. J Biol Chem
281:32344–32352
Rieder G, Merchant JL, Haas R (2005) Helicobacter pylori cag-type IV secretion system facilitates
corpus colonization to induce precancerous conditions in Mongolian gerbils. Gastroenterology
128:1229–1242
Rohde M, Püls J, Buhrdorf R, Fischer W, Haas R (2003) A novel sheathed surface organelle of the
Helicobacter pylori cag type IV secretion system. Mol Microbiol 49:219–234
Saadat I, Higashi H, Obuse C, Umeda M, Murata-Kamiya N, Saito Y, Lu H, Ohnishi N, Azuma T,
Suzuki A, Ohno S, Hatakeyama M (2007) Helicobacter pylori CagA targets PAR1/MARK
kinase to disrupt epithelial cell polarity. Nature 447:330–333
Saha A, Backert S, Hammond CE, Gooz M, Smolka AJ (2010) Helicobacter pylori CagL activates
ADAM17 to induce repression of the gastric H, K-ATPase alpha subunit. Gastroenterology
139:239–248
Segal ED, Cha J, Lo J, Falkow S, Tompkins LS (1999) Altered states: involvement of phosphor-
ylated CagA in the induction of host cellular growth changes by H. pylori. Proc Natl Acad Sci
U S A 96:14559–14564
Selbach M, Moese S, Hurwitz R, Hauck CR, Meyer TF, Backert S (2003) The Helicobacter pylori
CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactiva-
tion. EMBO J 22:515–528
Selbach M, Moese S, Backert S, Jungblut PR, Meyer TF (2004) The Helicobacter pylori CagA
protein induces tyrosine dephosphorylation of ezrin. Proteomics 4:2961–2968
Selbach M, Paul FE, Brandt S, Guye P, Daumke O, Backert S, Dehio C, Mann M (2009) Host cell
interactome of tyrosine-phosphorylated bacterial proteins. Cell Host Microbe 5:397–403
Shaffer CL, Gaddy JA, Loh JT, Johnson EM, Hill S, Hennig EE, McClain MS, McDonald WH,
Cover TL (2011) Helicobacter pylori exploits a unique repertoire of type IV secretion system
components for pilus assembly at the bacteria-host cell interface. PLoS Pathog 7:e1002237
Snider JL, Cardelli JA (2009) Helicobacter pylori induces cancer cell motility independent of the
c-Met receptor. J Carcinog 8:7
Sokolova O, Bozko PM, Naumann M (2008) Helicobacter pylori suppresses glycogen synthase
kinase 3beta to promote beta-catenin activity. J Biol Chem 283:29367–29374
Sokolova O, Maubach G, Naumann M (2014) MEKK3 and TAK1 synergize to activate IKK
complex in H. pylori infection. Biochim Biophys Acta 1843:715–724
Suzuki M, Mimuro H, Suzuki T, Park M, Yamamoto T, Sasakawa C (2005) Interaction of CagA
with Crk plays an important role in H. pylori-induced loss of gastric epithelial cell adhesion. J
Exp Med 202:1235–1247
Suzuki M, Mimuro H, Kiga K, Fukumatsu M, Ishijima N, Morikawa H, Nagai S, Koyasu S,
Gilman RH, Kersulyte D, Berg DE, Sasakawa C (2009) Helicobacter pylori CagA
phosphorylation-independent function in epithelial proliferation and inflammation. Cell Host
Microbe 5:23–34
Tammer I, Brandt S, Hartig R, K€onig W, Backert S (2007) Activation of Abl by Helicobacter
pylori: a novel kinase for CagA and crucial mediator of host cell scattering. Gastroenterology
132:1309–1319
Tan S, Noto JM, Romero-Gallo J, Peek RM Jr, Amieva MR (2011) H. pylori perturbs iron
trafficking in the epithelium to grow on the cell surface. PLoS Pathog 7:e1002050
Tanaka J, Suzuki T, Mimuro H, Sasakawa C (2003) Structural definition on the surface of H. pylori
type IV secretion apparatus. Cell Microbiol 5:395–404
Tegtmeyer N, Backert S (2011) Role of Abl and Src family kinases in actin-cytoskeletal
rearrangements induced by the Helicobacter pylori CagA protein. Eur J Cell Biol 90:880–890
Tegtmeyer N, Hartig R, Delahay RM, Rohde M, Brandt S, Conradi J, Takahashi S, Smolka AJ,
Sewald N, Backert S (2010) A small fibronectin-mimicking protein from bacteria induces cell
spreading and focal adhesion formation. J Biol Chem 285:23515–23526
Tegtmeyer N, Wessler S, Backert S (2011) Role of the cag-pathogenicity island encoded type IV
secretion system in Helicobacter pylori pathogenesis. FEBS J 278(8):1190–1202
Tegtmeyer N, Lind J, Schmid B, Backert S (2014) Helicobacter pylori CagL Y58/E59 mutation
turns-off type IV secretion-dependent delivery of CagA into host cells. PLoS One 9:e97782
Terradot L, Waksman G (2011) Architecture of the Helicobacter pylori Cag-type IV secretion
system. FEBS J 278:1213–1222
Tsang YH, Lamb A, Romero-Gallo J, Huang B, Ito K, Peek RM Jr, Ito Y, Chen LF (2010)
Helicobacter pylori CagA targets gastric tumor suppressor RUNX3 for proteasome-mediated
degradation. Oncogene 29:5643–5650
Tsutsumi R, Higashi H, Higuchi M, Okada M, Hatakeyama M (2003) Attenuation of Helicobacter
pylori CagA x SHP-2 signaling by interaction between CagA and C-terminal Src kinase. J Biol
Chem 278:3664–3670
Tsutsumi R, Takahashi A, Azuma T, Higashi H, Hatakeyama M (2006) Focal adhesion kinase is a

substrate and downstream effector of SHP-2 complexed with Helicobacter pylori CagA. Mol
Cell Biol 26:261–276
Umeda M, Murata-Kamiya N, Saito Y, Ohba Y, Takahashi M, Hatakeyama M (2009)
Helicobacter pylori CagA causes mitotic impairment and induces chromosomal instability. J
Biol Chem 284:22166–22172
Viala J, Chaput C, Boneca IG, Cardona A, Girardin SE, Moran AP, Athman R, Mémet S, Huerre
MR, DiStefano PS, Sansonetti PJ, Labigne A, Bertin J, Philpott DJ, Ferrero RL (2004) Nod1
responds to peptidoglycan delivered by the H. pylori cag pathogenicity island. Nat Immunol
5:1166–1174
Waksman G, Orlova EV (2014) Structural organisation of the type IV secretion systems. Curr
Opin Microbiol 17:24–31
Wandler AM, Guillemin K (2012) Transgenic expression of the Helicobacter pylori virulence
factor CagA promotes apoptosis or tumorigenesis through JNK activation in Drosophila. PLoS
Pathog 8:e1002939
Wei J, Noto JM, Zaika E, Romero-Gallo J, Piazuelo MB, Schneider B, El-Rifai W, Correa P, Peek
RM, Zaika AI (2015) Bacterial CagA protein induces degradation of p53 protein in a p14ARF-
dependent manner. Gut 64:1040–1108
Wessler S, Backert S (2008) Molecular mechanisms of epithelial-barrier disruption by
Helicobacter pylori. Trends Microbiol 16:397–405
Wiedemann T, Hofbaur S, Tegtmeyer N, Huber S, Sewald N, Wessler S, Backert S, Rieder G
(2012) Helicobacter pylori CagL dependent induction of gastrin expression via a novel αvβ5-
integrin linked kinase signaling complex. Gut 61:986–996
Yamaoka Y (2010) Mechanisms of disease: Helicobacter pylori virulence factors. Nat Rev
Gastroenterol Hepatol 7:629–641
Yeh YC, Chang WL, Yang HB, Cheng HC, Wu JJ, Sheu BS (2011) H. pylori cagL sequence
polymorphism Y58E59 induces a corpus shift of gastric integrin α5β1 related with gastric
carcinogenesis. Mol Carcinog 50:751–759
Yeo HJ, Savvides SN, Herr AB, Lanka E, Waksman G (2000) Crystal structure of the hexameric
ATPase from H. pylori. Mol. Cell 6:1461–1472
Yokoyama K, Higashi H, Ishikawa S, Fujii Y, Kondo S, Kato H, Azuma T, Wada A, Hirayama T,
Aburatani H, Hatakeyama M (2005) Functional antagonism between Helicobacter pylori
CagA and vacuolating toxin VacA in control of the NFAT signaling pathway in gastric
epithelial cells. Proc Natl Acad Sci U S A 102:9661–9666
Zeaiter Z, Cohen D, Müsch A, Bagnoli F, Covacci A, Stein M (2008) Analysis of detergent-
resistant membranes of Helicobacter pylori infected gastric adenocarcinoma cells reveals a
role for MARK2/Par1b in CagA-mediated disruption of cellular polarity. Cell Microbiol
10:781–794
Zhang X, Perez-Perez G, Tegtmeyer N, Sticht H, Backert S, Blaser M (2015) A specific A/T
polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori
CagA epithelial cell interactions. PLoS Pathog 11:e1004621
Chapter 5
Helicobacter pylori Vacuolating Toxin
Timothy L. Cover, Robin L. Holland, and Steven R. Blanke
Abstract The vacuolating cytotoxin (VacA) was one of the first H. pylori viru-
lence factors identified. All H. pylori strains contain a vacA gene, but there is
variation among H. pylori strains in the levels of VacA secretion and activity of
VacA proteins. Strains containing allelic types of vacA that produce more active
forms of the toxin are associated with human gastroduodenal disease. Experiments
in animal models suggest that VacA may contribute to H. pylori colonization of the
stomach, and the toxin has been linked to gastric epithelial damage. VacA induces a
variety of effects in cultured epithelial cells, including alterations in membrane
trafficking within the endolysosomal system, mitochondrial dysfunction, and cell
death. Most VacA effects on cells are a consequence of intracellular toxin activities.
Unlike most intracellular-acting bacterial toxins that enzymatically modify target
molecules within eukaryotic cells, VacA causes cellular alterations primarily
through the formation of intracellular ion-conducting channels. In this chapter,
we review the structure and function of VacA, along with the roles of VacA in
the pathogenesis of H. pylori-associated diseases.
5.1 Introduction
Helicobacter pylori colonizes the human stomach, and persistent infection is a risk
factor for peptic ulcer disease and gastric cancer. The development of these diseases
is dependent on the actions of specific H. pylori virulence factors and is also
T.L. Cover (*)

Department of Medicine, Department of Pathology, Microbiology and Immunology,
Vanderbilt University School of Medicine and Veterans Affairs Tennessee Valley Healthcare
System, Nashville, TN, USA
R.L. Holland
Department of Pathobiology, School of Veterinary Medicine, University of Illinois, Urbana,
IL, USA
S.R. Blanke (*)
Department of Microbiology, Department of Pathobiology, Institute for Genomic Biology,
School of Molecular and Cellular Biology, University of Illinois, Urbana, IL, USA
© Springer Japan (outside the USA) 2016 113

DOI 10.1007/978-4-431-55936-8_5
114 T.L. Cover et al.
influenced by host and environmental factors. One of the important H. pylori

virulence factors is the vacuolating cytotoxin (VacA). This toxin was originally
identified based on its ability to cause vacuolation of cultured host cells (Leunk
et al. 1988; Cover and Blaser 1992), but is now known to have many additional
activities. In this chapter, we discuss progress in our understanding of the structure-
function relationships that underlie the actions of VacA, the unusual manner by
which VacA is taken up into and trafficked within host cells, and the ability of VacA
to form ion-conducting channels in the membranes of intracellular organelles. In
addition, we discuss the role of VacA in the pathogenesis of H. pylori-associated
gastric disease.
5.2 The vacA Gene vacA Transcription
A single chromosomal copy of vacA is present in all sequenced H. pylori strains

(Cover et al. 1994; Telford et al. 1994; Schmitt and Haas 1994). VacA orthologues
are present in H. cetorum (a gastric Helicobacter found in dolphins and whales)
(Kersulyte et al. 2013), and a degenerated vacA gene has been detected in
H. acinonychis (found in large cats) (Dailidiene et al. 2004). The vacA transcript
is monocistronic, with a transcriptional start point about 120 nucleotides upstream
from the ATG start site (Schmitt and Haas 1994; Forsyth et al. 1998). There is
considerable variation in vacA transcription levels among H. pylori strains (Forsyth
et al. 1998).
5.3 Secretion and Proteolytic Processing of VacA
In H. pylori strain 60190, vacA encodes a 1287 amino acid (140 kDa) protoxin
(Cover et al. 1994), which undergoes distinct proteolytic processing steps (Fig. 5.1).
First, the 33 amino acid amino-terminal signal sequence is cleaved, presumably as
the toxin is transported across the cytoplasmic membrane into the periplasmic
space. Further processing results in an 88 kDa mature secreted form of the toxin,
a 12 kDa secreted peptide, and a ~33 kDa carboxyl-terminal domain that remains
associated with the bacterial cell (Cover and Blaser 1992; Nguyen et al. 2001;
Bumann et al. 2002; Telford et al. 1994). The process of VacA secretion is thought
to occur by a type V (or autotransporter) secretion pathway, and the secreted 88 kDa
toxin is considered to be the “passenger domain” (Fischer et al. 2001). Analogous to
other autotransporter proteins, the VacA carboxyl-terminal domain is predicted to
have a β-barrel structure and is required for secretion of the VacA passenger domain
(Schmitt and Haas 1994).
The 88 kDa VacA toxin is released from the bacteria into the extracellular space
as a soluble protein (Cover and Blaser 1992) or as a component of membrane blebs
(Fiocca et al. 1999). VacA can also remain on the surface of H. pylori, spatially
5 Helicobacter pylori Vacuolating Toxin 115
Fig. 5.1 VacA structure. Proteolytic cleavage of the VacA protoxin yields a signal peptide, an
88 kDa secreted protein, and the autotransporter domain. The secreted 88 kDa protein is composed
of two domains (p33 and p55), and the autotransporter domain is processed to yield a 12 kDa
secreted peptide and a 33 kDa cell-associated protein. Three main regions of vacA diversity are
currently recognized: the signal region (s1 and s2 alleles), intermediate region (i1 and i2), and
mid-region (m1 and m2 alleles)
organized into distinct toxin-rich domains (Ilver et al. 2004), and found predomi-
nantly in a unipolar location (nonflagellar pole) (Radin et al. 2013). The mecha-
nisms that govern release of VacA as a soluble protein or retention on the bacterial
surface have not been elucidated. Surface-bound VacA may promote H. pylori
adherence to gastric epithelial cells, and upon bacterial contact with host cells,
VacA can be transferred directly to host cells (Ilver et al. 2004).
5.4 Properties of the 88 kDa Secreted VacA Protein
The secreted 88 kDa toxin can undergo limited proteolysis to yield two fragments,
designated as p33 and p55 (Telford et al. 1994; Willhite et al. 2002; Ye et al. 1999;
Nguyen et al. 2001; Torres et al. 2005) (Fig. 5.1). The p55 domain has a predom-
inantly β-helical structure (Gangwer et al. 2007), which is a feature shared by many
bacterial autotransporter proteins. The structure of the p33 domain has not been
determined, but it contains a strongly hydrophobic region near the amino-terminus
that is required for membrane channel formation and many of the toxin’s cell-
modulating effects (see Sect. 6) (de Bernard et al. 1998; Ye and Blanke 2000;
Vinion-Dubiel et al. 1999; McClain et al. 2003).
Neither p33 nor p55 alone has detectable effects on eukaryotic cells. In contrast,
a mixture of p33 and p55, when added exogenously to host cells or co-expressed
intracellularly within cells, can reconstitute VacA cellular activity (Ye et al. 1999;
Gonzalez-Rivera et al. 2010). Both the p55 domain and the p33 domain have
important roles in binding of VacA to host cells (see Sect. 7) (Garner and Cover
1996; Wang et al. 2001; Reyrat et al. 1999; Pagliaccia et al. 1998; Torres
et al. 2005). About 422 amino acids at the amino-terminal end of VacA (which
includes the p33 domain and about 100 amino acids of the p55 domain) are
sufficient to induce cell vacuolation if VacA is expressed intracellularly in tran-

siently transfected cells (Ye et al. 1999; de Bernard et al. 1998).
VacA monomers can assemble into large water-soluble oligomeric structures
(Cover and Blaser 1992). Multiple types of oligomeric VacA structures have been
visualized, including single layered forms (containing 6–9 subunits), bilayered
forms (containing 12 or 14 subunits), and two-dimensional crystals (Lupetti
et al. 1996; Cover et al. 1997; Adrian et al. 2002; Czajkowsky et al. 1999; Chambers
et al. 2013). When exposed to either acidic or alkaline pH, VacA oligomers
dissociate into monomeric components, which can reassemble into oligomeric
structures following neutralization (Cover et al. 1997; Yahiro et al. 1999). VacA
oligomers have relatively little effect on cultured human cells in vitro, whereas the
monomers generated by acidification or alkalinization of oligomers are highly
active (de Bernard et al. 1995). As discussed further in Sect. 6, the water-soluble
oligomeric structures are likely to be structurally similar to membrane pores formed
by VacA.
5.5 VacA Allelic Diversity and Association of vacA

Genotypes with Disease
The activity of VacA in culture filtrates from different H. pylori strains is highly
variable (Leunk et al. 1988; Cover and Blaser 1992), due to nonsense mutations,
internal duplications, deletions, or 1-bp insertions within the vacA gene (Ito
et al. 1998), as well as amino acid sequence variation (Atherton et al. 1995).
Variation among strains in the levels of vacuolating activity in culture filtrates
has also been linked to differences in vacA transcription or differences in VacA
secretion efficiency (Forsyth et al. 1998). Several allelic families of vacA have been
described, based on nucleotide sequence variation in specific regions of vacA
(Fig. 5.1). The emergence of sequence variation may be due to a strong selective
pressure on vacA as H. pylori colonizes and adapts to new hosts and different host
environments (Gangwer et al. 2010).
The “m (for middle) region” of vacA is about 800 nucleotides in length and
encodes part of the p55 domain (Atherton et al. 1995). Two main families of vacA
alleles, designated as m1 and m2, can be differentiated based on diversity within the
m-region (Atherton et al. 1995). Type m1 and m2 forms of VacA differ in ability to
cause alterations in specific cell types, due at least in part to distinct cell-binding
properties (Pagliaccia et al. 1998; Wang et al. 2001). The VacA determinants that
influence the cell-type specificities of m1 and m2 forms of VacA have been mapped
to a 148-residue region within p55 (Skibinski et al. 2006).
Sequence diversity also exists within the “s-region,” which encodes the signal
sequence and the amino-terminus of the processed mature toxin. Two major allelic
clusters in this region are designated as s1 and s2 (Atherton et al. 1995). In contrast
to s1 forms of VacA, s2 forms of VacA fail to induce cellular vacuolation (Atherton
et al. 1995). The signal sequences of s1 and s2 VacA proteins are processed at
different sites, resulting in a mature form of s2 VacA with a 12 amino acid
extension that inactivates the toxin (Letley et al. 2003; McClain et al. 2001).
A third approach for classifying vacA alleles is based on diversity within a region
between the s- and m-regions (encoding part of the p33 domain), known as the “i
(for intermediate) region.” Two main families of alleles, designated as i1 and i2, are
recognized (Rhead et al. 2007; Basso et al. 2008). Type i1 VacA proteins typically
exhibit increased activity in gastric epithelial or T-cell culture assays compared to
type i2 forms of VacA (Rhead et al. 2007; Gonzalez-Rivera et al. 2012).
Epidemiologic studies have revealed strong associations between specific vacA
i-, m-, and s-region allelic types and the occurrence of disease in H. pylori-infected
humans. There is a higher incidence of peptic ulcer disease and gastric cancer in
individuals infected with H. pylori strains possessing s1 vacA alleles than in persons
infected with strains harboring s2 vacA alleles (Fig. 5.2) (Atherton et al. 1995,
1997; van Doorn et al. 1998; Figueiredo et al. 2002). The demonstration that s2
forms of VacA lack vacuolating toxin activity in cell culture assays (Atherton
et al. 1995; McClain et al. 2001; Letley et al. 2003) provides a possible functional
explanation for these epidemiologic observations. Several studies have reported
that, in comparison to strains harboring m2 vacA alleles, strains harboring m1 vacA
alleles are associated with a higher risk of gastric carcinoma (as well as gastric
Fig. 5.2 Role of VacA in H. pylori-associated gastric disease. Humans harboring H. pylori strains
that contain cagA and s1, i1, or m1 vacA alleles are at a greater risk for gastric disease, compared to
humans harboring H. pylori strains that are cagA negative and contain s2, i2, or m2 vacA alleles
histologic precursors of gastric cancer, such as epithelial damage, atrophic gastritis,

and intestinal metaplasia) (Figueiredo et al. 2002; Atherton et al. 1997). Strains
carrying s1/m1 vacA alleles are associated with increased bacterial load and
neutrophil infiltration within the human gastric mucosa (van Doorn et al. 1998;
Atherton et al. 1997; Figueiredo et al. 2002). The i-region has been reported to be a
more reliable predictor of severe gastric disease than the s- or m-regions of vacA
(Rhead et al. 2007; Basso et al. 2008; Winter et al. 2014).
Interpreting the results of epidemiologic studies can be complicated by
confounding factors, including the effects of acid-suppressive agents, aspirin or
other nonsteroidal anti-inflammatory agents, and the gender or age of patients.
Further challenges arise due to difficulty in culturing H. pylori strains from indi-
viduals with gastric cancer. Evaluating an association between vacA alleles and
disease risk can also be complicated by the existence of other strain-specific
bacterial factors that contribute to disease. One of the best understood examples
is a chromosomal region known as the cag pathogenicity island (cag PAI), which
encodes the CagA effector protein and a type IV secretion system (T4SS). The cag
PAI can be either present or absent in individual H. pylori strains. H. pylori isolates
associated with high disease risk frequently harbor a functional cag PAI and
produce the most active forms of VacA (Atherton et al. 1995; van Doorn
et al. 1998), making it difficult to assess the independent contributions of VacA
and the cag PAI. In addition to the cag PAI, several other genetic markers of
virulence, including an adhesin known as BabA, in-frame oipA (hopH) alleles, and
type I hopQ alleles, are present in H. pylori strains containing type s1 vacA alleles
more frequently than in H. pylori strains containing type s2 vacA alleles (Atherton
et al. 1995; van Doorn et al. 1998). For more details on the latter factors, please
refer to Chaps. 3, 4 and 6.
5.6 Membrane Channel Formation by VacA
VacA can insert into the plasma membrane of human cells or planar lipid bilayer
systems to form anion-selective membrane channels (Czajkowsky et al. 1999;
Iwamoto et al. 1999; Tombola et al. 1999a, b). Low-resolution images of
membrane-associated VacA indicate that the toxin forms hexagonal ring-shaped
structures (Czajkowsky et al. 1999; Adrian et al. 2002), similar in appearance to
water-soluble VacA oligomers (Czajkowsky et al. 1999). VacA channel formation
probably results from interaction of VacA monomers with the membrane, followed
by subsequent oligomerization and membrane insertion (Czajkowsky et al. 1999;
Iwamoto et al. 1999).
The mature 88 kDa VacA toxin is predicted to contain only one strongly
hydrophobic region, located near the amino-terminus of p33 (Vinion-Dubiel
et al. 1999). This region contains three tandem GXXXG motifs (defined by glycines
at positions 14, 18, 22, and 26) (McClain et al. 2003), which are characteristic of
transmembrane dimerization sequences. Mutagenesis of several residues within this

region (including G14 and G18) abolishes the capacity of VacA to form membrane
channels in planar lipid bilayers (McClain et al. 2003) and abolishes vacuolating
cytotoxin activity (McClain et al. 2003; Ye and Blanke 2000; de Bernard
et al. 1998), suggesting that the amino-terminal hydrophobic region of VacA inserts
into the membrane. Additional regions of VacA also may insert into the membrane
(Wang et al. 2000).
VacA can permeabilize the plasma membrane of epithelial cells, resulting in
leakage of ions and other small molecules (Tombola et al. 2001; Szabo et al. 1999),
presumably as a consequence of VacA insertion into the plasma membrane and
membrane channel formation. VacA-induced cell vacuolation has been attributed to
VacA channel formation in the membranes of late endocytic compartments
(Montecucco and Rappuoli 2001). VacA-induced alterations in mitochondrial
membrane permeability may be due to formation of VacA channels in mitochon-
drial membranes (Willhite et al. 2003; Willhite and Blanke 2004). Although many
cellular effects of VacA are attributable to membrane channel formation, several
VacA effects are probably the consequences of channel-independent actions (see
Sect. 8).
5.7 VacA Interactions with Host Cells: Binding, Uptake,

and Trafficking
5.7.1 Intracellular Actions of VacA
VacA is internalized into cultured cells (Garner and Cover 1996; Gauthier
et al. 2004, 2005; McClain et al. 2000), and inhibition of VacA uptake into cells
results in attenuated cellular activity of the toxin (Patel et al. 2002; Schraw
et al. 2002). Ectopic expression of functional VacA directly within the cytosol of
several mammalian cell lines results in both vacuole biogenesis and modulation of
mitochondrial function (de Bernard et al. 1997; Willhite and Blanke 2004; Willhite
et al. 2003), which provides evidence that VacA functions within the cytosol.
Despite nearly 20 years of study, the mechanism by which VacA is trafficked to
intracellular sites of toxin action (late endosomes and mitochondria) remains one
the most poorly understood aspects of VacA biology. Most studies of VacA
internalization and intracellular trafficking have been carried out using immortal-
ized cells of epithelial origin. These studies are relevant for understanding VacA
interactions with gastric epithelial cells, but it remains to be seen whether VacA
binds and is taken up into immune cells by similar mechanisms.
5.7.2 Interactions of VacA with the Epithelial Cell Surface
Both the p33 and p55 fragments of VacA bind to liposomes, suggesting that both
may contribute to VacA interactions with the plasma membrane of target cells
(Moll et al. 1995; Pagliaccia et al. 2000; Wang et al. 2000; Czajkowsky et al. 1999).
Correspondingly, a mixture of p33 and p55 recombinant fragments binds to target
cells to a much greater extent than either fragment individually (Torres et al. 2005).
Early studies using radiolabeled VacA suggested that the toxin binds to the
plasma membrane of sensitive cells nonspecifically or, alternatively, binds to an
abundant, low-affinity receptor (McClain et al. 2000; Ricci et al. 2000). In contrast,
indirect immunofluorescence and flow cytometry-based studies indicated that
VacA binding to HeLa cells is saturable (Massari et al. 1998), and competitive
binding studies (Wang et al. 2001) suggested that the toxin might bind specifically
to a component on the surface of host cells. Cross-linking studies revealed three
interacting proteins on the surface of human-derived AZ-521 gastric cells: receptor
protein tyrosine phosphatase β (RPTP-β, also known as Ptprz or PTP-zeta), receptor
protein tyrosine phosphatase α (RPTP-α), and low-density lipoprotein receptor-
related protein (LRP1) (Yahiro et al. 1999, 2003, 2012) (Fig. 5.3). Each of these
proteins influences the susceptibility of specific cell types to VacA (Padilla
et al. 2000; Fujikawa et al. 2003; Yahiro et al. 2004), and RPTP-β is required for
VacA-induced epithelial damage in a mouse model (Fujikawa et al. 2003).
More recently, the abundant plasma membrane sphingolipid, sphingomyelin
(SM), was demonstrated to confer sensitivity to VacA across a number of different
cell lines (Gupta et al. 2008, 2010). SM is important for binding VacA to the cell
surface and interacts with VacA, indicating that SM functions as a VacA receptor.
Moreover, SM was demonstrated to be important for VacA binding to the cell
surface, uptake, and intracellular trafficking in a manner relevant for toxin activity
(Gupta et al. 2010).
The relative contributions of RPTP-β, RPTP-α, LRP1, and SM to VacA cellular
binding, uptake, and trafficking are not fully clear. Membrane lipid rafts are
important for VacA cellular activity (Patel et al. 2002; Schraw et al. 2002; Gauthier
et al. 2004; Ricci et al. 2000), and SM has an important role in VacA association
with membrane rafts (Gupta et al. 2008, 2010). Based on the idea that membrane
rafts may function as specialized signaling platforms on the cell surface, one
plausible idea is that VacA binding to SM-enriched rafts provides a nucleating
center for other cell-associated factors, including RPTP-β, RPTP-α, or LRP1, to
assemble into a functional complex required for toxin uptake into cells.
5.7.3 Uptake of VacA into an Intracellular Compartment
VacA is taken up by cells and trafficked by an unusual pinocytic mechanism, not

previously described for other intracellular-acting bacterial exotoxins (Fig. 5.3)
Fig. 5.3 Intracellular trafficking of VacA. Upon interacting with the cell surface, VacA mono-
mers oligomerize into pore structures that are associated with lipid-enriched microdomains on the
plasma membrane. After binding to sphingomyelin in the microdomains, VacA is internalized by
an actin-dependent mechanism into GPI-anchored protein-enriched endosomal compartments
(GEECs). After internalization, VacA traffics to the mitochondria by a currently unknown
mechanism. There are three proposed models: (1) the VacA-containing vesicles (VCVs) associate
with mitochondria, allowing for direct transfer, (2) VacA is released into the cytosol and is
translocated through mitochondrial transporters, or (3) the VCVs fuse with the mitochondrial
membrane
(Ricci et al. 2000; Gauthier et al. 2005, 2006). Sites of VacA binding to the plasma
membrane are localized above F-actin structures, which are regulated by the small
GTPase Rac1 (Gauthier et al. 2005). Following uptake from the cell surface, VacA
enters VacA-containing vesicles (VCVs) that are similar to previously described
glycosylphosphatidylinositol-anchored protein-enriched endosomal compartments
(GEECs) (Gauthier et al. 2005, 2007). It is hypothesized that these noncanonical
early endosomal compartments promote VacA trafficking to intracellular sites of
toxin action (late endosomes and mitochondria).
GPI-anchored proteins within GEECs are typically recycled back to the plasma
membrane, but VacA within GEECs transitions to compartments enriched in
markers characteristic of early endosomal compartments and then ultimately to
late endosomes. The trafficking of VacA from GEECs requires polymerized actin
structures, which contact early VCVs (Gauthier et al. 2007). Although little is
known about this form of actin-dependent intracellular toxin trafficking, the capac-
ity of VacA to exploit such a pathway suggests unusual requirements for intracel-
lular VacA transport.
5.7.4 VacA Trafficking to Mitochondria
Ectopic intracellular expression of VacA results in localization of the p33 domain

of VacA to mitochondria (Galmiche et al. 2000), suggesting that VacA might
translocate from within the cytosol of intoxicated cells across mitochondrial outer
membranes (Willhite and Blanke 2004; Domanska et al. 2010). Conceptually, the
translocation of VacA to mitochondria from the cytosol would require that VacA
exit from the endolysosomal system, which is consistent with the mode of action of
many other intracellular-acting bacterial exotoxins.
Nonetheless, there is a striking dearth of direct evidence to indicate that VacA
translocates to the cytosol prior to mitochondrial localization. Recently, VacA-
containing vesicles (VCVs) possessing several canonical endolysosomal markers
were demonstrated to align in close juxtaposition with the mitochondria (Calore
et al. 2010), suggesting the possibility that VacA, rather than escaping to the cytosol
prior to targeting mitochondria, might instead be transferred directly from
endosomal compartments to the mitochondria (Fig. 5.4). The amino-terminal p33
domain of VacA promotes toxin import across the mitochondrial outer membrane
and insertion into the inner membrane by a mechanism requiring the import channel
of the mitochondrial TOM20 complex (Domanska et al. 2010). These findings
suggest that VacA is transferred from intracellular trafficking vesicles directly to
mitochondria by a mechanism requiring p33-mediated toxin interactions with
existing mitochondrial protein import machinery and that VacA reaches the mito-
chondria via VCV-mediated transfer of the toxin from the endolysosomal system.
The functional and molecular properties of VCVs required for targeting to mito-
chondria have not been identified.
5.7.5 VacA Uptake and Trafficking in Immune Cells
Relatively little is known about the binding, uptake, and trafficking of VacA in
immune cells. Studies of VacA interactions with primary human T cells indicate
that VacA binds to the CD18 receptor (β2 integrin) and is then internalized through
a clathrin-independent process that requires Ser/Thr kinases of the protein kinase C
(PKC) family (Sewald et al. 2008, 2011).
Fig. 5.4 VacA effects on mitochondria. Upon reaching the mitochondria, VacA exerts three
prominent effects. (1) VacA activates the proapoptotic protein Bax to translocate to the mitochon-
dria, where it forms a pore with another proapoptotic protein Bak, releasing cytochrome c into the
cytosol, (2) VacA induces mitochondrial fragmentation by recruiting the mitochondrial fission
protein Drp1 to the mitochondria, and (3) VacA dissipates the transmembrane potential of the
inner mitochondrial membrane
5.8 Effects of VacA on Host Cells In Vitro
VacA effects on host cells in vitro have been studied using viable intact H. pylori,
H. pylori culture supernatants containing VacA, VacA purified from H. pylori
culture supernatants, or recombinant VacA produced by E. coli. The oligomeric
form of VacA has relatively little activity and therefore is typically activated by
exposure to low pH or high pH prior to contact with cells (de Bernard et al. 1995). A
wide range of cell types, including gastric epithelial cells and several types of
immune cells, are susceptible to the effects of VacA. VacA can cause an array of
different effects within an individual cell type, ranging from subtle morphologic or
functional alterations to cell death (Fig. 5.5).
5.8.1 VacA as a Modulator of Epithelial Cell Function
5.8.1.1 Alterations in Endosomal Compartments
Transformed cell lines derived from multiple tissue types and from multiple
mammalian species undergo vacuolation in response to VacA (Leunk et al. 1988;
Fig. 5.5 Cellular alterations caused by VacA. Exposure of cultured cells to VacA results in
endocytic alterations, the induction of autophagy, and in some cases cell death due to VacA-
induced mitochondrial alterations (cytochrome c release, dissipation of the mitochondrial trans-
membrane potential, and mitochondrial fragmentation). Additionally, VacA disrupts epithelial
cell-cell junctions, allowing the toxin to interact with immune cells in the lamina propria. VacA
has both proinflammatory and anti-inflammatory effects and may facilitate adherence of H. pylori
to gastric epithelial cells
Pagliaccia et al. 1998). Primary human gastric epithelial cells are also susceptible
(Smoot et al. 1996). The intraluminal pH of VacA-induced vacuoles is acidic
(Cover et al. 1992; Papini et al. 1994), and the membranes of these vacuoles are
enriched in Rab7 and other markers for late endocytic compartments (Papini
et al. 1994, 1997; Li et al. 2004; Molinari et al. 1997). VacA-dependent vacuole
formation is enhanced by the presence of weak bases such as ammonium chloride
(Cover and Blaser 1992; Li et al. 2004).
Intracellular expression of VacA results in cell vacuolation (de Bernard
et al. 1997; Ye et al. 1999), and when added externally to cultured cells, VacA
localizes to membranes of VacA-induced vacuoles (Fiocca et al. 1999; Ricci
et al. 1997). A current model posits that VacA is internalized into cells and forms
anion-selective channels in the membranes of late endocytic compartments, and in
the presence of permeant weak bases, vacuoles arise due to swelling of late
endosomal compartments (Fig. 5.5) (Montecucco and Rappuoli 2001). VacA
mutant toxins that are deficient in membrane channel formation lack the capacity
to cause cell vacuolation, regardless of whether they are added to the surface of
cells or expressed intracellularly (Vinion-Dubiel et al. 1999; Ye and Blanke 2000;
McClain et al. 2003), and chemical inhibitors of anion channel function block
VacA-induced cellular effects (Tombola et al. 1999b). Correspondingly, wild-type

VacA causes swelling of isolated endosomes and enhances the v-ATPase proton
pump activity of endosomes in a chloride-dependent manner, whereas mutant
toxins lacking channel activity do not induce swelling of isolated endosomes
(Genisset et al. 2007). Multiple cellular factors, including vacuolar ATPase,
Rab7, Rac1, syntaxin 7, dynamin, and GPI-anchored proteins and connexin
43 (Papini et al. 1997; Hotchin et al. 2000; Li et al. 2004; Suzuki et al. 2001,
2003; Radin et al. 2014), are required for VacA-induced vacuolation and may have
roles in VacA internalization or vesicle swelling.
Underlying the process of VacA-mediated cell vacuolation, it remains unclear
whether VacA inserts directly into endosomal membranes to form channels,
whether the VacA channels form in the plasma membrane prior to endocytosis,
or whether both of these processes occur. Another area of uncertainty involves the
source (or sources) of membrane required for the formation of intracellular vacuole
compartments. One model proposes that VacA stimulates progressive enlargement
of pre-existing vesicular compartments, and another proposes that VacA stimulates
fusion of multiple smaller endocytic compartments. It has been suggested that
vacuoles could arise from late endosomes without a requirement for fusion of
different compartments, via a process involving fusion of late endosomal internal
membranes with the late endosomal limiting membrane (de Bernard et al. 2002;
Genisset et al. 2007; Suzuki et al. 2003).
VacA can cause detectable alterations in late endocytic compartments even in
the absence of visible vacuolation. For example, VacA can inhibit the intracellular
degradation of epidermal growth factor (Satin et al. 1997) and can inhibit
procathepsin D maturation (associated with mis-targeting of pro-cathepsin D out-
side the cell) (Satin et al. 1997). In addition, VacA can cause apical mislocalization
of the transferrin receptor, resulting in perturbation of transferrin recycling (Tan
et al. 2011).
5.8.1.2 Autophagy
In addition to inducing intracellular vacuoles, VacA stimulates the formation of

autophagosomes (Terebiznik et al. 2006, 2009). Low-density lipoprotein receptor-
related protein-1 (LRP1) is reported to be a cell surface receptor that mediates
VacA-induced autophagy (Yahiro et al. 2012). VacA-induced autophagy is associ-
ated with decreased intracellular glutathione levels, accumulation of reactive spe-
cies, and activation of AKT kinase (Kimura et al. 2001; Tsugawa et al. 2012). It has
been proposed that autophagy is a host mechanism to limit toxin-induced damage
(Terebiznik et al. 2009) and may also promote intracellular survival of H. pylori in
gastric epithelial cells (Terebiznik et al. 2006). Although acute exposure of cells to
VacA triggers autophagy, chronic exposure to VacA may lead to a disruption of
autophagy (Raju et al. 2012).
5.8.1.3 Cell Death
Vacuolated cells exclude trypan blue (Leunk et al. 1988), and if VacA is removed
from the medium overlying vacuolated cells, the cells continue to proliferate.
Conversely, VacA can inhibit cell proliferation and cause cell death following
exposure of cells to high doses of the toxin for prolonged time intervals (Kuck
et al. 2001; Cover et al. 2003). VacA-induced cell death was initially thought to be
an apoptotic phenomenon, but more recent studies have shown that cell death may
also occur through programmed necrosis (Radin et al. 2011), which, unlike apo-
ptosis, stimulates an inflammatory response. VacA-induced cell death potentially
occurs through multiple mechanisms involving autophagy, mitochondrial alter-
ations, activation of signal transduction pathways, and endoplasmic reticulum
stress, as discussed further below.
5.8.1.4 Alterations in Mitochondria
VacA induces reduction of the mitochondrial transmembrane potential and release

of cytochrome c, both of which reflect alterations in mitochondrial membrane
permeability (Kimura et al. 1999; Galmiche et al. 2000; Willhite et al. 2003;
Willhite and Blanke 2004). These changes result in a reduction in cellular ATP
levels and impaired cell cycle progression (Kimura et al. 1999), impairment of
glutathione metabolism, impaired resistance of cells against oxidative stress
(Kimura et al. 2001), activation of caspase 3, and cleavage of PARP (Galmiche
et al. 2000). In addition, VacA causes mitochondrial network fragmentation
through a Drp1-dependent process (Jain et al. 2011). Since release of cytochrome
c from mitochondria and activation of caspase 3 are proapoptotic phenomena,
VacA-induced mitochondrial alterations are thought to have an important role in
VacA-dependent cell death (Kuck et al. 2001; Cover et al. 2003; Galmiche
et al. 2000).
Both p33 and p55 expressed within mammalian cells and full-length VacA
applied to the surface of cells localize to the mitochondria (Galmiche et al. 2000;
Willhite and Blanke 2004; Calore et al. 2010; Foo et al. 2010). Similarly, when
VacA is added to isolated mitochondria in cell-free systems, the toxin is
translocated into mitochondria and associates with the mitochondrial inner mem-
brane (Galmiche et al. 2000; Domanska et al. 2010; Calore et al. 2010; Foo
et al. 2010). Mutant forms of VacA defective in the capacity to form membrane
channels fail to cause cytochrome c release, and chemical inhibitors of VacA
channel formation inhibit VacA-induced cytochrome c release (Willhite
et al. 2003; Willhite and Blanke 2004), suggesting that VacA can act to form
channels in mitochondrial membranes. Alternatively, the observed changes in
mitochondrial membrane permeability may result from indirect processes, includ-
ing the activation of endogenous channels found in mitochondria, through activa-
tion of the proapoptotic proteins Bax and Bak (Yamasaki et al. 2006; Calore
et al. 2010; Jain et al. 2011), through reduced expression of pro-survival Bcl2
proteins, or through endoplasmic reticulum stress (Akazawa et al. 2013).
5.8.1.5 Effects of VacA on Cellular Signal Transduction Pathways
Two classes of mitogen-activated protein (MAP) kinases (p38 and ERK1/2) and
the activating transcription factor 2 (ATF-2) signaling pathway are activated in
gastric epithelial cells in response to VacA (Nakayama et al. 2004; Hisatsune
et al. 2007). VacA-induced activation of the p38/ATF-2 signal pathway is likely to
be independent of VacA effects on late endocytic compartments and mitochondria
(Nakayama et al. 2004). One potential consequence of VacA-induced p38 activa-
tion is upregulation of cyclooxygenase-2 (COX-2) expression, leading to increased
prostaglandin E2 (PGE2) production (Hisatsune et al. 2007). Other cellular effects
attributed to activation of signal transduction pathways include activation of
G protein-coupled receptor kinase interactor (Git1) (Fujikawa et al. 2003),
upregulated expression of vascular endothelial growth factor through an epidermal
growth factor receptor-dependent pathway (Caputo et al. 2003), and activation of a
PI3K-dependent signaling pathway that leads to phosphorylation of protein kinase
B (AKT) and glycogen synthase kinase-3β (GSK3β) and subsequent translocation
of β-catenin to the nucleus (Nakayama et al. 2009). These effects occur relatively
rapidly, suggesting that they are the consequences of VacA interactions with
specific cell surface components, without a requirement for internalization of the
toxin (Fujikawa et al. 2003; Caputo et al. 2003).
5.8.1.6 Effects of VacA on Epithelial Cell Permeability

and the Cytoskeleton
VacA lowers the transepithelial electric resistance (TER) of polarized epithelial

cells due to increased paracellular epithelial permeability of the monolayers
(Papini et al. 1998). The mechanisms by which VacA alters paracellular perme-
ability are not understood, but VacA-induced alterations in actin filaments and the
microtubule network within intoxicated cells (Pai et al. 1999; Hennig et al. 2005;
Tabel et al. 2003) may be contributory (Wang et al. 2005). In addition to changes
in paracellular permeability, VacA increases the transepithelial flux of certain
molecules, including urea and bicarbonate (Szabo et al. 1999; Tombola et al. 2001;
Debellis et al. 2001; Guarino et al. 1998), by a mechanism attributed to the formation
of VacA channels in the plasma membrane (Szabo et al. 1999). Release of small
molecules may have a favorable effect on survival of H. pylori within the acidic
gastric mucus layer (Debellis et al. 2001), and release of factors such as Fe3+, Ni2+,
sugars, and amino acids may support the growth of H. pylori (Papini et al. 1998).
5.8.2 VacA as a Modulator of Immune Cell Function
VacA can cause alterations in many types of immune cells in vitro. VacA can
potentially gain access to immune cells in the lamina propria in vivo through
disruptions in the gastric epithelial layer. In addition, H. pylori residing within
the gastric mucus layer may have direct access to intraepithelial T lymphocytes and
dendritic cells (DCs).
5.8.2.1 Effects of VacA on T and B Lymphocytes
VacA causes alterations in both CD4+ T cells and CD8+ T cells (Gebert et al. 2003;
Boncristiano et al. 2003; Sundrud et al. 2004; Oswald-Richter et al. 2006; Torres
et al. 2007). When added to Jurkat T cells, VacA inhibits the production of
interleukin 2 (IL-2) (a factor required for T-cell viability and proliferation) and
downregulates surface expression of the IL-2 receptor (Gebert et al. 2003;
Boncristiano et al. 2003; Sundrud et al. 2004). VacA treatment of primary human
CD4+ T cells results in inhibition of activation-induced proliferation, mitochondrial
depolarization, ATP depletion, and cell cycle arrest (Sundrud et al. 2004; Oswald-
Richter et al. 2006). The inhibitory effects of VacA on IL-2 secretion are much
more prominent in Jurkat cells than in primary cells, whereas the inhibitory effects
of VacA on T-cell proliferation are more prominent in primary human CD4+ T cells
(Sundrud et al. 2004; Oswald-Richter et al. 2006; Sewald et al. 2008).
VacA can inhibit activation of nuclear factor of T cells (NFAT) (Gebert
et al. 2003; Boncristiano et al. 2003), a transcription factor that globally regulates
of immune response genes required for optimal T-cell activation. Within Jurkat T
cells, VacA alters the expression of numerous genes, including the Ca2+-calmod-
ulin-dependent phosphatase calcineurin (an enzyme that dephosphorylates NFAT)
(Gebert et al. 2003). It has been proposed that VacA blocks calcium influx into cells
from the extracellular milieu, thereby inhibiting the activity of calcineurin (Gebert
et al. 2003; Boncristiano et al. 2003). VacA can also activate MAP kinases (p38 and
MKK3/6) and the Rac-specific nucleotide exchange factor Vav in T cells
(Boncristiano et al. 2003; Oswald-Richter et al. 2006). All of these effects can
occur without a substantial increase in apoptosis or cell death.
Some effects on T cells are dependent on the formation of VacA channels in cell
membranes (Boncristiano et al. 2003; Sundrud et al. 2004; Oswald-Richter
et al. 2006), and other effects are the result of activation of altered signaling in T
cells via a channel-independent mechanism (Boncristiano et al. 2003). Most of the
known effects of VacA on T cells are expected to result in localized immunosup-
pression, but VacA also stimulates expression of COX-2 in T cells, which is
expected to have a proinflammatory effect (Boncristiano et al. 2003).
VacA interferes with antigen presentation by B lymphocytes. In one model
system, VacA interfered with proteolytic processing of tetanus toxoid and inhibited
the invariant chain (Ii)-dependent pathway of antigen presentation mediated by

newly synthesized major histocompatibility complex (MHC) class II molecules
(Molinari et al. 1998). This effect is likely due to VacA-induced alterations on
endocytic compartments, resulting in alterations in endocytic trafficking. VacA also
inhibits activation-induced proliferation of B cells (Torres et al. 2007).
5.8.2.2 Effects of VacA on Other Types of Immune Cells
Within macrophages, VacA disrupts proper vesicular maturation by inducing the

formation of large vesicular compartments (termed megasomes) (Allen et al. 2000;
Zheng and Jones 2003) and the recruitment and retention of the tryptophan
aspartate-containing coat protein (TACO or coronin 1) to phagosomes (Zheng
and Jones 2003). VacA is reported to stimulate activation of p38 MAP kinase,
cause increased expression of COX-2 (Boncristiano et al. 2003), and block cytokine
production in macrophages (Weiss et al. 2013). Finally, it has been reported that
VacA can activate various signal transduction pathways in macrophages (Hisatsune
et al. 2008) and can stimulate macrophages to undergo apoptosis (Menaker
et al. 2004).
VacA stimulates both mast cell chemotaxis and production of proinflammatory
cytokines by mast cells (Supajatura et al. 2002; de Bernard et al. 2005). Binding of
VacA to a mast cell line induces an oscillation in levels of cytosolic calcium and
exocytosis of secretory granules (de Bernard et al. 2005). VacA can cause
upregulated expression of chemokines in eosinophils (Kim et al. 2007) and apo-
ptosis of eosinophils as a later event (Kim et al. 2010). The former effect is reported
to occur via a pathway involving calcium influx, mitochondrial generation of
reactive oxygen intermediates, and NF-κB activation (Kim et al. 2007).
Neutrophils and DCs are also susceptible to VacA. VacA induces the activation
of p38 MAP kinase and increased expression of COX-2 in neutrophils
(Boncristiano et al. 2003; Brest et al. 2006) and inhibits DC maturation (Kim
et al. 2011). Isogenic H. pylori mutants lacking VacA are unable to prevent LPS-
induced DC maturation and fail to drive DC tolerization as assessed by induction of
Treg properties in cocultured naı̈ve T cells (Oertli et al. 2013). Thus, VacA
contributes to a tolerizing effect of H. pylori on DCs.
5.8.3 Effects of VacA on Parietal Cells and Acid/Base

Balance
When added to gastric glands or cultured parietal cells, VacA inhibits acid secretion
by blocking the recruitment of H,K-ATPase-containing tubulovesicles to the apical
membrane through a mechanism linked to an influx of extracellular calcium,
activation of calpain, and proteolysis of ezrin (Kobayashi et al. 1996; Wang

et al. 2008). VacA-induced alterations of parietal cells may contribute to the
hypochlorhydria sometimes observed in H. pylori-infected persons. VacA also
increases bicarbonate efflux from gastric epithelial cells (Debellis et al. 2001) and
inhibits duodenal bicarbonate secretion, through a histamine-dependent process
(Tuo et al. 2009).
5.9 Synergistic and Antagonistic Associations Between

VacA and CagA
VacA and CagA are strain-specific H. pylori virulence factors that contribute to
the pathogenesis of gastric disease associated with H. pylori infection. Like
VacA, CagA modulates host cell function in several ways, primarily by disrupting
signal transduction within intoxicated cells. There are several fundamental dif-
ferences in the processes by which VacA and CagA modulate host epithelial cells.
CagA, as a T4SS effector delivered to the eukaryotic cytosol, modulates the
functional properties of cells with which H. pylori has direct physical contact.
In contrast, secreted VacA can act on cells to which H. pylori is directly attached
as well as epithelial cells at distal sites (see Chap. 4 for more details). Thus,
gastric epithelial cells are predicted to be subject to the modulating effects of both
VacA and CagA.
The cell modulatory effects of VacA and CagA are in some cases synergistic.
For example, studies of H. pylori colonizing the apical surface of polarized epithe-
lial monolayers indicate that both VacA and CagA facilitate iron acquisition (Tan
et al. 2011). However, most studies to date indicate that the cellular activities of
VacA and CagA are primarily antagonistic. VacA and CagA inhibit each other’s
effects on epithelial cells, with CagA downregulating cellular vacuolation and
VacA downregulating CagA-induced cell alterations (Argent et al. 2008;
Tegtmeyer et al. 2009). CagA activates the NFAT pathway via activation of
calcineurin, whereas VacA blocks calcineurin activation through decreased cal-
cium influx, thereby downregulating the NFAT pathway (Yokoyama et al. 2005).
Additionally, CagA is degraded through an autophagic process; therefore, VacA-
induced autophagy leads to degradation of CagA (Tsugawa et al. 2012). Strikingly,
the capacity of VacA to induce the death of epithelial cells is blocked by CagA
(Oldani et al. 2009), and CagA further inhibits VacA-dependent apoptosis by
blocking the cellular uptake of VacA from the cell surface (Akada et al. 2010).
Overall, these findings are consistent with the idea that VacA and CagA promote
H. pylori persistence by functioning together to remodel the gastric niche occupied
by the bacterium and at the same time limit the degree to which the gastric mucosa
is damaged.
5.10 Role of VacA In Vivo
5.10.1 Role of VacA in H. pylori Colonization

of the Stomach
Mice, gerbils, and gnotobiotic piglets can be colonized by H. pylori vacA knockout
mutant strains (Eaton et al. 1997; Ogura et al. 2000; Salama et al. 2001; Wirth
et al. 1998), which indicates that, in these animal models, VacA production is not an
absolute requirement for gastric colonization. However, wild-type H. pylori strains
colonize better than vacA mutant strains, based on both competition experiments
and experiments using individual strains (Salama et al. 2001; Oertli et al. 2013). In
addition, VacA-immunized mice are protected against challenges with H. pylori
(Marchetti et al. 1998; Ghiara et al. 1997). H. pylori strains producing less active
s2/i2 forms of VacA colonize mice more efficiently than vacA knockout mutant
strains or strains that produce more active forms of VacA (Winter et al. 2014).
These studies provide evidence that VacA may contribute to an improved ability of
H. pylori to colonize the stomach.
Since murine T cells are resistant to VacA (Algood et al. 2007), there are
limitations in the use of mouse experiments for evaluating the role of VacA in
long-term H. pylori infection. Nevertheless, one study reported that a vacA mutant
strain induced stronger Th1 and Th17 responses and triggered more severe gastric
pathology in mice than did a wild-type strain; this was attributed to an ability of
VacA to promote the induction of Tregs (Oertli et al. 2013). Therefore, it seems
plausible that VacA might have a role in enabling H. pylori to persistently colonize
human hosts.
5.10.2 Role of VacA in Gastroduodenal Disease
As discussed in Sect. 5, strains of H. pylori that contain certain allelic forms of vacA
are associated with an increased risk of symptomatic gastroduodenal disease
(Fig. 5.2), but drawing conclusions from these epidemiologic studies is limited by
the possibility of confounding variables. Caution must be used when evaluating the
role of VacA in animal models, since some of the cellular components with which
VacA interacts are found exclusively in human cells (Algood et al. 2007). Never-
theless, direct administration of VacA protein into the stomachs of mice results in
gastric mucosal injury and gastric inflammation (Fujikawa et al. 2003; Supajatura
et al. 2002; Telford et al. 1994). A gastric mucosal inflammatory response may
occur as a consequence of either VacA-mediated damage to the gastric epithelium
or direct proinflammatory effects of VacA on various types of intoxicated cells
(Boncristiano et al. 2003; Hisatsune et al. 2007; Supajatura et al. 2002; Kim
et al. 2007).
Comparing the histologic alterations in animals infected with isogenic wild-type

or vacA-null mutant strains revealed that VacA may have a small but detectable
effect on the development of gastric ulceration in gerbils (Ogura et al. 2000).
Strains producing more active forms of VacA induced more severe and extensive
metaplasia and inflammation in mice than did strains producing less active s2/i2
forms of VacA (Winter et al. 2014). Other studies have not detected an effect of
VacA on gastric histology in animal models (Eaton et al. 1997; Ogura et al. 2000;
Salama et al. 2001) or reported that VacA has an anti-inflammatory effect (Oertli
et al. 2013).
While the vast majority of intracellular-acting bacterial toxins function as enzymes

to covalently modify and alter the properties of host intracellular targets, VacA
forms intracellular membrane channels, leading to alterations in membrane traf-
ficking within the endolysosomal system and altered dynamics and function of the
intracellular mitochondrial network. Exciting areas for future research will be to
ascertain how VacA forms and regulates toxin channels at discrete sites within the
cell and how intracellular channel formation contributes to host cell dysfunction.
One of the most difficult experimental challenges is to understand how VacA
contributes to H. pylori colonization, persistence, and gastroduodenal disease. For
bacteria that are largely human specific, such as H. pylori, animal models have
inherent limitations (see Chap. 10 for more details). In future studies, it will be
important to elucidate the contributions of the toxin to the long-term remodeling of
the gastric microenvironment, which may ultimately contribute to the development
of disease.
References
Adrian M, Cover TL, Dubochet J, Heuser JE (2002) Multiple oligomeric states of the Helicobacter
pylori vacuolating toxin demonstrated by cryo-electron microscopy. J Mol Biol 318:121–133
Akada JK, Aoki H, Torigoe Y, Kitagawa T, Kurazono H, Hoshida H, Nishikawa J, Terai S,
Matsuzaki M, Hirayama T, Nakazawa T, Akada R, Nakamura K (2010) Helicobacter pylori
CagA inhibits endocytosis of cytotoxin VacA in host cells. Dis Model Mech 3:605–617
Akazawa Y, Isomoto H, Matsushima K, Kanda T, Minami H, Yamaghchi N, Taura N,
Shiozawa K, Ohnita K, Takeshima F, Nakano M, Moss J, Hirayama T, Nakao K (2013)
Endoplasmic reticulum stress contributes to Helicobacter pylori VacA-induced apoptosis.
PLoS One 8:e82322
Allen LA, Schlesinger LS, Kang B (2000) Virulent strains of Helicobacter pylori demonstrate
delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages. J Exp Med
191:115–128
Argent RH, Thomas RJ, Letley DP, Rittig MG, Hardie KR, Atherton JC (2008) Functional
association between the Helicobacter pylori virulence factors VacA and CagA. J Med
Basso D, Zambon CF, Letley DP, Stranges A, Marchet A, Rhead JL, Schiavon S, Guariso G,
Ceroti M, Nitti D, Rugge M, Plebani M, Atherton JC (2008) Clinical relevance of Helicobacter
pylori cagA and vacA gene polymorphisms. Gastroenterology 135:91–99
Boncristiano M, Paccani SR, Barone S, Ulivieri C, Patrussi L, Ilver D, Amedei A, D’elios MM,
Telford JL, Baldari CT (2003) The Helicobacter pylori vacuolating toxin inhibits T cell
activation by two independent mechanisms. J Exp Med 198:1887–1897
Brest P, Hofman V, Lassalle S, Cesaro A, Ricci V, Selva E, Auberger P, Hofman P (2006) Human
polymorphonuclear leukocytes are sensitive in vitro to Helicobacter pylori VacA toxin.
Helicobacter 11:544–555
Calore F, Genisset C, Casellato A, Rossato M, Codolo G, Esposti MD, Scorrano L, De Bernard M
(2010) Endosome-mitochondria juxtaposition during apoptosis induced by Helicobacter pylori
VacA. Cell Death Differ 17:1707–1716
Caputo R, Tuccillo C, Manzo BA, Zarrilli R, Tortora G, Blanco Cdel V, Ricci V, Ciardiello F,
Romano M (2003) Helicobacter pylori VacA toxin up-regulates vascular endothelial growth
factor expression in MKN 28 gastric cells through an epidermal growth factor receptor-,
cyclooxygenase-2-dependent mechanism. Clin Cancer Res 9:2015–2021
Chambers MG, Pyburn TM, Gonzalez-Rivera C, Collier SE, Eli I, Yip CK, Takizawa Y, Lacy DB,
Cover TL, Ohi MD (2013) Structural analysis of the oligomeric states of Helicobacter pylori
VacA toxin. J Mol Biol 425:524–535
Cover TL, Blaser MJ (1992) Purification and characterization of the vacuolating toxin from
Helicobacter pylori. J Biol Chem 267:10570–10575
Cover TL, Vaughn SG, Cao P, Blaser MJ (1992) Potentiation of Helicobacter pylori vacuolating
toxin activity by nicotine and other weak bases. J Infect Dis 166:1073–1078
Cover TL, Tummuru MKR, Cao P, Thompson SA, Blaser MJ (1994) Divergence of genetic
sequences for the vacuolating cytotoxin among Helicobacter pylori strains. J Biol Chem
269:10566–10573
Cover TL, Hanson PI, Heuser JE (1997) Acid-induced dissociation of VacA, the Helicobacter
pylori vacuolating cytotoxin, reveals its pattern of assembly. J Cell Biol 138:759–769
Czajkowsky DM, Iwamoto H, Cover TL, Shao Z (1999) The vacuolating toxin from Helicobacter
pylori forms hexameric pores in lipid bilayers at low pH. Proc Natl Acad Sci U S A
96:2001–2006
Dailidiene D, Dailide G, Ogura K, Zhang M, Mukhopadhyay AK, Eaton KA, Cattoli G, Kusters
JG, Berg DE (2004) Helicobacter acinonychis: genetic and rodent infection studies of a
Helicobacter pylori-like gastric pathogen of cheetahs and other big cats. J Bacteriol
186:356–365
De Bernard M, Papini E, De Filippis V, Gottardi E, Telford J, Manetti R, Fontana A, Rappuoli R,
Montecucco C (1995) Low pH activates the vacuolating toxin of Helicobacter pylori, which
becomes acid and pepsin resistant. J Biol Chem 270:23937–23940
De Bernard M, Arico B, Papini E, Rizzuto R, Grandi G, Rappuoli R, Montecucco C (1997)

Helicobacter pylori toxin VacA induces vacuole formation by acting in the cell cytosol. Mol
De Bernard M, Burroni D, Papini E, Rappuoli R, Telford J, Montecucco C (1998) Identification of
the Helicobacter pylori VacA toxin domain active in the cell cytosol. Infect Immun
66:6014–6016
De Bernard M, Moschioni M, Habermann A, Griffiths G, Montecucco C (2002) Cell vacuolization
induced by Helicobacter pylori VacA cytotoxin does not depend on late endosomal SNAREs.
Cell Microbiol 4:11–18
De Bernard M, Cappon A, Pancotto L, Ruggiero P, Rivera J, Del Giudice G, Montecucco C (2005)
The Helicobacter pylori VacA cytotoxin activates RBL-2H3 cells by inducing cytosolic
calcium oscillations. Cell Microbiol 7:191–198
Debellis L, Papini E, Caroppo R, Montecucco C, Curci S (2001) Helicobacter pylori cytotoxin
VacA increases alkaline secretion in gastric epithelial cells. Am J Physiol Gastrointest Liver
Physiol 281:G1440–G1448
Domanska G, Motz C, Meinecke M, Harsman A, Papatheodorou P, Reljic B, Dian-Lothrop EA,
Galmiche A, Kepp O, Becker L, Gunnewig K, Wagner R, Rassow J (2010) Helicobacter pylori
VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.
Eaton KA, Cover TL, Tummuru MK, Blaser MJ, Krakowka S (1997) Role of vacuolating
cytotoxin in gastritis due to Helicobacter pylori in gnotobiotic piglets. Infect Immun
65:3462–3464
Figueiredo C, Machado JC, Pharoah P, Seruca R, Sousa S, Carvalho R, Capelinha AF, Quint W,
Caldas C, Van Doorn LJ, Carneiro F, Sobrinho-Simoes M (2002) Helicobacter pylori and
interleukin 1 genotyping: an opportunity to identify high-risk individuals for gastric carcinoma.
J Natl Cancer Inst 94:1680–1687
Fiocca R, Necchi V, Sommi P, Ricci V, Telford J, Cover TL, Solcia E (1999) Release of
Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding
of outer membrane vesicles. Uptake of released toxin and vesicles by gastric epithelium. J
Pathol 188:220–226
Fischer W, Buhrdorf R, Gerland E, Haas R (2001) Outer membrane targeting of passenger proteins
by the vacuolating cytotoxin autotransporter of Helicobacter pylori. Infect Immun
69:6769–6775
Foo JH, Culvenor JG, Ferrero RL, Kwok T, Lithgow T, Gabriel K (2010) Both the p33 and p55
subunits of the Helicobacter pylori VacA toxin are targeted to mammalian mitochondria. J Mol
Biol 401:792–798
Forsyth MH, Atherton JC, Blaser MJ, Cover TL (1998) Heterogeneity in levels of vacuolating
cytotoxin gene (vacA) transcription among Helicobacter pylori strains. Infect Immun
66:3088–3094
Fujikawa A, Shirasaka D, Yamamoto S, Ota H, Yahiro K, Fukada M, Shintani T, Wada A,
Aoyama N, Hirayama T, Fukamachi H, Noda M (2003) Mice deficient in protein tyrosine
phosphatase receptor type Z are resistant to gastric ulcer induction by VacA of Helicobacter
pylori. Nat Genet 33:375–381
Galmiche A, Rassow J, Doye A, Cagnol S, Chambard JC, Contamin S, De Thillot V, Just I,
Ricci V, Solcia E, Van Obberghen E, Boquet P (2000) The N-terminal 34 kDa fragment of
Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c
release. EMBO J 19:6361–6370
Gangwer KA, Mushrush DJ, Stauff DL, Spiller B, Mcclain MS, Cover TL, Lacy DB (2007)
Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain. Proc Natl Acad Sci
U S A 104:16293–16298
Gangwer KA, Shaffer CL, Suerbaum S, Lacy DB, Cover TL, Bordenstein SR (2010) Molecular
evolution of the Helicobacter pylori vacuolating toxin gene vacA. J Bacteriol 192:6126–6135
Garner JA, Cover TL (1996) Binding and internalization of the Helicobacter pylori vacuolating
cytotoxin by epithelial cells. Infect Immun 64:4197–4203
Gauthier NC, Ricci V, Gounon P, Doye A, Tauc M, Poujeol P, Boquet P (2004)
Glycosylphosphatidylinositol-anchored proteins and actin cytoskeleton modulate chloride
transport by channels formed by the Helicobacter pylori vacuolating cytotoxin VacA in
HeLa cells. J Biol Chem 279:9481–9489
Gauthier NC, Monzo P, Kaddai V, Doye A, Ricci V, Boquet P (2005) Helicobacter pylori VacA
cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed
to late endosomes. Mol Biol Cell 16:4852–4866
Gauthier NC, Ricci V, Landraud L, Boquet P (2006) Helicobacter pylori VacA toxin: a tool to
study novel early endosomes. Trends Microbiol 14:292–294
Gauthier NC, Monzo P, Gonzalez T, Doye A, Oldani A, Gounon P, Ricci V, Cormont M, Boquet P
(2007) Early endosomes associated with dynamic F-actin structures are required for late
trafficking of H. pylori VacA toxin. J Cell Biol 177:343–354
Gebert B, Fischer W, Weiss E, Hoffman R, Haas R (2003) Helicobacter pylori vacuolating
Genisset C, Puhar A, Calore F, De Bernard M, Dell’antone P, Montecucco C (2007) The concerted
action of the Helicobacter pylori cytotoxin VacA and of the v-ATPase proton pump induces
swelling of isolated endosomes. Cell Microbiol 9:1481–1490
Ghiara P, Rossi M, Marchetti M, Di Tommaso A, Vindigni C, Ciampolini F, Covacci A, Telford
JL, De Magistris MT, Pizza M, Rappuoli R, Del Giudice G (1997) Therapeutic intragastric
vaccination against Helicobacter pylori in mice eradicates an otherwise chronic infection and
confers protection against reinfection. Infect Immun 65:4996–5002
Gonzalez-Rivera C, Gangwer KA, Mcclain MS, Eli IM, Chambers MG, Ohi MD, Lacy DB, Cover
TL (2010) Reconstitution of Helicobacter pylori VacA toxin from purified components.
Biochemistry 49:5743–5752
Gonzalez-Rivera C, Algood HM, Radin JN, Mcclain MS, Cover TL (2012) The intermediate
region of Helicobacter pylori VacA is a determinant of toxin potency in a Jurkat T cell assay.
Guarino A, Bisceglia M, Canani RB, Boccia MC, Mallardo G, Bruzzese E, Massari P, Rappuoli R,
Telford J (1998) Enterotoxic effect of the vacuolating toxin produced by Helicobacter pylori in
Caco-2 cells. J Infect Dis 178:1373–1378
Gupta VR, Patel HK, Kostolansky SS, Ballivian RA, Eichberg J, Blanke SR (2008)
Sphingomyelin functions as a novel receptor for Helicobacter pylori VacA. PLoS Pathog 4:
e1000073
Gupta VR, Wilson BA, Blanke SR (2010) Sphingomyelin is important for the cellular entry and
intracellular localization of Helicobacter pylori VacA. Cell Microbiol 12:1517–1533
Hennig EE, Godlewski MM, Butruk E, Ostrowski J (2005) Helicobacter pylori VacA cytotoxin
interacts with fibronectin and alters HeLa cell adhesion and cytoskeletal organization in vitro.
FEMS Immunol Med Microbiol 44:143–150
Hisatsune J, Yamasaki E, Nakayama M, Shirasaka D, Kurazono H, Katagata Y, Inoue H, Han J,
Sap J, Yahiro K, Moss J, Hirayama T (2007) Helicobacter pylori VacA enhances PGE2
production through induction of COX-2 expression via a p38 MAP kinase/ATF-2 cascade in
AZ-521 cells. Infect Immun 75:4472–4481
Hisatsune J, Nakayama M, Isomoto H, Kurazono H, Mukaida N, Mukhopadhyay AK, Azuma T,
Yamaoka Y, Sap J, Yamasaki E, Yahiro K, Moss J, Hirayama T (2008) Molecular character-
ization of Helicobacter pylori VacA induction of IL-8 in U937 cells reveals a prominent role
for p38MAPK in activating transcription factor-2, cAMP response element binding protein,
and NF-kappaB activation. J Immunol 180:5017–5027
Hotchin NA, Cover TL, Akhtar N (2000) Cell vacuolation induced by the VacA cytotoxin of
Helicobacter pylori is regulated by the Rac1 GTPase. J Biol Chem 275:14009–14012
Ilver D, Barone S, Mercati D, Lupetti P, Telford JL (2004) Helicobacter pylori toxin VacA is
transferred to host cells via a novel contact-dependent mechanism. Cell Microbiol 6:167–174
Ito Y, Azuma T, Ito S, Suto H, Miyaji H, Yamazaki Y, Kohli Y, Kuriyama M (1998) Full-length
sequence analysis of the vacA gene from cytotoxic and noncytotoxic Helicobacter pylori. J
Infect Dis 178:1391–1398
Iwamoto H, Czajkowsky DM, Cover TL, Szabo G, Shao Z (1999) VacA from Helicobacter pylori:
a hexameric chloride channel. FEBS Lett 450:101–104
Jain P, Luo ZQ, Blanke SR (2011) Helicobacter pylori vacuolating cytotoxin A (VacA) engages
the mitochondrial fission machinery to induce host cell death. Proc Natl Acad Sci U S A
108:16032–16037
Kersulyte D, Rossi M, Berg DE (2013) Sequence divergence and conservation in genomes of
Helicobacter cetorum strains from a dolphin and a whale. PLoS One 8:e83177
Kim JM, Kim JS, Lee JY, Kim YJ, Youn HJ, Kim IY, Chee YJ, Oh YK, Kim N, Jung HC, Song IS
(2007) Vacuolating cytotoxin in Helicobacter pylori water-soluble proteins upregulates che-
mokine expression in human eosinophils via Ca2+ influx, mitochondrial reactive oxygen
intermediates, and NF-kappaB activation. Infect Immun 75:3373–3381
Kim JM, Kim JS, Lee JY, Sim YS, Kim YJ, Oh YK, Yoon HJ, Kang JS, Youn J, Kim N, Jung HC,
Kim S (2010) Dual effects of Helicobacter pylori vacuolating cytotoxin on human eosinophil
apoptosis in early and late periods of stimulation. Eur J Immunol 40:1651–1662
Kim JM, Kim JS, Yoo DY, Ko SH, Kim N, Kim H, Kim YJ (2011) Stimulation of dendritic cells
with Helicobacter pylori vacuolating cytotoxin negatively regulates their maturation via the
restoration of E2F1. Clin Exp Immunol 166:34–45
Kimura M, Goto S, Wada A, Yahiro K, Niidome T, Hatakeyama T, Aoyagi H, Hirayama T, Kondo
T (1999) Vacuolating cytotoxin purified from Helicobacter pylori causes mitochondrial
damage in human gastric cells. Microb Pathog 26:45–52
Kimura M, Goto S, Ihara Y, Wada A, Yahiro K, Niidome T, Aoyagi H, Hirayama T, Kondo T
(2001) Impairment of glutathione metabolism in human gastric epithelial cells treated with
vacuolating cytotoxin from Helicobacter pylori. Microb Pathog 31:29–36
Kobayashi H, Kamiya S, Suzuki T, Kohda K, Muramatsu S, Kurumada T, Ohta U, Miyazawa M,
Kimura N, Mutoh N, Shirai T, Takagi A, Harasawa S, Tani N, Miwa T (1996) The effect of
Helicobacter pylori on gastric acid secretion by isolated parietal cells from a guinea pig.
Association with production of vacuolating toxin by H. pylori. Scand J Gastroenterol
31:428–433
Kuck D, Kolmerer B, Iking-Konert C, Krammer PH, Stremmel W, Rudi J (2001) Vacuolating
cytotoxin of Helicobacter pylori induces apoptosis in the human gastric epithelial cell line
AGS. Infect Immun 69:5080–5087
Letley DP, Rhead JL, Twells RJ, Dove B, Atherton JC (2003) Determinants of non-toxicity in the
gastric pathogen Helicobacter pylori. J Biol Chem 278:26734–26741
Leunk RD, Johnson PT, David BC, Kraft WG, Morgan DR (1988) Cytotoxic activity in broth-
culture filtrates of Campylobacter pylori. J Med Microbiol 26:93–99
Li Y, Wandinger-Ness A, Goldenring JR, Cover TL (2004) Clustering and redistribution of late
endocytic compartments in response to Helicobacter pylori vacuolating toxin. Mol Biol Cell
15:1946–1959
Lupetti P, Heuser JE, Manetti R, Massari P, Lanzavecchia S, Bellon PL, Dallai R, Rappuoli R,
Telford JL (1996) Oligomeric and subunit structure of the Helicobacter pylori vacuolating
cytotoxin. J Cell Biol 133:801–807
Marchetti M, Rossi M, Giannelli V, Giuliani MM, Pizza M, Censini S, Covacci A, Massari P,
Pagliaccia C, Manetti R, Telford JL, Douce G, Dougan G, Rappuoli R, Ghiara P (1998)
Protection against Helicobacter pylori infection in mice by intragastric vaccination with
H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin
(LT) as adjuvant. Vaccine 16:33–37
Massari P, Manetti R, Burroni D, Nuti S, Norais N, Rappuoli R, Telford JL (1998) Binding of the
Helicobacter pylori vacuolating cytotoxin to target cells. Infect Immun 66:3981–3984
Mcclain MS, Schraw W, Ricci V, Boquet P, Cover TL (2000) Acid-activation of Helicobacter

pylori vacuolating cytotoxin (VacA) results in toxin internalization by eukaryotic cells. Mol
Mcclain MS, Cao P, Iwamoto H, Vinion-Dubiel AD, Szabo G, Shao Z, Cover TL (2001) A
12-amino-acid segment, present in type s2 but not type s1 Helicobacter pylori VacA proteins,
abolishes cytotoxin activity and alters membrane channel formation. J Bacteriol
183:6499–6508
Mcclain MS, Iwamoto H, Cao P, Vinion-Dubiel AD, Li Y, Szabo G, Shao Z, Cover TL (2003)
Essential role of a GXXXG motif for membrane channel formation by Helicobacter pylori
vacuolating toxin. J Biol Chem 278:12101–12108
Menaker RJ, Ceponis PJ, Jones NL (2004) Helicobacter pylori induces apoptosis of macrophages
in association with alterations in the mitochondrial pathway. Infect Immun 72:2889–2898
Molinari M, Galli C, Norais N, Telford JL, Rappuoli R, Luzio JP, Montecucco C (1997) Vacuoles
induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers. J
Biol Chem 272:25339–25344
Molinari M, Salio M, Galli C, Norais N, Rappuoli R, Lanzavecchia A, Montecucco C (1998)
Selective inhibition of Ii-dependent antigen presentation by Helicobacter pylori toxin VacA. J
Exp Med 187:135–140
Moll G, Papini E, Colonna R, Burroni D, Telford J, Rappuoli R, Montecucco C (1995) Lipid
interaction of the 37-kDa and 58-kDa fragments of the Helicobacter pylori cytotoxin. Eur J
Biochem 234:947–952
Montecucco C, Rappuoli R (2001) Living dangerously: how Helicobacter pylori survives in the
human stomach. Nat Rev Mol Cell Biol 2:457–466
Nakayama M, Kimura M, Wada A, Yahiro K, Ogushi KI, Niidome T, Fujikawa A, Shirasaka D,
Aoyama N, Kurazono H, Noda M, Moss J, Hirayama T (2004) Helicobacter pylori VacA
activates the p38/ATF-2-mediated signal pathway in AZ-521 cells. J Biol Chem
279:7024–7028
Nakayama M, Hisatsune J, Yamasaki E, Isomoto H, Kurazono H, Hatakeyama M, Azuma T,
Yamaoka Y, Yahiro K, Moss J, Hirayama T (2009) Helicobacter pylori VacA-induced
inhibition of GSK3 through the PI3K/Akt signaling pathway. J Biol Chem 284:1612–1619
Nguyen VQ, Caprioli RM, Cover TL (2001) Carboxy-terminal proteolytic processing of
Helicobacter pylori vacuolating toxin. Infect Immun 69:543–546
(2013) Helicobacter pylori gamma-glutamyl transpeptidase and vacuolating cytotoxin pro-
mote gastric persistence and immune tolerance. Proc Natl Acad Sci U S A 110:3047–3052
(2000) Virulence factors of Helicobacter pylori responsible for gastric diseases in mongolian
gerbil. J Exp Med 192:1601–1610
Oldani A, Cormont M, Hofman V, Chiozzi V, Oregioni O, Canonici A, Sciullo A, Sommi P,
Fabbri A, Ricci V, Boquet P (2009) Helicobacter pylori counteracts the apoptotic action of its
VacA toxin by injecting the CagA protein into gastric epithelial cells. PLoS Pathog 5:e1000603
Oswald-Richter K, Torres VJ, Sundrud MS, Vancompernolle SE, Cover TL, Unutmaz D (2006)
Helicobacter pylori VacA toxin inhibits human immunodeficiency virus infection of primary
human T cells. J Virol 80:11767–11775
Padilla PI, Wada A, Yahiro K, Kimura M, Niidome T, Aoyagi H, Kumatori A, Anami M,
Hayashi T, Fujisawa J, Saito H, Moss J, Hirayama T (2000) Morphologic differentiation of
HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori
VacA sensitivity. J Biol Chem 275:15200–15206
Pagliaccia C, De Bernard M, Lupetti P, Ji X, Burroni D, Cover TL, Papini E, Rappuoli R, Telford
JL, Reyrat JM (1998) The m2 form of the Helicobacter pylori cytotoxin has cell type-specific
vacuolating activity. Proc Natl Acad Sci U S A 95:10212–10217
Pagliaccia C, Wang XM, Tardy F, Telford JL, Ruysschaert JM, Cabiaux V (2000) Structure and
interaction of VacA of Helicobacter pylori with a lipid membrane. Eur J Biochem
267:104–109
Pai R, Cover TL, Tarnawski AS (1999) Helicobacter pylori vacuolating cytotoxin (VacA)
disorganizes the cytoskeletal architecture of gastric epithelial cells. Biochem Biophys Res
Commun 262:245–250
Papini E, De Bernard M, Milia E, Bugnoli M, Zerial M, Rappuoli R, Montecucco C (1994)
Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compart-
ments. Proc Natl Acad Sci U S A 91:9720–9724
Papini E, Satin B, Bucci C, De Bernard M, Telford JL, Manetti R, Rappuoli R, Zerial M,
Montecucco C (1997) The small GTP binding protein rab7 is essential for cellular vacuolation
induced by Helicobacter pylori cytotoxin. EMBO J 16:15–24
Papini E, Satin B, Norais N, De Bernard M, Telford JL, Rappuoli R, Montecucco C (1998)
Selective increase of the permeability of polarized epithelial cell monolayers by Helicobacter
pylori vacuolating toxin. J Clin Invest 102:813–820
Patel HK, Willhite DC, Patel RM, Ye D, Williams CL, Torres EM, Marty KB, Macdonald RA,
Blanke SR (2002) Plasma membrane cholesterol modulates cellular vacuolation induced by the
Helicobacter pylori vacuolating cytotoxin. Infect Immun 70:4112–4123
Radin JN, Gonzalez-Rivera C, Ivie SE, Mcclain MS, Cover TL (2011) Helicobacter pylori VacA
induces programmed necrosis in gastric epithelial cells. Infect Immun 79:2535–2543
Radin JN, Gaddy JA, Gonzalez-Rivera C, Loh JT, Algood HM, Cover TL (2013) Flagellar
localization of a Helicobacter pylori autotransporter protein. MBio 4:e00613-12
Radin JN, Gonzalez-Rivera C, Frick-Cheng AE, Sheng J, Gaddy JA, Rubin DH, Algood HM,
Mcclain MS, Cover TL (2014) Role of connexin 43 in Helicobacter pylori VacA-induced cell
death. Infect Immun 82:423–432
Raju D, Hussey S, Ang M, Terebiznik MR, Sibony M, Galindo-Mata E, Gupta V, Blanke SR,
Delgado A, Romero-Gallo J, Ramjeet MS, Mascarenhas H, Peek RM, Correa P, Streutker C,
Hold G, Kunstmann E, Yoshimori T, Silverberg MS, Girardin SE, Philpott DJ, El Omar E,
Jones NL (2012) Vacuolating cytotoxin and variants in Atg16L1 that disrupt autophagy
promote Helicobacter pylori infection in humans. Gastroenterology 142:1160–1171
Reyrat JM, Lanzavecchia S, Lupetti P, De Bernard M, Pagliaccia C, Pelicic V, Charrel M,
Ulivieri C, Norais N, Ji X, Cabiaux V, Papini E, Rappuoli R, Telford JL (1999) 3D imaging
of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin. J Mol Biol
290:459–470
region, is associated with gastric cancer. Gastroenterology 133:926–936
Ricci V, Sommi P, Fiocca R, Romano M, Solcia E, Ventura U (1997) Helicobacter pylori
vacuolating toxin accumulates within the endosomal- vacuolar compartment of cultured
gastric cells and potentiates the vacuolating activity of ammonia. J Pathol 183:453–459
Ricci V, Galmiche A, Doye A, Necchi V, Solcia E, Boquet P (2000) High cell sensitivity to
Helicobacter pylori VacA toxin depends on a GPI-anchored protein and is not blocked by
inhibition of the clathrin- mediated pathway of endocytosis. Mol Biol Cell 11:3897–3909
Salama NR, Otto G, Tompkins L, Falkow S (2001) Vacuolating cytotoxin of Helicobacter pylori
plays a role during colonization in a mouse model of infection. Infect Immun 69:730–736
Satin B, Norais N, Telford J, Rappuoli R, Murgia M, Montecucco C, Papini E (1997) Effect of
Helicobacter pylori vacuolating toxin on maturation and extracellular release of procathepsin
D and on epidermal growth factor degradation. J Biol Chem 272:25022–25028
Schmitt W, Haas R (1994) Genetic analysis of the Helicobacter pylori vacuolating cytotoxin:
structural similarities with the IgA protease type of exported protein. Mol Microbiol
12:307–319
Schraw W, Li Y, Mcclain MS, Van Der Goot FG, Cover TL (2002) Association of Helicobacter
pylori vacuolating toxin (VacA) with lipid rafts. J Biol Chem 277:34642–34650
Sewald X, Gebert-Vogal B, Prassl S, Barwig I, Weiss E, Fabbri M, Osicka R, Schiemann M, Busch

DH, Semmrich M, Holzmann B, Sebo P, Haas R (2008) CD18 is the T-lymphocyte receptor of
the Helicobacter pylori vacuolating cytotoxin. Cell Host Microbe 3:20–29
Sewald X, Jimenez-Soto L, Haas R (2011) PKC-dependent endocytosis of the Helicobacter pylori
vacuolating cytotoxin in primary T lymphocytes. Cell Microbiol 13:482–496
Skibinski DA, Genisset C, Barone S, Telford JL (2006) The cell-specific phenotype of the
polymorphic vacA midregion is independent of the appearance of the cell surface receptor
protein tyrosine phosphatase beta. Infect Immun 74:49–55
Smoot DT, Resau JH, Earlington MH, Simpson M, Cover TL (1996) Effects of Helicobacter
pylori vacuolating cytotoxin on primary cultures of human gastric epithelial cells. Gut
39:795–799
Supajatura V, Ushio H, Wada A, Yahiro K, Okumura K, Ogawa H, Hirayama T, Ra C (2002)
Cutting edge: VacA, a vacuolating cytotoxin of Helicobacter pylori, directly activates mast
cells for migration and production of proinflammatory cytokines. J Immunol 168:2603–2607
Suzuki J, Ohnishi H, Shibata H, Wada A, Hirayama T, Iiri T, Ueda N, Kanamaru C, Tsuchida T,
Mashima H, Yasuda H, Fujita T (2001) Dynamin is involved in human epithelial cell
vacuolation caused by the Helicobacter pylori-produced cytotoxin VacA. J Clin Invest
107:1329
Suzuki J, Ohnishi H, Wada A, Hirayama T, Ohno H, Ueda N, Yasuda H, Iiri T, Wada Y, Futai M,
Mashima H (2003) Involvement of syntaxin 7 in human gastric epithelial cell vacuolation
induced by the Helicobacter pylori-produced cytotoxin VacA. J Biol Chem 278:25585–25590
Szabo I, Brutsche S, Tombola F, Moschioni M, Satin B, Telford JL, Rappuoli R, Montecucco C,
Papini E, Zoratti M (1999) Formation of anion-selective channels in the cell plasma membrane
by the toxin VacA of Helicobacter pylori is required for its biological activity. EMBO J
18:5517–5527
Tabel G, Hoa NT, Tarnawski A, Chen J, Domek M, Ma TY (2003) Helicobacter pylori infection
inhibits healing of the wounded duodenal epithelium in vitro. J Lab Clin Med 142:421–430
Tan S, Noto JM, Romero-Gallo J, Peek RM Jr, Amieva MR (2011) Helicobacter pylori perturbs
iron trafficking in the epithelium to grow on the cell surface. PLoS Pathog 7:e1002050
Tegtmeyer N, Zabler D, Schmidt D, Hartig R, Brandt S, Backert S (2009) Importance of EGF
receptor, HER2/Neu and Erk1/2 kinase signalling for host cell elongation and scattering
induced by the Helicobacter pylori CagA protein: antagonistic effects of the vacuolating
cytotoxin VacA. Cell Microbiol 11:488–505
Telford JL, Ghiara P, Dell’orco M, Comanducci M, Burroni D, Bugnoli M, Tecce MF, Censini S,
Covacci A, Xiang Z et al (1994) Gene structure of the Helicobacter pylori cytotoxin and
evidence of its key role in gastric disease. J Exp Med 179:1653–1658
Terebiznik MR, Vazquez CL, Torbicki K, Banks D, Wang T, Hong W, Blanke SR, Colombo MI,
Jones NL (2006) Helicobacter pylori VacA toxin promotes bacterial intracellular survival in
gastric epithelial cells. Infect Immun 74:6599–6614
Terebiznik MR, Raju D, Vazquez CL, Torbricki K, Kulkarni R, Blanke SR, Yoshimori T,
Colombo MI, Jones NL (2009) Effect of Helicobacter pylori’s vacuolating cytotoxin on the
autophagy pathway in gastric epithelial cells. Autophagy 5:370–379
Tombola F, Carlesso C, Szabo I, De Bernard M, Reyrat JM, Telford JL, Rappuoli R,
Montecucco C, Papini E, Zoratti M (1999a) Helicobacter pylori vacuolating toxin forms
anion-selective channels in planar lipid bilayers: possible implications for the mechanism of
cellular vacuolation. Biophys J 76:1401–1409
Tombola F, Oregna F, Brutsche S, Szabo I, Del Giudice G, Rappuoli R, Montecucco C, Papini E,
Zoratti M (1999b) Inhibition of the vacuolating and anion channel activities of the VacA toxin
of Helicobacter pylori. FEBS Lett 460:221–225
Tombola F, Morbiato L, Del Giudice G, Rappuoli R, Zoratti M, Papini E (2001) The Helicobacter
pylori VacA toxin is a urea permease that promotes urea diffusion across epithelia. J Clin
Invest 108:929–937
Torres VJ, Ivie SE, Mcclain MS, Cover TL (2005) Functional properties of the p33 and p55
domains of the Helicobacter pylori vacuolating cytotoxin. J Biol Chem 280:21107–21114
Torres VJ, Vancompernolle SE, Sundrud MS, Unutmaz D, Cover TL (2007) Helicobacter pylori
Tsugawa H, Suzuki H, Saya H, Hatakeyama M, Hirayama T, Hirata K, Nagano O, Matsuzaki J,
Hibi T (2012) Reactive oxygen species-induced autophagic degradation of Helicobacter pylori
CagA is specifically suppressed in cancer stem-like cells. Cell Host Microbe 12:764–777
Tuo B, Song P, Wen G, Sewald X, Gebert-Vogl B, Haas R, Manns M, Seidler U (2009)
Helicobacter pylori vacuolating cytotoxin inhibits duodenal bicarbonate secretion by a
histamine-dependent mechanism in mice. J Infect Dis 199:505–512
Van Doorn LJ, Figueiredo C, Sanna R, Plaisier A, Schneeberger P, De Boer W, Quint W (1998)
Clinical relevance of the cagA, vacA, and iceA status of Helicobacter pylori. Gastroenterology
115:58–66
Vinion-Dubiel AD, Mcclain MS, Czajkowsky DM, Iwamoto H, Ye D, Cao P, Schraw W, Szabo G,
Blanke SR, Shao Z, Cover TL (1999) A dominant negative mutant of Helicobacter pylori
vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation. J Biol Chem
274:37736–37742
Wang X, Wattiez R, Paggliacia C, Telford JL, Ruysschaert J, Cabiaux V (2000) Membrane
topology of VacA cytotoxin from H. pylori. FEBS Lett 481:96–100
Wang W-C, Wang H-J, Kuo C-H (2001) Two distinctive cell binding patterns by vacuolating toxin
fused with glutathione S-transferase: one high-affinity m1-specific binding and the other lower-
affinity binding for variant m forms. Biochemistry 40:11887–11896
Wang HT, Li ZH, Yuan JP, Zhao W, Shi XD, Tong SQ, Guo XK (2005) Effect of Helicobacter
pylori VacA on gene expression of gastric cancer cells. World J Gastroenterol 11:109–113
Wang F, Xia P, Wu F, Wang D, Wang W, Ward T, Liu Y, Aikhionbare F, Guo Z, Powell M, Liu B,
Bi F, Shaw A, Zhu Z, Elmoselhi A, Fan D, Cover TL, Ding X, Yao X (2008) Helicobacter
pylori VacA disrupts apical membrane-cytoskeletal interactions in gastric parietal cells. J Biol
Chem 283:26714–26725
Weiss G, Forster S, Irving A, Tate M, Ferrero RL, Hertzog P, Frokiaer H, Kaparakis-Liaskos M
(2013) Helicobacter pylori VacA suppresses Lactobacillus acidophilus-induced interferon
beta signaling in macrophages via alterations in the endocytic pathway. MBio 4:e00609–
e00612
Willhite DC, Blanke SR (2004) Helicobacter pylori vacuolating cytotoxin enters cells, localizes to
the mitochondria, and induces mitochondrial membrane permeability changes correlated to
toxin channel activity. Cell Microbiol 6:143–154
Willhite DC, Ye D, Blanke SR (2002) Fluorescence resonance energy transfer microscopy of the
Helicobacter pylori vacuolating cytotoxin within mammalian cells. Infect Immun
70:3824–3832
Willhite DC, Cover TL, Blanke SR (2003) Cellular vacuolation and mitochondrial cytochrome c
release are independent outcomes of Helicobacter pylori vacuolating cytotoxin activity that are
each dependent on membrane channel formation. J Biol Chem 278:48204–48209
Winter JA, Letley DP, Cook KW, Rhead JL, Zaitoun AM, Ingram RJM, Amilon KR, Croxall NJ,
Kaye PV, Robinson K, Atherton JC (2014) A role for the vacuolating cytotoxin, VacA, in
colonization and Helicobacter pylori – induced metaplasia in the stomach. J Infect Dis
210:954–963
Wirth HP, Beins MH, Yang M, Tham KT, Blaser MJ (1998) Experimental infection of Mongolian
gerbils with wild-type and mutant Helicobacter pylori strains. Infect Immun 66:4856–4866
Yahiro K, Niidome T, Kimura M, Hatakeyama T, Aoyagi H, Kurazono H, Imagawa K, Wada A,
Moss J, Hirayama T (1999) Activation of Helicobacter pylori VacA toxin by alkaline or acid
conditions increases its binding to a 250-kDa receptor protein-tyrosine phosphatase beta. J Biol
Chem 274:36693–36699
Yahiro K, Wada A, Nakayama M, Kimura T, Ogushi K, Niidome T, Aoyagi H, Yoshino K,
Yonezawa K, Moss J, Hirayama T (2003) Protein-tyrosine phosphatase alpha, RPTP alpha, is a
Helicobacter pylori VacA receptor. J Biol Chem 278:19183–19189
Yahiro K, Wada A, Yamasaki E, Nakayama M, Nishi Y, Hisatsune J, Morinaga N, Sap J, Noda M,
Moss J, Hirayama T (2004) Essential domain of receptor tyrosine phosphatase beta (RPTPbeta)
for interaction with Helicobacter pylori vacuolating cytotoxin. J Biol Chem 279:51013–51021
Yahiro K, Satoh M, Nakano M, Hisatsune J, Isomoto H, Sap J, Suzuki H, Nomura F, Noda M,
Moss J, Hirayama T (2012) Low-density lipoprotein receptor-related protein-1 (LRP1) medi-
ates autophagy and apoptosis caused by Helicobacter pylori VacA. J Biol Chem
287:31104–31115
Yamasaki E, Wada A, Kumatori A, Nakagawa I, Funao J, Nakayama M, Hisatsune J, Kimura M,
Moss J, Hirayama T (2006) Helicobacter pylori vacuolating cytotoxin induces activation of the
proapoptotic protein Bax and Bak, leading to cytochrome c release and cell death, independent
of vacuolation. J Biol Chem 281:11250–11259
Ye D, Blanke SR (2000) Mutational analysis of the Helicobacter pylori vacuolating toxin amino
terminus: identification of amino acids essential for cellular vacuolation. Infect Immun
68:4354–4357
Ye D, Willhite DC, Blanke SR (1999) Identification of the minimal intracellular vacuolating
domain of the Helicobacter pylori vacuolating toxin. J Biol Chem 274:9277–9282
Yokoyama K, Higashi H, Ishikawa S, Fujii Y, Kondo S, Kato H, Azuma T, Wada A, Hirayama T,
Aburatani H, Hatakeyama M (2005) Functional antagonism between Helicobacter pylori
CagA and vacuolating toxin VacA in control of the NFAT signaling pathway in gastric
epithelial cells. Proc Natl Acad Sci U S A 102:9661–9666
Zheng PY, Jones NL (2003) Helicobacter pylori strains expressing the vacuolating cytotoxin
interrupt phagosome maturation in macrophages by recruiting and retaining TACO (coronin 1)
protein. Cell Microbiol 5:25–40
Chapter 6
Roles of the BabA and the SabA Adhesins
in Gastroduodenal Diseases
Anna Arnqvist
Abstract Adhesion is an important prerequisite for colonization and it is the initial

step in infections with pathogenic bacteria. Adherence to host epithelial surfaces is
the result of bacterial surface proteins, called adhesins, and their specific interaction
with cognate protein- or glycoconjugate receptors on the host cells. Often, the
bacteria have a set of complementary adhesins that are specific for different host
receptors. Alternative mechanism has been suggested to mediate H. pylori adhe-
sion, and this chapter will focus on the two well-characterized adhesins BabA and
SabA. In the healthy gastric mucosa, the Lewis b antigen (Leb) is present in the
gastric epithelial lining of blood group O (H-antigen), B, and A individuals.
H. pylori binding to ABO/Leb is mediated by the blood group antigen-binding
BabA adhesin. As the inflammation develops, Leb is downregulated and the levels
of sialylated antigens increase. Sialyl-Lewis x/a antigens (sLex/a) are specifically
recognized by the H. pylori sialic acid-binding adhesin SabA. Even though bacte-
rial adherence per se cannot cause disease, adherence is considered as a crucial step
in pathogenesis since it is needed for bacterial delivery of effector molecules into
the host cell. The presence of receptors and host-immune responses are two factors
that differently affect adhesion. To achieve long-term colonization, H. pylori must
regulate the expression of a cognate adhesin to fit the available receptors. Adhesion
to the gastric epithelial cells promotes gain of nutrients, but too tight adhesion may
be intimidating because of the risk of clearance by the bacteria for life-threatening
immune responses. Thus, expression levels of the adhesins must be fine-tuned in
accord to host receptor expression levels. This chapter will also discuss H. pylori
adhesion in relation to severe gastric diseases.
Keywords Helicobacter pylori • Adhesion • Blood group antigen-binding adhesin

BabA • Sialic acid binding adhesin SabA • ABO blood group antigen/Lewis b
antigen (ABO/Leb) • Sialyl-Lewis x antigen (sLex) • Homologous recombination •
Slipped-strand mispairing
A. Arnqvist (*)
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden

DOI 10.1007/978-4-431-55936-8_6
144 A. Arnqvist
6.1 Introduction
Bacterial colonization of the human host is the initial step to establish and maintain
an infection. Bacterial adhesion to host cells is mediated via ligand-receptor
interactions. Bacteria recognize and bind to certain receptors, including proteins,
glycoproteins, or glycolipids on the host cell mucosal surface. Oligosaccharides
present on the highly glycosylated secreted and membrane-bound mucins and to
glycoproteins and glycosphingolipids on the host cell surfaces act as receptors for
H. pylori attachment. Proteins on the bacterial cell surface that facilitated binding to
the host receptor are called adhesins. A given bacterium often carries multiple
adhesins. Adhesins interact with their cognate receptors with high specificity,
which contributes to host and tissue tropism. Some adhesins are polymorphic and
recognize several receptor moieties. The affinities of adhesin-receptor interactions
are variable, from very high-affinity interactions to those of low affinity. Clustering
of adhesins and/or receptors can cause multivalency effects and increase the
binding. Expression of the host cell receptor repertoire exhibits a wide individual
variation due to genetic predisposition, as well as variation in relation to healthy or
inflamed mucosa.
In gastric biopsy material, the majority of H. pylori are found in the mucus layer
but a subset is firmly attached to the epithelial cell surface (Hessey et al. 1990).
H. pylori has also been identified intracellularly (Aspholm et al. 2006b; Semino-
Mora et al. 2003). For H. pylori, it is a delicate balance to on the one hand stay close
to the host cell to gain nutrients and on the other hand have the capacity to loosen
the grip when the host cellular response becomes too vigorous or upon host cell
shedding. Adhesion of H. pylori to the gastric mucosa also aids the bacteria to resist
the shear force during the peristaltic movements. To persistently colonize the
gastric mucosa for the lifetime is challenging, but obviously H. pylori have the
ability to adapt to the continuous changes that occur in the stomach environment.
The capacity to attach to gastric epithelial cells contributes to bacterial delivery of
effector molecules to the host and subsequent host responses, events that sometimes
lead to the development of disease. Therefore, adhesion is a significant step in the
pathogenesis of H. pylori-related peptic ulcer disease and gastric cancer. This
chapter discusses the two best-characterized H. pylori adhesins, the BabA adhesin
that mediate binding to fucosylated blood group antigens in healthy gastric mucosa
and the SabA adhesin that recognizes sialic acid sialyl-Lewis x antigen in inflamed
mucosa, how their respective expression is regulated, and their role in gastric
diseases.
6 Roles of the BabA and the SabA Adhesins in Gastroduodenal Diseases 145
6.2 The Blood Group Antigen-Binding Adhesin BabA
6.2.1 Identification of the Blood Group Antigen-Binding

Adhesin BabA
Fucosylated H antigens are complex carbohydrates with a α1.2 fucose residue

linked to a terminal Galβ (Fig. 6.1). The H-antigen forms the base for the ABO
blood group antigens. Addition of a fucose residue to the H-antigen forms the
difucosylated Lewis b antigen (Leb) (Fig. 6.1). In blood group A individuals, the H
and Leb antigens are extended with terminal N-acetylgalactosamine (GalNAc),
whereas in the blood group B individuals, H or Leb have instead been extended
with galactose (Gal) (Fig. 6.1). Individuals with a so-called positive secretor status
also expresses the secretor (fucosyl) transferase, in saliva, tears, milk and gastro-
intestinal mucus secretions, and epithelium, thus adding the α1.2 fucose residue to
form the H-antigen that then can be extended into the ABO blood group antigens
(reviewed by (Clausen and Hakomori 1989). Individuals of nonsecretor phenotype
do not express the fucosyl transferase in gastric mucus and epithelium. The first
functional host receptor for H. pylori to be identified was the fucosylated H and
Lewis b antigens (Leb) (Borén et al. 1993). Since cell lines are transformed cells,
the ABO/Lewis antigens are usually not expressed at all or only in limited amount.
Therefore, the finding of Leb as a functional receptor for H. pylori was dependent
on the usage of a newly developed in vitro adherence assay (Falk et al. 1993).
Fluorescently labeled H. pylori were overlaid on to human gastric histo-tissue
sections, which showed that H. pylori is bound to gastric surface mucous cells.
Colostrum samples reflect the individual blood group phenotype. Later the same
year, Borén and co-workers (1993) used the same in vitro adherence assay in
combination with colostrum samples from individuals with different Lewis blood
group antigens to show that the H. pylori receptor on human gastric surface mucous
cells contains Leb (Borén et al. 1993). H. pylori is a human- and primate-specific
pathogen and the Leb antigen is not expressed in mice. However, H. pylori mediates
specific adhesion to the gastric and intestinal tract of transgenic mice that specif-
ically expressed the human α-1,3/4 fucosyl transferase (Falk et al. 1995). When
these transgenic mice were infected with H. pylori, it was demonstrated that Leb
binding was associated with the development of chronic gastritis (Guruge
et al. 1998).
Fig. 6.1 Composition of fucosylated H1 and Leb ABO blood group antigens. Addition of a fucose
residue to the H1 antigen forms the Leb antigen. In blood group A and B individuals, the H1 or
corresponding Leb antigens are either extended with an N-acetylgalactosamine (GalNAc) or a
galactose (Gal)
146 A. Arnqvist
The first study to analyze the prevalence of Leb-receptor binding activity in a

collection of strains showed that 66 % of them exhibited Leb binding (Ilver
et al. 1998). Receptor-binding activity was measured using 125I-labeled Leb semi-
synthetic HSA glycoconjugate (Aspholm et al. 2006a; Ilver et al. 1998). Affinity
analysis using Scatchard assays, in which the binding affinity, the association
constant Ka, is calculated, showed that Leb binding in strain CCUG17875 was of
high affinity (the Ka ¼ 1 1010) (method is described in (Aspholm et al. 2006a).
Based on using electron microscopy and Leb-receptor conjugate labeled with gold
particles, the number of Leb-binding adhesins was estimated to be approximately
500 per bacterial cell surface. Far-western blot analysis, i.e., receptor overlay
analysis with soluble Leb-receptor conjugate, was used to identify a Leb-binding
protein. To isolate the Leb-binding protein, an affinity purification method called
receptor activity-directed affinity tagging (retagging) was established (Ilver
et al. 1998). An N-terminal amino acid sequence of the Leb-binding adhesin was
determined and used to design degenerated PCR primers for PCR amplification of a
DNA fragment, which then was used as a probe for the screening of a plasmid
library. Initially, two sets of alleles were identified that encoded for proteins that
displayed identical N- and C-terminal domains but contained different central
domains. An additional, extended N-terminal sequence made it possible to distin-
guish the clones and determine which that encoded for the Leb-binding protein. The
corresponding protein was named the blood group antigen-binding adhesin BabA,
and the gene was consequently named babA (Ilver et al. 1998). The gene
corresponding to the second set of clones was named babB. The function of the
BabB protein is yet elusive.
At the time it was not known that the H. pylori strain that was used to identify the
Leb receptor expressed a BabA adhesin with a specialist phenotype, meaning that
its BabA adhesin specifically recognized H1 and Leb of blood group O individuals
but not A-Leb or B-Leb. This strain, P466, originates from South America, where a
majority of the population is of blood group O phenotype (Aspholm-Hurtig
et al. 2004). Binding of strain CCUG17875 to gastric mucosa could be blocked
with Leb, A-Leb, or B-Leb, while binding of strain P466 only was blocked with
Leb. Close to 400 H. pylori strains from different geographic regions (Sweden,
Germany, Spain, Japan, and Alaska) were analyzed for their receptor-binding
phenotypes, and it then became evident that 95 % of them bound to A-Leb,
B-Leb, and Leb. In contrast, strains from South America (Peru, Venezuela Ama-
zons, and Colombian mestizo) displayed different binding patterns since 60 % of
them only recognized Leb and not A-Leb or B-Leb (Aspholm-Hurtig et al. 2004).
Strains that only bound Leb were named “specialists,” and strains that exhibited the
ABO/Leb phenotype were called “generalists.” Receptor-overlay analysis
(far-western probed with receptor glycoconjugate) and a DNA transformation
approach to shuffle specialist and generalist babA alleles both showed that the
BabA protein alone determines the “specialist”- or the “generalist”-binding modes.
Moreover, phylogenetic analysis of babA sequences from specialist and generalist
strains suggested that long-term adaptation of the babA alleles occurs in relation to
the types of receptors that are available in the local population (Aspholm-Hurtig
et al. 2004).
Besides binding to the host epithelial cells, the BabA adhesin mediates binding
to Lewis b antigen expressed on MUC5AC, MUC5B, and MUC1 mucins (Lindén
et al. 2002, 2004). BabA also exhibits binding to MUC5B in saliva and the
glycoprotein gp-340 (Walz et al. 2005, 2009). An update of BabA carbohydrate-
binding specificities to further explore the structural requirements for carbohydrate
recognitions was recently published (Benktander et al. 2012).
6.2.2 Location of the babA Gene
The BabA adhesin was first described for strain CCUG17875 (Ilver et al. 1998). To
identify its genomic location, screening of an ordered cosmid library constructed
from the NCTC11638 strain (Bukanov and Berg 1994) identified two babA alleles
and one babB gene (Ilver et al. 1998). DNA sequence analysis showed that the
babA1 allele in comparison with the babA2 allele carried a 10 bp frameshifting
deletion in its very 50 end that causes a premature stop. Knockout deletion analysis
confirmed that the babA2 allele encoded for the BabA adhesin and that the babA1
allele was silent. The results of DNA sequence analyses showed that the babA and
the babB genes encode for proteins with a high level of amino acid identity in their
N-terminal domains, display unique central domains, and share the ~300 most
C-terminal amino acids. Both the BabA adhesin and the BabB protein belong to
the Hop (H. pylori outer membrane porins) family that is characterized by a
conserved N-terminal signal peptide, a variable middle domain, and a conserved
C-terminal membrane-spanning β-sheet motif, of which the middle domain deter-
mines the specificity of the protein (Alm et al. 2000). Thus, the middle domain of
BabA is likely to contain the Leb-binding domain.
Today, when the babA gene has been identified and sequenced in a large number
of strains, it is evident that most strains carry only one babA allele. Comparison of
the two first genome sequences of the 26695 and J99 strains showed that the babA
and the babB alleles are located in reciprocal loci (Alm et al. 1999; Tomb
et al. 1997). Then, the babB gene was found to be located in a third, alternative
locus. The gene encoding for this third bab paralog has been named babC (Colbeck
et al. 2006; Oh et al. 2006). The three loci where the bab genes are found are now
called locus A, B, and C, respectively (Hennig et al. 2006). The babA gene is most
often found in the A locus (Hennig et al. 2006; Oh et al. 2006). Some occasional
strains have multiple copies of the babA allele, while some strains lack the gene.
Noteworthy is that the majority of H. pylori strains seem to carry a babB gene.
148 A. Arnqvist
6.2.3 Mechanisms That Switches BabA Expression On

and Off
When the first genome sequences were published, high levels of homologous
sequences in the 50 and 30 ends of the hop genes were found, indicating the
possibility for homologous recombination events (Alm et al. 1999, 2000). In
particular, Pride and Blaser reported about chimeric babA/B genes in 2 out of
42 strains (Pride and Blaser 2002). The same study suggested that H. pylori
homologous recombination events were RecA dependent and DNase insensitive,
which suggested that they probably were of intragenomic origin, i.e., gene conver-
sion (Pride and Blaser 2002). Intragenomic changes of the babA gene were later
found when rhesus macaques were experimentally infected with the BabA-
expressing J166 H. pylori strain (Solnick et al. 2004). Output clones recovered
17 weeks postinfection had lost BabA expression, either by homologous recombi-
nation with babB creating a babA/B chimeric gene or by insertion or deletion of
nucleotides in the dinucleotide CT repeat tract, 50 of babA. One or two nucleotide
changes in the CT repeat tract via slipped-strand mispairing cause frameshifts and
thus a truncated protein. The opposite event, recombination of babA into the babB
gene that results in a Lewis-binding babB/A chimera, was found in another study
om et al. 2004). Here, homologous recombination between the bab genes
(Bäckstr€
was demonstrated when Leb-binding clones were identified and isolated from an
H. pylori strain carrying a silent babA allele. Clones of the Leb-binding phenotype
were enriched by using biotinylated Leb conjugate and streptavidin-magnetic
beads, and they were then identified using colony screening (Bäckstr€om
et al. 2004). The acquired Leb-binding clones were the result of homologous
recombination of the silent babA allele into the babB gene (located in B locus).
This chimeric babB/A gene carried a dinucleotide CT repeat tract in the 50 end and
was thus subjected to frequent reversible on/off phase shift variations. Phase shift
variation via slipped-strand mispairing is a faster process than phase shift via
homologous recombination. Homologous recombination of babA into the B locus
in the 17875 strain was determined to occur with a frequency of 1 105
(Bäckstr€
om et al. 2004). The frequency for homologous recombination in the
opposite direction, recombination of the babB gene into the A locus, occurs with
similar frequency in strain J166, e.g., 3 105 or 3 106 per cell division
(Amundsen et al. 2008). The same study showed that bab recombinations are
promoted by RecA and the double break repair enzyme AddA. Phase shift via
slipped strand occurs with a higher frequency. The babA gene situated in the B
locus turns expression on and off with a frequency of 5 103. Additional studies
have also described homologous recombination event and slipped-strand
mispairing between the bab genes (Colbeck et al. 2006; Hennig et al. 2006).
Besides gene conversion and slipped-strand mispairing, BabA expression can be
switched off via mutations. Experimental infection of rhesus monkeys with strain
J99 showed that BabA expression is turned off via single base-pair mutations (Styer
et al. 2010). Another study where Mongolian gerbils were long-term infected with
H. pylori also showed that BabA expression was switched off (Ohno et al. 2011).
Here output clones had switched off BabA expression due to either one base-pair
insertions or deletions that introduced a premature stop codon. In addition, clones
with larger deletions (31, 70, 84 bp, respectively) were also identified (Ohno
et al. 2011).
Other recombination events can also switch off Leb binding. Output clones from
Mongolian gerbils infected with strain 7.13 displayed BabA proteins with Leb-non-
binding phenotype (Styer et al. 2010). Genetic analysis showed that the 7.13 strain
carry two babA alleles, similar to strain CCUG17875, where a babA2 allele encodes
for a Leb-binding adhesin and the babA1 allele is silent. In the Leb-non-binding
7.13 output clones, a DNA fragment encoding for six amino acids was replaced
resulting in the expression of Leb-non-binding BabA proteins.
6.2.4 Regulation of BabA Expression Levels
The first H. pylori genome analyses showed that there are a few RNA polymerase
(RNAP) sigma (σ) factors and a few genes that encode for transcriptional regulatory
proteins (Alm et al. 1999; Tomb et al. 1997). It was also shown that the H. pylori
genomes contain many simple sequence repeats, which are typical hot spots for
slipped-strand mispairing and thus to act as contingency loci that are known to
contribute to generation of heterologous populations. BabA expression levels and
Leb-binding activity vary between strains, but a few studies concerning regulatory
mechanisms have been conducted. BabA expression in strain CCUG17875 was
found to be higher when BabA was expressed from the A locus than when
expressed from the B locus (Bäckstr€om et al. 2004). The transcriptional start sites
for the babA gene (located in the A locus) and the babB gene (located in the B
locus) were determined with primer extension analysis. The 10 promoter region
of babA (50 -TATAAT) had a perfect match to the 10 consensus sequence of
E. coli σ70 housekeeping promoters (50 -TATAAT) compared to the 10 promoter
region of the babB (50 -GATAAG) (Bäckstr€om et al. 2004). Even though a consen-
sus sequence for H. pylori 35 promoter element has not been determined, the 35
region of the A locus (50 -ATGACA) in CCUG17875 have an almost perfect match
to the E. coli 35 promoter region (50 -TTGACA). Besides the differences in
nucleotide composition of the binding sequences for the RNAP σ-factor (i.e., the
35 and the 10 regions), the distance between these motifs are known to affect
transcription initiation. The second babA allele of strain CCUG17875, babA1, has
five extra As between the 10 and the 35 promoter regions. It seems possible that
the length of this A tract affects promoter activity, but it has not been confirmed
experimentally. Hennig and co-workers (2006) analyzed BabA expression levels
relative the spacing between the ribosomal binding site and the babA ATG trans-
lational start codon in 35 strains. Although there was a variation, it did not correlate
to the BabA expression levels (Hennig et al. 2006). They also did not find any
sequence variations in the promoter regions that were associated with the BabA
150 A. Arnqvist
expression levels. The recent mapping of the H. pylori transcriptome showed the
presence of many small RNAs (sRNAs) and a massive antisense transcription,
which suggested that H. pylori uses riboregulation to regulate gene expression
(Sharma et al. 2010). However, no role of riboregulation on BabA expression has
so far been demonstrated.
6.2.5 BabA Expression and Gastric Disease
There is a continuous interest in the BabA adhesin and its role in disease outcome.
Over the years, a series of papers have reported about BabA and its association to
severe mucosal inflammation and increased risk of peptic ulcer disease and gastric
cancer (Aspholm-Hurtig et al. 2004; Colbeck et al. 2006; Fujimoto et al. 2007;
Gerhard et al. 1999; Hennig et al. 2004; Ilver et al. 1998; Lehours et al. 2004;
Odenbreit et al. 2009; Olfat et al. 2005; Oliveira et al. 2003; Sheu et al. 2006; Song
et al. 2014; Yamaoka et al. 2002; Yu et al. 2002). Different approaches have been
used to evaluate the association between BabA and disease. A series of studies
applied PCR to detect the presence of the babA gene. Often, primers that amplify
the babA2 allele are used. Using such approach, babA located in other locus than the
A locus will not be found unless additional primer pairs are used. Other studies have
analyzed BabA expression by reverse transcription quantitative real-time polymer-
ase chain reaction (RT-qPCR), immunoblots, or assays that detect for BabA protein
expression with Leb-binding activity such when RIA assay with 125I-labeled
Leb-receptor conjugate is used (Aspholm et al. 2006a; Ilver et al. 1998) or when
enzyme-linked immunosorbent assay (ELISA) is used (Solnick et al. 2004). The
collection of clinical isolates that has been assayed for BabA expression and
binding to Leb has demonstrated differences in BabA expression levels as well as
differences in binding affinity to the Leb receptor among strains. These differences
probably mirror the ability of H. pylori to adapt local environmental conditions
during the lifelong persistent infection (Aspholm-Hurtig et al. 2004; Fujimoto
et al. 2007; Ilver et al. 1998; Yamaoka et al. 2002).
Already in 1999, Gerhard and co-workers reported that there was an epidemio-
logic association between cagA, vacAs1, and babA2 genotypes and higher inci-
dence of ulcer disease and gastric cancer (Gerhard et al. 1999). Later, BabA
expression in combination with CagA- and VacAs1-expressing strains has been
confirmed to be associated with severe gastric disease (Azevedo et al. 2008;
Ishijima et al. 2011). Moreover, BabA-mediated binding to Leb plays an important
role for the initiation of contact-dependent signaling mediated by the type IV
secretion system (T4SS). When Leb-transfected cells were infected with a wild-
type H. pylori strain and isogenic BabA and T4SS mutants, it was shown that
BabA-Leb-binding induced T4SS-dependent host cell signaling, which increased
mRNA levels of genes coding for pro-inflammatory cytokines as well as
precancerous-related factors. These data were also supported by in vivo data from
experimental H. pylori infection of Mongolian gerbils (Ishijima et al. 2011).
It was recently suggested that H. pylori has the capacity to jeopardize the host
genome integrity via direct bacterial-host cell contact. H. pylori adhesion, via
BabA-Leb binding, induced higher levels of double-strand breaks in the chromo-
somal DNA of infected host cells than a babA deletion mutant (Toller et al. 2011).
Similarly, host cells, pre-incubated with soluble Leb prior to infection, exhibited
reduced double-strand break induction. Other virulence-associated factors such as
the VacA cytotoxin, the γ-glutamyl transpeptidase (GGT), and the cag pathogenic-
ity island (cagPAI) were tested but did not promote double-strand breaks in host
target cells (Toller et al. 2011). DNA damages that are not precisely repaired may
increase carcinogenesis.
For experimental infection, the rhesus macaque is a highly relevant animal
model since they are naturally infected by H. pylori and because it mimics the
clinical outcome observed in humans. Several studies have focused on BabA
expression and Leb binding (Mahdavi et al. 2002; Styer et al. 2010). Although
not naturally infected by H. pylori, the Mongolian gerbil is an attractive alternative
experimental model for studying H. pylori infection, and it has been used for studies
of BabA (Ohno et al. 2011; Styer et al. 2010). Mongolian gerbils were infected with
the BabA-expressing strain TN2GF4 for 18 months (Ohno et al. 2011). Besides
BabA expression, the degree of inflammation was followed by histological exam-
ination and by scoring mononuclear cell (MNC) and polymorphonuclear cell
(PMN) infiltration during the same time span. Cellular infiltration increased after
1 month and gradually increased to reach a peak after 6 months. Gastric ulcers
developed to varying degree. Output clones were examined for BabA expression
and Leb binding. Similar to what has been reported for rhesus macaques and gerbils
previously, BabA expression was lost during the course of infection. After 1 month,
80 % of the output clones displayed BabA expression, but the levels declined to
33 % after 3 months. No output clones expressed BabA after 6 and 18 months
postinfection. Among the 1-month output clones, BabA expression levels had
increased significantly. The changes in expression levels could not be explained
by any changes in DNA sequences in the promoter regions nor in the babA open
reading frame (ORF). There was also a correlation between output clones isolated
from gerbils with gastric ulcer, which displayed no BabA expression. In the same
study, it was suggested that the BabA expression levels directly or indirectly
contribute to cellular inflammation (Ohno et al. 2011). Gerbils were infected with
isolated output clones with high or low expression levels. No differences in cellular
infiltration occurred after 1 month, but after 3 months, the level of cellular infiltra-
tion of gerbils infected with output clones with low BabA expression levels was
lower compared to those infected with output clones with high BabA expression
levels (Ohno et al. 2011). Thus, the differences in BabA expression levels as well as
in Leb-binding affinity among strains are likely to affect disease outcome.
152 A. Arnqvist
6.3 The Sialic Acid-Binding Adhesin SabA
6.3.1 H. pylori Binding to the Inflammation-Associated

Sialyl-Lewis x/a Antigen Receptor
A babA knockout deletion mutant was found to adhere to gastric histo-tissue

sections from an H. pylori-infected patient, and the binding could not be fully
blocked with soluble Leb-receptor conjugate (Mahdavi et al. 2002). In contrast, no
binding to histo-tissue sections from healthy individuals was detected. Together
these results suggested that H. pylori exhibit an additional receptor-adhesin inter-
action for binding to the gastric mucosa. Instead of the ABO/Leb as receptors, it
was shown that H. pylori recognizes sialyl-Lewis x and a antigens (sLex/a) in
inflamed gastric mucosa (Mahdavi et al. 2002). The sLex receptor was identified
using thin-layer chromatography (TLC), mass spectrometry (MS), and 1H nuclear
magnetic resonance (1H-NMR). First, glycosphingolipids were separated using
TLC, which thereafter were overlaid with H. pylori. Binding to the sialyl-Lewis x
(sLex) and sialyl-diLewis x (sdiLex) was confirmed with monoclonal antibodies.
Stronger binding was obtained to sdiLex. The glycosphingolipid bound to H. pylori
was confirmed to be sdiLex using MS and 1H-NMR (Mahdavi et al. 2002). Only
minute levels of sLex are found in healthy gastric mucosa, and there is a reciprocal
regulation of the fucosylated ABO/Leb and sialylated sLex expression during
H. pylori infection (Lindén et al. 2008; Mahdavi et al. 2002). An H. pylori infection
alters expression of several genes involved in glycan biosynthesis and in particular
upregulation of the gene encoding the N-Acetylglucosamine (GlcNAc) transferase
β3GlcNacT5 (Marcos et al. 2008). Biosynthesis of Lewis antigens is dependent on
β3GlcNacT5, and when β3GlcNacT5 was overexpressed, the expression levels of
sLex increased. It has also been shown that H. pylori colonization density increases
in patients with high expression levels of sLex (Sheu et al. 2006).
6.3.2 Identification of the Sialic Acid-Binding Adhesin SabA
The retagging technique was once again used for identification and purification of
an H. pylori adhesin. The sLex-receptor conjugate was used in affinity purification
of the sialic acid-binding adhesin, SabA (Mahdavi et al. 2002). Using mass spec-
trometry, four peptides were found to match one gene (JHP662 in strain J99), and
two of these peptides also matched a related gene (JHP659 in strain J99). Deletion
mutagenesis verified that JHP662 encoded the SabA adhesin since binding to sLex
was abolished. The JHP659 gene was called sabB. Similar to the BabA and the
BabB proteins, the SabA adhesin and the SabB protein share a high degree of
homologies in their N- and C-terminal domains. The SabA and SabB proteins both
belong to the Hop family of proteins. Mapping of the receptor epitope revealed that
the NeuAcα2-3Gal-disaccharide is the minimal sialylated binding epitope that is
required for SabA-mediated binding (Aspholm et al. 2006b). Interestingly, when

clinical SabA-expressing isolates were tested for binding to a series of sialylated
glycans such as sialyl-di-Lex, sLea, and sialyl-lactosamine, it was found that the
majority exhibited polymorphism in sialyl-binding properties and that it is an
inherent property of the SabA adhesin itself (Aspholm et al. 2006b).
6.3.3 The SabA Adhesin Is the H. pylori Hemagglutinin
H. pylori was early described to hemagglutinate human erythrocytes (Emody

et al. 1988). It was for many years a debate about the H. pylori hemagglutinin, i.
e., the protein that is responsible for sialic acid-dependent hemagglutination of
erythrocytes. Initially, the HpaA protein was described as the H. pylori hemagglu-
tinin (Evans et al. 1993), but construction of a deletion mutant did not abolish the
hemagglutination activity (O’Toole et al. 1995). However, when a sabA deletion
mutant strain was tested for its function in sialic acid-dependent hemagglutination
of erythrocytes, no agglutination was observed, which suggested that the SabA
adhesin is the H. pylori hemagglutinin (Aspholm et al. 2006b). An additional role of
SabA is its role in neutrophil activation via binding to sialic acid-containing
receptors and thus to mediate nonopsonic activation of human neutrophils and
oxidative burst (Unemo et al. 2005). Besides being a key player for oxidative
metabolism, SabA has also been suggested to be important for the induction of
phagocytosis (Petersson et al. 2006).
6.3.4 Mechanisms for Regulation of SabA Expression
Screening of single clones for sLex-binding activity showed that approximately

1 % switched off their binding (Mahdavi et al. 2002). The analysis of the sabA DNA
sequence identified a simple dinucleotide CT sequence repeat, a typical hot spot for
slipped-strand mispairing and thus phase variation. sLex-binding versus sLex-non-
binding clones exhibited differences in the number of CTs which generated frame-
shifts that either put the sabA ORF in or out of frame (Mahdavi et al. 2002).
Variation in the number of CT repeats and frameshifts were later confirmed with
immunoblot analysis and α-SabA antibodies (Lehours et al. 2004; Yamaoka
et al. 2006). Similar to the babA gene, sabA can also be located in alternative
genomic loci without the CT repeats in the 50 end of the sabA ORF (Sheu
et al. 2006). Recently, the exact location of the sabA and the sabB genes was
mapped in a thorough analysis of some fifty North American clinical strains
(Talarico et al. 2012). Strain J99, in which the sabA gene corresponds to
JHP0622 (sabA locus), the sabB gene corresponds to JHP0659 (sabB locus), and
a third gene omp27 (hopQ) corresponds to JHP1103, was used as reference. In this
set of strains, 84 % of the sabA gene was located in the sabA locus and the
154 A. Arnqvist
remaining was found in the hopQ locus. A significant number of strains lacked the
sabB gene, while there seemed to be a selection to maintain the sabA gene. The
same study showed that gene conversion events occur at the sabA, sabB, and hopQ
loci. Similar as for babA and babB gene conversions, the sabA gene conversion
events were affected by RecA, the nuclease-helicase AddA that is involved in
double-strand break repair and RecG (Amundsen et al. 2008; Talarico
et al. 2012). Thus, expression of the SabA adhesin can be switch on and off via
phase variation, often by slipped-strand mispairing during replication of the CT
nucleotide repeat tract but also via gene conversions.
There is a variation in SabA expression levels among strains (Aspholm
et al. 2006b; Sheu et al. 2006; Yamaoka et al. 2006). Differences in SabA expres-
sion levels can often be explained by differences in the promoter strength (Åberg
et al. 2014). In addition, a simple thymine (T) nucleotide repeat tract is located in
close proximity to the 35 region of the sabA promoter and was suggested to affect
promoter strength (Goodwin et al. 2008; Kao et al. 2012). There are wide differ-
ences in the length of the T-tract, from T5 to T28 where T13 to T19 are to the most
common length (Kao et al. 2012; Åberg et al. 2014). Variation of T-tract length
changes along the course of infection. Output pools from mice as well as from
human antrum, and corpus stomach regions displayed variation in sLex-binding
phenotype as well as T-tract length (Åberg et al. 2014).
The actual effect of changes in the T-tract length was elucidated using site-
directed in vitro mutagenesis to create a series of isogenic mutants with varying
numbers of Ts (Åberg et al. 2014). The analysis of promoter strength, mRNA
levels, SabA protein expression, as well as sLex-binding, both to receptor conjugate
and human gastric histo-tissue sections, showed a multiphasic expression pattern.
The maximum and minimum mRNA and protein levels were observed with a
T-tract length interval of approximately ten base pairs, which corresponds to one
turn of the DNA helix (Åberg et al. 2014). The effect of T-tract length variations
and sabA mRNA levels has been confirmed by Harvey et al. (2014).
Simple sequence repeats between the 10 and 35 promoter elements can
affect the docking of the RNAP σ-factor. The sabA T-tract has a promoter proximal
location, and the effect of the T-tract length was likely to operate by an alternative
mechanism. Exchange of specific lengths of the T-tract with adenine and guanine
showed that the T-tract does not only work as a spacer to adjust the size and hence
expression, but it probably affects DNA topology (Harvey et al. 2014; Åberg
et al. 2014). In silico and functional biochemical analysis showed the T-tracts affect
the local DNA structure and thus binding of the RNAP. A model where changes in
the T-tract length affect the axial alignment between the core promoter and UP-like
elements has been suggested (Åberg et al. 2014). Thus, without input from known
trans-acting regulators, changes in the T-tract length act as a promoter rheostat to
fine-tune SabA expression. Clones with optimal SabA expression levels, which best
fit the host prerequisites, survive and continue colonization. In addition to sabA,
other genes with simple sequence repeat motifs located at similar positions in other
genes were also shown to be affect promoter output in a similar way (Åberg
et al. 2014).
6.3.5 SabA Expression and Regulation by Acidic Conditions
The majority of H. pylori reside in the mucus layer, and a minor population (about
20 %) is situated close to the gastric epithelium (Hessey et al. 1990). There is a pH
gradient from very acidic conditions in the lumen through the mucus layer to be
close to neutral at the epithelium. Thus, pH is an important environmental factor
that H. pylori have to sense and respond to. Using DNA arrays and RT-qPCR, it was
shown that sabA expression decreases as a response to acidic conditions and that the
repression is regulated via the acid responsive regulon ArsRS (Bury-Moné
et al. 2004; Merrell et al. 2003; Pflock et al. 2006; Yamaoka et al. 2006). The
ArsR response regulatory protein probably acts to regulated SabA expression via its
direct binding to the sabA promoter region located 20 to +38 relative the tran-
scriptional start site (Harvey et al. 2014). Considering the neutral pH close to the
epithelium where the bacteria make use of adhesion properties, it seems logical to
downregulate adhesion properties in order to loosen the grip from the shedding host
gastric epithelial cell when it reaches the lumen. Alternatively, when ArsRS senses
acidic pH, downregulation of binding aids the bacteria to avoid attachment to
receptor moieties present on mucins where the pH is acidic and instead promotes
motility and thus the ability to swim toward the environment of neutral pH.
6.3.6 SabA and Gastroduodenal Diseases
The first study to assay for sLex binding among clinical H. pylori isolates showed
that 37 % among 95 European isolates bound to sLex. sLex-positive strains often
exhibited binding to Leb as well. Among the sLex-binding strains, almost half of
them showed binding to sLea (Mahdavi et al. 2002). The analysis of SabA expres-
sion in a collection of 200 clinical isolates from the USA and Colombia diagnosed
with either gastritis, duodenal ulcer, or gastric cancer showed that 66 % of the
gastritis isolates, 88 % of the duodenal ulcer isolates, and 89 % of the strains
isolated from patients with gastric cancer exhibited SabA expression (Yamaoka
et al. 2006). A study based on 145 Taiwanese clinical isolates showed that all
isolates expressed BabA and 31 % expressed SabA. Here, no differences in SabA
expression and outcome of disease were found (Sheu et al. 2006).
Upon H. pylori-induced gastritis, the gastric mucosa is infiltrated with neutro-
phils. The SabA adhesin seems to have an essential function in adherence of
H. pylori to sialylated neutrophils. SabA-mediated binding to neutrophils was a
156 A. Arnqvist
prerequisite for nonopsonic activation of neutrophils and is thus likely to have a role
in phagocytosis of H. pylori (Unemo et al. 2005).
6.4 BabA- and SabA-Mediated Binding to Mucins
The majority of individuals are of positive secretor phenotype, i.e., express

ABO/Leb antigens in the gastric mucus. H. pylori that resides in the stomach
mucus layer binds to secreted highly glycosylated mucins (Lindén et al. 2002).
The secreted MUC5AC and the MUC6 mucins are the two major mucins expressed
in the stomach mucus layer (Lindén et al. 2002; Van den Brink et al. 2000). The
BabA adhesin mediates binding of H. pylori to MUC5AC mucin in individuals of
positive secretor phenotype that expresses Leb glycans (Lindén et al. 2002). In
addition, H. pylori mediate binding to mucins carrying sLex by the SabA adhesin
(Lindén et al. 2008). Similar as in humans, the glycosylation and spatial distribution
of mucins change in rhesus macaques as a consequence of the H. pylori infection
(Cooke et al. 2009; Lindén et al. 2008). Experimental infection of rhesus monkeys
showed that individuals that display a weak-secretor phenotype had more stable
levels of fucosylation, lower degree of inflammation, and lower bacterial infection
load, whereas individuals with a positive secretor phenotype displayed increased
levels of inflammation-associated (sialylated) glycans and a transient decrease in
fucosylated (Leb) glycans. This suggested that the secretor phenotype determines
the dynamics of mucosal glycosylation in response to H. pylori infection and that
H. pylori infection is associated with an increase in sialylated (sLex/a) mucosal
antigens and a reciprocal decrease in fucosylated (Leb) mucosal antigens (Lindén
et al. 2008). At acidic conditions, the BabA-mediated binding to Leb-containing
glycans on the mucins is abolished (Lindén et al. 2004). Moreover, the bacterial-
mucin interplay seems to co-regulate both babA and sabA expression as well as
bacterial growth. The effects were host specific because mucins from different
individuals with different disease state caused different responses (Skoog
et al. 2012). The MUC1 mucin is present in the gastric glands. Adherence to the
MUC1 mucin may limit the H. pylori infection and protect the gastric glands from
H. pylori colonization (Lindén et al. 2009). Thus, MUC1 act as a decoy due to the
release of the MUC1 extracellular domain upon H. pylori binding (Lindén
et al. 2009). Mice that carried a deletion in MUC1 exhibited increased H. pylori
colonization and increased inflammation relative wild-type mice (McGuckin
et al. 2007).
6.5 BabA- and SabA-Mediated Adhesion of H. pylori Outer

Membrane Vesicles to the Gastric Mucosa
Gram-negative bacteria shed outer membrane vesicles (OMVs) (Kulp and Kuehn
2010). The exact role of OMVs is yet elusive but it is clear that they function as
vehicles to deliver bacterial components to host cells. Since the composition of the
OMVs represents the outer membrane of the bacteria and also contain additional
components from mainly the periplasmic space, they are carrier of host-effector
molecules and thus disease-promoting factors (Kulp and Kuehn 2010). H. pylori
OMVs carrying the VacA cytotoxin are present in human gastric biopsy specimens
(Fiocca et al. 1999). Two-dimensional 31P1H NMR correlation spectra have been
used to determine the phospholipid composition of H. pylori OMVs, and compre-
hensive mass spectrometry analyses have been used to identify their protein
composition (Olofsson et al. 2010). Phosphatidylethanolamine and cardiolipin
were the dominating phospholipids (Olofsson et al. 2010), and the majority of
outer membrane proteins were found in the OMVs and among them the BabA and
the SabA adhesins (Mullaney et al. 2009; Olofsson et al. 2010). Adhesion is a key
step for the delivery of toxins and effector molecules to target tissues. Using
electron microscopy in combination with soluble Leb- and sLex-receptor conju-
gates and gold particles confirmed that H. pylori OMVs carry the BabA and the
SabA adhesins and that both bound their respective receptor. Receptor displace-
ment assay showed that BabA on intact H. pylori bacterial cells and OMV-BabA
are bound to the Leb receptor with the same affinity, which suggested that the BabA
adhesins exhibit similar folding in the outer membrane as in the OMVs (Olofsson
et al. 2010). Moreover, the same study showed that both the BabA and the SabA
adhesins on OMVs mediate receptor-specific adhesion to human gastric mucosa.
6.6 Conclusion and Outlook
For H. pylori the life in the human stomach is similar to a roller coaster ride. Close
attachment to the epithelium offers a nutrient-rich, replicative niche but with a high
risk of eradication by host-immune responses or clearance by shear forces caused
by the peristaltic movement. Life further out in the mucus layer offers a famine
lifestyle and a high risk of acid exposure. The glycans on mucins and on the gastric
epithelial cells that function as receptors for H. pylori vary from person to person.
The glycans that are expressed depend on the individual expression of the transfer-
ase enzymes involved in glycan biosynthesis. In addition to this, the H. pylori
infection per se alters regulation of the glycan transferases, which results in variable
expression levels in different locations in the stomach. Therefore, it is absolutely
essential for H. pylori to continuously adapt its adhesion properties to fit the local
gastric environment in order to stay colonized (Fig. 6.2). Detailed knowledge about
the molecular terms that operate to fine-tune the expression of the BabA and SabA
158 A. Arnqvist
Fig. 6.2 Role of H. pylori adhesion in sickness and in health. The so far best-characterized
H. pylori adhesins are the blood group antigen-binding BabA adhesin and the sialic acid-binding
adhesin SabA. In the healthy gastric mucosa, the BabA adhesin binds to the fucosylated ABO/Leb
antigens, while adhesion to inflamed gastric mucosa mainly is mediated by the binding of the SabA
adhesin to the sLex and sLea antigens. The glycosylation of the gastric mucosa varies during the
persistent H. pylori infection, and, therefore, the bacterium needs to adapt its adhesion properties
accordingly. Expression of the BabA and the SabA adhesins can be switch on and off via
homologous recombination or SSM. SSM also plays an important role in fine-tuning the expres-
sion levels of the SabA adhesin. A thymine (T ) nucleotide repeat tract located close to the sabA
35 promoter element is a target for SSM, and changes in the T-tract length affect promoter
strength and thus transcription initiation. Altogether, homologous recombination and SSM gener-
ate heterogeneous populations that include best-fit clones ready to adapt to any host changes such
as changes in the glycosylation pattern and available receptor structures
adhesins in relation to continuously changes in the local environment during the

persistent H. pylori infection remains to be solved.
Successful colonization is the first step for the development of chronic gastritis,
peptic ulcer disease, and gastric cancer. Blocking attachment is an attractive target
for future design of new drugs. Even though a large number of strains have been
assayed for their Leb-binding activity and the nucleotide composition of the babA
gene has been determined, no consensus for Leb binding in BabA has yet been
determined (Aspholm-Hurtig et al. 2004). Further research to gain deep knowledge
about the molecular details for ligand-receptor binding is crucial for design of drugs
that can inhibit adhesion to the gastric mucosa. Therefore, structural determination
will be essential to identify the Leb-receptor-binding domain. Recently, a crystal
structure of a recombinantly expressed extracellular domain of the SabA protein
(1–460 amino acids) was described to have a “club shape.” A conserved cavity in
the SabA head domain that may function as binding site was identified. The authors
made attempts to co-crystallize the protein together with sLex but did not succeed.
However, upon testing the expressed SabA protein in surface resonance experi-
ments, they could detect binding to sLex but not to Leb, Lea, or Ley. Some binding
of low affinity was also detected in Lex. This difference in binding specificity of the
recombinantly expressed SabA and native SabA may be explained by differences in
steric blocking, hydrophobicity, or charges in the local environment of the SabA
protein (Pang et al. 2014). Based on the analysis of the SabA primary and tertiary
structure of the suggested binding pocket, four highly conserved amino acid
residues were mutated. A Q159A substitution reduced biding to sLex, while the
Y148A and Q162A substitutions showed reduced binding to Lex, and the K152A
substitution did not result affect binding to neither of sLex or Lex (Pang et al. 2014).
The SabA carbohydrate-binding domain contains amino acid residues that are
conserved both between SabA orthologs but also to the BabA protein (Pang
et al. 2014). Further research is needed to fully determine the relation between
structure and function.
Besides the BabA and the SabA adhesins, H. pylori have other adhesion prop-
erties that aid in attachments and probably pathogenesis, but their function is
beyond the scope of this chapter. These additional attachment mechanisms have
to be taken into account to fully describe the adhesion process as well as in design of
future drugs.
References
Åberg A, Gideonsson P, Vallstrom A, Olofsson A, Ohman C, Rakhimova L, Boren T, Engstrand L,

Brannstrom K, Arnqvist A (2014) A repetitive DNA element regulates expression of the
Helicobacter pylori sialic acid binding adhesin by a rheostat-like mechanism. PLoS Pathog
10:e1004234
Alm RA, Ling LS, Moir DT, King BL, Brown ED, Doig PC, Smith DR, Noonan B, Guild BC,
deJonge BL, Carmel G, Tummino PJ, Caruso A, Uria-Nickelsen M, Mills DM, Ives C,
Gibson R, Merberg D, Mills SD, Jiang Q, Taylor DE, Vovis GF, Trust TJ (1999) Genomic-
sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter
pylori. Nature 397:176–180
Helicobacter pylori: analysis of the outer membrane protein families. Infect Immun
68:4155–4168
Amundsen SK, Fero J, Hansen LM, Cromie GA, Solnick JV, Smith GR, Salama NR (2008)
Helicobacter pylori AddAB helicase-nuclease and RecA promote recombination-related DNA
repair and survival during stomach colonization. Mol Microbiol 69:994–1007
Aspholm M, Kalia A, Ruhl S, Schedin S, Arnqvist A, Linden S, Sjostrom R, Gerhard M, Semino-
Mora C, Dubois A, Unemo M, Danielsson D, Teneberg S, Lee WK, Berg DE, Boren T (2006a)
Helicobacter pylori adhesion to carbohydrates. Methods Enzymol 417:293–339
Aspholm M, Olfat FO, Norden J, Sonden B, Lundberg C, Sjostrom R, Altraja S, Odenbreit S,
Haas R, Wadstrom T, Engstrand L, Semino-Mora C, Liu H, Dubois A, Teneberg S, Arnqvist A,
Boren T (2006b) SabA is the H. pylori hemagglutinin and is polymorphic in binding to
sialylated glycans. PLoS Pathog 2:e110
Aspholm-Hurtig M, Dailide G, Lahmann M, Kalia A, Ilver D, Roche N, Vikstr€ om S, Sj€
ostr€
om R,
Lindén S, Bäckstr€om A, Lundberg C, Arnqvist A, Mahdavi J, Nilsson UJ, Velapatino B,
Gilman RH, Gerhard M, Alarcon T, Lopez-Brea M, Nakazawa T, Fox JG, Correa P,
160 A. Arnqvist
Dominguez-Bello MG, Perez-Perez GI, Blaser MJ, Normark S, Carlstedt I, Oscarson S,

Teneberg S, Berg DE, Borén T (2004) Functional adaptation of BabA, the H. pylori ABO
blood group antigen binding adhesin. Science 305:519–522
Azevedo M, Eriksson S, Mendes N, Serpa J, Figueiredo C, Resende LP, Ruvoen-Clouet N, Haas R,
Boren T, Le Pendu J, David L (2008) Infection by Helicobacter pylori expressing the BabA
adhesin is influenced by the secretor phenotype. J Pathol 215:308–316
Bäckstr€
om A, Lundberg C, Kersulyte D, Berg DE, Borén T, Arnqvist A (2004) Metastability of
Helicobacter pylori bab adhesin genes and dynamics in Lewis b antigen binding. Proc Natl
Acad Sci U S A 101:16923–16928
Benktander J, Angstrom J, Breimer ME, Teneberg S (2012) Redefinition of the carbohydrate
binding specificity of Helicobacter pylori BabA adhesin. J Biol Chem 287:31712–31724
Borén T, Falk P, Roth KA, Larson G, Normark S (1993) Attachment of Helicobacter pylori to
human gastric epithelium mediated by blood group antigens. Science 262:1892–1895
Bukanov NO, Berg DE (1994) Ordered cosmid library and high-resolution physical-genetic map
of Helicobacter pylori strain NCTC11638. Mol Microbiol 11:509–523
Bury-Moné S, Thiberge JM, Contreras M, Maitournam A, Labigne A, De Reuse H (2004)
Clausen H, Hakomori S (1989) ABH and related histo-blood group antigens; immunochemical
differences in carrier isotypes and their distribution. Vox Sang 56:1–20
Colbeck JC, Hansen LM, Fong JM, Solnick JV (2006) Genotypic profile of the outer membrane
proteins BabA and BabB in clinical isolates of Helicobacter pylori. Infect Immun
74:4375–4378
Cooke CL, An HJ, Kim J, Canfield DR, Torres J, Lebrilla CB, Solnick JV (2009) Modification of
gastric mucin oligosaccharide expression in rhesus macaques after infection with Helicobacter
pylori. Gastroenterology 137:1061–1071, 1071 e1061-1068
Emody L, Carlsson A, Ljungh A, Wadstrom T (1988) Mannose-resistant haemagglutination by
Campylobacter pylori. Scand J Infect Dis 20:353–354
Evans DG, Karjalainen TK, Evans DJ Jr, Graham DY, Lee CH (1993) Cloning, nucleotide
sequence, and expression of a gene encoding an adhesin subunit protein of Helicobacter pylori.
Falk P, Roth KA, Boren T, Westblom TU, Gordon JI, Normark S (1993) An in vitro adherence
assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human
gastric epithelium. Proc Natl Acad Sci U S A 90:2035–2039
Falk PG, Bry L, Holgersson J, Gordon JI (1995) Expression of a human alpha-1,3/4-
fucosyltransferase in the pit cell lineage of FVB/N mouse stomach results in production of
Leb-containing glycoconjugates: a potential transgenic mouse model for studying
Helicobacter pylori infection. Proc Natl Acad Sci U S A 92:1515–1519
Fiocca R, Necchi V, Sommi P, Ricci V, Telford J, Cover TL, Solcia E (1999) Release of
Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding
of outer membrane vesicles. Uptake of released toxin and vesicles by gastric epithelium. J
Pathol 188:220–226
Fujimoto S, Olaniyi Ojo O, Arnqvist A, Wu JY, Odenbreit S, Haas R, Graham DY, Yamaoka Y
(2007) Helicobacter pylori BabA expression, gastric mucosal injury, and clinical outcome.
Clin Gastroenterol Hepatol 5:49–58
Gerhard M, Lehn N, Neumayer N, Borén T, Rad R, Schepp W, Miehlke S, Classen M, Prinz C
adhesin. Proc Natl Acad Sci U S A 96:12778–12783
Goodwin AC, Weinberger DM, Ford CB, Nelson JC, Snider JD, Hall JD, Paules CI, Peek RM Jr,
Forsyth MH (2008) Expression of the Helicobacter pylori adhesin SabA is controlled via phase
variation and the ArsRS signal transduction system. Microbiology 154:2231–2240
Guruge JL, Falk PG, Lorenz RG, Dans M, Wirth HP, Blaser MJ, Berg DE, Gordon JI (1998)
Epithelial attachment alters the outcome of Helicobacter pylori infection. Proc Natl Acad Sci
U S A 95:3925–3930
Harvey VC, Acio CR, Bredehoft AK, Zhu L, Hallinger DR, Quinlivan-Repasi V, Harvey SE,
Forsyth MH (2014) Repetitive sequence variations in the promoter region of the adhesin
encoding gene sabA of Helicobacter pylori affect transcription. J Bacteriol 196:3421–3429
Hennig EE, Mernaugh R, Edl J, Cao P, Cover TL (2004) Heterogeneity among Helicobacter pylori
strains in expression of the outer membrane protein BabA. Infect Immun 72:3429–3435
Hennig EE, Allen JM, Cover TL (2006) Multiple chromosomal loci for the babA gene in
Helicobacter pylori. Infect Immun 74:3046–3051
Hessey SJ, Spencer J, Wyatt JI, Sobala G, Rathbone BJ, Axon AT, Dixon MF (1990) Bacterial
adhesion and disease activity in Helicobacter associated chronic gastritis. Gut 31:134–138
Ilver D, Arnqvist A, Ögren J, Frick IM, Kersulyte D, Incecik ET, Berg DE, Covacci A,
Engstrand L, Borén T (1998) Helicobacter pylori adhesin binding fucosylated histo-blood
group antigens revealed by retagging. Science 279:373–377
Ishijima N, Suzuki M, Ashida H, Ichikawa Y, Kanegae Y, Saito I, Borén T, Haas R, Sasakawa C,
Mimuro H (2011) BabA-mediated adherence is a potentiator of the Helicobacter pylori type IV
secretion system activity. J Biol Chem 286:25256–25264
Kao CY, Sheu SM, Sheu BS, Wu JJ (2012) Length of thymidine homopolymeric repeats modu-
lates promoter activity of sabA in Helicobacter pylori. Helicobacter 17:203–209
Kulp A, Kuehn MJ (2010) Biological functions and biogenesis of secreted bacterial outer
membrane vesicles. Annu Rev Microbiol 64:163–184
Lehours P, Menard A, Dupouy S, Bergey B, Richy F, Zerbib F, Ruskone-Fourmestraux A,
Delchier JC, Megraud F (2004) Evaluation of the association of nine Helicobacter pylori
virulence factors with strains involved in low-grade gastric mucosa-associated lymphoid tissue
lymphoma. Infect Immun 72:880–888
Lindén S, Nordman H, Hedenbro J, Hurtig M, Boren T, Carlstedt I (2002) Strain- and blood group-
dependent binding of Helicobacter pylori to human gastric MUC5AC glycoforms. Gastroen-
terology 123:1923–1930
Lindén S, Mahdavi J, Hedenbro J, Boren T, Carlstedt I (2004) Effects of pH on Helicobacter pylori
binding to human gastric mucins: identification of binding to non-MUC5AC mucins. Biochem
J 384:263–270
Lindén S, Mahdavi J, Semino-Mora C, Olsen C, Carlstedt I, Boren T, Dubois A (2008) Role of
ABO secretor status in mucosal innate immunity and H. pylori infection. PLoS Pathog 4:e2
Lindén SK, Sheng YH, Every AL, Miles KM, Skoog EC, Florin TH, Sutton P, McGuckin MA
(2009) MUC1 limits Helicobacter pylori infection both by steric hindrance and by acting as a
releasable decoy. PLoS Pathog 5:e1000617
Mahdavi J, Sondén B, Hurtig M, Olfat FO, Forsberg L, Roche N, Ångstr€ om J, Larsson T,
Teneberg S, Karlsson KA, Altraja S, Wadstr€om T, Kersulyte D, Berg DE, Dubois A,
Hammarstrom L, Borén T (2002) Helicobacter pylori SabA adhesin in persistent infection
Marcos NT, Magalhaes A, Ferreira B, Oliveira MJ, Carvalho AS, Mendes N, Gilmartin T, Head
SR, Figueiredo C, David L, Santos-Silva F, Reis CA (2008) Helicobacter pylori induces
beta3GnT5 in human gastric cell lines, modulating expression of the SabA ligand sialyl-
Lewis x. J Clin Invest 118:2325–2336
McGuckin MA, Every AL, Skene CD, Lindén SK, Chionh YT, Swierczak A, McAuley J,
Harbour S, Kaparakis M, Ferrero R, Sutton P (2007) Muc1 mucin limits both Helicobacter
pylori colonization of the murine gastric mucosa and associated gastritis. Gastroenterology
133:1210–1218
162 A. Arnqvist
Mullaney E, Brown PA, Smith SM, Botting CH, Yamaoka YY, Terres AM, Kelleher DP, Windle
HJ (2009) Proteomic and functional characterization of the outer membrane vesicles from the
gastric pathogen Helicobacter pylori. Proteomics Clin Appl 3:785–796
Odenbreit S, Swoboda K, Barwig I, Ruhl S, Boren T, Koletzko S, Haas R (2009) Outer membrane
protein expression profile in Helicobacter pylori clinical isolates. Infect Immun 77:3782–3790
Oh JD, Kling-Backhed H, Giannakis M, Xu J, Fulton RS, Fulton LA, Cordum HS, Wang C,
Elliott G, Edwards J, Mardis ER, Engstrand LG, Gordon JI (2006) The complete genome
sequence of a chronic atrophic gastritis Helicobacter pylori strain: evolution during disease
progression. Proc Natl Acad Sci U S A 103:9999–10004
Ohno T, Vallstr€ om A, Rugge M, Ota H, Graham DY, Arnqvist A, Yamaoka Y (2011) Effects of
blood group antigen-binding adhesin expression during Helicobacter pylori infection of
Mongolian gerbils. J Infect Dis 203:726–735
Olfat FO, Zheng Q, Oleastro M, Voland P, Boren T, Karttunen R, Engstrand L, Rad R, Prinz C,
Gerhard M (2005) Correlation of the Helicobacter pylori adherence factor BabA with duodenal
ulcer disease in four European countries. FEMS Immunol Med Microbiol 44:151–156
Oliveira AG, Santos A, Guerra JB, Rocha GA, Rocha AM, Oliveira CA, Cabral MM, Nogueira
AM, Queiroz DM (2003) babA2- and cagA-positive Helicobacter pylori strains are associated
with duodenal ulcer and gastric carcinoma in Brazil. J Clin Microbiol 41:3964–3966
Olofsson A, Vallstrom A, Petzold K, Tegtmeyer N, Schleucher J, Carlsson S, Haas R, Backert S,
Wai SN, Grobner G, Arnqvist A (2010) Biochemical and functional characterization of
Helicobacter pylori vesicles. Mol Microbiol 77:1539–1555
O’Toole PW, Janzon L, Doig P, Huang J, Kostrzynska M, Trust TJ (1995) The putative
neuraminyllactose-binding hemagglutinin HpaA of Helicobacter pylori CCUG 17874 is a
lipoprotein. J Bacteriol 177:6049–6057
Pang SS, Nguyen ST, Perry AJ, Day CJ, Panjikar S, Tiralongo J, Whisstock JC, Kwok T (2014)
The three-dimensional structure of the extracellular adhesion domain of the sialic acid-binding
adhesin SabA from Helicobacter pylori. J Biol Chem 289:6332–6340
Petersson C, Forsberg M, Aspholm M, Olfat FO, Forslund T, Borén T, Magnusson KE (2006)
Helicobacter pylori SabA adhesin evokes a strong inflammatory response in human neutro-
phils which is down-regulated by the neutrophil-activating protein. Med Microbiol Immunol
195:195–206
Pflock M, Finsterer N, Joseph B, Mollenkopf H, Meyer TF, Beier D (2006) Characterization of the
ArsRS regulon of Helicobacter pylori, involved in acid adaptation. J Bacteriol 188:3449–3462
Pride DT, Blaser MJ (2002) Concerted evolution between duplicated genetic elements in
Helicobacter pylori. J Mol Biol 316:629–642
Semino-Mora C, Doi SQ, Marty A, Simko V, Carlstedt I, Dubois A (2003) Intracellular and
interstitial expression of Helicobacter pylori virulence genes in gastric precancerous intestinal
metaplasia and adenocarcinoma. J Infect Dis 187:1165–1177
Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S, Sittka A, Chabas S, Reiche K,
Hackermuller J, Reinhardt R, Stadler PF, Vogel J (2010) The primary transcriptome of the
major human pathogen Helicobacter pylori. Nature 464:250–255
Sheu BS, Odenbreit S, Hung KH, Liu CP, Sheu SM, Yang HB, Wu JJ (2006) Interaction between
host gastric Sialyl-Lewis X and H. pylori SabA enhances H. pylori density in patients lacking
gastric Lewis B antigen. Am J Gastroenterol 101:36–44
Skoog EC, Sjoling A, Navabi N, Holgersson J, Lundin SB, Linden SK (2012) Human gastric
mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions
with host cells. PLoS One 7:e36378
Solnick JV, Hansen LM, Salama NR, Boonjakuakul JK, Syvanen M (2004) Modification of
Helicobacter pylori outer membrane protein expression during experimental infection of
rhesus macaques. Proc Natl Acad Sci U S A 101:2106–2111
Song H, Michel A, Nyren O, Ekstrom AM, Pawlita M, Ye W (2014) A CagA-independent cluster
of antigens related to the risk of noncardia gastric cancer: associations between Helicobacter
pylori antibodies and gastric adenocarcinoma explored by multiplex serology. Int J Cancer
134:2942–2950
Styer CM, Hansen LM, Cooke CL, Gundersen AM, Choi SS, Berg DE, Benghezal M, Marshall BJ,
Peek RM Jr, Boren T, Solnick JV (2010) Expression of the BabA adhesin during experimental
infection with Helicobacter pylori. Infect Immun 78:1593–1600
Talarico S, Whitefield SE, Fero J, Haas R, Salama NR (2012) Regulation of Helicobacter pylori
adherence by gene conversion. Mol Microbiol 84:1050–1061
Toller IM, Neelsen KJ, Steger M, Hartung ML, Hottiger MO, Stucki M, Kalali B, Gerhard M,
Sartori AA, Lopes M, Muller A (2011) Carcinogenic bacterial pathogen Helicobacter pylori
triggers DNA double-strand breaks and a DNA damage response in its host cells. Proc Natl
Acad Sci U S A 108:14944–14949
Unemo M, Aspholm-Hurtig M, Ilver D, Bergstr€om J, Borén T, Danielsson D, Teneberg S (2005)
The sialic acid binding SabA adhesin of Helicobacter pylori is essential for nonopsonic
activation of human neutrophils. J Biol Chem 280:15390–15397
Van den Brink GR, Tytgat KM, Van der Hulst RW, Van der Loos CM, Einerhand AW, Buller HA,
Dekker J (2000) H pylori colocalises with MUC5AC in the human stomach. Gut 46:601–607
Walz A, Odenbreit S, Mahdavi J, Boren T, Ruhl S (2005) Identification and characterization of
binding properties of Helicobacter pylori by glycoconjugate arrays. Glycobiology 15:700–708
Walz A, Odenbreit S, Stuhler K, Wattenberg A, Meyer HE, Mahdavi J, Boren T, Ruhl S (2009)
Identification of glycoprotein receptors within the human salivary proteome for the lectin-like
BabA and SabA adhesins of Helicobacter pylori by fluorescence-based 2-D bacterial overlay.
Proteomics 9:1582–1592
Yamaoka Y, Souchek J, Odenbreit S, Haas R, Arnqvist A, Borén T, Kodama T, Osato MS,
Gutierrez O, Kim JG, Graham DY (2002) Discrimination between cases of duodenal ulcer
and gastritis on the basis of putative virulence factors of Helicobacter pylori. J Clin Microbiol
40:2244–2246
odenal disease. Gut 55:775–781
Yu J, Leung WK, Go MY, Chan MC, To KF, Ng EK, Chan FK, Ling TK, Chung SC, Sung JJ
(2002) Relationship between Helicobacter pylori babA2 status with gastric epithelial cell
turnover and premalignant gastric lesions. Gut 51:480–484
Chapter 7
Emerging Novel Virulence Factors
of Helicobacter pylori
Silja Wessler
Abstract Through the expression of a wide range of differently functioning

virulence factors, Helicobacter pylori possesses multiple possibilities to interact
with host target cells that in combination determine the development and progress
of diseases. Prominent H. pylori factors such as the type IV secretion system
(T4SS), cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA),
and blood-group antigen-binding adhesin (BabA)/sialic acid-binding adhesin
(SabA) are discussed in other chapters. Besides those well-established pathogenic
and virulence factors, a series of other bacterial factors exist, including neutrophil-
activating protein (NapA), γ-glutamyl transpeptidase (GGT), tumor necrosis
factor-alpha-inducing protein alpha (Tipα), the cell-translocating serine/threonine
kinase (CtkA), and bacterial proteases, such as the serine protease high-temperature
requirement A (HtrA) or the collagenase Hp0169. These factors are described as
secreted H. pylori proteins that might be implicated in pathogenesis as well. As
additional novel disease-associated factors, the Helicobacter outer membrane pro-
teins (Hop) HopQ and HopZ and the duodenal ulcer-promoting gene A (DupA) are
also currently under intensive investigation. Since these H. pylori factors are either
secreted or surface exposed, they can directly interfere with host cell functions to
modulate cellular responses. Research on these bacterial structures is still at the
beginning; however, they represent novel target molecules for supporting thera-
peutic intervention strategies.
Keywords CtkA • DupA • GGT • HopQ • HopZ • H. pylori • HtrA • NapA • Tipα
7.1 Introduction
As a highly dynamic and complex bacterial organism, H. pylori encodes multiple

different factors and structures, which have the capability to modulate host cell
functions. The molecular mechanisms of the type IV secretion system (T4SS) that
translocates the bacterial effect protein CagA, the secreted pore-forming protein
S. Wessler (*)
Department of Microbiology, Paris Lodron University, Billrothstr. 11, 5020 Salzburg, Austria

DOI 10.1007/978-4-431-55936-8_7
166 S. Wessler
VacA, or different adhesins, such as BabA or SabA, are subject of many studies,
and most of them are well characterized of how they interact with host cell proteins.
The cellular and molecular mechanisms of these proteins are discussed in other
chapters (see Chaps. 3, 4, 5, and 6). Indeed, additional factors might contribute to
bacterial pathogenesis of H. pylori. Obviously, H. pylori-induced pathogenesis is a
multistep process reflecting many different modes of interference with host cell
functions (Backert and Clyne 2011; Posselt et al. 2013; Wessler and Backert 2008).
In particular, GGT, NapA, Tipα, CtkA, HopQ, HopZ, and HtrA attracted much
attention as bacterial compounds in the last years that are implicated in many
different mechanisms to induce cellular responses (Table 7.1), hence acting as
emerging novel factors of H. pylori. These factors share their extracellular locali-
zation allowing direct contact with molecules of host cells to interfere with their
unique functions (Fig. 7.1). However, it is still of debate whether those H. pylori
V. Tipα
HopZ
I. nucleolin
HopQ binding
changes in cell
CagA IV. CtkA
II. HtrA morphology
gastric epithelial cells
injection V. Tipα
E-cadherin
cleavage nuclear
cytokines localization nuclear TNF-α
apoptosis localization
trans- CagA
migration injection V.
Tcf-Lef
Tipα
regulation? CtkA
I.
IV.
HopZ
HopQ
fibronectin
cleavage DC VII.
ECM
ECM NapA
cleavage
proliferation VI.
III. inhibition attraction
II. HtrA GGT tolerizing
T-cell neutrophil
Fig. 7.1 Emerging novel factors with possible functions in H. pylori pathogenesis. The adhesins
HopQ and HopZ (I ) contribute to H. pylori adherence on gastric epithelial cells. HopQ expression
promotes CagA translocation via an unknown host receptor. HtrA (II) is secreted by H. pylori and
cleaves the cell adhesion molecule and tumor suppressor E-cadherin allowing transmigration of
H. pylori. If E-cadherin cleavage affects Tcf-Lef signaling is unknown. HtrA also targets fibro-
nectin, a component of the extracellular matrix (ECM). Hp0169 (III) functions as a collagenase
with possible substrates in the ECM. CtkA (IV) is a dual-specific bacterial serine/threonine and
tyrosine kinase, which enters the cell and translocates into the nucleus, where it is involved in gene
regulation leading to cytokine release and apoptosis. Tipα (V ) binds to nucleolin on the cell surface
leading to changes of the gastric cell morphology and/or adhesion. Tipα also translocates into the
nucleus to induce TNF-α expression. The enzyme GGT (VI) has multiple effects by inhibition of T
cell and epithelial cell proliferation. Dendritic cells (DCs) are tolerized in response to GGT.
Neutrophils are attracted by NapA (VII)
7 Emerging Novel Virulence Factors of Helicobacter pylori 167
structures represent pathogenic or virulence factors or if they can facilitate the

molecular interaction of other bacterial factors with host cells, which are discussed
in this chapter.
7.2 The Effects of GGT on Epithelial Cells and Immune

Cells
H. pylori commonly encode the gamma-glutamyl transpeptidase (GGT) as a con-

stitutively expressed enzyme (Chevalier et al. 1999) that converts glutamine into
glutamate and ammonia and glutathione into glutamate and cysteinylglycine. GGT
is expressed as a 61 kDa protein that contains an N-terminally located signal
peptide composed of 26 amino acids. After cleavage of the signal peptide,
pro-GGT is further processed into a 40 kDa N-terminal polypeptide and a 21 kDa
C-terminal domain (Chevalier et al. 1999). The 20 kDa variant of GGT has been
identified in the soluble fraction of H. pylori culture supernatants, and the 38 kDa
subunit was found in the structure bound and soluble fractions of H. pylori (Backert
et al. 2005), indicating that GGT is actively secreted by H. pylori. GGT expression
and activity have been correlated with the development of peptic ulcer disease
(Gong et al. 2010). Initially, GGT was suggested as an essential factor for H. pylori
colonization (Chevalier et al. 1999), but this statement has been softened a few
years later in a study describing that GGT-negative H. pylori were able to colonize
piglets and mice to a significant lower extent compared to isogenic wild-type strains
(McGovern et al. 2001). The fact that H. pylori GGT knockout mutants can
colonize animals was then repeatedly published (Chevalier et al. 1999; McGovern
et al. 2001; Oertli et al. 2013) implying that GGT is not essential per se but
obviously an important factor in the establishment of H. pylori infection. The
reason for the reduced colonization of GGT-negative bacteria is still not clear as
GGT obviously does not function as an adhesin or virtually contributes to bacterial
survival. However, if GGT activity alters, bacterial growth is contradictory in the
literature. Gong and co-workers reported that H. pylori growth correlated with the
activity of GGT and that higher GGT activity favors bacterial growth in vitro (Gong
and Ho 2004). Other publications indicated that GGT-negative H. pylori strains
grew equally well as compared to the corresponding wild-type strain (Chevalier
et al. 1999; McGovern et al. 2001; Rossi et al. 2012).
GGT from H. pylori and H. bilis inhibited AGS cell proliferation (Fig. 7.1), even
though higher concentrations were required and involved an apoptosis-independent
mechanism (Rossi et al. 2012). However, apoptosis as another cellular function for
H. pylori GGT was also described (Shibayama et al. 2003). Similar effects on host
cell death have also been observed for H. suis GGT (Flahou et al. 2011). Increasing
concentrations of GGT, which was enriched from H. pylori membrane fractions,
induced apoptosis in AGS cells, while an inhibitor targeting GGT activity
decreased apoptosis significantly (Shibayama et al. 2003). This observation has
168 S. Wessler
been confirmed by other studies. Recombinant GGT purified from Escherichia coli
triggered apoptosis in AGS cells. Activation of caspase-3 and caspase-9 was
induced by GGT stimulation, and a possible regulation of the apoptosis-related
factors Bax and Bcl-2 was suggested by expression analyses (Boonyanugomol
et al. 2012; Kim et al. 2007). GGT-dependent regulation of the inhibitor of
apoptosis protein (IAP) survivin was recently also detected. The protein level of
survivin is decreased in the mucosa of H. pylori-infected patients with gastritis
(Valenzuela et al. 2010) and in H. pylori-colonized MKN-45 and AGS cells in vitro
(Valenzuela et al. 2013). Further analysis revealed that the protein level of survivin
is downregulated via a proteasomal pathway (Valenzuela et al. 2013). Many studies
describing apoptosis-inducing properties of GGT have been performed using
recombinant GGT. However, a GGT knockout mutant still induced T-cell apoptosis
to a similar extent (Beigier-Bompadre et al. 2011), indicating that GGT might not
play a major role in H. pylori-mediated induction of apoptosis in T cells.
Along with its apoptosis-inducing properties, enzymatically active GGT acts as
a novel immunosuppressive factor by inhibiting T-cell proliferation (Fig. 7.1),
which could contribute to the persistence and/or immune evasion of H. pylori
(Schmees et al. 2007). T-cell inhibition resulted from depriving the extracellular
space of glutamine (Wustner et al. 2015). Interestingly, GGT-mediated apoptosis
was not observed in T cells (Schmees et al. 2007). Mechanistically, H. pylori GGT
can induce a cell cycle arrest at the G1/S phase transition in T cells (Schmees
et al. 2007) and in gastric epithelial cells (Kim et al. 2010). In epithelial cells,
downregulation of the cell cycle-associated proteins cyclin E, cyclin A, cyclin-
dependent kinase (Cdk) Cdk 4, and Cdk 6 and upregulated Cdk inhibitors p27 and
p21 led to cell cycle arrest and apoptosis (Kim et al. 2010). In cultured AGS and
MKN-28 cells, GGT-mediated upregulation of COX-2 and HB-EGF was observed
(Busiello et al. 2004). Even though the functional consequences of enhanced
COX-2 and HB-EGF expression were not associated to a cellular phenotype, the
authors speculated that GGT influences the cell cycle via this pathway (Busiello
et al. 2004). If those effects also occur in T cells leading to the inhibition of
proliferation has not been analyzed in detail yet (Schmees et al. 2007). T-cell
inhibition was initially reported as a VacA-mediated effect (Gebert et al. 2003);
hence, GGT is an additional important factor in T-cell inhibition underlining the
importance of secreted factors in H. pylori pathogenesis. Furthermore, in a follow-
ing publication, it was shown that both GGT and VacA contributed to H. pylori’s
tolerizing effects on murine dendritic cells (DCs) in vitro and in vivo (Fig. 7.1)
(Oertli et al. 2013), which can also contribute to the prevention of asthma in vivo
(Engler et al. 2014). This effect was independent of T cells indicating important
functions of GGT in manipulating the immune system and establishing persistent
infection. These kinds of combined effects of VacA and GGT were also described
for the induction of miRNA-155 (Fassi Fehri et al. 2010). GGT/VacA-induced
miRNA-155 directly targets the protein kinase A inhibitor α (PKIα) mRNA in a
cAMP-Foxp3-dependent manner in T cells (Fassi Fehri et al. 2010). However,
whether this pathway contributes to the T-cell inhibition or other responses is yet
unknown.
The research on GGT is still at the beginning as reflected by the small but
accumulating number of publications. However, these studies on the relatively
diverse pathways and cellular responses are steadily increasing and will more and
more complete the picture of GGT as a novel and important factor in H. pylori
pathogenesis.
7.3 NapA Affects the Host Immune System
The effects of neutrophil-activating factor A (NapA) on H. pylori-infected host

cells appear to be restricted on immune cells, while functions on gastric epithelial
cells were not yet described. Initially, H. pylori NapA was identified as an extra-
cellular 150 kDa multimeric protein complex in water extracts of H. pylori (Evans
et al. 1995). It was suggested that NapA is released from the cytosol by autolysis,
but the exact pathway of NapA delivery into the supernatant is yet unknown.
It was consistently described that NapA can attract neutrophils (Fig. 7.1) via
chemotaxis and promotes the adherence of neutrophils to endothelial cells. In
principle, recombinantly expressed NapA from Bacillus subtilis and E. coli was
used for these studies (Table 7.1), but also NapA enriched from H. pylori water
extracts has been associated with increased adherence of neutrophils to endothelial
cells, which could be decreased by adding a polyclonal anti-NapA antibody (Evans
et al. 1995). LPS-free NapA produced in B. subtilis significantly induced chemo-
taxis of neutrophils and monocytes (Satin et al. 2000). Furthermore, recombinant
NapA resulted in an increased transendothelial migration of neutrophils, which was
slightly reduced using a NapA-deletion mutant of H. pylori without exerting a
direct effect on endothelial cells (Brisslert et al. 2005). When rat mesenteric venules
were directly exposed to NapA, leukocytes were stimulated to adhere to the
endothelium. The authors then indicated that NapA has the capability to cross the
endothelial layer to promote leukocyte adherence directly through the activation of
β2-integrins. They excluded a paracellular route of NapA delivery but suggested a
mechanism that involves a NapA transport within the endothelial cells (Polenghi
et al. 2007). In transwell filter assays using Caco-2 cells as a polarized cell culture
model, it was repeatedly shown that NapA itself could translocate from the apical to
the basolateral compartment. The mechanism of NapA transmigration in this model
remained unknown (Montemurro et al. 2002). However, these data consistently
indicate that NapA plays a prominent role in the attraction of neutrophils to the site
of infection.
In expression analyses of mononuclear cells (MNC), NapA induced the expres-
sion of tissue factor (TF) and PAI-1 (plasminogen activator inhibitor-2)
(Montemurro et al. 2001). In peritoneal mast cells (PMC) from rats indicated that
NapA induced the release of β-hexosaminidase and interleukin-6 (IL-6). Both could
be blocked using pertussis toxin suggesting the involvement of heterotrimeric
G proteins (Montemurro et al. 2002). It was further implied that recombinant
NapA induced the production of reactive oxygen intermediates (ROI) in the host
170 S. Wessler
(Evans et al. 1995), which involved activation of the plasma membrane NADPH
oxidase via phosphatidylinositol 3-kinase (PI3-K) and Src family tyrosine kinases
(Satin et al. 2000). This signaling pathway might be upstream of the activated
MAPK (mitogen-activated protein kinase) ERK1/2 and p38 in NapA-treated neu-
trophils, which was shown by Nishioka and colleagues a few years later (Nishioka
et al. 2003). Inhibition of ERK1/2 and p38 significantly inhibited chemotaxis in
these cells (Nishioka et al. 2003). These studies were mainly performed using
recombinant NapA protein. In contrast, using a deletion mutant of H. pylori, a
NapA-dependent increase of oxidative burst was observed (Petersson et al. 2006)
making more experiments necessary to investigate oxidative burst in response
to NapA.
NapA forms dodecameric multimers that bind iron in a ferroxidase site (Tonello
et al. 1999; Yokoyama et al. 2012; Zanotti et al. 2002). A functional ferroxidase site
appears to be important for the formation of dodecamers to protect DNA from
oxidative damage without direct binding (Kottakis et al. 2008). As potential host
cell binding partners for NapA, sulfated oligosaccharide structures (Namavar
et al. 1998) and acid glycosphingolipid fraction on neutrophils (Teneberg
et al. 1997) were identified. Later it was speculated that NapA represents a toll-
like receptor-2 (TLR2) agonist to induce NF-κB-mediated IL-12 and IL-23 release
in neutrophils and monocytes (Amedei et al. 2006). Among the induced cytokines,
NapA-mediated IL-1β release from monocytes appears to trigger an increase in cell
survival through preventing apoptosis (Cappon et al. 2010). The possible NapA
function as a TLR2 ligand was investigated in tumor growth in a bladder cancer
mouse model. Interestingly, local administration of NapA decreased tumor growth
by triggering tumor necrosis (Codolo et al. 2012) suggesting that recombinant
NapA could be applied in anticancer strategies. The assumption that NapA func-
tions as a therapeutic agent has been confirmed and expanded by further reports. In
vivo recombinant NapA induced Th1 polarization and decreased T-cell-specific
allergic responses, which might be a novel strategy of prevention and treatment of
allergic diseases or asthma (Amedei et al. 2006; Codolo et al. 2008). A similar
beneficial effect of NapA on Trichinella spiralis-mediated pathogenesis was also
detected (Del Prete et al. 2008). NapA is highly immunogenic as many H. pylori-
infected patients have antibodies against NapA and produced a significant
immunoprotective response in mice (Satin et al. 2000). Using attenuated measles
virus (MV) vectors, animals immunized with MV strains expressing the secretory
NapA antigen developed strong humoral immunity against NapA (Iankov
et al. 2011). This model was further developed for the therapeutic efficacy of
oncolytic MV strains expressing NapA as a therapeutic transgene for the treatment
of metastatic breast cancer pleural effusion (Iankov et al. 2012). According to the
measles virus model, oncolytic adenovirus harboring napA as an immunomodula-
tory gene exhibited therapeutic effects on neuroendocrine tumors (Ramachandran
et al. 2013). It was further demonstrated that MV-encoded NapA-tagged chimeric
antigen can induce significantly stronger immune response (Iankov et al. 2013)
indicating that NapA can enhance immunogenicity of poor immunogens as a novel
approach in vaccine development and immunotherapy. In summary, these results
identify NapA as a virulence factor relevant for the pathogenic effects of H. pylori
at the sites of infection and point to NapA as a therapeutic agent in immunotherapy
and cancer-related disorders.
7.4 Tipα: A Multifunctional Factor?
The induction of cytokines in epithelial cells in response to H. pylori infection is

dependent on a functional T4SS (Backert et al. 2010; Fischer et al. 2001). In
addition to these well-established data, the tumor necrosis factor-alpha-inducing
protein alpha (Tipα) was suggested as a novel bacterial factor that activated nuclear
factor kappa B (NF-κB) and TNF-α in Bhas 42 (BALB/3T3 cells transfected with v-
H-ras gene) and MGT-40 cells (mouse gastric epithelial cell line) exhibiting
transforming activities (Suganuma et al. 2005). This unexpected observation was
confirmed in macrophages (Godlewska et al. 2008), a study indicating that Tipα
also has an influence on mice colonization (Godlewska et al. 2008), which might
account for the NF-κB activation. Because TNF-α acts as a promoter of tumor
progression (Bauer et al. 2012; Vendramini-Costa and Carvalho 2012; Walczak
2011), Tipα has consequently been suggested as a carcinogenic factor that might
trigger inflammation and/or carcinogenesis in patients infected with cagPAI-nega-
tive H. pylori strains. In this context, future studies are required to investigate the
pathophysiological role of Tipα in vivo.
Structurally, Tipα contains an N-terminal signal peptide and forms a homodimer
via one or two interchain disulfide bonds mediated by cysteine residues C25 and
C27 at low pH values and hydrophobic interactions (Jang et al. 2009; Tosi
et al. 2009; Tsuge et al. 2009). It might also share some homologies with
penicillin-binding proteins of Gram-positive bacteria (Kuzuhara et al. 2005) imply-
ing that the tipα gene was acquired by horizontal gene transfer. Tipα was also
described as a highly immunogenic factor. In vivo, recombinant Tipα can stimulate
the production of specific antibodies (Voland et al. 2002), which could induce a
partially protective response against H. pylori colonization (Inoue et al. 2009). In
DNA microarray and ELISA analyses, several chemokines and cytokines (e.g.,
Ccl2, Ccl7, Cxcl1, etc.) were upregulated in Tipα-treated cells (Kuzuhara
et al. 2007a; Tang et al. 2013). Mechanistically, it was further suggested that
Tipα can be internalized by epithelial cells to translocate into the nucleus
(Fig. 7.1), where it binds DNA (Suganuma et al. 2008). Supporting this assumption,
in DNA-binding experiments, recombinant Tipα bound different elements of the
TNF-α promoter (Kuzuhara et al. 2007b) leading to the hypothesis that Tipα could
directly transactivate gene promoters to trigger cytokine/chemokine synthesis and
carcinogenesis. Hence, it is unclear to which extent Tipα-induced NF-κB or Tipα
itself activates cytokine synthesis. Recently, nucleolin was described as a cell
surface receptor for Tipα (Watanabe et al. 2010a, b) proposed as the origin of
signal transduction leading to TNF-α transactivation. The main function of
nucleolin as chromatin-associated protein containing histone chaperone activities
172 S. Wessler
involves the regulation of many different aspects of gene transcription and DNA
metabolism (Storck et al. 2007). On cancer cells, nucleolin was also detected on the
cell surface, where it is implicated in signal transduction events and also in viral and
bacterial infections (Abdelmohsen and Gorospe 2012). Tipα-nucleolin interaction
was described to change the cell morphology and adhesion (Fig. 7.1) which was
interpreted as “epithelial-mesenchymal transition” (EMT) (Watanabe et al. 2014).
Generally, EMT is considered as a morphogenetic reprogramming of epithelial
cells leading to the loss of typical epithelial properties and the increase of a highly
motile, mesenchymal morphology (Lamouille et al. 2014; Nieto 2013). H. pylori-
infected AGS cells strongly develop an EMT-like phenotype as the consequence of
CagA injection and tyrosine phosphorylation (Backert et al. 2001; Moese
et al. 2004). Indeed, the authors observed an upregulation of vimentin in Tip-
α-treated cells representing a protein marker for mesenchymal cells and an increase
in cell adhesion and spreading (Watanabe et al. 2014). Although a Tipα-knockout
H. pylori mutant exists (Godlewska et al. 2008), a large number of publications
describe effects of recombinant Tipα (Table 7.1). In summary, function of Tipα in
H. pylori pathogenesis needs to be validated since an in vivo proof with a Tip-
α-deletion mutant of H. pylori is still missing in most reports.
7.5 JHP0940 Encodes the Bacterial Kinase CtkA
Comparing the genomes of the H. pylori strain Hp26695 with J99 revealed various
strain-specific open reading frames (ORFs). One of these ORFs, JHP940, is located
within a plasticity region of J99 but is absent in Hp26695. Based on the observation
that JHP940 was more frequently expressed in gastric cancer strains, JHP940 was
suggested as a potential pathogenicity marker (Occhialini et al. 2000; Yakoob
et al. 2010). This correlation between genetic jhp0940 variation and H. pylori-
associated disease was further expanded on patients with chronic gastritis and
duodenal and gastric ulceration (Armitano et al. 2013; Yakoob et al. 2010). In
contrast to these studies, jhp0940-positive H. pylori isolates were not significantly
associated with gastric ulcer, duodenal ulcer, or gastric carcinoma in other reports
(Romo-Gonzalez et al. 2009; Santos et al. 2003; Sugimoto et al. 2012). The reasons
for these contradictory studies are not fully clear but might reflect geographic
differences in H. pylori isolates.
Although JHP940 is not expressed in all isolates, it could exert cellular responses
during infection with H. pylori. JHP940 is expressed as a 36 kDa protein, which
stimulated NF-κB-mediated TNF-α and IL-8 secretion in human macrophages
(Rizwan et al. 2008). Functionally, JHP940 acts as an active, autophosphorylating
serine/threonine kinase, which enters, as a recombinant protein, the nucleus of
human cells to induce NF-κB activity and cytokine secretion (Fig. 7.1). Therefore,
JHP940 was renamed cell-translocating kinase A (CtkA) (Kim do et al. 2010).
Recently, CtkA secretion was demonstrated to be involved in the induction of host
Table 7.1 Proposed functions of emerging H. pylori pathogenicity factors

Physiological Cellular
Namea Function role phenotype Methoda References
CtkA Kinase Apoptosis Rec. CtkA Tenguria
et al. (2014)
Inflammation Induction of Rec. CtkA Kim do et al. (2010),
cytokines Rizwan
et al. (2008), and
Tenguria
et al. (2014)
Nuclear Rec. CtkA (Kim do et al. 2010)
localization
GGT Hydrolase Supports col- Supports Knockout Chevalier
onization/ adherence et al. (1999),
persistence McGovern
et al. (2001), and
Oertli et al. (2013)
Inhibits T cells Knockout Beigier-Bompadre
et al. (2011) and
Schmees
et al. (2007)
Rec. GGT Boonyanugomol
et al. (2012) and
Schmees
et al. (2007)
GGT Schmees
inhibitor et al. (2007)
DC tolerization Knockout Oertli et al. (2013)
GGT Oertli et al. (2013)
inhibitor
Epithelial cell Induces Knockout Boonyanugomol
survival apoptosis et al. (2012), and
Kim et al. (2007),
Shibayama
et al. (2003), and
Valenzuela
et al. (2013)
Enriched Shibayama
GGT et al. (2003)
GGT Shibayama
inhibitor et al. (2003)
Rec. GGT Flahou et al. (2011)
and Kim
et al. (2007, 2010)
Inhibits Rec. GGT Rossi et al. (2012)
Prevents proliferation Knockout Engler et al. (2014)
asthma Rec. GGT Engler et al. (2014)
(continued)
174 S. Wessler

HopQ Adhesin CagA Knockout Belogolova
translocation et al. (2013) and
Jimenez-Soto
et al. (2013)
HopZ Adhesin Binding to epi- Knockout Peck et al. (1999)
thelial cells and Yamaoka
et al. (2002)
Hp0169 Protease Supports col- Degrades com- Knockout Kavermann
onization of ponents of the et al. (2003)
H. pylori ECM Rec. Kavermann
Hp0169 et al. (2003)
HtrA Protease Disruption of Cleaves Rec. HtrA Hoy et al. (2010,
adherence E-cadherin 2012)
junctions Inhibitor of Hoy et al. (2010,
HtrA 2012)
Cleaves Rec. HtrA Boehm et al. (2012)
fibronectin and Hoy
et al. (2010)
NapA – Effects on Chemotaxis Rec. NapA Nishioka
neutrophils et al. (2003) and
Satin et al. (2000)
Adhesion Rec. NapA Evans et al. (1995),
Nishioka
et al. (2003), and
Polenghi
et al. (2007)
Blocking Evans et al. (1995)
antibody
Water Evans et al. (1995)
extract
Transendothelial Knockout Brisslert
migration et al. (2005)
Rec. NapA Brisslert
et al. (2005)
Cell cul- Brisslert
ture et al. (2005)
supernatant
Induction of oxi- Rec. NapA Nishioka
dative burst et al. (2003) and
Satin et al. (2000)
Enriched Evans et al. (1995)
NapA and Satin
et al. (2000)
Decrease of oxi- Knockout Petersson
dative burst et al. (2006)
(continued)

Effects on Induction of Rec. NapA Amedei et al. (2006)
monocytes cytokines and Cappon
and DCs et al. (2010)
Knockout Amedei et al. (2006)
Anti-apoptosis Rec. NapA Cappon et al. (2010)
IP NapA Cappon et al. (2010)
TLR2 ligand Rec. NapA Amedei et al. (2006)
Further Prevention of Rec. NapA Amedei et al. (2006)
applications allergic and Codolo
disorders/asthma et al. (2008)
Antitumor Rec. NapA Codolo et al. (2012)
(immune) Rec. Iankov et al. (2012)
response viruses and Ramachandran
et al. (2013)
Enhances vacci- Rec. mea- Iankov et al. (2011,
nation against sles virus 2013)
viruses
Tipα – Involved in Knockout Godlewska
colonization et al. (2008)
Modulation of DNA binding Rec. Tipα Jang et al. (2009)
the immune and Kuzuhara
system et al. (2007b)
Induction of Rec. Tipα Kuzuhara
cytokines and et al. (2007a),
chemokines Suganuma
et al. (2005, 2008),
and Tang
et al. (2013)
Induction of Induction of cell Rec. Tipα Watanabe
“EMT” migration/ et al. (2014)
spreading
a
Abbreviations: CtkA cell-translocating serine/threonine kinase A, EMT epithelial-mesenchymal
transition, GGT γ-glutamyl transpeptidase, Hop outer membrane protein, HtrA high-temperature
requirement A, IP immunoprecipitation, NapA neutrophil-activating protein, rec. recombinant,
Tipα tumor necrosis factor-alpha-inducing protein alpha, TLR2 toll-like receptor 2
cell apoptosis (Fig. 7.1) (Tenguria et al. 2014). Furthermore, it could be shown that
CtkA is also an autophosphorylating tyrosine kinase (Tenguria et al. 2014) with
unknown substrates. Although the implication of bacterial tyrosine kinases in
bacterial physiology became more accepted in the last decade, CtkA is the first
identified tyrosine kinase in H. pylori. Together with its translocating capacity and
its threonine/serine and tyrosine kinase activity, CtkA is an attractive novel factor in
the research of H. pylori pathogenesis.
176 S. Wessler
7.6 Helicobacter pylori Secretes Proteases That Target Host

Cell Proteins with Important Functions in Pathogenesis
Comparable to secreted NapA, GGT, or Tipα, various proteases represent interest-

ing virulence factors if they are cell surface presented or secreted. The first
identified active protease secreted or exposed by H. pylori with a putative function
in H. pylori pathogenesis was Hp0169, which acts as a calcium-dependent colla-
genase that degrades components of the extracellular matrix of host cells
(Kavermann et al. 2003). In vitro, type I collagen was used as a substrate to
demonstrate proteolytic activity of Hp0169 (Fig. 7.1). Together with the observa-
tion that genomic deletion of the hp0169 gene led to a drastic defect in stomach
colonization of mice indicated that Hp0169-targeted ECM components are cru-
cially necessary for colonization (Kavermann et al. 2003).
In fact, H. pylori express a wide range of different (hypothetical) proteases with
unidentified functions (Lower et al. 2008). For instance, H. pylori sheds a protease
that degrades PDGF (platelet-derived growth factor) and TGF-β (transforming
growth factor beta) (Piotrowski et al. 1997; Slomiany et al. 1996). The identity of
this protease is of high interest since this protease might directly influence distinct
signal transduction pathways, which are under direct control of H. pylori. Gener-
ally, pharmacologically interesting proteases have an extracellular localization for
accessible druggability. In a comprehensive study, the whole genome of H. pylori
was analyzed to predict hypothetical proteases exhibiting an extracellular localiza-
tion. Among 14 proteins with hypothesized proteolytic activities (e.g., Hp1012,
Hp0506, etc.), HtrA was identified as a secreted active serine protease (Lower
et al. 2008). HtrA has previously been localized in supernatants of H. pylori
cultures, but activity and biological significant substrates were unknown (Bumann
et al. 2002). In organisms, HtrA proteases are widely expressed, and its functions as
a serine protease and chaperone in bacterial physiology have been well described in
Escherichia coli and some other bacteria (Clausen et al. 2011; Hansen and
Hilgenfeld 2013). In E. coli, three different types of HtrAs are synthesized
(DegP, DegQ, and DegS). The closest homologue to H. pylori HtrA in E. coli is
DegP, which is active as multiple trimers (Clausen et al. 2011). The structure of
HtrA is highly conserved. Its N-terminal signal peptide is necessary for periplasmic
localization. After a stretch of approximately 200 amino acids, there is a serine
protease domain containing a classical catalytic triad composing an asparagine,
histidine, and serine. The protease domain is followed by two PDZ domains which
are important for protein-protein interaction and promote the formation of trimer,
hexamer, dodecamers, etc. (Clausen et al. 2011). The proteolytic activity of
H. pylori HtrA has initially been demonstrated in 2008 by the detection of HtrA
in casein zymography analysis. In this study, HtrA was identified as an active
monomer of ~50 kDa and as a multimer of >170 kDa (Lower et al. 2008). These
data were consistent with an earlier report describing a metalloproteinase-like
protease secreted with a native molecular size of approximately 200 kDa, which
was detected by casein zymography (Windle and Kelleher 1997). Although the
identity of this protease remained unknown in this report, it appears likely that this
is the first description of active H. pylori HtrA. The authors suggested that this
surface-exposed metalloprotease activity might be involved in proteolysis of a
variety of host proteins in vivo and thereby contribute to gastric pathology (Windle
and Kelleher 1997).
If H. pylori HtrA exhibits a role for pathogenesis remained speculative for a long
time. From other pathogens, it has been proposed that HtrA proteases increase the
viability of bacteria under stress conditions (Ingmer and Brondsted 2009). This role
was mainly attributed to the observation that HtrA acts a chaperone in the protein
quality control of microbes and degrades misfolded proteins. Eventually, H. pylori
HtrA increases viability under stress conditions since it tolerated extreme temper-
atures, pH, and ion concentrations (Hoy et al. 2013). However, data are accumu-
lating that there is also a more direct role of HtrA in bacterial pathogenesis.
7.7 HtrA Can Affect H. pylori Pathogenesis via Direct

Cleavage of E-Cadherin and Fibronectin
Besides its function as a chaperone to degrade misfolded proteins in the periplasm,

HtrA can directly interfere with proteins exposed on the surface of host cells.
E-cadherin and fibronectin were identified as the first substrates with biological
functions for H. pylori HtrA (Fig. 7.1) (Hoy et al. 2010). The role of HtrA-mediated
fibronectin cleavage is not well understood. Importantly, H. pylori CagL targets β1-
integrins to inject CagA (Kwok et al. 2007). This interaction occurs via the RGD
motif, a well-characterized integrin-binding motif in fibronectin. Hence, it might be
interesting to investigate if HtrA-mediated fibronectin cleavage facilitated CagL
binding to β1-integrin to promote CagA delivery in host cells.
The molecular and functional mechanism of HtrA-mediated E-cadherin cleav-
age is better understood. The gastric epithelium forms an effective first protection
barrier against intruding bacteria, which requires a highly structured architecture
establishing and maintaining an apical-basolateral domain structure. As important
intercellular structures, gastric epithelial cells express different junctions mediating
cell-to-cell adhesion and intercellular communication. Among them, the functional
integrity of adherence junctions plays an important role. The transmembrane
protein E-cadherin is the key molecule in the establishment and maintenance of
adherence junctions (van Roy 2014). The extracellular E-cadherin domain forms
homophilic interactions between the extracellular E-cadherin domains of two
adjacent cells. The intracellular domain of E-cadherin recruits signaling proteins
into a multiprotein complex. Among them, p120ctn binds to the juxtamembrane,
while another catenin, β-catenin, binds to the intracellular E-cadherin domain and
bridges E-cadherin to the actin cytoskeleton via binding to α-catenin (van Roy
2014). HtrA cleaves off the extracellular E-cadherin domain resulting in the
disruption of adherence junctions allowing H. pylori to enter the intercellular
178 S. Wessler
space of gastric epithelial cells (Fig. 7.1). This mode of transmigration can be
blocked by a specific HtrA inhibitor that prevents E-cadherin shedding (Hoy
et al. 2010). HtrA-triggered E-cadherin cleavage and consequent disruption of
adherence junctions are not unique for H. pylori. A similar mechanism has been
observed for Campylobacter jejuni and has been suggested for other Gram-negative
pathogens of the gastrointestinal tracts indicating that the interference of HtrA with
host cells functions is rather a prevalent mechanism in bacterial pathogenesis
(Boehm et al. 2012; Hoy et al. 2012).
In contrast to other pathogens, functional investigation of HtrA in H. pylori
infections is rather challenging because it was not possible to create a genomic
deletion or mutations in the htrA gene of H. pylori (Hoy et al. 2010; Salama
et al. 2004). Similar to the reports on Tipα or NapA, most data were obtained
from studies using recombinant HtrA protein (Table 7.1). Therefore, small mole-
cule inhibitors have been designed via structure-based virtual screening based on a
comprehensive prediction of ligand-binding sites on a computational protein model
(Hoy et al. 2010; Lower et al. 2011). A preliminary X-ray structure of H. pylori
HtrA confirmed the computation model (Perna et al. 2014). In vitro and in cell
culture studies, pharmacological inhibition of HtrA activity using defined lead
structures decreased E-cadherin ectodomain shedding and bacterial transmigration
through the intercellular space (Hoy et al. 2010, 2012).
The functional integrity of adherence junctions attracted much attention to the
research community since it has been discovered that p120ctn and β-catenin have an
additional role in tumor development (van Roy 2014). Besides contributing to the
integrity and stability of the E-cadherin-mediated AJ, β-catenin and p120ctn can be
released form E-cadherin and can translocate into the nucleus where they act as
important cofactors for TCF/Lef transcription factors of cancer-related target genes.
Therefore, functional E-cadherin-based AJs are also considered as important tumor
suppressors (van Roy 2014). Hence, it might be interesting if HtrA-mediated
E-cadherin cleavage might also promote H. pylori-dependent gastric carcinogene-
sis through the disruption of intercellular adhesions and the induction of E-
cadherin-originated signal transduction pathways.
7.8 The Helicobacter Outer Membrane Proteins HopQ

and HopZ Contribute to Bacterial Adherence
H. pylori express a large family of hop genes encoding outer membrane proteins
(Alm et al. 2000). Among this large family, HopZ and HopQ acquired increased
attention as reports are accumulating indicating that these factors play important
roles in H. pylori-host interactions.
HopZ is encoded by two alleles, which were differentially distributed in
H. pylori strains. The intact HopZ protein functions as an adhesion in vitro and
in vivo (Peck et al. 1999; Yamaoka et al. 2002) but had no influence on CagA
translocation (Odenbreit et al. 2002). The on/off expression status of the phase-
variable hopZ gene could not consistently be associated with H. pylori diseases
(de Jonge et al. 2004a, b; Lehours et al. 2004). These data might indicate that HopZ
function is redundant and can be compensated by other adhesins.
Corresponding to HopZ, HopQ is also expressed from two hopQ alleles that
share 75–80 % identical nucleotide sequences and could be classified as type I and
type II hopQ alleles according to their relatedness in different H. pylori strains (Cao
and Cover 2002). Type I hopQ alleles were stronger associated with cag+/type
s1-vacA strains from patients with peptic ulcer disease (Cao and Cover 2002).
While type I hopQ alleles were found in Western and Asian H. pylori strains,
type II hopQ alleles were mainly identified in Western H. pylori (Cao et al. 2005).
HopQ is localized on the surface of H. pylori (Sabarth et al. 2005) implying that
it could represent a functional adhesin. H. pylori hopQ mutants showed different
phenotypes in adherence dependent on the H. pylori strain. In Hp26695 and J178,
hopQ mutants were hyperadherent, but no alteration were observed in J99 and
87-29. Correspondingly, CagA injection and tyrosine phosphorylation were
enhanced in AGS cells infected with hyperadherent hopQ mutants, but IL-8 secre-
tion was not affected (Loh et al. 2008). In coinfection experiments of two different
H. pylori isolates, HopQ expression in one competing strain was involved in the
restriction of CagA translocation by the other strain. Although CagA translocation
of the hopQ-negative H. pylori was not investigated in this study, these data suggest
that HopQ expression may play a role in CagA translocation by a yet unknown
mechanism (Jimenez-Soto et al. 2013). However, HopQ was recently identified as
an important factor promoting CagA translocation in a study employing H. pylori
hopQ knockout and complemented mutants (Fig. 7.1). Here, deletion of hopQ also
decreased NF-κB and MAPK activity and exhibited negative effects on IL-8
secretion. The hopQ mutant did not show a defect in bacterial adhesion to host
cells (Belogolova et al. 2013). In summary, HopQ is an interesting novel candidate
with signal transduction-inducing capacities, which needs to be investigated in
more detail.
7.9 DupA as a Marker for H. pylori-Associated Disorders
The first description of dupA as a duodenal ulcer-promoting gene revealed that it

was the only gene in 500 H. pylori strains from East Asia and South America that
was significantly associated with the induction of duodenal ulceration with neutro-
phil infiltration, while DupA exhibited protective effects on atrophy, intestinal
metaplasia, and gastric cancer (Lu et al. 2005; Zhang et al. 2008). Many different
reports followed and described an association between the presence of dupA and
H. pylori-dependent diseases in different geographic populations. The correlation
between DupA and duodenal ulcer was not confirmed in other reports (Argent
et al. 2007; Douraghi et al. 2008; Gomes et al. 2008). These inconsistent data could
be explained by the finding that dupA genes are polymorphic and most H. pylori
180 S. Wessler
strains contain a longer dupA allele (Hussein et al. 2010) suggesting that dupA has
two genotypes, which are responsible for the conflicting data. Underlining this
consideration, H. pylori containing the long dupA variant induced a significant
higher IL-12p40 level in PBMCs but not in epithelial cells (Hussein et al. 2010).
This could also point to a cell-type-specific response. Aforementioned studies
mainly base on the comparison of dupA-positive versus dupA-negative H. pylori
strains but not on the genes sequence. A more extensive analysis indicated that not
dupA alone but a dupA gene cluster determines the risk of developing duodenal
ulcers (Jung et al. 2012). Since DupA shares high homologies to the VirB4 ATPase,
it was speculated that DupA participates in T4SS-dependent processes. The exis-
tence of additional vir genes around the dupA gene could indicate an addition T4SS
system resembling the cagPAI and the ComB system. However, as long as neither
DupA protein expression nor a biochemical function in H. pylori could be demon-
strated experimentally, it remains a theoretical debate.
The interaction of VacA and the T4SS-delivered CagA with host cells has been
intensively investigated and, hence, is relatively well understood how they manip-
ulate host cell signaling contributing to H. pylori-associated disorders. Investigation
of GGT, NapA, CtkA, HopQ, HopZ, Tipα, or HtrA in H. pylori pathogenesis is a
comparatively young field leading to the accumulation of novel interesting data,
which might add important pieces into puzzle of the multiple mechanisms of
H. pylori factors interfering with host cell functions (Table 7.1). More efforts are
necessary to identify receptors or target molecules in host cells to increase our
understanding of the complex network of H. pylori-host interactions. However,
most of these factors share their extracellular localization making them attractive
candidates for pharmacological inhibition or vaccination strategies.
References
Abdelmohsen K, Gorospe M (2012) RNA-binding protein nucleolin in disease. RNA Biol 9

(6):799–808. doi:10.4161/rna.19718
Helicobacter pylori: analysis of the outer membrane protein families. Infect Immun 68
(7):4155–4168
Amedei A, Cappon A, Codolo G, Cabrelle A, Polenghi A, Benagiano M, Tasca E, Azzurri A,
D’Elios MM, Del Prete G, de Bernard M (2006) The neutrophil-activating protein of
Helicobacter pylori promotes Th1 immune responses. J Clin Invest 116(4):1092–1101.
doi:10.1172/jci27177
Argent RH, Burette A, Miendje Deyi VY, Atherton JC (2007) The presence of dupA in
Helicobacter pylori is not significantly associated with duodenal ulceration in Belgium,
South Africa, China, or North America. Clin Infect Dis: Off Publ Infect Dis Soc Am 45
(9):1204–1206. doi:10.1086/522177
Armitano RI, Matteo MJ, Goldman C, Wonaga A, Viola LA, De Palma GZ, Catalano M (2013)
Helicobacter pylori heterogeneity in patients with gastritis and peptic ulcer disease. Infect
Genet Evol J Mol Epidemiol Evol Genet Infect Dis 16:377–385. doi:10.1016/j.meegid.2013.
02.024
Backert S, Clyne M (2011) Pathogenesis of Helicobacter pylori infection. Helicobacter 16(Suppl
1):19–25. doi:10.1111/j.1523-5378.2011.00876.x
Backert S, Moese S, Selbach M, Brinkmann V, Meyer TF (2001) Phosphorylation of tyrosine
972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype
in gastric epithelial cells. Mol Microbiol 42(3):631–644
Backert S, Kwok T, Schmid M, Selbach M, Moese S, Peek RM Jr, Konig W, Meyer TF, Jungblut
PR (2005) Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed
by two-dimensional gel electrophoresis and mass spectrometry. Proteomics 5(5):1331–1345.
doi:10.1002/pmic.200401019
Backert S, Tegtmeyer N, Selbach M (2010) The versatility of Helicobacter pylori CagA effector
protein functions: the master key hypothesis. Helicobacter 15(3):163–176. doi:10.1111/j.1523-
5378.2010.00759.x
Bauer J, Namineni S, Reisinger F, Zoller J, Yuan D, Heikenwalder M (2012) Lymphotoxin,
NF-kB, and cancer: the dark side of cytokines. Dig Dis (Basel, Switzerland) 30(5):453–468.
doi:10.1159/000341690
Beigier-Bompadre M, Moos V, Belogolova E, Allers K, Schneider T, Churin Y, Ignatius R, Meyer
TF, Aebischer T (2011) Modulation of the CD4+ T-cell response by Helicobacter pylori
depends on known virulence factors and bacterial cholesterol and cholesterol alpha-glucoside
content. J Infect Dis 204(9):1339–1348. doi:10.1093/infdis/jir547
Belogolova E, Bauer B, Pompaiah M, Asakura H, Brinkman V, Ertl C, Bartfeld S, Nechitaylo TY,
Haas R, Machuy N, Salama N, Churin Y, Meyer TF (2013) Helicobacter pylori outer
membrane protein HopQ identified as a novel T4SS-associated virulence factor. Cell Microbiol
15(11):1896–1912. doi:10.1111/cmi.12158
Boehm M, Hoy B, Rohde M, Tegtmeyer N, Baek KT, Oyarzabal OA, Brondsted L, Wessler S,
Backert S (2012) Rapid paracellular transmigration of Campylobacter jejuni across polarized
epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but
not fibronectin. Gut Pathog 4(1):3. doi:10.1186/1757-4749-4-3
Boonyanugomol W, Chomvarin C, Song JY, Kim KM, Kim JM, Cho MJ, Lee WK, Kang HL,
Rhee KH, Sripa B, Hahnvajanawong C, Baik SC (2012) Effects of Helicobacter pylori gamma-
glutamyltranspeptidase on apoptosis and inflammation in human biliary cells. Dig Dis Sci 57
(10):2615–2624. doi:10.1007/s10620-012-2216-2
Brisslert M, Enarsson K, Lundin S, Karlsson A, Kusters JG, Svennerholm AM, Backert S,
Quiding-Jarbrink M (2005) Helicobacter pylori induce neutrophil transendothelial migration:
role of the bacterial HP-NAP. FEMS Microbiol Lett 249(1):95–103. doi:10.1016/j.femsle.
2005.06.008
Infect Immun 70(7):3396–3403
Busiello I, Acquaviva R, Di Popolo A, Blanchard TG, Ricci V, Romano M, Zarrilli R (2004)
Helicobacter pylori gamma-glutamyltranspeptidase upregulates COX-2 and EGF-related pep-
tide expression in human gastric cells. Cell Microbiol 6(3):255–267
Cao P, Cover TL (2002) Two different families of hopQ alleles in Helicobacter pylori. J Clin
Microbiol 40(12):4504–4511
Cao P, Lee KJ, Blaser MJ, Cover TL (2005) Analysis of hopQ alleles in East Asian and Western
strains of Helicobacter pylori. FEMS Microbiol Lett 251(1):37–43. doi:10.1016/j.femsle.2005.
07.023
182 S. Wessler
Cappon A, Babolin C, Segat D, Cancian L, Amedei A, Calzetti F, Cassatella MA, D’Elios MM, de
Bernard M (2010) Helicobacter pylori-derived neutrophil-activating protein increases the
lifespan of monocytes and neutrophils. Cell Microbiol 12(6):754–764. doi:10.1111/j.1462-
5822.2010.01431.x
gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice. Mol
Microbiol 31(5):1359–1372
Clausen T, Kaiser M, Huber R, Ehrmann M (2011) HTRA proteases: regulated proteolysis in
protein quality control. Nature reviews. Mol Cell Biol 12(3):152–162. doi:10.1038/nrm3065
Codolo G, Mazzi P, Amedei A, Del Prete G, Berton G, D’Elios MM, de Bernard M (2008) The
neutrophil-activating protein of Helicobacter pylori down-modulates Th2 inflammation in
ovalbumin-induced allergic asthma. Cell Microbiol 10(11):2355–2363. doi:10.1111/j.1462-
5822.2008.01217.x
Codolo G, Fassan M, Munari F, Volpe A, Bassi P, Rugge M, Pagano F, D’Elios MM, de Bernard
M (2012) HP-NAP inhibits the growth of bladder cancer in mice by activating a cytotoxic Th1
response. Cancer Immunol Immunother CII 61(1):31–40. doi:10.1007/s00262-011-1087-2
de Jonge R, Durrani Z, Rijpkema SG, Kuipers EJ, van Vliet AH, Kusters JG (2004a) Role of the
Helicobacter pylori outer-membrane proteins AlpA and AlpB in colonization of the guinea pig
stomach. J Med Microbiol 53(Pt 5):375–379
de Jonge R, Pot RG, Loffeld RJ, van Vliet AH, Kuipers EJ, Kusters JG (2004b) The functional
status of the Helicobacter pylori sabB adhesin gene as a putative marker for disease outcome.
Helicobacter 9(2):158–164. doi:10.1111/j.1083-4389.2004.00213.x
Del Prete G, Chiumiento L, Amedei A, Piazza M, D’Elios MM, Codolo G, de Bernard M,
Masetti M, Bruschi F (2008) Immunosuppression of TH2 responses in Trichinella spiralis
infection by Helicobacter pylori neutrophil-activating protein. J Allergy Clin Immunol 122
(5):908–913.e905. doi:10.1016/j.jaci.2008.08.016
Douraghi M, Mohammadi M, Oghalaie A, Abdirad A, Mohagheghi MA, Hosseini ME, Zeraati H,
Ghasemi A, Esmaieli M, Mohajerani N (2008) dupA as a risk determinant in Helicobacter
pylori infection. J Med Microbiol 57(Pt 5):554–562. doi:10.1099/jmm.0.47776-0
Engler DB, Reuter S, van Wijck Y, Urban S, Kyburz A, Maxeiner J, Martin H, Yogev N,
Waisman A, Gerhard M, Cover TL, Taube C, Muller A (2014) Effective treatment of allergic
airway inflammation with Helicobacter pylori immunomodulators requires BATF3-dependent
dendritic cells and IL-10. Proc Natl Acad Sci U S A 111(32):11810–11815. doi:10.1073/pnas.
1410579111
Evans DJ Jr, Evans DG, Takemura T, Nakano H, Lampert HC, Graham DY, Granger DN, Kvietys
PR (1995) Characterization of a Helicobacter pylori neutrophil-activating protein. Infect
Immun 63(6):2213–2220
Fassi Fehri L, Koch M, Belogolova E, Khalil H, Bolz C, Kalali B, Mollenkopf HJ, Beigier-
Bompadre M, Karlas A, Schneider T, Churin Y, Gerhard M, Meyer TF (2010) Helicobacter
pylori induces miR-155 in T cells in a cAMP-Foxp3-dependent manner. PLoS One 5(3):e9500.
doi:10.1371/journal.pone.0009500
cells and induction of interleukin-8. Mol Microbiol 42(5):1337–1348
Flahou B, Haesebrouck F, Chiers K, Van Deun K, De Smet L, Devreese B, Vandenberghe I,
Favoreel H, Smet A, Pasmans F, D’Herde K, Ducatelle R (2011) Gastric epithelial cell death
caused by Helicobacter suis and Helicobacter pylori gamma-glutamyl transpeptidase is mainly
glutathione degradation-dependent. Cell Microbiol 13(12):1933–1955. doi:10.1111/j.1462-
5822.2011.01682.x
cytotoxin inhibits T lymphocyte activation. Science (New York, NY) 301(5636):1099–1102.
doi:10.1126/science.1086871
Godlewska R, Pawlowski M, Dzwonek A, Mikula M, Ostrowski J, Drela N, Jagusztyn-Krynicka

EK (2008) Tip-alpha (hp0596 gene product) is a highly immunogenic Helicobacter pylori
protein involved in colonization of mouse gastric mucosa. Curr Microbiol 56(3):279–286.
doi:10.1007/s00284-007-9083-7
Gomes LI, Rocha GA, Rocha AM, Soares TF, Oliveira CA, Bittencourt PF, Queiroz DM (2008)
Lack of association between Helicobacter pylori infection with dupA-positive strains and
gastroduodenal diseases in Brazilian patients. Int J Med Microbio: IJMM 298(3–4):223–230.
doi:10.1016/j.ijmm.2007.05.006
Gong M, Ho B (2004) Prominent role of gamma-glutamyl-transpeptidase on the growth of
Helicobacter pylori. World J Gastroenterol WJG 10(20):2994–2996
Gong M, Ling SS, Lui SY, Yeoh KG, Ho B (2010) Helicobacter pylori gamma-glutamyl
transpeptidase is a pathogenic factor in the development of peptic ulcer disease. Gastroenter-
ology 139(2):564–573. doi:10.1053/j.gastro.2010.03.050
Hansen G, Hilgenfeld R (2013) Architecture and regulation of HtrA-family proteins involved in
protein quality control and stress response. Cell Mol Life Sci 70(5):761–775. doi:10.1007/
s00018-012-1076-4
Hoy B, Lower M, Weydig C, Carra G, Tegtmeyer N, Geppert T, Schroder P, Sewald N, Backert S,
Schneider G, Wessler S (2010) Helicobacter pylori HtrA is a new secreted virulence factor that
cleaves E-cadherin to disrupt intercellular adhesion. EMBO Rep 11(10):798–804. doi:10.1038/
embor.2010.114
Hoy B, Geppert T, Boehm M, Reisen F, Plattner P, Gadermaier G, Sewald N, Ferreira F, Briza P,
Schneider G, Backert S, Wessler S (2012) Distinct roles of secreted HtrA proteases from gram-
negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin. J Biol
Chem 287(13):10115–10120. doi:10.1074/jbc.C111.333419
Hoy B, Brandstetter H, Wessler S (2013) The stability and activity of recombinant Helicobacter
pylori HtrA under stress conditions. J Basic Microbiol 53(5):402–409. doi:10.1002/jobm.
201200074
Hussein NR, Argent RH, Marx CK, Patel SR, Robinson K, Atherton JC (2010) Helicobacter pylori
dupA is polymorphic, and its active form induces proinflammatory cytokine secretion by
mononuclear cells. J Infect Dis 202(2):261–269. doi:10.1086/653587
Iankov ID, Haralambieva IH, Galanis E (2011) Immunogenicity of attenuated measles virus
engineered to express Helicobacter pylori neutrophil-activating protein. Vaccine 29
(8):1710–1720. doi:10.1016/j.vaccine.2010.12.020
Iankov ID, Allen C, Federspiel MJ, Myers RM, Peng KW, Ingle JN, Russell SJ, Galanis E (2012)
Expression of immunomodulatory neutrophil-activating protein of Helicobacter pylori
enhances the antitumor activity of oncolytic measles virus. Mol Ther J Am Soc Gene Ther
20(6):1139–1147. doi:10.1038/mt.2012.4
Iankov ID, Federspiel MJ, Galanis E (2013) Measles virus expressed Helicobacter pylori
neutrophil-activating protein significantly enhances the immunogenicity of poor immunogens.
Vaccine 31(42):4795–4801. doi:10.1016/j.vaccine.2013.07.085
Ingmer H, Brondsted L (2009) Proteases in bacterial pathogenesis. Res Microbiol 160(9):704–710.
doi:10.1016/j.resmic.2009.08.017
Inoue K, Shiota S, Yamada K, Gotoh K, Suganuma M, Fujioka T, Ahmed K, Iha H, Nishizono A
(2009) Evaluation of a new tumor necrosis factor-alpha-inducing membrane protein of
Helicobacter pylori as a prophylactic vaccine antigen. Helicobacter 14(5):135–143. doi:10.
1111/j.1523-5378.2009.00713.x
Jang JY, Yoon HJ, Yoon JY, Kim HS, Lee SJ, Kim KH, Kim do J, Jang S, Han BG, Lee BI, Suh
SW (2009) Crystal structure of the TNF-alpha-inducing protein (tipalpha) from Helicobacter
pylori: insights into its DNA-binding activity. J Mol Biol 392(1):191–197. doi:10.1016/j.jmb.
2009.07.010
Jimenez-Soto LF, Clausen S, Sprenger A, Ertl C, Haas R (2013) Dynamics of the Cag-type IV
secretion system of Helicobacter pylori as studied by bacterial co-infections. Cell Microbiol 15
(11):1924–1937. doi:10.1111/cmi.12166
184 S. Wessler
Jung SW, Sugimoto M, Shiota S, Graham DY, Yamaoka Y (2012) The intact dupA cluster is a
more reliable Helicobacter pylori virulence marker than dupA alone. Infect Immun 80
(1):381–387. doi:10.1128/iai.05472-11
Kavermann H, Burns BP, Angermuller K, Odenbreit S, Fischer W, Melchers K, Haas R (2003)
Identification and characterization of Helicobacter pylori genes essential for gastric coloniza-
tion. J Exp Med 197(7):813–822. doi:10.1084/jem.20021531
Kim do J, Park KS, Kim JH, Yang SH, Yoon JY, Han BG, Kim HS, Lee SJ, Jang JY, Kim KH, Kim
MJ, Song JS, Kim HJ, Park CM, Lee SK, Lee BI, Suh SW (2010) Helicobacter pylori
proinflammatory protein up-regulates NF-kappaB as a cell-translocating Ser/Thr kinase. Proc
Natl Acad Sci U S A 107(50):21418–21423. doi:10.1073/pnas.1010153107
Kim KM, Lee SG, Park MG, Song JY, Kang HL, Lee WK, Cho MJ, Rhee KH, Youn HS, Baik SC
(2007) Gamma-glutamyltranspeptidase of Helicobacter pylori induces mitochondria-mediated
apoptosis in AGS cells. Biochem Biophys Res Commun 355(2):562–567. doi:10.1016/j.bbrc.
2007.02.021
Kim KM, Lee SG, Kim JM, Kim DS, Song JY, Kang HL, Lee WK, Cho MJ, Rhee KH, Youn HS,
Baik SC (2010) Helicobacter pylori gamma-glutamyltranspeptidase induces cell cycle arrest at
the G1-S phase transition. J Microbiol (Seoul, Korea) 48(3):372–377. doi:10.1007/s12275-
010-9293-8
Kottakis F, Papadopoulos G, Pappa EV, Cordopatis P, Pentas S, Choli-Papadopoulou T (2008)
Helicobacter pylori neutrophil-activating protein activates neutrophils by its C-terminal region
even without dodecamer formation, which is a prerequisite for DNA protection – novel
approaches against Helicobacter pylori inflammation. FEBS J 275(2):302–317. doi:10.1111/
j.1742-4658.2007.06201.x
Kuzuhara T, Suganuma M, Tsuge H, Fujiki H (2005) Presence of a motif conserved between
Helicobacter pylori TNF-alpha inducing protein (Tipalpha) and penicillin-binding proteins.
Biol Pharm Bull 28(11):2133–2137
Kuzuhara T, Suganuma M, Kurusu M, Fujiki H (2007a) Helicobacter pylori-secreting protein
Tipalpha is a potent inducer of chemokine gene expressions in stomach cancer cells. J Cancer
Res Clin Oncol 133(5):287–296. doi:10.1007/s00432-006-0169-6
Kuzuhara T, Suganuma M, Oka K, Fujiki H (2007b) DNA-binding activity of TNF-alpha inducing
protein from Helicobacter pylori. Biochem Biophys Res Commun 362(4):805–810. doi:10.
1016/j.bbrc.2007.08.058
activation. Nature 449(7164):862–866. doi:10.1038/nature06187
Lamouille S, Xu J, Derynck R (2014) Molecular mechanisms of epithelial-mesenchymal transi-
tion. Nature reviews. Mol Cell Biol 15(3):178–196. doi:10.1038/nrm3758
Lehours P, Menard A, Dupouy S, Bergey B, Richy F, Zerbib F, Ruskone-Fourmestraux A,
Delchier JC, Megraud F (2004) Evaluation of the association of nine Helicobacter pylori
virulence factors with strains involved in low-grade gastric mucosa-associated lymphoid tissue
lymphoma. Infect Immun 72(2):880–888
Loh JT, Torres VJ, Algood HM, McClain MS, Cover TL (2008) Helicobacter pylori HopQ outer
membrane protein attenuates bacterial adherence to gastric epithelial cells. FEMS Microbiol
Lett 289(1):53–58
Lower M, Weydig C, Metzler D, Reuter A, Starzinski-Powitz A, Wessler S, Schneider G (2008)
Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals
proteolytic activity of the Hp1018/19 protein HtrA. PLoS One 3(10):e3510. doi:10.1371/
journal.pone.0003510
Lower M, Geppert T, Schneider P, Hoy B, Wessler S, Schneider G (2011) Inhibitors of
Helicobacter pylori protease HtrA found by ‘virtual ligand’ screening combat bacterial inva-
sion of epithelia. PLoS One 6(3):e17986. doi:10.1371/journal.pone.0017986
Lu H, Hsu PI, Graham DY, Yamaoka Y (2005) Duodenal ulcer promoting gene of Helicobacter
pylori. Gastroenterology 128(4):833–848
McGovern KJ, Blanchard TG, Gutierrez JA, Czinn SJ, Krakowka S, Youngman P (2001) gamma-
Glutamyltransferase is a Helicobacter pylori virulence factor but is not essential for coloniza-
tion. Infect Immun 69(6):4168–4173. doi:10.1128/iai.69.6.4168-4173.2001
Moese S, Selbach M, Kwok T, Brinkmann V, Konig W, Meyer TF, Backert S (2004) Helicobacter
pylori induces AGS cell motility and elongation via independent signaling pathways. Infect
Immun 72(6):3646–3649. doi:10.1128/iai.72.6.3646-3649.2004
Montemurro P, Barbuti G, Dundon WG, Del Giudice G, Rappuoli R, Colucci M, De Rinaldis P,
Montecucco C, Semeraro N, Papini E (2001) Helicobacter pylori neutrophil-activating protein
stimulates tissue factor and plasminogen activator inhibitor-2 production by human blood
mononuclear cells. J Infect Dis 183(7):1055–1062. doi:10.1086/319280
Montemurro P, Nishioka H, Dundon WG, de Bernard M, Del Giudice G, Rappuoli R, Montecucco
C (2002) The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a potent
stimulant of mast cells. Eur J Immunol 32(3):671–676. doi:10.1002/1521-4141(200203)
32:3<671::aid-immu671>3.0.co;2-5
Namavar F, Sparrius M, Veerman EC, Appelmelk BJ, Vandenbroucke-Grauls CM (1998)
Neutrophil-activating protein mediates adhesion of Helicobacter pylori to sulfated carbohy-
drates on high-molecular-weight salivary mucin. Infect Immun 66(2):444–447
Nieto MA (2013) Epithelial plasticity: a common theme in embryonic and cancer cells. Science
(New York, NY) 342(6159):1234850. doi:10.1126/science.1234850
Nishioka H, Baesso I, Semenzato G, Trentin L, Rappuoli R, Del Giudice G, Montecucco C (2003)
The neutrophil-activating protein of Helicobacter pylori (HP-NAP) activates the MAPK
pathway in human neutrophils. Eur J Immunol 33(4):840–849. doi:10.1002/eji.200323726
Occhialini A, Marais A, Alm R, Garcia F, Sierra R, Megraud F (2000) Distribution of open reading
frames of plasticity region of strain J99 in Helicobacter pylori strains isolated from gastric
carcinoma and gastritis patients in Costa Rica. Infect Immun 68(11):6240–6249
Odenbreit S, Kavermann H, Puls J, Haas R (2002) CagA tyrosine phosphorylation and interleukin-
8 induction by Helicobacter pylori are independent from alpAB, HopZ and bab group outer
membrane proteins. Int J Med Microbiol IJMM 292(3–4):257–266. doi:10.1078/1438-4221-
00205
mote gastric persistence and immune tolerance. Proc Natl Acad Sci U S A 110(8):3047–3052.
doi:10.1073/pnas.1211248110
Peck B, Ortkamp M, Diehl KD, Hundt E, Knapp B (1999) Conservation, localization and
expression of HopZ, a protein involved in adhesion of Helicobacter pylori. Nucleic Acids
Res 27(16):3325–3333
Perna AM, Reisen F, Schmidt TP, Geppert T, Pillong M, Weisel M, Hoy B, Simister PC, Feller
SM, Wessler S, Schneider G (2014) Inhibiting Helicobacter pylori HtrA protease by
addressing a computationally predicted allosteric ligand binding site. Chem Sci 5
(9):3583–3590. doi:10.1039/C4SC01443J
Petersson C, Forsberg M, Aspholm M, Olfat FO, Forslund T, Boren T, Magnusson KE (2006)
Helicobacter pylori SabA adhesin evokes a strong inflammatory response in human neutro-
phils which is down-regulated by the neutrophil-activating protein. Med Microbiol Immunol
195(4):195–206. doi:10.1007/s00430-006-0018-x
Piotrowski J, Slomiany A, Slomiany BL (1997) Suppression of Helicobacter pylori protease
activity towards growth factors by sulglycotide. J Physiol Pharmacol Off J Pol Physiol Soc
48(3):345–351
Polenghi A, Bossi F, Fischetti F, Durigutto P, Cabrelle A, Tamassia N, Cassatella MA,
Montecucco C, Tedesco F, de Bernard M (1950) The neutrophil-activating protein of
Helicobacter pylori crosses endothelia to promote neutrophil adhesion in vivo. J Immunol
(Baltimore, MD) 178(3):1312–1320
186 S. Wessler
Posselt G, Backert S, Wessler S (2013) The functional interplay of Helicobacter pylori factors
with gastric epithelial cells induces a multi-step process in pathogenesis. Cell Commun Signal
CCS 11:77. doi:10.1186/1478-811x-11-77
Ramachandran M, Yu D, Wanders A, Essand M, Eriksson F (2013) An infection-enhanced
oncolytic adenovirus secreting H. pylori neutrophil-activating protein with therapeutic effects
on neuroendocrine tumors. Mol Ther J Am Soc Gene Ther 21(11):2008–2018. doi:10.1038/mt.
2013.153
Rizwan M, Alvi A, Ahmed N (2008) Novel protein antigen (JHP940) from the genomic plasticity
region of Helicobacter pylori induces tumor necrosis factor alpha and interleukin-8 secretion
by human macrophages. J Bacteriol 190(3):1146–1151. doi:10.1128/jb.01309-07
Romo-Gonzalez C, Salama NR, Burgeno-Ferreira J, Ponce-Castaneda V, Lazcano-Ponce E,
Camorlinga-Ponce M, Torres J (2009) Differences in genome content among Helicobacter
pylori isolates from patients with gastritis, duodenal ulcer, or gastric cancer reveal novel
disease-associated genes. Infect Immun 77(5):2201–2211. doi:10.1128/iai.01284-08
Rossi M, Bolz C, Revez J, Javed S, El-Najjar N, Anderl F, Hyytiainen H, Vuorela P, Gerhard M,
Hanninen ML (2012) Evidence for conserved function of gamma-glutamyltranspeptidase in
Helicobacter genus. PLoS One 7(2):e30543. doi:10.1371/journal.pone.0030543
Sabarth N, Hurvitz R, Schmidt M, Zimny-Arndt U, Jungblut PR, Meyer TF, Bumann D (2005)
Identification of Helicobacter pylori surface proteins by selective proteinase K digestion and
antibody phage display. J Microbiol Methods 62(3):345–349. doi:10.1016/j.mimet.2005.04.
030
Salama NR, Shepherd B, Falkow S (2004) Global transposon mutagenesis and essential gene
analysis of Helicobacter pylori. J Bacteriol 186(23):7926–7935. doi:10.1128/jb.186.23.7926-
7935.2004
Santos A, Queiroz DM, Menard A, Marais A, Rocha GA, Oliveira CA, Nogueira AM, Uzeda M,
Megraud F (2003) New pathogenicity marker found in the plasticity region of the Helicobacter
pylori genome. J Clin Microbiol 41(4):1651–1655
Satin B, Del Giudice G, Della Bianca V, Dusi S, Laudanna C, Tonello F, Kelleher D, Rappuoli R,
Montecucco C, Rossi F (2000) The neutrophil-activating protein (HP-NAP) of Helicobacter
pylori is a protective antigen and a major virulence factor. J Exp Med 191(9):1467–1476
Gerhard M (2007) Inhibition of T-cell proliferation by Helicobacter pylori gamma-glutamyl
transpeptidase. Gastroenterology 132(5):1820–1833. doi:10.1053/j.gastro.2007.02.031
Shibayama K, Kamachi K, Nagata N, Yagi T, Nada T, Doi Y, Shibata N, Yokoyama K, Yamane K,
Kato H, Iinuma Y, Arakawa Y (2003) A novel apoptosis-inducing protein from Helicobacter
pylori. Mol Microbiol 47(2):443–451
Slomiany BL, Piotrowski J, Slomiany A (1996) Susceptibility of growth factors to degradation by
Helicobacter pylori protease: effect of ebrotidine and sucralfate. Biochem Mol Biol Int 40
(1):209–215
Storck S, Shukla M, Dimitrov S, Bouvet P (2007) Functions of the histone chaperone nucleolin in
diseases. Subcell Biochem 41:125–144
Suganuma M, Kurusu M, Suzuki K, Nishizono A, Murakami K, Fujioka T, Fujiki H (2005) New
tumor necrosis factor-alpha-inducing protein released from Helicobacter pylori for gastric
cancer progression. J Cancer Res Clin Oncol 131(5):305–313. doi:10.1007/s00432-004-0652-x
Suganuma M, Yamaguchi K, Ono Y, Matsumoto H, Hayashi T, Ogawa T, Imai K, Kuzuhara T,
Nishizono A, Fujiki H (2008) TNF-alpha-inducing protein, a carcinogenic factor secreted from
H. pylori, enters gastric cancer cells. Int J Cancer 123(1):117–122. doi:10.1002/ijc.23484
Sugimoto M, Watada M, Jung SW, Graham DY, Yamaoka Y (2012) Role of Helicobacter pylori
plasticity region genes in development of gastroduodenal diseases. J Clin Microbiol 50
(2):441–448. doi:10.1128/jcm.00906-11
Tang CL, Hao B, Zhang GX, Shi RH, Cheng WF (2013) Helicobacter pylori tumor necrosis
factor-alpha inducing protein promotes cytokine expression via nuclear factor-kappaB. World
J Gastroenterol WJG 19(3):399–403. doi:10.3748/wjg.v19.i3.399
Teneberg S, Miller-Podraza H, Lampert HC, Evans DJ Jr, Evans DG, Danielsson D, Karlsson KA
(1997) Carbohydrate binding specificity of the neutrophil-activating protein of Helicobacter
pylori. J Biol Chem 272(30):19067–19071
Tenguria S, Ansari SA, Khan N, Ranjan A, Devi S, Tegtmeyer N, Lind J, Backert S, Ahmed N
(2014) Helicobacter pylori cell translocating kinase (CtkA/JHP0940) is pro-apoptotic in
mouse macrophages and acts as auto-phosphorylating tyrosine kinase. Int J Med Microbiol
304(8):1066–1076. doi:10.1016/j.ijmm.2014.07.017
Tonello F, Dundon WG, Satin B, Molinari M, Tognon G, Grandi G, Del Giudice G, Rappuoli R,
Montecucco C (1999) The Helicobacter pylori neutrophil-activating protein is an iron-binding
protein with dodecameric structure. Mol Microbiol 34(2):238–246
Tosi T, Cioci G, Jouravleva K, Dian C, Terradot L (2009) Structures of the tumor necrosis factor
alpha inducing protein Tipalpha: a novel virulence factor from Helicobacter pylori. FEBS Lett
583(10):1581–1585. doi:10.1016/j.febslet.2009.04.033
Tsuge H, Tsurumura T, Utsunomiya H, Kise D, Kuzuhara T, Watanabe T, Fujiki H, Suganuma M
(2009) Structural basis for the Helicobacter pylori-carcinogenic TNF-alpha-inducing protein.
Biochem Biophys Res Commun 388(2):193–198. doi:10.1016/j.bbrc.2009.07.121
Valenzuela M, Perez-Perez G, Corvalan AH, Carrasco G, Urra H, Bravo D, Toledo H, Quest AF
(2010) Helicobacter pylori-induced loss of the inhibitor-of-apoptosis protein survivin is linked
to gastritis and death of human gastric cells. J Infect Dis 202(7):1021–1030. doi:10.1086/
656143
Valenzuela M, Bravo D, Canales J, Sanhueza C, Diaz N, Almarza O, Toledo H, Quest AF (2013)
Helicobacter pylori-induced loss of survivin and gastric cell viability is attributable to secreted
bacterial gamma-glutamyl transpeptidase activity. J Infect Dis 208(7):1131–1141. doi:10.
1093/infdis/jit286
van Roy F (2014) Beyond E-cadherin: roles of other cadherin superfamily members in cancer.
Nature reviews. Cancer 14(2):121–134. doi:10.1038/nrc3647
Vendramini-Costa DB, Carvalho JE (2012) Molecular link mechanisms between inflammation
and cancer. Curr Pharm Des 18(26):3831–3852
Voland P, Weeks DL, Vaira D, Prinz C, Sachs G (2002) Specific identification of three low
molecular weight membrane-associated antigens of Helicobacter pylori. Aliment Pharmacol
Ther 16(3):533–544
Walczak H (2011) TNF and ubiquitin at the crossroads of gene activation, cell death, inflamma-
tion, and cancer. Immunol Rev 244(1):9–28. doi:10.1111/j.1600-065X.2011.01066.x
Watanabe T, Hirano K, Takahashi A, Yamaguchi K, Beppu M, Fujiki H, Suganuma M (2010a)
Nucleolin on the cell surface as a new molecular target for gastric cancer treatment. Biol Pharm
Bull 33(5):796–803
Watanabe T, Tsuge H, Imagawa T, Kise D, Hirano K, Beppu M, Takahashi A, Yamaguchi K,
Fujiki H, Suganuma M (2010b) Nucleolin as cell surface receptor for tumor necrosis factor-
alpha inducing protein: a carcinogenic factor of Helicobacter pylori. J Cancer Res Clin Oncol
136(6):911–921. doi:10.1007/s00432-009-0733-y
Watanabe T, Takahashi A, Suzuki K, Kurusu-Kanno M, Yamaguchi K, Fujiki H, Suganuma M
(2014) Epithelial-mesenchymal transition in human gastric cancer cell lines induced by TNF-
alpha-inducing protein of Helicobacter pylori. J Int Cancer 134(10):2373–2382. doi:10.1002/
ijc.28582
Wessler S, Backert S (2008) Molecular mechanisms of epithelial-barrier disruption by
Helicobacter pylori. Trends Microbiol 16(8):397–405. doi:10.1016/j.tim.2008.05.005
Windle HJ, Kelleher D (1997) Identification and characterization of a metalloprotease activity
from Helicobacter pylori. Infect Immun 65(8):3132–3137
Wustner S, Mejias-Luque R, Koch MF, Rath E, Vieth M, Sieber SA, Haller D, Gerhard M (2015)
Helicobacter pylori gamma-glutamyltranspeptidase impairs T-lymphocyte function by
compromising metabolic adaption through inhibition of cMyc and IRF4 expression. Cell
Microbiol 17(1):51–61. doi:10.1111/cmi.12335
188 S. Wessler
Yakoob J, Abbas Z, Naz S, Islam M, Abid S, Jafri W (2010) Associations between the plasticity
region genes of Helicobacter pylori and gastroduodenal diseases in a high-prevalence area. Gut
Liver 4(3):345–350. doi:10.5009/gnl.2010.4.3.345
Yamaoka Y, Kita M, Kodama T, Imamura S, Ohno T, Sawai N, Ishimaru A, Imanishi J, Graham
DY (2002) Helicobacter pylori infection in mice: role of outer membrane proteins in coloni-
zation and inflammation. Gastroenterology 123(6):1992–2004. doi:10.1053/gast.2002.37074
Yokoyama H, Tsuruta O, Akao N, Fujii S (2012) Crystal structure of Helicobacter pylori
neutrophil-activating protein with a di-nuclear ferroxidase center in a zinc or cadmium-
bound form. Biochem Biophys Res Commun 422(4):745–750. doi:10.1016/j.bbrc.2012.05.073
Zanotti G, Papinutto E, Dundon W, Battistutta R, Seveso M, Giudice G, Rappuoli R, Montecucco
C (2002) Structure of the neutrophil-activating protein from Helicobacter pylori. J Mol Biol
323(1):125–130
Zhang Z, Zheng Q, Chen X, Xiao S, Liu W, Lu H (2008) The Helicobacter pylori duodenal ulcer
promoting gene, dupA in China. BMC Gastroenterol 8:49. doi:10.1186/1471-230x-8-49
Chapter 8
The Primary Transcriptome and Noncoding
RNA Repertoire of Helicobacter pylori
Sandy R. Pernitzsch, Fabien Darfeuille, and Cynthia M. Sharma
Abstract The intense study of Helicobacter pylori, one of the most prevalent
human pathogens, has contributed much to understanding of bacterial virulence
mechanisms. While genome sequencing revealed a high genetic diversity among
Helicobacter strains, its transcriptional organization has been less understood. The
H. pylori genome encodes for only a small number of transcriptional regulators, and
little is known about the role of posttranscriptional gene regulation and the mech-
anisms and functions of small regulatory RNAs (sRNAs) in Epsilonproteobacteria.
Until recently, Helicobacter was even regarded as a bacterium without RNA-based
regulation (riboregulation). However, the development of deep sequencing tech-
nology and its application to massively parallel high-throughput cDNA analysis
(RNA-seq) has revolutionized transcriptome analysis of pro- and eukaryotes. A
differential RNA-seq (dRNA-seq) study of H. pylori strain 26695 selective for
primary transcriptome analysis allowed for genome-wide mapping of transcrip-
tional start sites. This study also revealed an unexpectedly complex and compact
transcriptional output from the small H. pylori genome. Besides an extensive
antisense transcription, more than 60 sRNA candidates were identified, including
potential regulators of cis- and trans-encoded target mRNAs. This indicates
that posttranscriptional regulation represents an extensive layer of gene
expression control in Helicobacter. In this chapter we review how RNA-seq has
facilitated transcriptome annotation and identification of novel regulatory features
in H. pylori and what is currently known about sRNAs in this widespread gastric
pathogen.
Keywords Helicobacter pylori • RNA sequencing (RNA-seq) • Deep sequencing •

Terminator exonuclease • Differential RNA-seq (dRNA-seq) • Small regulatory
RNAs • Posttranscriptional regulation • Transcriptional start sites • Primary
transcriptome
S.R. Pernitzsch • C.M. Sharma (*)

Research Center for Infectious Diseases (ZINF), University of Würzburg, Josef-Schneider-
Straße 2/D15, 97080 Würzburg, Germany
F. Darfeuille
INSERM U869, University of Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux, France

DOI 10.1007/978-4-431-55936-8_8
190 S.R. Pernitzsch et al.
8.1 Introduction
The human stomach is a dynamic and hostile environment, in which the gastric
pathogen Helicobacter pylori encounters a variety of environmental stressors.
These include pH fluctuations, nutrient limitations, ever changing availabilities of
metal ions, and oxidative stress caused by the host’s immune system (Sachs
et al. 2003). Despite being very well adapted to the human gastric niche (Salama
et al. 2013; Suerbaum and Josenhans 2007), H. pylori requires regulatory systems
for rapidly changing its gene expression to adapt to different colonization condi-
tions. Gene expression changes induced by environmental stimuli or mutation of
transcriptional regulators have been previously analyzed in H. pylori mainly using
microarrays in combination with qRT-PCR, primer extension, and in vitro
promoter-binding assays (reviewed in Danielli et al. 2010; Josenhans et al. 2007).
These studies have provided important insight into gene expression control at the
transcriptional level. However, until recently, little was known about the transcrip-
tional organization at single-nucleotide resolution and posttranscriptional gene
regulation in H. pylori. Small regulatory RNAs (sRNAs) are an emerging class of
posttranscriptional gene expression regulators that act during bacterial stress
response or virulence control in pathogens (Papenfort and Vogel 2010; Storz
et al. 2011). While H. pylori was previously regarded as an organism without
riboregulation (Mitarai et al. 2007), deep sequencing-based transcriptome analysis
has revealed a wealth of sRNAs in this pathogen (Sharma et al. 2010).
The development of high-throughput DNA sequencing technology and its appli-
cation to sequencing of cDNA libraries, so-called RNA sequencing (RNA-seq), has
provided a powerful method for both annotating and quantifying the transcriptional
output from an organism’s genome. RNA-seq has revolutionized our view of the
extent and complexity of both eukaryotic and prokaryotic transcriptomes (Wang
et al. 2009; Croucher and Thomson 2010; Barquist and Vogel 2015). It has been
successfully used to improve genome annotations and has revealed a wealth of
novel noncoding transcripts (ncRNAs). In this chapter, we summarize the analysis
of the primary transcriptome of H. pylori using a novel differential RNA-seq
(dRNA-seq) approach (Sharma et al. 2010). This approach allows for the specific
mapping of primary transcripts and has defined a genome-wide map of transcrip-
tional start sites (TSS) in H. pylori. While H. pylori was the first organism to be
studied by dRNA-seq, this approach has been subsequently applied to many other
pro- and eukaryotes (Sharma and Vogel 2014). In addition to a very complex and
tightly packed transcriptional output, an extensive antisense transcription from the
opposite strand of protein-coding genes as well as more than 60 potential sRNAs
were uncovered in the small H. pylori genome. We describe how these H. pylori
transcriptome characteristics uncovered by dRNA-seq have revealed new regula-
tory features in this important human pathogen. This approach has opened a new
perspective on the extent of RNA-mediated regulation not only in H. pylori but also
in several other bacterial pathogens (Sharma and Vogel 2014), including the related
food-borne pathogen Campylobacter jejuni.
8 The Primary Transcriptome and Noncoding RNA Repertoire of Helicobacter pylori 191
8.2 Primary Transcriptome Analysis of Helicobacter pylori
8.2.1 Bacterial Transcriptome Analysis Using RNA-Seq
The transcriptome describes the identity and abundance of all cellular transcripts
that are expressed under a specific condition. In contrast to genomes,
transcriptomes can be highly dynamic, and transcript abundances can rapidly
change in response to changing cell states, environments, or stress conditions.
Throughout their life cycle, RNAs can be modified or cleaved for maturation or
function, and processing by diverse RNA degradation enzymes mediates transcript
stabilization or degradation. In the last two decades, hybridization-based methods
such as microarrays and tiling arrays that rely on PCR probes or oligonucleotide
probes were commonly used for transcriptome analyses in diverse organisms,
including bacteria such as H. pylori (Josenhans et al. 2007). These include studies
on transcriptome changes in response to important stress conditions for H. pylori
such as pH fluctuations (Merrell et al. 2003; Wen et al. 2003; Bury-Mone
et al. 2004) or metal ion availabilities (Danielli et al. 2010). Regardless of the
power of array-based approaches for transcriptome analyses and determining gene
expression profiles, they have major limitations. For example, probe design of
microarrays is mostly restricted to regions of known or predicted genes, excluding
not only ncRNAs such as sRNAs but also 50 and 30 untranslated regions (UTRs) of
open reading frames (ORFs). Even though tiling arrays cover intergenic regions
(IGRs) and other regulatory sequences (e.g., UTRs), disadvantages such as cross-
hybridizations and a limited detection of low abundant transcripts still exist for
array-based approaches. The development of RNA-seq technology has greatly
facilitated transcriptome analyses by allowing for transcript identification and
quantification at single-nucleotide resolution over a large dynamic range (Wang
et al. 2009; Croucher and Thomson 2010; Sorek and Cossart 2010). Compared to
microarrays, RNA-seq is more sensitive, provides absolute quantity levels, is not
affected by on-chip sequence biases, and gives additional information on variations
in transcript processing. Furthermore, RNA-seq does not rely on specific probe
design and allows for the detection of known and novel transcripts.
In a typical bacterial RNA-seq workflow, either total RNA or selectively frac-
tionated RNA, e.g., size-selected RNA or RNA from a co-immunoprecipitation
experiment, is first converted into a cDNA library. Upon PCR amplification, the
cDNA library is analyzed by one of the currently availiable next-generation
sequencing platforms, such as Solexa (Illumina), 454 (454 Life Sciences) or Solid
(ABI), resulting in millions of short cDNA sequences (“reads”). These reads are
then aligned (“mapped”) to the respective reference genome and gene expression
can be visualized as cDNA coverage plots at single-nucleotide resolution in a
genome browser. The cDNA read patterns and counts can then be used to annotate
transcripts or to determine gene expression changes. Different protocols have been
developed to construct cDNA libraries for specific and general applications. To
perform strand-specific RNA-seq, which is important to distinguish between sense
and antisense transcription, cDNA library preparation protocols have been designed
that are based on ligation of a 50 RNA linker combined with either 30 poly(A)-tailing
of RNAs and oligo(dT)-priming for reverse transcription or cDNA synthesis from a
ligated 30 linker (see references in Borries et al. 2010; Passalacqua et al. 2012;
Heidrich et al. 2015). The first bacterial RNA-seq studies included depletion steps
for ribosomal RNA (rRNA) and transfer RNA (tRNA), which comprise 95 % of the
cellular RNA pool, to enrich for sequencing of mRNAs and sRNAs (reviewed in
Sorek and Cossart 2010). However, since the sequencing depth and affordability of
the next-generation sequencing platforms is constantly improving, depletion steps
are no longer required. This reduces library preparation biases associated with such
depletion steps.
Besides the annotation of transcriptome features, including 50 and 30 transcript
boundaries or identification of novel transcripts such as sRNAs or small ORFs,
RNA-seq is nowadays also widely used for gene expression profiling and is
replacing microarray approaches (Croucher and Thomson 2010; Sorek and Cossart
2010). Several recent RNA-seq-based transcriptome studies in bacteria, including
Epsilonproteobacteria (Chaudhuri et al. 2011; Butcher and Stintzi 2013; Taveirne
et al. 2013), indicate that this method can be used to perform comprehensive as well
as quantitative expression profiling of the transcriptome under various stress con-
ditions or between mutant and wild-type strains.
8.2.2 Differential RNA-Seq for Primary Transcriptome

Analysis
Several RNA-seq approaches have been developed for annotation of transcript 50

ends on a genome-wide scale (Wurtzel et al. 2010; Mendoza-Vargas et al. 2009;
Cho et al. 2009; Singh and Wade 2014; Lin et al. 2013; Salgado et al. 2013). Many
of these approaches cannot distinguish between primary and processed transcripts.
In contrast, the differential RNA-seq approach allows for a selective sequencing of
primary transcripts and, in turn, global identification of transcriptional start sites
(TSS) and associated promoter sequences (Sharma et al. 2010; Sharma and Vogel
2014; Bischler et al. 2015). Primary transcripts carry a 50 triphosphate end (50 PPP),
whereas processed transcripts, such as the abundant ribosomal RNAs or tRNAs,
carry a 50 monophosphate (50 P) or, less frequently, a 50 hydroxyl (50 OH) group
(Fig. 8.1a). The dRNA-seq approach is based on a differential treatment of RNA
with 50 P-dependent terminator exonuclease (TEX) that specifically degrades 50 P
RNAs, whereas primary transcripts are not affected (Fig. 8.1b). Thus, TEX treat-
ment results in a relative enrichment of primary transcripts and depletes processed
RNAs. To distinguish between primary and processed RNAs by dRNA-seq, two
differential cDNA libraries are sequenced and compared: one library (TEX-) is
generated from untreated, total RNA and the other (TEX+) from RNA enriched for
primary transcripts by TEX treatment (see Borries et al. 2010; Heidrich et al. 2015;
a
Cellular RNA (rRNA, tRNA, mRNA, sRNA)
Cell lysis
Total RNA extraction PPP
P
PPP P
H. pylori 26695 OH
b -/+TEX treatment
Total RNA (TEX-) Enrichment for primary transcripts (TEX+)
PPP PPP
P P
PPP P TEX PPP P
TAP treatment
cDNA library preparation RNA linker ligation and poly(A)-tailing
Reverse transcription
Deep sequencing
c
+1 +1 +1
100
mapped cDNA reads
TEX+
10
100 Determination of TSS

mapped cDNA reads
Analysis of expression profiles

TEX-
10
AUG
small ORF/sRNA open reading frame tRNA
RNase P cleavage site
Fig. 8.1 Workflow of the RNA-seq approach exemplified for H. pylori strain 26695. (a) Total
cellular RNA consists of primary (green) transcripts with a 50 triphosphate (PPP) and processed
transcripts (black) with a 50 monophosphate (P) or, less frequently, a 50 hydroxyl group OH. (b) For
generation of dRNA-seq cDNA libraries, RNA samples are split into two halves. One is left
untreated (TEX-), whereas the other one is subjected to 50 -dependent terminator exonuclease
(TEX) treatment (TEX+). TEX (blue) specifically degrades RNAs with a 50 P. Following differ-
ential TEX treatment, 50 PPP ends will be trimmed to 50 P by tobacco acid pyrophosphatase (TAP),
allowing RNA linker ligation. RNAs with a 50 OH are not accessible for linker ligation and, thus,
are not represented in the final cDNA library. Upon poly(A)-tailing of the 30 end using poly
(A) polymerase and reverse transcription, cDNA libraries are analyzed by deep sequencing. (c)
Sequenced cDNA reads of the TEX /+libraries are mapped to the reference genome and are
visualized as cDNA coverage plots representing the number of reads (left scale) per nucleotide in a
genome browser. TEX treatment leads to a characteristic enrichment pattern at the 50 ends of
primary transcripts, thereby allowing for mapping of transcriptional start sites (TSS) (black
arrows). In contrast, 50 ends of processing sites are not enriched (dotted arrow)
Bischler et al. 2015 for a detailed protocol). Since TEX also removes abundant
processed RNAs including 16S and 23S rRNAs, no additional rRNA depletion
steps are required, and in contrast to many other bacterial RNA-seq studies, dRNA-
seq is typically performed on total RNA.
After the differential TEX treatment, the RNA 50 PPP ends are converted into 50 P
ends by tobacco acid pyrophosphatase (TAP) treatment to allow for RNA linker
ligation. Upon poly(A)-tailing of the RNA and conversion into cDNA by reverse
transcription using oligo(dt)-priming, the resulting cDNA libraries are amplified
and sequenced on one of the next-generation sequencing platforms. While the first
dRNA-seq study to reveal the primary transcriptome of H. pylori strain 26695
employed 454 pyrosequencing (Sharma et al. 2010), nowadays dRNA-seq mainly
employs Illumina sequencing, allowing for higher sequencing depth (Sharma and
Vogel 2014). Sequencing of the dRNA-seq libraries results in a characteristic,
sawtooth-like enrichment pattern: in the TEX+ sample cDNA reads cluster toward
the 50 ends of primary transcripts, whereas those from the TEX- sample are
randomly distributed along transcripts (Fig. 8.1c). These enrichment patterns
allow for globally annotating TSS for all expressed genes under the condition
(s) examined. While TSS were manually annotated in the dRNA-seq studies of
H. pylori and diverse other organisms, several algorithms have been developed that
now allow for an automated TSS annotation (Bischler et al. 2015 and reviewed in
Sharma and Vogel 2014).
8.3 Helicobacter Transcriptome Features Identified by

dRNA-Seq
8.3.1 Global Transcriptional Start Site Maps
Previously, the 50 ends of transcripts and respective promoters were mapped on a

gene-by-gene basis using laborious RNase S1 protection experiments (Berk and
Sharp 1977), primer extension assays (Thompson et al. 1979), or RACE (rapid
amplification of cDNA ends) approaches (Argaman et al. 2001; Vogel et al. 2003;
Bensing et al. 1996). For example, the TSS of several abundantly transcribed
H. pylori genes, such as ureA or cagA, were mapped using these methods (Spohn
et al. 1997; Spohn and Scarlato 1999; Shirai et al. 1999). The dRNA-seq-based
primary transcriptome analysis of H. pylori strain 26695 allowed, for the first time,
mapping of TSS in a bacterium on a genome-wide scale (Sharma et al. 2010).
dRNA-seq analysis of H. pylori 26695 grown under five different growth condi-
tions, combined with 454 pyrosequencing at a depth of 200,000–500,000 cDNA
reads per library, revealed about 1,900 unique TSS. This dense TSS map exceeded
the number of ~1,700 annotated genes and uncovered an unexpectedly complex
transcriptional output from the small H. pylori genome. Identified TSS were
classified according to their location relative to flanking genes into five different
Fig. 8.2 Annotation of transcriptional start sites and 50 UTRs in H. pylori strain 26695. (a) (Top)
Representation of TSS categories based on gene expression and location according to flanking
genes: primary (p), secondary (s), internal (i), antisense (as) and orphan (o) TSS. (Bottom) Pie
chart showing the distribution of TSS to the different TSS categories for H. pylori strain 26695. (b)
Bioinformatics-based motif searches upstream of the 1,906 identified H. pylori TSS revealed an
extended 10 promoter element (tgnTAtaAT) and a periodic A/T-rich pattern as the consensus
promoter motif for the housekeeping sigma factor, σ80. (c) Frequency of 50 UTR lengths based on
annotated primary and secondary TSS. More than 30 leaderless mRNAs with a 50 UTR length
<10 nt (read bars) were identified. A commonly identified ribosome binding site motif
(SD sequence, AAGGag) is shown in the inset. (d) cDNA reads of mid-log growth (ML/+)
and acid stress (AS/+) libraries were mapped to the cheY-HP1074 operon (black arrow). In
addition to the pTSS upstream of cheY, two iTSS, upstream of ftsH (cell division) and copP
(copper transport) were detected using dRNA-seq. This suggests transcription of suboperons
(green arrows) that uncouple expression of these downstream genes. Gray arrows represent the
annotated ORFs and +1 denotes identified TSS in H. pylori 26695 (Sharma et al. 2010). The figure
was adapted from Sharma et al. 2010
categories: (I) primary (pTSS) and (II) secondary TSS (sTSS) located <500 bp
upstream of ORFs that correspond to main and alternative promoters of mRNA and
rRNA/tRNA genes, respectively; (III) internal TSS (iTSS) that are transcribed
sense within genes but initiate after the annotated start codon; (IV) antisense TSS
(asTSS) that are located antisense either within or 100 nt up- or downstream of
annotated genes; and (V) orphan TSS (oTSS) that have no annotation in close
proximity and might correspond to novel sRNAs or mRNAs (Fig. 8.2a). Besides
more than 800 pTSS and 110 sTSS, a wealth of novel noncoding transcripts was
detected. The discovery of more than 960 asTSS, including at least one asTSS for
more than half of all H. pylori ORFs, indicated that antisense RNA-mediated
regulation might be a major level of gene expression control in H. pylori.
The H. pylori genome encodes three sigma factors: the housekeeping sigma
factor RpoD (σ80) and the two alternative sigma factors RpoN (σ54) and FliA (σ28),
which control the expression of motility genes (Lertsethtakarn et al. 2011). In
Escherichia coli, the RNA polymerase recognizes, upon assembly with one of the
sigma factors, specific promoter sequences such as the 10 (50 -TATAAT consen-
sus) and 35 (50 -TTGACA consensus) boxes of the housekeeping RpoD-driven
promoters (Burgess and Anthony 2001). Analysis of the upstream regions of the
1,900 TSS mapped in H. pylori provided the first global evidence that promoters of
RpoD in Epsilonproteobacteria consist of an extended 10 box (50 -tgnTAtaAT)
preceded by a periodic A/T-rich pattern but lack a 35 box (Fig. 8.2b). Such a
promoter motif was previously suggested based on the analysis of a small number
of H. pylori genes (Forsyth and Cover 1999) and bioinformatics-based promoter
predictions in C. jejuni (Petersen et al. 2003; Wosten et al. 1998). Different dRNA-
seq analyses of C. jejuni have further confirmed this global promoter consensus for
the pathogenic Epsilonproteobacteria (Dugar et al. 2013; Porcelli et al. 2013).
Genomes are mainly annotated in an automated fashion, which can be error
prone through selection for the longest possible open reading frames. The genome-
wide TSS mapping can improve genome annotations by providing global informa-
tion about 50 transcript boundaries. Furthermore, transcripts that may contain
smaller ORFs missed by genome annotations are also identified. The dRNA-seq
analysis of H. pylori revealed several genes where transcription started downstream
of the annotated start codon. Further examination of these genes and incorporation
of information about start codon conservation in different H. pylori strains allowed
for re-annotation of the start codons for at least 18 genes. In general, a combined
transcriptome and genome sequencing will increase the accuracy of genome anno-
tations of novel strains and species, especially those that utilize different promoter
consensus sequences from those of the model enterobacteria.
8.3.2 50 UTR Lengths
Until recently, the full repertoire of 50 UTRs for a bacterium was largely unknown.
The annotation of primary and secondary TSS upstream of open reading frames
allowed for the exact mapping of 825 50 UTRs in H. pylori (Fig. 8.2c, Sharma
et al. 2010). Analysis of the 50 UTR length distribution showed that at least 50 % of
them are 20–40 nt in length (Fig. 8.2c). A decrease in frequency for shorter 50 UTRs
was observed, since these leaders are too short to harbor sufficient sequence
information required for ribosome binding and translation initiation (Ramakrishnan
2002). A classical consensus AAGGag sequence was identified as a Shine–
Dalgarno (SD) motif for most of the H. pylori mRNAs. However, a surprising
number of genes (2.2 %) have a 50 UTR length <10 nt and correspond to leaderless
mRNAs, lacking an SD sequence. Leaderless mRNAs are typically translated by
specialized ribosomes without SD interaction (Moll et al. 2002, 2004) and tran-
scriptional initiation occurs at the start codon. Besides these putative leaderless
transcripts, 337 long 50 UTRs (>60 nt) were detected that could contain posttran-
scriptional control elements such as cis-encoded transcription attenuators or
riboswitches (Serganov and Nudler 2013). For example, a search for conserved
structural RNA motifs in the 50 UTRs revealed a potential thiamine pyrophosphate
(TPP) riboswitch upstream of the pnuC gene encoding a nicotinamide mononucle-
otide transporter in H. pylori, which is highly conserved in bacteria, archaea, and
plants and has been suggested to lead to transcription attenuation in the presence of
TPP (Rodionov et al. 2002). Future studies will show the function and mechanism
of this potential H. pylori riboswitch, and examination of other long 50 UTRs might
reveal novel regulatory RNA elements and/or RNA thermometers (Kortmann and
Narberhaus 2012).
8.3.3 Operon and Suboperon Structure
In bacteria, many genes, especially those encoding for functionally related proteins
or components of the same enzymatic pathway or structural complex, are encoded
in operons and are transcribed as polycistronic transcripts. It was previously
assumed that H. pylori lacks an extensive operon organization (Tomb et al. 1997;
Thompson et al. 2003). However, dRNA-seq analysis indicated that there are
multiple iTSS within open reading frames, including genes in predicted operons
(Mao et al. 2009). Combination of the dRNA-seq-derived TSS map with conven-
tional RNA-seq that covers full-length transcripts provided a genome-wide operon
map of H. pylori strain 26695 and revealed that 87.5 % of all genes are located
within 337 primary operons. In addition, 126 alternative operons and 66 single
genes overlapping the 30 part of polycistronic transcripts were detected. For exam-
ple, in the eight-gene operon composed of cheY-HP1074 (Fig. 8.2d), two iTSS were
identified, one within prmA (HP1068) and another within copA (HP1072), which
lead to uncoupling of the ftsH-HP1074 and copP-HP1074 suboperons from the
primary operon cheY-HP1074. Bacteria might use such suboperons to uncouple
and differentially regulate certain genes from the rest of the primary operon under
certain stress or growth conditions and thereby increase their transcriptome com-
plexity. Global transcriptome studies have also reported transcription of suboperons
in other bacterial species such as Mycoplasma (Guell et al. 2009).
8.3.4 Noncoding RNAs
Posttranscriptional regulation constitutes an important layer of gene expression

control in the cell. In bacteria, the 50–400-nt-long small regulatory RNAs are
posttranscriptional regulators that control gene expression during stress responses

and virulence control (Papenfort and Vogel 2010; Storz et al. 2011). While some
sRNAs can directly bind proteins and modulate their activity, most of the function-
ally characterized sRNAs act as antisense RNAs by base-pairing interactions on
either cis- or trans-encoded mRNAs (Waters and Storz 2009). Whereas cis-encoded
sRNAs originate from the opposite DNA strand and share full complementarity with
their target transcripts, trans-encoded sRNAs are encoded at distinct genomic
locations and regulate target mRNAs by short and imperfect base-pairing interac-
tions. Most of the functionally characterized trans-encoded sRNAs repress transla-
tion of their target mRNAs by base-pairing near to, or directly at, the ribosome
binding site (RBS) and start codon. This translational repression is often coupled to
transcript degradation, but several mechanisms of activation of gene expression have
also been reported (Frohlich and Vogel 2009). It is now also clear that sRNAs can
regulate multiple genes of functionally related pathways by either direct binding to
multiple target mRNAs or by regulation of transcription factors and thereby act as
global regulators in response to environmental stress conditions (Storz et al. 2011).
The RNA chaperone Hfq is a key player in sRNA-mediated regulation in
enterobacteria such as E. coli and Salmonella and is required for sRNA stabilization
and/or facilitation of sRNA–mRNA interactions (Vogel and Luisi 2011). Despite its
crucial roles in enterobacteria, an obvious Hfq homolog is absent in about 50 % of all
bacteria, and it seems to be dispensable for sRNA-mediated gene regulation in
Gram-positive bacteria (Chao and Vogel 2010), indicating that additional
RNA-binding proteins might be involved in posttranscriptional regulation.
Even though the genome of H. pylori was sequenced nearly 20 years ago, little is
known about its posttranscriptional gene regulation including sRNAs and
RNA-binding proteins. None of the enterobacterial sRNAs, except for the house-
keeping RNAs, transfer-messenger RNA (tmRNA), signal recognition particle
RNA (SRP/4.5S RNA), and M1 RNA (RNase P), are conserved at the sequence
level in H. pylori (Sharma et al. 2010). Bioinformatics-based prediction and a
small-scale cDNA cloning approach identified only a few candidates for natural
antisense transcripts and sRNAs in Helicobacter (Livny et al. 2006; Xiao
et al. 2009a, b). The limited knowledge of riboregulation in H. pylori might be
also due to the traditional probe design of microarrays used for transcriptome
studies, which were so far mainly restricted to known or predicted mRNAs.
Although sRNAs have been intensively investigated during the last years,
knowledge of their regulatory potential and mechanisms is mainly based on work
in the model organisms E. coli, Salmonella, and other Gammaproteobacteria.
Systematic searches for sRNAs using experimental methods or biocomputational
predictions have greatly facilitated the identification of sRNA genes on a genome-
wide scale in various bacteria (Backofen and Hess 2010; Sharma and Vogel 2009).
In particular, RNA-seq-based approaches have revealed a wealth of potential
sRNAs in diverse bacterial species (Croucher and Thomson 2010; Sorek and
Cossart 2010; Barquist and Vogel 2015). The abovementioned dRNA-seq analysis
of H. pylori strain 26695 not only helped to define a global map of TSS but also
revealed many noncoding transcripts. Diverse candidates were identified that are
a antisense sense IGR
HP0513 HP0660 rnhA HP1043

HP1043 HP1044
HP1044
HPnc2450 HPnc3320 RepG
20
RepG
Log normalized expression
antisense sRNA small ORF sense in ORF 6S RNA
10
10
6S RNA
20
Mbp
0 0.5 1.0 1.5 1.67
b c
trans-encoded sRNA cis-encoded sRNA
UreA UreB
acidic neutral
tlpB HP0102 HP1043
HP1043 HP1044
HP1044 conditions conditions
5' 3'
ArsS PP ArsR
P
P + ArsS
5' 3'
ureA ureB
ArsR
5' 3'
RepG 5'ureB-sRNA +
3' 5'
3' 5'
RepG
RNA polymerase
U UC
UU A
C C UCCCCCUCC tlpB-HP0102 mRNA ureA ureB
RBS
5' AUG 3'
Premature
ureAB mRNA transcription
5' termination
3' 5'
Repression of tlpB Activation of tlpB
(8-12G) (>14G) 5'ureB-sRNA
UreA UreB
Decreased urease activity
Fig. 8.3 Examples of cis- and trans-acting sRNAs in H. pylori. (a) (Upper panel) Examples for
sRNA candidates that were discovered in intergenic regions or that are located sense or antisense
to open reading frames. (Lower panel) Relative expression levels for newly detected transcripts
(noncoding RNAs and novel small mRNAs) along the H. pylori 26695 chromosome. The figure
was adapted from Sharma et al. 2010. (b) Regulation of expression of the chemotaxis receptor
TlpB by the trans-encoded RepG sRNA (Pernitzsch et al. 2014). The 87-nt-long RepG sRNA is
encoded in the intergenic region between the orphan response regulator HP1043 and the hypo-
thetical protein HP1044. The C/U-rich terminator loop of RepG directly binds to a G-repeat in the
50 UTR of the tlpB-HP0102 mRNA. Dependent on the G-repeat length, RepG mediates repression
(8–12 G) or activation (14 G) of TlpB. (c) Regulation of urease expression by the cis-encoded
antisense 50 ureB-sRNA (Wen et al. 2011, 2013). (Left) Expression of the ureAB operon, which
encodes for the urease apoenzyme, is induced under acidic conditions by the phosphorylated ArsR
response regulator of the acid-responsive ArsSR two-component system. (Right) Expression of the
cis-encoded antisense transcript, 50 ureB-sRNA, is induced by unphosphorylated ArsR under
neutral conditions. An interaction between 50 ureB-sRNA and the 50 region of ureB leads to
premature transcription termination of the ureAB mRNA. Truncation of ureB mRNA represses
its translation, resulting in a reduced amount of UreB and decreased urease activity
either expressed as single genes from intergenic regions or antisense/sense to

coding genes (Fig. 8.3a), suggesting that H. pylori has the potential for extensive
riboregulation.
8.3.4.1 Housekeeping RNAs
The housekeeping RNAs of H. pylori, tmRNA, M1 RNA, and SRP/4.5S RNA were
found to be highly expressed in the dRNA-seq study. These conserved RNAs are
essential for a variety of cellular processes such as mRNA translation, tRNA
maturation, as well as protein translocation. The housekeeping tmRNA, which is
involved in trans-translation (i.e., rescue of stalled ribosomes and degradation of
truncated, potentially toxic proteins), was shown to be essential and required for
stress response as well as natural competence in H. pylori (Thibonnier et al. 2008).
While tmRNA and its protein cofactor, SmpB, have similar functions in H. pylori
and E. coli, tmRNA possesses certain sequence constrains that are required to
achieve ribosome rescue in a given organism (Thibonnier et al. 2010). Thus,
while such housekeeping RNAs and their functions are ubiquitous in eubacteria,
they have coevolved with the translation machinery of their host organisms and,
thus, possess specialized characteristics.
One of the most abundant transcripts in the dRNA-seq study of H. pylori was
found to be a homolog of the housekeeping RNA, 6S RNA, which was missed in
previous bioinformatics predictions (Barrick et al. 2005; Sharma et al. 2010). 6S
RNA is an abundant and ubiquitous riboregulator that mimics an open promoter
complex and thereby sequesters RNA polymerase (RNAP) bound to the house-
keeping sigma factor (σ70), resulting in transcriptional activation of σS-dependent
genes (Cavanagh and Wassarman 2014). Despite only little sequence conservation
to the E. coli 6S RNA, the 180-nt-long 6S RNA from H. pylori folds into the same
characteristic long hairpin structure which was shown to be essential for its binding
to RNA polymerase in E. coli and Bacillius subtilis. The dRNA-seq data also
detected 14–20-nt-long “product RNAs” (pRNAs), which are transcribed using
6S RNA as a template and are important for the recycling of RNAP during
outgrowth or supply of new nutrients such as NTPs (Wassarman and Saecker
2006), and thus provides further evidence that this RNA is a functional 6S RNA
homolog. However, detection of pRNAs arising from both strands of 6S RNA, as
well as the absence of RpoS in H. pylori, suggests that, like tmRNA, the H. pylori
system has slightly diverged from the E. coli paradigm. Whether 6S RNA has a role
during stress response or stationary phase growth in H. pylori or, like in Legionella,
impacts on its virulence (Faucher et al. 2010) still needs to be investigated.
8.3.4.2 Mechanisms and Functions of Antisense/Base-Pairing RNAs

in H. pylori
Trans-Encoded Antisense RNAs
First insight into sRNA-mediated posttranscriptional regulation of gene expression

in Helicobacter was gained based on the expression of an artificial antisense RNA
(asRNA) with full complementarity to the 50 UTR and start codon of the essential
alkyl hydroperoxide reductase gene ahpC (Croxen et al. 2007). This antisense RNA
has been shown to repress expression of this abundant enzyme, indicating that
sequestration of the ribosome binding site and thus abrogation of target mRNA
translation might be a feature of sRNA-mediated regulation in Helicobacter.
Although several of the dRNA-seq-identified H. pylori sRNAs are candidates for
putative trans-encoded base-pairing RNAs, the mechanisms and functions for most
are still unknown. The dRNA-seq approach revealed a highly abundant and con-
served 87-nt-long sRNA, RepG (Regulator of polymeric G-repeats), which is
encoded between genes of an orphan response regulator (HP1043) and a protein
of unknown function (HP1044) (Fig. 8.3b). This sRNA represents the first example
of a trans-acting antisense RNA in Helicobacter and represses expression of one of
the four chemotaxis receptors, TlpB, in H. pylori strain 26695 (Sharma et al. 2010).
TlpB has been suggested to sense protons and plays a role in pH-taxis, quorum
sensing, and colonization and inflammation of the gastric mucosa in mice and
Mongolian gerbils (McGee et al. 2005; Williams et al. 2007; Croxen et al. 2006;
Rader et al. 2011). RepG regulates tlpB expression at the posttranscriptional level
by direct interaction between the C/U-rich terminator loop of the sRNA and a
homopolymeric G-repeat in the 50 UTR of tlpB mRNA (Pernitzsch et al. 2014).
Whereas RepG is highly conserved, the tlpB G-repeat length varies among diverse
H. pylori strains, resulting in a strain-specific tlpB regulation. The G-repeat corre-
sponds to one of the variable simple sequence repeats of H. pylori, which usually
affect transcription or coding potential of genes when they are located in promoter
regions or within open reading frames, respectively (Moxon et al. 2006). Modifi-
cation of the G-repeat in the tlpB 50 UTR within H. pylori strain 26695 demon-
strated that the G-repeat length determines the outcome of posttranscriptional
regulation (activation or repression) of tlpB by RepG, mainly at the translational
level (Pernitzsch et al. 2014). This represents an unexpected connection of pheno-
typic variation through variable simple sequence repeats and sRNA-mediated
regulation and shows that studying sRNAs in H. pylori can reveal novel twists of
gene regulation. In addition, expression profiling of other sRNA candidates under
various growth conditions, as well as target gene identification by whole proteome
and transcriptome analysis of sRNA mutants, together with bioinformatics-based
predictions, will help to understand the roles of sRNAs during stress response
and/or virulence regulation of H. pylori.
Cis-Encoded Antisense RNAs
Naturally occurring antisense transcription is a common phenomenon in all king-

doms of life and has been considered as an important feature in creating transcrip-
tional and thus, phenotypic complexity. Cis-encoded asRNAs are encoded in the
chromosome or on mobile genetic elements, e.g., plasmids, transposons, or phages,
and have been shown to affect gene expression by translation inhibition, transcrip-
tion interference and attenuation, transcript stabilization, or degradation (Wagner
et al. 2002; Brantl 2007; Waters and Storz 2009). Using tiling arrays or RNA-seq,
recent genome-wide transcriptome studies of several Gram-negative and -positive

bacteria suggested that antisense transcription from the chromosome is more
widespread than anticipated and revealed a wealth of cis-encoded antisense
RNAs (reviewed in Sorek and Cossart 2010; Georg and Hess 2011; Thomason
and Storz 2010). Although increasing numbers of asRNAs have been reported in
numerous studies, it is still under debate whether all of these transcripts are
functional or are simply the result of spurious transcription (Wade and Grainger
2014). In Gram-positive bacteria, long asRNAs derived from divergently tran-
scribed genes have been reported to span full genes or even operons (Sesto
et al. 2013). Using such an “excludon” paradigm, divergently transcribed genes
of related or opposing functions can control each other’s expression.
Consistent with the increasing number of studies that report widespread anti-
sense transcription in bacteria, more than 900 cis-encoded asRNAs were identified
in H. pylori strain 26695 using dRNA-seq (Sharma et al. 2010). These include bona
fide asRNAs, as well as overlapping 50 or 30 regions of mRNAs from contiguous
genes that are transcribed in opposite directions. Antisense transcription occurs
across the entire genome of H. pylori independent of local GC content, and no
general bias toward core or variable genes was observed. With at least one antisense
TSS associated with about half of all open reading frames (46 %), the fraction of
genes associated with asRNAs identified by RNA-seq in H. pylori is among the
highest compared to other bacteria (reviewed in Thomason and Storz 2010; Georg
and Hess 2011). Since overlapping transcription can affect the expression of the
gene on the complementary strand by various mechanisms, posttranscriptional
regulation by cis-encoded antisense RNAs and, for example, the double-strand-
specific ribonuclease RNase III might mediate widespread gene expression control
in Helicobacter.
Among the wealth of antisense transcripts, a new class of asRNA was discov-
ered. These asRNAs are complementary to stable RNAs, such as ribosomal RNAs
(23S and 16S rRNAs) or antisense to about one third of all H. pylori tRNAs.
Antisense RNA targeting of stable RNA has also been reported in the chloroplast
of the plant Arabidopsis thaliana, where a asRNA to the 5S rRNA seems to inhibit
its maturation (Sharwood et al. 2011). In H. pylori, two small cis-encoded asRNAs,
HPnc1880 and HPnc0260/0270, were identified that are expressed antisense to the
50 end precursor of the 23S-5S ribosomal RNAs and to the 30 end of tRNA-Val,
respectively. Both asRNAs are induced in response to low pH, indicating that they
may have a regulatory function under specific stress conditions. However, future
studies will be required to investigate whether they are involved in rRNA matura-
tion and/or degradation.
The recent characterization of a naturally occurring 292-nt-long cis-encoded
asRNA, 50 ureB-sRNA, from the opposite strand of the urease operon (ureAB),
further demonstrated functionality of asRNAs in Helicobacter and their potential
role in acid adaptation (Wen et al. 2011). In H. pylori strain 43504, transcription of
the 50 ureB-sRNA is induced under neutral pH conditions by the unphosphorylated
ArsR response regulator of the acid-sensing ArsRS two-component system, which
activates the expression of the ureAB operon in its phosphorylated form in response
to acid (Fig. 8.3c). The antisense 50 ureB-sRNA represses expression of the urease
apoenzyme by interacting with the 50 end of ureB coding region, resulting in
premature transcription termination of the polycistronic ureAB mRNA by tran-
scription attenuation (Wen et al. 2013). Whether the truncated ureAB transcript is
subsequently degraded or if the reduced amount of UreB protein leads to a decrease
in urease activity is still unclear. The 50 ureB-sRNA was not detected in the dRNA-
seq study of H. pylori strain 26695, which might be due to low expression levels
under the conditions examined or strain-specific antisense RNA expression. Dif-
ferent H. pylori strains might have evolved strain-specific sRNA/asRNA repertoires
as observed in a comparative dRNA-seq analysis of multiple strains of C. jejuni
(Dugar et al. 2013). Such unique cis- and trans-encoded RNAs could mediate
strain-specific regulation and thereby determine phenotypic differences among
strains and facilitate colonization of different hosts of niches.
8.3.5 Class I Toxin–Antitoxin Loci
In addition to novel sRNAs, the dRNA-seq study revealed several new small ORFs
encoding hydrophobic proteins of less than 50 amino acids which had been missed
during genome annotation (Sharma et al. 2010). Several of these predicted proteins
resemble small toxins or antimicrobial peptides and have cis-encoded antisense
RNAs, indicating that they may represent class I toxin–antitoxin (TA) loci in
H. pylori. Class I TA systems are composed of a protein toxin whose translation is
inhibited by an antisense RNA (Fozo et al. 2008a). A family of six structurally
related ~80-nt-long asRNAs, termed IsoA1-6 (RNA inhibitor of small ORF family
A), are expressed antisense to small ORFs of homologous 30 amino acid-long
AapA1-6 (antisense RNA-associated peptide family A) peptides. Although there
can be multiple loci encoding members of these peptide families within one
H. pylori strain (one to nine copies per genome), the peptides belonging to family
A are only conserved in two closely related Helicobacter species: H. cetorum and
H. acinonychis. Three additional peptide families (AapB, AapC, and AapD) were
identified in the H. pylori genome, two of which were also conserved in other
bacterial species. The AapD family, for instance, displays strong homology to the
Ibs type I toxin, which was first identified in E. coli (Fozo et al. 2008b). The
corresponding peptides for each family (A, B, C, and D) were not particularly
hydrophobic compared to other small hypothetical proteins previously annotated
in the H. pylori genome. For at least one of the peptides of family A (AapA), recent
work has shown that (I) overexpression of this peptide is toxic for the bacterium and
(II) asRNAs expressed in antisense orientation (IsoA) prevent their synthesis in vitro
and in vivo (Arnion and Darfeuille unpublished; Sharma et al. 2010). Thus, the
aapA–isoA loci might represent first examples of class I TA systems in H. pylori.
The physiological role of these TA loci is currently unknown and whether they
might be involved in persister cell formation or antibiotics resistance as recently
shown for the TisB peptide from E. coli (reviewed in Wagner and Unoson 2012).
8.4 Protein Factors Involved in Posttranscriptional

Regulation
Throughout their life cycle, RNAs are subjected to multiple processing and regu-
latory steps. During their regulatory or structural function, they can interact with a
variety of RNA-binding proteins (RBPs). These include ribonucleases (RNases)
that govern sRNA and/or target mRNA maturation or turnover, auxiliary protein
factors that mediate RNA–RNA interactions and/or stabilize sRNAs, proteins that
mediate intracellular transport of RNAs, or those whose functions are modulated by
the sRNA. Concordantly, sRNAs act in concert with RBPs and RNases to elicit
posttranscriptional regulation of gene expression. In H. pylori, analysis of the
urease operon transcript revealed pH-dependent transcript stabilities and processing
into multiple species, indicating an extensive posttranscriptional regulation of this
operon in response to acid stress (Akada et al. 2000). However, it is still unclear
which sRNAs or RPBs are required for processing of this mRNA. Only few RNA–
protein interactions have been studied in H. pylori so far. For example, the essential
tmRNA has been shown to interact with its protein cofactor SmpB, and both are
required for trans-translation and translational control (Thibonnier et al. 2008,
2010). The identification and subsequent characterization of novel H. pylori ribo-
nucleoprotein complexes will identify novel RBPs and will expand our knowledge
about cellular regulators and the underlying mechanisms of riboregulation in these
bacteria.
8.4.1 RNA-Binding Proteins
The Sm-like RNA chaperone Hfq is considered to be a key player in posttranscrip-

tional regulation of gene expression in many bacteria (Vogel and Luisi 2011).
Deletion of hfq causes pleiotropic phenotypes including impaired stress response
as well as reduced virulence in several bacterial pathogens (Chao and Vogel 2010).
In contrast to Gram-negative bacteria, Hfq seems to be dispensable for sRNA-
mediated regulation in Gram-positive bacteria (Jousselin et al. 2009). The lack of
an apparent hfq homolog in Epsilonproteobacteria suggests two things: either a
so-far unidentified RNA-binding protein replaces the role of Hfq, or sRNAs in these
bacteria act independently of an RNA chaperone by novel mechanisms compared to
enterobacterial Hfq-binding sRNAs.
Besides Hfq, the ubiquitous family of the CsrA (carbon storage regulator)/RsmA
(repressor of secondary metabolites) proteins/regulators have been shown to act as
global posttranscriptional regulators of gene expression and also virulence in
bacterial pathogens (Seyll and Van Melderen 2013; Heroven et al. 2012). The
CsrA protein mainly acts as a translational repressor by direct binding to
GGA-rich sequences in the 50 UTR of bacterial mRNAs and, thereby, controls
global physiological phenomena such as central carbon metabolism, motility,
biofilm formation, quorum sensing, and virulence (Romeo et al. 2013). A hallmark
of these systems in enterobacteria is the regulation of CsrA activity by two sRNAs,
CsrB/CsrC, which mimic the RNA substrate of CsrA and thereby sequester the
protein away from target RNAs like a sponge (Babitzke and Romeo 2007). The
CrsA homolog of Helicobacter is required for oxidative stress response, virulence
in mice infections, and full motility (Barnard et al. 2004). Reduced protein and
mRNA levels of the FlaA and FlaB flagellins were also observed in an a
nonflagellated csrA deletion mutant of H. pylori strain J99 (Kao et al. 2014).
However, different effects on morphology and flagellin expression have been
reported for other H. pylori strains, indicating that there might be a strain-specific
regulation. Neither target genes of CsrA nor homologs of the CsrA-antagonizing
sRNAs, CsrB/CsrC, have been identified in H. pylori so far. A global analysis
of RNA substrates of CsrA from C. jejuni using a combination of
co-immunoprecipitation of chromosomally epitope-tagged CsrA combined with
RNA-seq of co-purified transcripts revealed that many transcripts of the flagellum
or related to motility are bound by CsrA in C. jejuni (Dugar and Sharma
unpublished). This indicates CsrA might play a potential role in the regulation of
flagellar assembly. Moreover, CsrA was found to repress translation of the major
flagellin FlaA in C. jejuni.
In addition to CsrA, the zinc-ribbon domain-containing protein HP0958 (FlgZ)
has been described as a potential posttranscriptional regulator of motility genes in
H. pylori (Douillard et al. 2008; Ryan et al. 2005). HP0958 might act as a chaperone
for the alternative sigma factor RpoN, as it is required for its stability (Pereira and
Hoover 2005; Pereira et al. 2011). HP0958 is essential for flagellum biogenesis and
involved together with the sigma factor FliA in proper regulation of the hierarchical
expression of flagellar genes. This protein was shown to bind and stabilize the
major flagellin flaA mRNA, thereby modulating the amount of the transcript
available for translation (Douillard et al. 2008). However, the exact mechanism
and potential additional targets of HP0958 still need to be examined.
The recent findings that additional proteins with so far unknown functions or
those unrelated to RNA metabolism are involved in posttranscriptional regulation
indicate that we are far away from knowing all RNA-binding proteins and their
roles in bacteria (Pandey et al. 2011; Mitobe et al. 2011). For example, in H. pylori,
a direct interaction of the apoenzyme of aconitase (AcnB) with the 30 UTR of the
cell wall-modifying peptidoglycan deacetylase (pgdA) was shown to increase the
stability and expression of pgdA, resulting in altered in vivo survival (Austin and
Maier 2013). AcnB is a moonlighting protein in E. coli and a major enzyme of the
TCA cycle under iron-rich conditions. During iron limitation, its apo form
(apo-AcnB) favors binding to and stabilization of its own mRNA 30 UTR (Tang
and Guest 1999). Binding of AcnB to its own mRNA also prevents mRNA
degradation by the iron-regulated sRNA RyhB under low-iron conditions, despite
its repression of translation initiation through RyhB (Benjamin and Masse 2014).
Several strategies for the global investigation of RNA–protein complexes
have been developed in recent years. These include, for example,
co-immunoprecipitation of epitope-tagged RNA-binding proteins and identification
of RNA-binding partners by RNA sequencing (RIP-Seq). In Salmonella, RIP-seq

analysis of FLAG-tagged Hfq revealed various sRNAs and mRNAs that are
specifically bound and/or stabilized by this RNA chaperone (Sittka et al. 2008).
For an unbiased, genome-wide identification of novel RNA-binding proteins, an
orthogonal approach using aptamer-tagged RNAs and affinity chromatography can
be used, where recovered RNA–protein complexes are analyzed by mass spectrom-
etry (Windbichler and Schroeder 2006; Said et al. 2009). More recently, approaches
that use in vitro and in vivo ultraviolet (UV) cross-linking of RNA–protein inter-
actions and co-immunoprecipitation (CLIP) strategies have been developed that
allow for investigation of RNA–protein complexes at a high resolution and spec-
ificity (K€onig et al. 2012). In contrast to RIP-seq, these approaches are not limited
to very stable RNA–protein (RNP) complexes and less prone to nonspecific inter-
actions. In H. pylori, several strategies for the isolation and analysis of RNP
complexes have been established (Rieder et al. 2012). Using either affinity purifi-
cation of aptamer-tagged RNAs or RIP-seq, RNA–protein interactions between the
ribosomal proteins S1 and various mRNAs as well as sRNAs were identified in
H. pylori strain 26695. Ribosomal protein S1 might be a candidate protein that does
not only act to facilitate translation initiation but might also act as a general RNA
chaperone (Hajnsdorf and Boni 2012). Moreover, HP1334, a protein of unknown
function, was shown to specifically interact with HPnc6910 and stabilize expression
of this abundant sRNA (Rieder et al. 2012). Therefore, Helicobacter proteins of
unknown function or those associated with other functions such as bacterial mem-
brane binding, translation, cell cycle, or virulence could potentially bind RNA and
play a role in posttranscriptional regulation.
8.4.2 Ribonucleases
In enterobacteria, sRNA-mediated target-mRNA decay mainly depends on the

RNA degradosome, a protein complex composed of an endoribonuclease (RNase
E), a 30 –50 polynucleotide phosphorylase (PNPase), an enolase and an RNA helicase
(RhlB) (Morita et al. 2005; Caron et al. 2010). Although the exact mechanism of
repression is still unclear, both functionally characterized antisense RNAs in
Helicobacter (RepG and 50 ureB-sRNA; Fig. 8.3b, c) cause reduced protein levels,
most likely due to transcript destabilization or active recruitment of RNases upon
sRNA–target mRNA interaction. As all Epsilonproteobacteria appear to lack an
RNase E homolog, it is unclear whether and which endoribonuclease participates
in sRNA-mediated mRNA cleavage in these bacteria. Recent studies in B. subtilis
and Staphylococcus aureus identified two functional RNase E orthologs in the
RNA degradosome of Gram-positive bacteria, RNase J1 and RNAse J2 (Mathy
et al. 2010; Roux et al. 2011). Furthermore, in S. aureus the double-strand-specific
RNase III degrades, mainly in concert with the sRNA, RNAIII, several mRNAs
encoding virulence factors (Huntzinger et al. 2005; Chevalier et al. 2008; Boisset
et al. 2007; Lioliou et al. 2012). Only nine ribonucleases have been annotated in
Table 8.1 Homologs of ribonucleases and RNA degradation enzymes in Helicobacter pylori
Homolog in
H. pylori
RNase strain 26695 Activity or substrate Essentiala References
RNase Y HP0760 Endoribonuclease No
RNase R/II HP1248 30 –50 exonuclease No Tsao
et al. (2009)
YbeY HP1160 Putative endoribonuclease/ Yes
RNA chaperone
PNPase HP1213 30 –50 exonuclease No
RNase J HP1430 50 P RNA, exo- (50 –30 ) and Yes Redko
endoribonuclease et al. (2013)
RNase III HP0662 Double-stranded RNA No, but strain
specific
RNase H1 HP0661 RNA in RNA/DNA hybrid No
RNase H2 HP1323 RNA in RNA/DNA hybrid n.d.
RNase P HP1448 50 end of pre-tRNA n.d.
(protein)
HP0268 DNA nicking endonuclease/ Yes Lee
RNase activity et al. (2015)
RppH HP1228 putative RNA pyrophospho- No
hydrolase, 50 PPP RNA
No homolog has been identified so far for RNase E, G, T, Z, D, and PH
n.d. not determined
a
Based on unpublished results from the Darfeuille/Sharma labs if not indicated otherwise
H. pylori so far, including potential homologs of RNase J and RNase III (Table 8.1).
In Helicobacter, most of the mRNA degradation seems to be carried out by a
minimal RNA degradosome consisting of a homolog of RNase J and the only
DExD-box RNA helicase of H. pylori, RhpA (Redko et al. 2013). The biochemical
characterization of a purified recombinant enzyme showed that it contains both 50 –
30 exonucleolytic and endonucleolytic activity similar to its B. subtilis ortholog
(Dorleans et al. 2011). In addition to RNase J, H. pylori also contains a potential
homolog of the RNA pyrophosphohydrolase RppH also known as NudA, (Lundin
et al. 2003), which removes 50 -terminal phosphates to generate a full-length
mRNA intermediate that is monophosphorylated and therefore vulnerable to rapid
50 -exonucleolytic digestion by RNase J (Richards et al. 2011). In general, H. pylori
seems to possess the majority of the key enzymes (RNase J, RNase Y, PNPase, and
RNase III) that are known to be involved in mRNA degradation in the Gram-
positive B. subtilis (Condon 2010). In line with their important function in the
RNA metabolism of Gram-positive bacteria, H. pylori RNase J and RNase III are
essential in the majority of H. pylori strains (Redko et al. 2013; Iost and Darfeuille
unpublished). A large number of processes that occur during sRNA-mediated decay
and rRNA maturation await characterization and assignment to specific enzymes or
encoding genes. For example, the highly conserved bacterial protein, YbeY, may
play a major, previously unrecognized role in bacterial sRNA regulation (Pandey

et al. 2011, 2014), and its deletion has been described to sensitize cells to various
stresses and affect rRNA biosynthesis, translational fidelity, and ribosome assembly
in E. coli and Sinorhizobium meliloti (Davies et al. 2010; Rasouly et al. 2009, 2010;
Jacob et al. 2013; Grinwald and Ron 2013). Furthermore, there are strong genetic
interactions in functions of ybeY and other genes encoding exoribonucleases such as
RNase R and PNPase (Davies et al. 2010). Interestingly, the ybeY gene is essential
in H. pylori (Iost and Darfeuille unpublished). In contrast, the two 30 –50
exoribonucleases RNase R and PNPase are not essential, but they have been
found to play an important role in controlling virulent gene expression in
H. pylori (Rosenzweig and Chopra 2013; Tsao et al. 2009). Similarly, inactivation
of the pnp gene in C. jejuni significantly altered its ability to adhere and invade
gastrointestinal epithelial cells and, thus, affect chicken colonization (Haddad
et al. 2012). Surprisingly, deletion of this exoribonuclease only modified the
abundance of a specific set of proteins involved in virulence and motility. More-
over, the 30 –50 exoribonuclease RNase R has been shown to posttranscriptionally
downregulate six virulence-related genes of H. pylori (Tsao et al. 2009). Whether or
not RNase R is also involved in sRNA-mediated regulation needs to be clarified.
The dRNA-seq-based analysis of the H. pylori primary transcriptome revealed a

surprisingly complex transcriptional output from its small genome. The discovery
of an unexpected wealth of sRNAs, as well as extensive antisense transcription,
indicates a global level of posttranscriptional gene expression control in this
pathogenic Epsilonproteobacterium, which might complement its relatively modest
repertoire of protein transcriptional regulators. The ongoing functional characteri-
zation of cis- and trans-acting sRNA candidates along with the identification of
RNA-binding proteins and RNases will help to unveil their regulatory functions and
role in H. pylori virulence control and stress response. As shown for the trans-
acting RepG sRNA, such studies can reveal novel mechanisms of posttranscrip-
tional regulation independent of the RNA chaperone Hfq, and will expand our
knowledge about riboregulation in emerging human pathogens.
Besides mechanistic aspects of riboregulation, research on sRNAs and antisense
transcripts in H. pylori will help to understand phenotypes observed in previous
work, including genetic screens. Global transposon screens for virulence and
colonization factors revealed about 220 candidate mutants with a colonization
defect in mice (Baldwin et al. 2007). Several mutants showed a transposon hit at
the end of genes and intergenic regions, indicating that regulatory RNA elements
such as riboswitches or cis- and trans-encoded sRNAs might be important for
H. pylori virulence. Moreover, several virulence-associated genes could be under
posttranscriptional control.
The study of riboregulation in H. pylori will also help to understand common

themes of posttranscriptional regulation in pathogenic Epsilonproteobacteria,
including Campylobacter spp. First sRNA candidates have also been identified in
C. jejuni NCTC11168 based on RNA-seq (Chaudhuri et al. 2011; Porcelli
et al. 2013; Taveirne et al. 2013). Furthermore, a comparative dRNA-seq analysis
of multiple C. jejuni strains indicates that many of the sRNAs are conserved among
C. jejuni strains (Dugar et al. 2013). However, some of these conserved sRNAs
display distinct expression patterns in certain strains, indicating that they could
have varying, strain-specific functions. This comparative approach revealed also
strain-specific sRNAs and transcriptional output based on point mutations in pro-
moter regions, which might underlie phenotypic differences among strains. With
regard to the high genotypic and phenotypic diversity of H. pylori strains, such a
comparative approach between different H. pylori isolates might help to understand
differences among strains at the transcriptome level. A cross-species comparison of
the primary transcriptomes of C. jejuni and H. pylori revealed a lack of conserva-
tion of operon organization, position of intragenic and antisense promoters, and
leaderless mRNAs, indicating that regulatory features may have evolved after these
species split from a common ancestor (Porcelli et al. 2013). While Helicobacter and
Campylobacter seem to have evolved their own specific sRNAs, they harbor similar
capacity for riboregulation compared to E. coli, respective to their smaller genome
sizes.
Next-generation sequencing methods have proven to be an effective tool for
uncovering transcriptome structure and identifying novel transcripts in prokaryotes
as well as for single-nucleotide resolution gene expression profiling (Croucher and
Thomson 2010; Sorek and Cossart 2010; Sharma and Vogel 2014). A full under-
standing of the infection process requires the investigation of gene expression
changes in both the pathogen and the host. Parallel sequencing of infection samples
(in vivo or in vitro) without physical separation of host and bacterial RNA, followed
by mapping of cDNA reads to the host and pathogen genome (dual RNA-seq), will
allow for simultaneous analysis of gene expression changes in host and pathogen
(Westermann et al. 2012). Accordingly, RNA-seq could provide a powerful tool in
analyzing transcriptomes from biopsy samples of fresh clinical H. pylori isolates
from human patients with different clinical outcomes of infection. Furthermore, the
investigation of single-cell transcriptomes using RNA-seq is emerging as a power-
ful tool to profile cell-to-cell variations within bacterial or host cell populations
(Saliba et al. 2014). Overall, in-depth analysis of transcriptional output and its
functional consequences, including sRNA functions, in H. pylori, as well as its
cousin C. jejuni, together with parallel transcriptome analysis during the time
course of infection will help to shed light on posttranscriptional regulation and
virulence mechanisms not only in Epsilonproteobacteria but also other bacterial
pathogens, including species that lack Hfq.
References
Akada JK, Shirai M, Takeuchi H, Tsuda M, Nakazawa T (2000) Identification of the urease operon
in Helicobacter pylori and its control by mRNA decay in response to pH. Mol Microbiol
36:1071–1084
Argaman L, Hershberg R, Vogel J, Bejerano G, Wagner EG, Margalit H, Altuvia S (2001) Novel
small RNA-encoding genes in the intergenic regions of Escherichia coli. Curr Biol 11:941–950
Austin CM, Maier RJ (2013) Aconitase-mediated posttranscriptional regulation of Helicobacter
pylori peptidoglycan deacetylase. J Bacteriol 195:5316–5322
Babitzke P, Romeo T (2007) CsrB sRNA family: sequestration of RNA-binding regulatory pro-
teins. Curr Opin Microbiol 10:156–163
Backofen R, Hess WR (2010) Computational prediction of sRNAs and their targets in bacteria.
RNA Biol 7:33–42
Baldwin DN, Shepherd B, Kraemer P, Hall MK, Sycuro LK, Pinto-Santini DM, Salama NR (2007)
Identification of Helicobacter pylori genes that contribute to stomach colonization. Infect
Immun 75:1005–1016
Barnard FM, Loughlin MF, Fainberg HP, Messenger MP, Ussery DW, Williams P, Jenks PJ
(2004) Global regulation of virulence and the stress response by CsrA in the highly adapted
human gastric pathogen Helicobacter pylori. Mol Microbiol 51:15–32
Barrick JE, Sudarsan N, Weinberg Z, Ruzzo WL, Breaker RR (2005) 6S RNA is a widespread
regulator of eubacterial RNA polymerase that resembles an open promoter. RNA 11:774–784
Barquist L, Vogel J (2015) Accelerating discovery and functional analysis of small RNAs with
new technologies. Annu Rev Genet 49:367–394
Benjamin JA, Masse E (2014) The iron-sensing aconitase B binds its own mRNA to prevent
sRNA-induced mRNA cleavage. Nucleic Acids Res 42:10023–10036
Bensing BA, Meyer BJ, Dunny GM (1996) Sensitive detection of bacterial transcription initiation
sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer
system of Enterococcus faecalis. Proc Natl Acad Sci U S A 93:7794–7799
Berk AJ, Sharp PA (1977) Sizing and mapping of early adenovirus mRNAs by gel electrophoresis
of S1 endonuclease-digested hybrids. Cell 12:721–732
Bischler T, Tan HS, Nieselt K, Sharma CM (2015) Differential RNA-seq (dRNA-seq) for
annotation of transcriptional start sites and small RNAs in Helicobacter pylori. Methods
86:89–101
Boisset S, Geissmann T, Huntzinger E, Fechter P, Bendridi N, Possedko M, Chevalier C, Helfer
AC, Benito Y, Jacquier A, Gaspin C, Vandenesch F, Romby P (2007) Staphylococcus aureus
RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator
Rot by an antisense mechanism. Genes Dev 21:1353–1366
Borries A, Vogel J, Sharma CM (2010) Differential RNA sequencing (dRNA-seq): deep-sequenc-
ing based analysis of primary transcriptomes. In: Harbers M, Kahl G (eds) Tag-based
approaches for next-generation sequencing. Wiley-Blackwell-VCH, Hoboken
Brantl S (2007) Regulatory mechanisms employed by cis-encoded antisense RNAs. Curr Opin
Burgess RR, Anthony L (2001) How sigma docks to RNA polymerase and what sigma does. Curr
Opin Microbiol 4:126–131
Bury-Mone S, Thiberge JM, Contreras M, Maitournam A, Labigne A, De Reuse H (2004)
Butcher J, Stintzi A (2013) The transcriptional landscape of Campylobacter jejuni under iron
replete and iron limited growth conditions. PLoS One 8:e79475
Caron MP, Lafontaine DA, Masse E (2010) Small RNA-mediated regulation at the level of
transcript stability. RNA Biol 7:140–144
Cavanagh AT, Wassarman KM (2014) 6S RNA, a global regulator of transcription in Escherichia
coli, Bacillus subtilis, and beyond. Annu Rev Microbiol 68:45–60
Chao Y, Vogel J (2010) The role of Hfq in bacterial pathogens. Curr Opin Microbiol 13:24–33
Chaudhuri RR, Yu L, Kanji A, Perkins TT, Gardner PP, Choudhary J, Maskell DJ, Grant AJ (2011)
Quantitative RNA-seq analysis of the transcriptome of Campylobacter jejuni. Microbiology
157:2922–2932
Chevalier C, Huntzinger E, Fechter P, Boisset S, Vandenesch F, Romby P, Geissmann T (2008)
Staphylococcus aureus endoribonuclease III purification and properties. Methods Enzymol
447:309–327
Cho BK, Zengler K, Qiu Y, Park YS, Knight EM, Barrett CL, Gao Y, Palsson BO (2009) The
transcription unit architecture of the Escherichia coli genome. Nat Biotechnol 27:1043–1049
Condon C (2010) What is the role of RNase J in mRNA turnover? RNA Biol 7:316–321
Croucher NJ, Thomson NR (2010) Studying bacterial transcriptomes using RNA-seq. Curr Opin
Croxen MA, Sisson G, Melano R, Hoffman PS (2006) The Helicobacter pylori chemotaxis
receptor TlpB (HP0103) is required for pH taxis and for colonization of the gastric mucosa.
Croxen MA, Ernst PB, Hoffman PS (2007) Antisense RNA modulation of alkyl hydroperoxide
reductase levels in Helicobacter pylori correlates with organic peroxide toxicity but not
infectivity. J Bacteriol 189:3359–3368
Danielli A, Scarlato V (2010) Regulatory circuits in Helicobacter pylori: network motifs and
regulators involved in metal-dependent responses. FEMS Microbiol Rev 34:738–752
Danielli A, Amore G, Scarlato V (2010) Built shallow to maintain homeostasis and persistent
infection: insight into the transcriptional regulatory network of the gastric human pathogen
Helicobacter pylori. PLoS Pathog 6:e1000938
Davies BW, Kohrer C, Jacob AI, Simmons LA, Zhu J, Aleman LM, Rajbhandary UL, Walker GC
(2010) Role of Escherichia coli YbeY, a highly conserved protein, in rRNA processing. Mol
Dorleans A, Li de la Sierra-Gallay I, Piton J, Zig L, Gilet L, Putzer H, Condon C (2011) Molecular
basis for the recognition and cleavage of RNA by the bifunctional 50 –30 exo/endoribonuclease
RNase J. Structure 19:1252–1261
Douillard FP, Ryan KA, Caly DL, Hinds J, Witney AA, Husain SE, O’Toole PW (2008)
Posttranscriptional regulation of flagellin synthesis in Helicobacter pylori by the RpoN chap-
erone HP0958. J Bacteriol 190:7975–7984
Dugar G, Herbig A, Forstner KU, Heidrich N, Reinhardt R, Nieselt K, Sharma CM (2013) High-
resolution transcriptome maps reveal strain-specific regulatory features of multiple campylo-
bacter jejuni isolates. PLoS Genet 9:e1003495
Faucher SP, Friedlander G, Livny J, Margalit H, Shuman HA (2010) Legionella pneumophila 6S
RNA optimizes intracellular multiplication. Proc Natl Acad Sci U S A 107:7533–7538
Forsyth MH, Cover TL (1999) Mutational analysis of the vacA promoter provides insight into
gene transcription in Helicobacter pylori. J Bacteriol 181:2261–2266
Fozo EM, Hemm MR, Storz G (2008a) Small toxic proteins and the antisense RNAs that repress
them. Microbiol Mol Biol Rev 72:579–589, Table of Contents
Fozo EM, Kawano M, Fontaine F, Kaya Y, Mendieta KS, Jones KL, Ocampo A, Rudd KE, Storz G
(2008b) Repression of small toxic protein synthesis by the Sib and OhsC small RNAs. Mol
Frohlich KS, Vogel J (2009) Activation of gene expression by small RNA. Curr Opin Microbiol
12:674–682
Georg J, Hess WR (2011) cis-antisense RNA, another level of gene regulation in bacteria.
Microbiol Mol Biol Rev 75:286–300
Grinwald M, Ron EZ (2013) The Escherichia coli translation-associated heat shock protein YbeY
is involved in rRNA transcription antitermination. PLoS One 8:e62297
Guell M, van Noort V, Yus E, Chen WH, Leigh-Bell J, Michalodimitrakis K, Yamada T,
Arumugam M, Doerks T, Kuhner S, Rode M, Suyama M, Schmidt S, Gavin AC, Bork P,
Serrano L (2009) Transcriptome complexity in a genome-reduced bacterium. Science

326:1268–1271
Haddad N, Tresse O, Rivoal K, Chevret D, Nonglaton Q, Burns CM, Prevost H, Cappelier JM
(2012) Polynucleotide phosphorylase has an impact on cell biology of Campylobacter jejuni.
Front Cell Infect Microbiol 2:30
Hajnsdorf E, Boni IV (2012) Multiple activities of RNA-binding proteins S1 and Hfq. Biochimie
94:1544–1553
Heidrich N, Dugar G, Vogel J, Sharma CM (2015) Investigating CRISPR RNA biogenesis and
function using RNA-seq. Methods Mol Biol 1311:1–21
Heroven AK, Bohme K, Dersch P (2012) The Csr/Rsm system of Yersinia and related pathogens: a
post-transcriptional strategy for managing virulence. RNA Biol 9:379–391
Huntzinger E, Boisset S, Saveanu C, Benito Y, Geissmann T, Namane A, Lina G, Etienne J,
Ehresmann B, Ehresmann C, Jacquier A, Vandenesch F, Romby P (2005) Staphylococcus
aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO
J 24:824–835
Jacob AI, Kohrer C, Davies BW, RajBhandary UL, Walker GC (2013) Conserved bacterial RNase
YbeY plays key roles in 70S ribosome quality control and 16S rRNA maturation. Mol Cell
49:427–438
Josenhans C, Beier D, Linz B, Meyer TF, Suerbaum S (2007) Pathogenomics of helicobacter. Int J
Med Microbiol 297:589–600
Jousselin A, Metzinger L, Felden B (2009) On the facultative requirement of the bacterial RNA
chaperone, Hfq. Trends Microbiol 17:399–405
Kao CY, Sheu BS, Wu JJ (2014) CsrA regulates helicobacter pylori J99 motility and adhesion by
controlling flagella formation. Helicobacter 19:443–454
K€onig J, Zarnack K, Luscombe NM, Ule J (2012) Protein-RNA interactions: new genomic
technologies and perspectives. Nat Rev Genet 13:77–83
Kortmann J, Narberhaus F (2012) Bacterial RNA thermometers: molecular zippers and switches.
Nat Rev Microbiol 10:255–265
Lee KY, Lee KY, Kim JH, Lee IG, Lee SH, Sim DW, Won HS, Lee BJ (2015) Structure-based
functional identification of Helicobacter pylori HP0268 as a nuclease with both DNA nicking
and RNase activities. Nucleic Acids Res 43:5194–5207
Lertsethtakarn P, Ottemann KM, Hendrixson DR (2011) Motility and chemotaxis in Campylo-
bacter and Helicobacter. Annu Rev Microbiol 65:389–410
Lin YF, A DR, Guan S, Mamanova L, McDowall KJ (2013) A combination of improved
differential and global RNA-seq reveals pervasive transcription initiation and events in all
stages of the life-cycle of functional RNAs in Propionibacterium acnes, a major contributor to
wide-spread human disease. BMC Genomics 14:620
Lioliou E, Sharma CM, Caldelari I, Helfer AC, Fechter P, Vandenesch F, Vogel J, Romby P (2012)
Global regulatory functions of the Staphylococcus aureus endoribonuclease III in gene expres-
sion. PLoS Genet 8:e1002782
Livny J, Brencic A, Lory S, Waldor MK (2006) Identification of 17 Pseudomonas aeruginosa
sRNAs and prediction of sRNA-encoding genes in 10 diverse pathogens using the bioinfor-
matic tool sRNAPredict2. Nucleic Acids Res 34:3484–3493
Lundin A, Nilsson C, Gerhard M, Andersson DI, Krabbe M, Engstrand L (2003) The NudA protein
in the gastric pathogen Helicobacter pylori is an ubiquitous and constitutively expressed
dinucleoside polyphosphate hydrolase. J Biol Chem 278:12574–12578
Mao F, Dam P, Chou J, Olman V, Xu Y (2009) DOOR: a database for prokaryotic operons.
Nucleic Acids Res 37:D459–D463
Mathy N, Hebert A, Mervelet P, Benard L, Dorleans A, Li de la Sierra-Gallay I, Noirot P,
Putzer H, Condon C (2010) Bacillus subtilis ribonucleases J1 and J2 form a complex with
altered enzyme behaviour. Mol Microbiol 75:489–498
McGee DJ, Langford ML, Watson EL, Carter JE, Chen YT, Ottemann KM (2005) Colonization
and inflammation deficiencies in Mongolian gerbils infected by Helicobacter pylori chemo-
taxis mutants. Infect Immun 73:1820–1827
Mendoza-Vargas A, Olvera L, Olvera M, Grande R, Vega-Alvarado L, Taboada B, Jimenez-
Jacinto V, Salgado H, Juarez K, Contreras-Moreira B, Huerta AM, Collado-Vides J, Morett E
(2009) Genome-wide identification of transcription start sites, promoters and transcription
factor binding sites in E. coli. PLoS One 4:e7526
Mitarai N, Andersson AM, Krishna S, Semsey S, Sneppen K (2007) Efficient degradation and
expression prioritization with small RNAs. Phys Biol 4:164–171
Mitobe J, Yanagihara I, Ohnishi K, Yamamoto S, Ohnishi M, Ishihama A, Watanabe H (2011)
RodZ regulates the post-transcriptional processing of the Shigella sonnei type III secretion
system. EMBO Rep 12:911–916
Moll I, Grill S, Gualerzi CO, Blasi U (2002) Leaderless mRNAs in bacteria: surprises in ribosomal
recruitment and translational control. Mol Microbiol 43:239–246
Moll I, Hirokawa G, Kiel MC, Kaji A, Blasi U (2004) Translation initiation with 70S ribosomes:
an alternative pathway for leaderless mRNAs. Nucleic Acids Res 32:3354–3363
Morita T, Maki K, Aiba H (2005) RNase E-based ribonucleoprotein complexes: mechanical basis
of mRNA destabilization mediated by bacterial noncoding RNAs. Genes Dev 19:2176–2186
Moxon R, Bayliss C, Hood D (2006) Bacterial contingency loci: the role of simple sequence DNA
repeats in bacterial adaptation. Annu Rev Genet 40:307–333
Pandey SP, Minesinger BK, Kumar J, Walker GC (2011) A highly conserved protein of unknown
function in Sinorhizobium meliloti affects sRNA regulation similar to Hfq. Nucleic Acids Res
39:4691–4708
Pandey SP, Winkler JA, Li H, Camacho DM, Collins JJ, Walker GC (2014) Central role for RNase
YbeY in Hfq-dependent and Hfq-independent small-RNA regulation in bacteria. BMC Geno-
mics 15:121
Papenfort K, Vogel J (2010) Regulatory RNA in bacterial pathogens. Cell Host Microbe
8:116–127
Passalacqua KD, Varadarajan A, Weist C, Ondov BD, Byrd B, Read TD, Bergman NH (2012)
Strand-specific RNA-seq reveals ordered patterns of sense and antisense transcription in
Bacillus anthracis. PLoS One 7:e43350
Pereira L, Hoover TR (2005) Stable accumulation of sigma54 in Helicobacter pylori requires the
novel protein HP0958. J Bacteriol 187:4463–4469
Pereira LE, Tsang J, Mrazek J, Hoover TR (2011) The zinc-ribbon domain of Helicobacter pylori
HP0958: requirement for RpoN accumulation and possible roles of homologs in other bacteria.
Microb Inform Exp 1:1–10
Pernitzsch SR, Tirier SM, Beier D, Sharma CM (2014) A variable homopolymeric G-repeat
defines small RNA-mediated posttranscriptional regulation of a chemotaxis receptor in
Helicobacter pylori. Proc Natl Acad Sci U S A 111:E501–E510
Petersen L, Larsen TS, Ussery DW, On SL, Krogh A (2003) RpoD promoters in Campylobacter
jejuni exhibit a strong periodic signal instead of a -35 box. J Mol Biol 326:1361–1372
Porcelli I, Reuter M, Pearson BM, Wilhelm T, van Vliet AH (2013) Parallel evolution of genome
structure and transcriptional landscape in the Epsilonproteobacteria. BMC Genomics 14:616
Rader BA, Wreden C, Hicks KG, Sweeney EG, Ottemann KM, Guillemin K (2011) Helicobacter
pylori perceives the quorum-sensing molecule AI-2 as a chemorepellent via the chemoreceptor
TlpB. Microbiology 157:2445–2455
Ramakrishnan V (2002) Ribosome structure and the mechanism of translation. Cell 108:557–572
Rasouly A, Schonbrun M, Shenhar Y, Ron EZ (2009) YbeY, a heat shock protein involved in
translation in Escherichia coli. J Bacteriol 191:2649–2655
Rasouly A, Davidovich C, Ron EZ (2010) The heat shock protein YbeY is required for optimal
activity of the 30S ribosomal subunit. J Bacteriol 192:4592–4596
Redko Y, Aubert S, Stachowicz A, Lenormand P, Namane A, Darfeuille F, Thibonnier M, De

Reuse H (2013) A minimal bacterial RNase J-based degradosome is associated with translating
ribosomes. Nucleic Acids Res 41:288–301
Richards J, Liu Q, Pellegrini O, Celesnik H, Yao S, Bechhofer DH, Condon C, Belasco JG (2011)
An RNA pyrophosphohydrolase triggers 50 -exonucleolytic degradation of mRNA in Bacillus
subtilis. Mol Cell 43:940–949
Rieder R, Reinhardt R, Sharma CM, Vogel J (2012) Experimental tools to identify RNA-protein
interactions in Helicobacter pylori. RNA Biol 9:520–531
Rodionov DA, Vitreschak AG, Mironov AA, Gelfand MS (2002) Comparative genomics of
thiamin biosynthesis in procaryotes. New genes and regulatory mechanisms. J Biol Chem
277:48949–48959
Romeo T, Vakulskas CA, Babitzke P (2013) Post-transcriptional regulation on a global scale: form
and function of Csr/Rsm systems. Environ Microbiol 15:313–324
Rosenzweig JA, Chopra AK (2013) The exoribonuclease polynucleotide phosphorylase influences
the virulence and stress responses of yersiniae and many other pathogens. Front Cell Infect
Microbiol 3:81
Roux CM, DeMuth JP, Dunman PM (2011) Characterization of components of the Staphylococcus
aureus mRNA degradosome holoenzyme-like complex. J Bacteriol 193:5520–5526
Ryan KA, Karim N, Worku M, Moore SA, Penn CW, O’Toole PW (2005) HP0958 is an essential
motility gene in Helicobacter pylori. FEMS Microbiol Lett 248:47–55
Sachs G, Weeks DL, Melchers K, Scott DR (2003) The gastric biology of Helicobacter pylori.
Annu Rev Physiol 65:349–369
Said N, Rieder R, Hurwitz R, Deckert J, Urlaub H, Vogel J (2009) In vivo expression and
purification of aptamer-tagged small RNA regulators. Nucleic Acids Res 37:e133
Salama NR, Hartung ML, Muller A (2013) Life in the human stomach: persistence strategies of the
bacterial pathogen Helicobacter pylori. Nat Rev Microbiol 11:385–399
Salgado H, Peralta-Gil M, Gama-Castro S, Santos-Zavaleta A, Muniz-Rascado L, Garcia-Sotelo
JS, Weiss V, Solano-Lira H, Martinez-Flores I, Medina-Rivera A, Salgado-Osorio G,
Alquicira-Hernandez S, Alquicira-Hernandez K, Lopez-Fuentes A, Porron-Sotelo L, Huerta
AM, Bonavides-Martinez C, Balderas-Martinez YI, Pannier L, Olvera M, Labastida A,
Jimenez-Jacinto V, Vega-Alvarado L, Del Moral-Chavez V, Hernandez-Alvarez A,
Morett E, Collado-Vides J (2013) RegulonDB v8.0: omics data sets, evolutionary conserva-
tion, regulatory phrases, cross-validated gold standards and more. Nucleic Acids Res 41:D203–
D213
Saliba AE, Westermann AJ, Gorski SA, Vogel J (2014) Single-cell RNA-seq: advances and future
challenges. Nucleic Acids Res 42:8845–8860
Serganov A, Nudler E (2013) A decade of riboswitches. Cell 152:17–24
Sesto N, Wurtzel O, Archambaud C, Sorek R, Cossart P (2013) The excludon: a new concept in
bacterial antisense RNA-mediated gene regulation. Nat Rev Microbiol 11:75–82
Seyll E, Van Melderen L (2013) The ribonucleoprotein Csr network. Int J Mol Sci
14:22117–22131
Sharma CM, Vogel J (2009) Experimental approaches for the discovery and characterization of
regulatory small RNA. Curr Opin Microbiol 12:536–546
Sharma CM, Vogel J (2014) Differential RNA-seq: the approach behind and the biological insight
gained. Curr Opin Microbiol 19C:97–105
Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S, Sittka A, Chabas S, Reiche K,
Hackermuller J, Reinhardt R, Stadler PF, Vogel J (2010) The primary transcriptome of the
major human pathogen Helicobacter pylori. Nature 464:250–255
Sharwood RE, Hotto AM, Bollenbach TJ, Stern DB (2011) Overaccumulation of the chloroplast
antisense RNA AS5 is correlated with decreased abundance of 5S rRNA in vivo and inefficient
5S rRNA maturation in vitro. RNA 17:230–243
Shirai M, Fujinaga R, Akada JK, Nakazawa T (1999) Activation of Helicobacter pylori ureA
promoter by a hybrid Escherichia coli-H. pylori rpoD gene in E. coli. Gene 239:351–359
Singh N, Wade JT (2014) Identification of regulatory RNA in bacterial genomes by genome-scale

mapping of transcription start sites. Methods Mol Biol 1103:1–10
Sittka A, Lucchini S, Papenfort K, Sharma CM, Rolle K, Binnewies TT, Hinton JC, Vogel J (2008)
Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-
transcriptional regulator, Hfq. PLoS Genet 4:e1000163
Sorek R, Cossart P (2010) Prokaryotic transcriptomics: a new view on regulation, physiology and
pathogenicity. Nat Rev Genet 11:9–16
Spohn G, Scarlato V (1999) Motility of Helicobacter pylori is coordinately regulated by the
transcriptional activator FlgR, an NtrC homolog. J Bacteriol 181:593–599
Spohn G, Beier D, Rappuoli R, Scarlato V (1997) Transcriptional analysis of the divergent cagAB
genes encoded by the pathogenicity island of Helicobacter pylori. Mol Microbiol 26:361–372
Storz G, Vogel J, Wassarman KM (2011) Regulation by small RNAs in bacteria: expanding
frontiers. Mol Cell 43:880–891
Tang Y, Guest JR (1999) Direct evidence for mRNA binding and post-transcriptional regulation
by Escherichia coli aconitases. Microbiology 145(Pt 11):3069–3079
Taveirne ME, Theriot CM, Livny J, DiRita VJ (2013) The complete Campylobacter jejuni
transcriptome during colonization of a natural host determined by RNAseq. PLoS One 8:
e73586
Thibonnier M, Thiberge JM, De Reuse H (2008) Trans-translation in Helicobacter pylori: essen-
tiality of ribosome rescue and requirement of protein tagging for stress resistance and compe-
tence. PLoS One 3:e3810
Thibonnier M, Aubert S, Ecobichon C, De Reuse H (2010) Study of the functionality of the
Helicobacter pylori trans-translation components SmpB and SsrA in an heterologous system.
BMC Microbiol 10:91
Thomason MK, Storz G (2010) Bacterial antisense RNAs: how many are there, and what are they
doing? Annu Rev Genet 44:167–188
Thompson JA, Radonovich MF, Salzman NP (1979) Characterization of the 50 -terminal structure
of simian virus 40 early mRNA’s. J Virol 31:437–446
Thompson LJ, Merrell DS, Neilan BA, Mitchell H, Lee A, Falkow S (2003) Gene expression
profiling of Helicobacter pylori reveals a growth-phase-dependent switch in virulence gene
expression. Infect Immun 71:2643–2655
Tsao MY, Lin TL, Hsieh PF, Wang JT (2009) The 30 -to-50 exoribonuclease (encoded by HP1248)
of Helicobacter pylori regulates motility and apoptosis-inducing genes. J Bacteriol
191:2691–2702
Vogel J, Luisi BF (2011) Hfq and its constellation of RNA. Nat Rev Microbiol 9:578–589
Vogel J, Bartels V, Tang TH, Churakov G, Slagter-Jager JG, Huttenhofer A, Wagner EG (2003)
RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional
output in bacteria. Nucleic Acids Res 31:6435–6443
Wade JT, Grainger DC (2014) Pervasive transcription: illuminating the dark matter of bacterial
transcriptomes. Nat Rev Microbiol 12:647–653
Wagner EG, Unoson C (2012) The toxin-antitoxin system tisB-istR1: expression, regulation, and
biological role in persister phenotypes. RNA Biol 9:1513–1519
Wagner EG, Altuvia S, Romby P (2002) Antisense RNAs in bacteria and their genetic elements.
Adv Genet 46:361–398
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat
Rev Genet 10:57–63
Wassarman KM, Saecker RM (2006) Synthesis-mediated release of a small RNA inhibitor of RNA
polymerase. Science 314:1601–1603
Waters LS, Storz G (2009) Regulatory RNAs in bacteria. Cell 136:615–628
Wen Y, Marcus EA, Matrubutham U, Gleeson MA, Scott DR, Sachs G (2003) Acid-adaptive
genes of Helicobacter pylori. Infect Immun 71:5921–5939
Wen Y, Feng J, Scott DR, Marcus EA, Sachs G (2011) A cis-encoded antisense small RNA
regulated by the HP0165-HP0166 two-component system controls expression of ureB in
Helicobacter pylori. J Bacteriol 193:40–51
Wen Y, Feng J, Sachs G (2013) Helicobacter pylori 50 ureB-sRNA, a cis-encoded antisense small
RNA, negatively regulates ureAB expression by transcription termination. J Bacteriol
195:444–452
Westermann AJ, Gorski SA, Vogel J (2012) Dual RNA-seq of pathogen and host. Nat Rev
Williams SM, Chen YT, Andermann TM, Carter JE, McGee DJ, Ottemann KM (2007)
Helicobacter pylori chemotaxis modulates inflammation and bacterium-gastric epithelium
interactions in infected mice. Infect Immun 75:3747–3757
Windbichler N, Schroeder R (2006) Isolation of specific RNA-binding proteins using the
streptomycin-binding RNA aptamer. Nat Protoc 1:637–640
Wosten MM, Boeve M, Koot MG, van Nuenen AC, van der Zeijst BA (1998) Identification of
Campylobacter jejuni promoter sequences. J Bacteriol 180:594–599
Wurtzel O, Sapra R, Chen F, Zhu Y, Simmons BA, Sorek R (2010) A single-base resolution map
of an archaeal transcriptome. Genome Res 20:133–141
Xiao B, Li W, Guo G, Li B, Liu Z, Jia K, Guo Y, Mao X, Zou Q (2009a) Identification of small
noncoding RNAs in Helicobacter pylori by a bioinformatics-based approach. Curr Microbiol
58:258–263
Xiao B, Li W, Guo G, Li BS, Liu Z, Tang B, Mao XH, Zou QM (2009b) Screening and
identification of natural antisense transcripts in Helicobacter pylori by a novel approach
based on RNase I protection assay. Mol Biol Rep 36:1853–1858
Chapter 9
Genome Evolution: Helicobacter pylori
as an Extreme Model
Ichizo Kobayashi
Abstract Helicobacter pylori strains have quite diverse genome sequences likely
because of high mutation and recombination rates, and they may be a useful model
to study genetics and evolution. Here, I discuss some features of their evolution
that have emerged from comparative analyses of complete H. pylori genomes
and methylomes. Emphasis will be placed on the roles of various modes of
recombination.
The evolution of their chromosome synteny was reconstructed by analyzing
inversions via four mechanisms: recombination events involving long or short
sequence similarities, inversions adjacent to the insertion of a mobile element,
and DNA duplications associated with inversions, a novel process of DNA
duplication.
Phylogenetic trees of individual genes are often different from that of the core
genome in size and topology, partly due to homologous recombination between
lineages. The fine population structure of the species was inferred from an analysis
of homologous recombination using a method called chromosome painting. Gene
sequences often diverge between European strains and East Asian strains. The
evolution of Western-type CagA to East Asian-type CagA can be explained by
illegitimate recombination events, with the Amerindian type as an intermediate.
Massive decay of molybdenum-related genes was found in East Asian strains.
Whole methylome decoding at single-nucleotide resolution revealed that the
H. pylori methylome is highly variable because its many methyltransferases often
change sequence specificity. Their target recognition domains may move between
different genes, sometimes beyond species barriers, and they may even move
within a gene (domain movement). These extremely variable methylomes, as
opposed to variable genomes, might provide targets for natural selection in adaptive
evolution—a hypothesis that may be called epigenetics-driven adaptive evolution.
Keywords Genome comparison • Recombination • DNA duplication • Inversion •

CagA • Epigenetics • Methylome • DNA methylation
I. Kobayashi (*)
Department of Computational Biology and Medical Sciences, Graduate School of Frontier
Sciences & Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan

DOI 10.1007/978-4-431-55936-8_9
218 I. Kobayashi
9.1 Prologue
Helicobacter pylori may be regarded as a model organism to study genetics and

evolution. Long-standing questions in biology may be answered by studying this
eubacterial species that infects humans. These questions include the nature of
adaptive evolution, the units and nature of natural selection, the nature of the
information transferred to the next generation, the roles of epigenetics and recom-
bination in these processes, and the nature of species. H. pylori’s relatively specific
and defined niche (the stomach and duodenum), high mutation and mutual homol-
ogous recombination rates (Suerbaum and Josenhans 2007), and abundant DNA
methylation systems (Furuta et al. 2014) underlie the idea that they may provide
deep insights into the aforementioned issues.
Recent innovations in DNA sequencing and related technology now allow
decoding of many whole-genome sequences of this species, as well as other pro-
karyotes. We can study their complete genome sequences at single-nucleotide
resolution. Especially, we can study the roles of various forms of recombination
in genome-wide evolution: homologous recombination involving a pair of long
similar sequences, site-specific recombination involving specific sequences, and
illegitimate recombination involving very short and unpredictable sequences. They
can also be classified according to their relationship with a particular DNA region,
such as a mobile genetic element or a particular gene, as well as whether they result
in an insertion, deletion, inversion, duplication, decay, etc.
One recent advance in DNA sequencing, Single-Molecule Real-Time (SMRT)
sequencing, now allows decoding of a complete methylome at single-nucleotide
resolution in bacteria (Murray et al. 2012). This provides a unique occasion to
evaluate the roles of epigenetics in evolution. In H. pylori, which encodes many
DNA methyltransferases, such studies may lead to new concepts in epigenetics and
adaptive evolution.
This brief sketch of H. pylori evolution emphasizes the roles of recombination
and methylation in genome-wide evolution that have emerged from single-
nucleotide resolution comparisons of multiple complete genomes/methylomes in
this species. We often focus on differences between Western-type strains and
Eastern-type strains.
9.2 Phylogeny and Population Structure
The current standard method for the phylogenetic classification of H. pylori is

multi-locus sequence typing (MLST) based on concatenated sequences from
seven conserved genes (atpA, efp, mutY, ppa, trpC, ureI, and yphC). The results
are archived in a large and ever-expanding database (the H. pylori MLST database
[http://pubmlst.org/helicobacter/]), and phylogenetic trees are constructed from
these data. Population structure is inferred from these sequences according to
9 Genome Evolution: Helicobacter pylori as an Extreme Model 219
their clustering, as determined using STRUCTURE (Falush et al. 2003a, b). The
extant populations are hpAfrica2, hpAfrica1, hpNEAfrica, hpEurope, hpAsia2,
hpSahul, and hpEastAsia (Falush et al. 2003b; Moodley et al. 2012). Each of
these may be divided into subpopulations. For example, hpEastAsia is divided
into hspAmerind, hspMaori, and hspEastAsia.
9.2.1 Genome Trees vs. Gene Trees
Starting with multiple complete genome sequences, we can obtain more informa-
tion about their phylogeny. The common core genome structure of genomes can be
extracted (Uchiyama 2008). The concatenated sequence of conserved genes results
in a well-resolved phylogenetic tree, as shown in Fig. 9.1a (Yahara et al. 2013).
Such trees are much more robust than standard MLST trees primarily because the
tree is composed of a large quantity of sequence information and many more
(approximately 1000) genes.
When phylogenetic trees of individual genes are examined, they turn out to be
quite variable in size and topology (Fig. 9.2a) (Uchiyama 2008; Yahara et al. 2012).
The incongruence with the core tree is at least partly explained by high levels of
homologous recombination between lineages. Indeed, each of the global H. pylori
genomes appears to be a mosaic of sequences transferred from other lineages
(Yahara et al. 2012, 2013).
9.2.2 Inference of Population Structure from Mutual

Homologous Recombination
Phylogenetic tree construction is, in principle, based on the assumption that genome
evolution takes place primarily through nucleotide substitution. In organisms with
low levels of mutual homologous recombination, phylogenetic tree construction
will allow the identification of populations, which in turn will allow the detection of
rare recombination events between such populations. The high mutual homologous
recombination rate in H. pylori makes it difficult to infer population structure from
phylogenetic trees and to identify traces of past recombination events.
Recently, a method called chromosome painting in silico was developed
to overcome this problem in the human genome (Lawson et al. 2012), and this
method was applied to global strains of H. pylori (Fig. 9.1b) (Yahara et al. 2013).
This method detects the transfer of DNA sequence chunks between genomes
through homologous recombination throughout the genome. Based on this, the
chromosome-painting algorithm calculates the expected number of chunks
imported from a donor genome to a recipient genome and then summarizes
these values in a matrix (the “co-ancestry matrix”) (Fig. 9.1b) (Yahara et al. 2013).
220 I. Kobayashi
A.
hspEastAsia
hspAmerind
hpAsia2
hpEurope
hpAfrica1
B. (i)
B. (ii)
donor
EastAsia_2
EastAsia_3
EastAsia_4
EastAsia_1
Amerind_1
Amerind_2
Amerind_4
Amerind_3
Africa1_1
Europe_1
Europe_2
Asia2_1
recipient
No. chunks transferred
Africa1_1
Europe_1
Europe_2
Asia2_1
Amerind_1
Amerind_2
Amerind_3
Amerind_4
EastAsia_1
EastAsia_2
EastAsia_3
EastAsia_4
Fig. 9.1 Genome evolution in H. pylori. (a) A phylogenetic tree of H. pylori based on core
genome sequences. Numbers indicate bootstrap values. The scale bar indicates substitutions per
nucleotide. The color in front of a strain name indicates a subgroup identified with chromosome
Based on the matrix, individual strains were assigned to subgroups by the

fineSTRUCTURE clustering algorithm (the left part of Fig. 9.1b). This new method
revealed a finer population structure than a previous method that examined only
seven MLST genes (Yahara et al. 2013).
An examination of the genetic flux in the co-ancestry matrix (Fig. 9.1b) showed
some singleton strains to be hybrids of subgroups. For example, the 13th strain
(PeCan4) is a hybrid in that has received a considerable number of chunks from the
Amerindian, Africa1, and European strains. The co-ancestry matrix also revealed
evident signs of population admixture in Africa, Europe, and parts of Asia (Yahara
et al. 2013).
A modification of the chromosome-painting method allowed the detection of
regions that have frequently transferred between lineages in multiple bacterial
species (Yahara et al. 2014; Yahara et al. 2015).
9.3 Evolution of Individual Genes
The evolution of individual genes of H. pylori turned out to often involve various
modes of recombination. Many genes have diverged between Western (¼ African
and European) strains and East Asian strains (Kawai et al. 2011). These include
many virulence genes, for example, the cagA oncogene (Hatakeyama 2014).
9.3.1 The cagA Oncogene
The CagA gene product is injected into epithelial cells, undergoes phosphorylation
at a tyrosine (Y) residue in its EPIYA motif by host cell kinases, and perturbs host
signaling pathways. CagA is known for its geographical, structural, and functional
diversity in its C-terminal half, where the EPIYA host-interacting motif is repeated.
The Western version carries EPIYA segment types A, B, and C, while the East
Asian version carries types A, B, and D, which results in higher virulence
(Hatakeyama 2014).
⁄
Fig. 9.1 (continued) painting and the fineSTRUCTURE algorithm (Modified from Figure S2 of
Yahara et al. (2013)). (b) Chromosome painting/fineSTRUCTURE analysis. (i) Chromosome
painting in silico. Each lane indicates the chromosome of a strain shown on the right. The strains
are classified by fineSTRUCTURE into subgroups labeled by colors on the left. A color along the
chromosome indicates that the subgroup apparently donated a chunk of single nucleotide poly-
morphisms (SNPs) through homologous recombination. All genomic positions are transformed to
those of a reference strain (26695). (ii) Co-ancestry matrix with population structure and genetic
flux. The color of each cell of the matrix indicates the expected number of chunks imported from a
donor genome (column) to a recipient genome (row). The tree on the left shows the clustering of
the listed population subgroups (Modified from Figures 1 and 2 of Yahara et al. (2013))
222 I. Kobayashi
A. (ii) B.
(i)
0.1
zone 1 zone 2 (e) zone 3
db (f)
(c)(d)
da (a) (b)
2 × da*
0.01
da*
hspEAsia da
hpEurope 0.001
1e-04
0.01
0.001 0.005 0.01 0.02 0.05 0.1 0.2
db*
db
(iii)
(a) cheY (c) sotB (e) cagA
0.01 0.01
(d) vacA 0.01

(b) fixQ
(f) HP1250
0.01
0.01
0.01
C.
hspEastAsia
hpEurope
Fig. 9.2 Evolution of individual genes. (a) Phylogenetic trees of individual genes. (i) Diagram of
the analysis. Black dots, the last common ancestors of Eastern and Western strains. da, the length of
the branch separating the two groups; db, the average branch length of the Eastern strains. (ii) Plot
of gene trees based on the two distance values. Large green dot, the well-defined core tree; da*, da
for the well-defined core tree; db*, db for the well-defined core tree; inset box, the well-defined
core tree; zone 1, db <0.00550; zone 2, 0.00550 db 0.0231; zone 3, db >0.0231; red dot, genes
with positive selection resulting in amino acid changes and with da >2 da*, that is, da >0.02324.
N ¼ 692 genes. (iii) Representative gene trees with high divergence between hspEastAsia and
hpEurope strains. Their positions are indicated in (ii). Lowest common ancestor (LCA) of
hspEastAsia (red) and hpEurope (cyan) is marked (Modified from Figure 8 of Kawai
et al. (2011)). (b) CagA and its evolutionary pathway. Black box: the EPIYA sequence. Gray
box: the CM sequence (Modified by Yoshikazu Furuta and the author from Figure 1 of Furuta
et al. 2011c). (c) Decay of molybdenum-related genes in hspEastAsia strains. The left labels
indicate strain names (Modified by Mikihiko Kawai and the author from Figure 4 of Kawai
et al. (2011))
Insight into the relationships between cagA variants through various modes of
recombination was obtained by analyzing all known cagA variant sequences (over
1100) in the public database at single-nucleotide resolution (Furuta et al. 2011c).
The left half of the EPIYA-D segment characteristic of East Asian cagA was
derived from the Western-type EPIYA, with the Amerindian-type EPIYA as the
intermediate, through rearrangements of specific sequences within the gene
(Fig. 9.2b).
Many of their structural variants can be explained by: (i) homologous recombi-
nation between DNA sequences encoding the CM (CagA multimerization) domain;
(ii) site-specific recombination between DNA sequences in the EPIYA motif; and
(iii) illegitimate recombination between short similar DNA sequences (Furuta
et al. 2011c).
9.3.2 Decay of Molybdenum-Related Genes
Some genes decay, via point mutations and various types of recombination, as has
occurred in H. pylori. To closely follow such evolutionary processes, it is necessary
to thoroughly characterize the gene content in each of the multiple genomes. Such
phylogenetic profiling needs to determine the presence or absence of a domain,
rather than a gene (a coding region), and to detect split genes, partially deleted
genes, and partially duplicated genes (Uchiyama 2006). The analysis can then go to
the single-nucleotide level.
Such an analysis revealed that functions related to molybdenum (Mo) were lost
in hspEastAsia strains (Kawai et al. 2011). The trace element Mo is essential for
nearly all organisms. After transport into the cell as molybdate, it is incorporated
into metal cofactors for specific enzymes (molybdo-enzymes) that catalyze
reduction–oxidation reactions mediated by two-electron transfer. At least one
gene involved in each of these Mo-related functions decayed through point muta-
tion or recombination in all hspEastAsia strains analyzed. Some Amerindian strains
(B type) also lack Mo-related genes (Gressmann et al. 2005). The occurrence of
apparently independent multiple mutations suggests some selection against the use
of Mo.
9.3.3 Outer Membrane Proteins
Outer membrane proteins (OMPs), which are involved in host interactions, among
others, are numerous and variable in H. pylori (Leituti and Goldberg 2012). They
form many large families that show various modes of evolution via mutation and
recombination. These include decay, gene conversion (Kawai et al. 2011), and
duplication (see below). Some of their sequences are specific to phylogenetic
groups. There are, for example, C-terminal sequences in HopMN that are charac-
teristic of hpEastAsia strains (Kawai et al. 2011), while the horA gene is fragmented
in hspEastAsia strains (Kawai et al. 2011).
224 I. Kobayashi
9.4 Evolution of Chromosome Synteny
In general, inversion events, as well as insertion/deletion events, play an important

role in the evolution of chromosome synteny. Large insertion and deletion events
affecting synteny, especially those mediated by mobile elements such as trans-
posons, prophages, genomic islands, and restriction-modification (RM) systems,
are important but are beyond the scope of this article.
9.4.1 Inversion
An inversion relationship was identified by comparing two complete genome

sequences of H. pylori (Alm et al. 1999). More recently, all large inversion events
between ten complete genomes of global strains were identified (Fig. 9.3b) (Furuta
et al. 2011a). Sequence alignments in these studies revealed four underlying
mechanisms: (i) homologous recombination between long sequences of high iden-
tity in an opposite orientation; (ii) illegitimate recombination between short similar
sequences in an opposite orientation; (iii) inversion adjacent to the insertion of a
mobile element; and (iv) DNA duplication associated with inversion (DDAI).
9.4.2 DNA Duplication Associated with Inversion
In the last process (DDAI), a DNA segment at one chromosomal locus is copied and
inserted, in an inverted orientation, into a distant locus on the same chromosome,
while the entire region between these two loci is also inverted (Fig. 9.3a). DDAI
was found by comparing the complete genome sequences of Western strains and
East Asian strains: Gain and loss of genes (loci) for OMPs occurred at breakpoints
of chromosomal inversions (Fig. 9.3a). This mode of DNA duplication was also
found in other organisms (Ranz et al. 2007; Chen et al. 2013).
9.4.3 Reconstruction of Synteny Evolution
Recognition of these four modes of inversion allowed the reconstruction of synteny

evolution through inversions in H. pylori (Fig. 9.3c). The paths of synteny evolution
are related to, but not identical with, those of genome sequence evolution. For
example, closely related genomes of four Japanese strains take different forms
because of different inversion events.
A.
(i)
Europe
Inversion Inversion
East Asia
(ii)
Inversion
Duplication
B.
C.
Japanese
Fig. 9.3 Evolution of chromosome synteny through inversion. (a) DNA duplication associated
with inversion (DDAI). (i) Linkage of oipA gene duplication and hopN gene decay with
226 I. Kobayashi
9.5 Evolution of the Methylome
Epigenetic modifications, such as DNA methylation, have large effects on gene

expression and genome maintenance in prokaryotes and eukaryotes. Although
many of the epigenetic marks are reprogrammed in most cases in plants and
animals, there are increasing lines of evidence for trans-generation inheritance of
epigenetic status. H. pylori has a large number of DNA methyltransferase genes,
with different strains having unique repertoires.
9.5.1 Restriction–Modification Systems
Many of the DNA methyltransferases in prokaryotes are a part of RM systems

(Fig. 9.4a). In an RM system, a modification enzyme (a DNA methyltransferase)
transfers a methyl group to a specific DNA sequence. A paired restriction enzyme
will attack DNA lacking this ID of the self-epigenome.
RM systems behave as selfish mobile elements, just like transposons and viral
genomes (Furuta and Kobayashi 2013). They are sometimes linked to genome
rearrangements, such as inversion events. Because they increase genetic isolation
and affect gene expression (Furuta et al. 2014; Kumar et al. 2012), they may
contribute to adaptive evolution (Fig. 9.4b).
RM systems are classified into four types (Roberts et al. 2003). They differ in the
location of their target recognition domain (TRD) for methylation. In Type III
systems, the TRD is in the same polypeptide (Mod protein) as the DNA
methyltransferase (Rao et al. 2014). In Type I systems, the TRD is on the S
(specificity) subunit and consists of TRD1 and TRD2, each recognizing half of a
bipartite recognition sequence (Loenen et al. 2014). In some S subunits, the number
of repeats of an amino acid sequence measures the distance (the number of
nonspecific nucleotide Ns) between these two half sites. A thorough description
of the many RM systems in H. pylori is beyond the scope of this article (see
REBASE Genomes, http://rebase.neb.com/rebase/rebase.html).
Fig. 9.3 (continued) chromosomal inversions between typical Western and East Asian genomes.
The upper line corresponds to the P12 genome, and the lower line corresponds to an ancestral
structure of four Japanese genomes (Modified from Figure 1 of Furuta et al. 2011a with a
permission from PNAS). (ii) The concept of DDAI. Duplication of the black arrow region to a
new site in an opposite orientation is associated with inversion of the white arrow region between
its old site and its new site. (b) Inversions detected by genome comparisons. Large inversions
detected by comparing ten global genomes were mapped on a H. pylori genome. The outer circle
indicates the genome of P12, a European strain, whereas the inner circle indicates its origin and
coordinates. An arc outside the outer circle indicates an inversion in the East Asian strains and
Amerindian strain, whereas an inside arc indicates an inversion in European and/or West African
strains. A triangle indicates a region generated by a DDAI event (From Figure 2 of Furuta
et al. 2011a with a permission from PNAS). (c) Reconstruction of synteny evolution through
inversion. Triangles indicate regions duplicated through DDAI (Modified from Figure 5 of Furuta
et al. 2011a with a permission from PNAS)
A.
(i) (ii)
Microorganism DNA
Modification
M enzyme NON-SELF
Host A Host B Me (-CH3) Restriction
enzyme
SELF damage
(iii) NON-SELF
epigenome Genetic
isolation
Bacterial SELF
cell epigenome Adaptation?
rm
Gene expression Unique
properties
(i) (ii)
TRD (a)
(Target Recognition Domain)
(b)
(iii)
Fig. 9.4 Restriction-modification systems. (a) Action. (i) Adaptation of a microorganism to its
host. The microorganism may be a bacteriophage and the host may be a bacterium. (ii) Distinction
228 I. Kobayashi
9.5.2 Base-Excising Restriction Enzymes
All the restriction enzymes examined thus far hydrolyze phosphodiester bonds in
the DNA backbone, while a member of their family with half-pipe fold excises a
base from the recognition sequence (Miyazono et al. 2014). In other words, it is a
DNA glycosylase. The resulting apurinic/apyrimidinic (AP) site is sometimes
cleaved by the second activity of the enzyme, AP lyase (Fukuyo et al. 2015).
Restriction enzymes may be now classified into two basic classes: the phosphodi-
esterase class and the glycosylase class. Its family member, R.HpyAXII, present in
several H. pylori strains can limit transformation (Humbert and Salama 2008). The
PabI family of restriction-modification system behaves as a mobile genetic element,
similar to the other families of Type II restriction-modification systems. The
distribution of the hrgC gene replacing the PabI family in the subpopulations of
H. pylori, hspAmerind, hspEAsia and hpAsia2, corresponds to the two human
migration events, one from East Asia to Americas and the other from China to
Malaysia (Kojima and Kobayashi 2015) .
9.5.3 Sequence Specificity Variation in DNA Methylation

Systems
Genome sequence comparisons and methylome decoding (Furuta and Kobayashi

2012b; Furuta et al. 2011b; Furuta et al. 2014) revealed that these DNA
methyltransferases often change their sequence specificity, generating wide diver-
sity in the methylome.
In Type III RM systems, the changes may be mediated by the replacement of the
TRD repertoire by allelic homologous recombination (Fig. 9.4d) or by ectopic
homologous recombination (gene conversion) (Fig. 9.4d). The latter takes place
between distantly related genes at separate loci, taking advantage of the weakly
similar DNA sequences encoding methyltransferase motifs. Some of the TRD
sequences (homology groups) may move beyond the species boundary and spread
throughout the bacterial world (Furuta and Kobayashi 2012b).
Fig. 9.4 (continued) between the self and non-self-epigenome. A modification enzyme transfers a
methyl group to a specific DNA sequence. A cognate restriction enzyme will cleave DNA lacking
this self-epigenome identification. (iii) Possible dual roles in adaptive evolution. Methylation of a
specific sequence at many sites along the genome may provide a specific gene expression pattern,
among other unique properties. These lineages are isolated from each other. (b) Changes in the
sequence specificity of RM systems through various modes of recombination. (i) Allelic recom-
bination. The same gene (locus). (ii) Gene conversion. Different genes (loci). a General scheme.
b The case with Type III mod genes. (iii) Domain movement. Different sites in the same gene.
Recombination events at the marked sequences flanking target recognition domains (TRDs) are
responsible for the movement (From Figure 1 of Furuta et al. 2011b)
In the Type I S subunit, TRD sequences may replace each other by allelic
homologous recombination at the same site, move between different genes, and
move between different sites within a gene (DoMo ¼ domain movement)
(Fig. 9.4d). The movement between TRD1 and TRD2 likely involves shared
sequences flanking the TRDs. There is evidence that a change in the number of
repeats between TRD1 and TRD2 takes place within the bacteria during an infec-
tion (Andres et al. 2010).
The resulting changes in the methylome may lead to changes in gene expression
and phenotypes, and they may be subject to natural selection (Furuta and Kobayashi
2012a; Furuta et al. 2014). This hypothesis of epigenetics-driven adaptive evolution
awaits further experimental tests.
9.6 Epilogue
I have described how single-nucleotide resolution comparisons of complete

genomes and methylomes revealed the dynamics of genome and epigenome evo-
lution. I have not covered the emerging relationships of these genome/epigenome
changes with phenotype changes, which are relevant to host interactions and
adaptive evolution. Most of the description here is based on strain comparisons,
but there are also interesting studies of the evolution of individual lineages. I also
had to omit a recent description of the microbiome surrounding H. pylori.
I expect that current breakthroughs in OMICS analyses, aided by bioinformatic
analysis and other experimental approaches, will lead to the emergence of a more
complete picture of adaptive evolution in this model organism within a decade.
References
Alm RA, Linq LS, Moir DT, King BL, Brown ED, Doig PC, Smith DR, Noonan B, Guild BC,
deJonge BL, Carmel G, Tummino PJ, Caruso A, Uria-Nickelsen M, Mills DM, Ives C,
Gibson R, Merberg D, Mills SD, Jiang Q, Taylor DE, Vovis GF, Trust TJ (1999) Genomic-
sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter
pylori. Nature 397:176–180
Andres S, Skoglund A, Nilsson C, Krabbe M, Bj€orkholm B, Engstrand L (2010) Type I restriction-
modification loci reveal high allelic diversity in clinical Helicobacter pylori isolates.
Chen J, Huang Q, Gao D, Wang J, Lang Y, Liu T, Li B, Bai Z, Luis Goicoechea J, Liang C,
Chen C, Zhang W, Sun S, Liao Y, Zhang X, Yang L, Song C, Wang M, Shi J, Liu G, Liu J,
Zhou H, Zhou W, Yu Q, An N, Chen Y, Cai Q, Wang B, Liu B, Min J, Huang Y, Wu H, Li Z,
Zhang Y, Yin Y, Song W, Jiang J, Jackson SA, Wing RA, Wang J, Chen M (2013) Whole-
genome sequencing of Oryza brachyantha reveals mechanisms underlying Oryza genome
evolution. Nat Commun 4:1595
Falush D, Stephens M, Pritchard JK (2003a) Inference of population structure using multilocus
genotype data: linked loci and correlated allele frequencies. Genetics 164:1567–1587
230 I. Kobayashi
Falush D, Wirth T, Linz B, Pritchard JK, Stephens M, Kidd M, Blaser MJ, Graham DY, Vacher S,
Perez-Perez GI, Yamaoka Y, Mégraud F, Otto K, Reichard U, Katzowitsch E, Wang X,
Achtman M, Suerbaum S (2003b) Traces of human migrations in Helicobacter pylori
populations. Science 299:1582–1585
Fukuyo M, Nakano T, Zhang Y, Furuta Y, Ishikawa K, Watanabe-Matsui M, Yano H,
Hamakawa T, Ide H, Kobayashi I (2015) Restriction-modification system with methyl-
inhibited base excision and abasic-site cleavage activities. Nucleic Acids Res 43:2841–2852
Furuta Y, Kobayashi I (2012a) Mobility of DNA sequence recognition domains in DNA
methyltransferases suggests epigenetics-driven adaptive evolution. Mob Genet Elem
2:292–296
Furuta Y, Kobayashi I (2012b) Movement of DNA sequence recognition domains between
non-orthologous proteins. Nucleic Acids Res 40:9218–9232
Furuta Y, Kobayashi I (2013) Restriction-modification systems as mobile epigenetic elements. In:
Roberts A and Mullany P (eds) Bacterial integrative mobile genetic elements. Landes Biosci-
ence, Austin, Texas, pp 85–103
Furuta Y, Kawai M, Yahara K, Takahashi N, Handa N, Tsuru T, Oshima K, Yoshida M, Azuma T,
Hattori M, Uchiyama I, Kobayashi I (2011a) Birth and death of genes linked to chromosomal
inversion. Proc Natl Acad Sci U S A 108:1501–1506
Furuta Y, Kawai M, Uchiyama I, Kobayashi I (2011b) Domain movement within a gene: a novel
evolutionary mechanism for protein diversification. PLoS One 6:e18819
Furuta Y, Yahara K, Hatakeyama M, Kobayashi I (2011c) Evolution of cagA oncogene of
Helicobacter pylori through recombination. PLoS One 6:e23499
Furuta Y, Namba-Fukuyo H, Shibata TF, Nishiyama T, Shigenobu S, Suzuki Y, Sugano S,
Hasebe M, Kobayashi I (2014) Methylome diversification through changes in DNA
methyltransferase sequence specificity. PLoS Genet 10:e1004272
Gressmann H, Linz B, Ghai R, Pleissner KP, Schlapbach R, Yamaoka Y, Kraft C, Suerbaum S,
Meyer TF, Achtman M (2005) Gain and loss of multiple genes during the evolution of
Helicobacter pylori. PLoS Genet 1:e43
Hatakeyama M (2014) Helicobacter pylori CagA and gastric cancer: a paradigm for hit-and-run
carcinogenesis. Cell Host Microbe 15:306–316
Humbert O, Salama NR (2008) The Helicobacter pylori HpyAXII restriction-modification system
limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of
the DNA methyltransferase and restriction endonuclease components. Nucleic Acids Res
36:6893–6906
Kawai M, Furuta Y, Yahara K, Tsuru T, Oshima K, Handa N, Takahashi N, Yoshida M, Azuma T,
Hattori M, Uchiyama I, Kobayashi I (2011) Evolution in an oncogenic bacterial species with
extreme genome plasticity: Helicobacter pylori East Asian genomes. BMC Microbiol 11:104
Kojima KK, Kobayashi I (2015) Transmission of PabI family of restriction DNA glycosylase
genes: mobility and long-term inheritance. BMC Genomics 16:817
Kumar R, Mukhopadhyay AK, Ghosh P, Rao DN (2012) Comparative transcriptomics of H. pylori
strains AM5, SS1 and their hpyAVIBM deletion mutants: possible roles of cytosine methyl-
ation. PLoS One 7:e42303
Lawson DJ, Hellenthal G, Myers S, Falush D (2012) Inference of population structure using dense
haplotype data. PLoS Genet 8:e1002453
Leituti G, Goldberg JB (2012) Outer membrane biogenesis in Escherichia coli, Neisseria
meningitidis, and Helicobacter pylori: paradigm deviations in H. pylori. Front Cell Infect
Microbiol 2:29
Loenen WA, Dryden DT, Raleigh EA, Wilson GG (2014) Type I restriction enzymes and their
relatives. Nucleic Acids Res 42:20–44
Miyazono K, Furuta Y, Watanabe-Matsui M, Miyakawa T, Ito T, Kobayashi I, Tanokura M (2014)
A sequence-specific DNA glycosylase mediates restriction-modification in Pyrococcus abyssi.
Nat Commun 5:3178
Moodley Y, Linz B, Bond RP, Nieuwoudt M, Soodyall H, Schlebusch CM, Bernh€ oft S, Hale J,
Suerbaum S, Mugisha L, van der Merwe SW, Achtman M (2012) Age of the Association
between Helicobacter pylori and Man. PLoS Pathog 8:e1002693
Murray IA, Clark TA, Morgan RD, Boitano M, Anton BP, Luong K, Fomenkov A, Turner SW,
Korlach J, Roberts RJ (2012) The methylomes of six bacteria. Nucleic Acids Res
40:11450–11462
Ranz JM, Maurin D, Chan YS, von Grotthuss M, Hillier LW, Roote J, Ashburner M, Bergman CM
(2007) Principles of genome evolution in the Drosophila melanogaster species group. PLoS
Biol 5:e152
Rao DN, Dryden DT, Bheemanaik S (2014) Type III restriction-modification enzymes: a historical
perspective. Nucleic Acids Res 42:45–55
Roberts RJ, Belfort M, Bestor T, Bhagwat AS, Bickle TA, Bitinaite J, Blumenthal RM, Degtyarev
SK, Dryden DT, Dybvig K, Firman K, Gromova ES, Gumport RI, Halford SE, Hattman S,
Heitman J, Hornby DP, Janulaitis A, Jeltsch A, Josephsen J, Kiss A, Klaenhammer TR,
Kobayashi I, Kong H, Krüger DH, Lacks S, Marinus MG, Miyahara M, Morgan RD, Murray
NE, Nagaraja V, Piekarowicz A, Pingoud A, Raleigh E, Rao DN, Reich N, Repin VE, Selker
EU, Shaw PC, Stein DC, Stoddard BL, Szybalski W, Trautner TA, Van Etten JL, Vitor JM,
Wilson GG, Xu SY (2003) A nomenclature for restriction enzymes, DNA methyltransferases,
homing endonucleases and their genes. Nucleic Acids Res 31:1805–1812
Uchiyama I (2006) Hierarchical clustering algorithm for comprehensive orthologous-domain
classification in multiple genomes. Nucleic Acids Res 34:647–658
Uchiyama I (2008) Multiple genome alignment for identifying the core structure among moder-
ately related microbial genomes. BMC Genomics 9:515
Yahara K, Kawai M, Furuta Y, Takahashi N, Handa N, Tsuru T, Oshima K, Yoshida M, Azuma T,
Hattori M, Uchiyama I, Kobayashi I (2012) Genome-wide survey of mutual homologous
recombination in a highly sexual bacterial species. Genome Biol Evol 4:628–640
Yahara K, Furuta Y, Oshima K, Yoshida M, Azuma T, Hattori M, Uchiyama I, Kobayashi I (2013)
Chromosome painting in silico in a bacterial species reveals fine population structure. Mol Biol
Evol 30:1454–1464
Yahara K, Didelot X, Ansari MA, Sheppard SK, Falush D (2014) Efficient inference of recombi-
nation hot regions in bacterial genomes. Mol Biol Evol 31:1593–1605
Yahara K, Didelot X, Jolley KA, Kobayashi I, Maiden MCJ, Sheppard SK, Falush D (2015) The
landscape of realized homologous recombination in pathogenic bacteria. Mol Biol Evol.
doi:10.1093/molbev/msv237
Chapter 10
Non-Helicobacter pylori Helicobacter
Infections in Humans and Animals
Bram Flahou, Freddy Haesebrouck, and Annemieke Smet
Abstract Since the first description of the human pathogen Helicobacter pylori in
the early 1980s, the number of known species in the genus Helicobacter has
increased largely. Currently, 45 different Helicobacter species have been identified.
Bacteria belonging to this genus can roughly be divided into two major groups,
gastric and enterohepatic species. Gastric helicobacters express urease at a high
level which helps them to survive in the acidic environment of the stomach,
whereas most enterohepatic helicobacters do not. The best-known gastric
Helicobacter species is H. pylori. This chapter, however, deals with non-H. pylori
helicobacters (NHPH). Most NHPH are animal-associated bacteria, but some of
them are of zoonotic significance. First, gastric infections with these bacteria in
humans are considered. Thereafter, an overview of natural and experimental gastric
infections in animal hosts is given, with emphasis on the gastric helicobacters that
are mainly associated with dogs, cats, and pigs. Finally, enterohepatic Helicobacter
species are briefly discussed.
Keywords Gastric non-Helicobacter pylori helicobacters • Enterohepatic

helicobacters • Zoonotic significance • Gastric disease • Enterohepatic disease •
Animal models
10.1 Introduction
Since the original description of the human pathogen H. pylori in 1983, the number
of known species in the genus Helicobacter has increased largely (Warren and
Marshall 1983). Currently this genus includes 45 identified species. An overview is
shown in Fig. 10.1. The Helicobacter bacteria can roughly be divided into gastric
and enterohepatic species. Gastric Helicobacter species are able to survive the
acidic environment in the stomach by expressing high levels of urease (Pot
et al. 2007). Enterohepatic species do not normally colonize the gastric mucosa.
B. Flahou • F. Haesebrouck (*) • A. Smet

Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine,
Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

DOI 10.1007/978-4-431-55936-8_10
234 B. Flahou et al.
'Candidatus H. bovis' (bovine strain R2XA, AF127027)

Wolinella succinogenes (ATCC 29543, M88159)
H. fennelliae (canine strain ATCC 35684, M88154)
H. aurati (hamster strain MIT 97-5057, AF297868)
H. mastomyrinus (rodent strain MIT 97-5574, AY242307)
H. trogontum (rat strain LRB 8581, U65103)
H. equorum (horse strain LMG23362, DQ307735)
H. pullorum (poultry strain ATCC 51801, AY631956)
H. canadensis (human strain MIT 98-5491, AF262037)
H. muridarum (rodent strain ST1, M80205)
H. hepaticus (mouse strain ATCC 51448, U07574)
H. typlonius (mouse strain MIT 97-6810, AF127912)
H. rappini (porcine strain H157 S, AY034821)
H. westmaedii (human strain, HSU44756)
'Candidatus H. colifelis' (feline strain, AF142062)
H. canis (canine strain CIP104753, AY631945)
H. cinaedi (human strain CIP103752, M88150)
H. bilis (mouse strain Hb1, U18766)
H. magdeburgensis (mouse strain HM007, EF990624)
H. callitrichis (marmoset strain R-204, AY192526)
H. mesocricetorum (Syrian hamster strain MU97-1514, AF072471)
H. ganmani (mouse strain CMRI H02, AF000221)
H. rodentium (mouse strain MIT 95-1701, U96296)
H. winghamensis (human strain NLEP97-1090, AF246984)
H. valdiviensis (wild bird strain WBE19, KF549904)
H. pametensis (tern strain ATCC 51478, M88147)
H. brantae (Candian goose strain MIT 04-9366, DQ415546)
H. cholecystus (hamster strain CIP105596, AY686606)
H. anseris (goose strain MIT 04-9368, DQ415545)
H. mustelae (ferret strain ATCC 43772, M35048)
H. suncus (house musc screw strain Kaz-2, AB006148)
H. macacae (Rhesus monkey strain MIT99-10773, EF526074)
H. muricola (wild mouse strain w-06, AF284783)
H. marmotae (prairie dog strain MIT 04-8534, GU902715)
H. suis (human strain HU1, AF506784)
H. suis (porcine strain HS1, EF204589)
H. heilmannii (wild feline strain C3E, AF506776)
H. heilmannii (wild feline strain T1, AF506795)
H. bizzozeronii (canine strain CCUG 35546, AY366429)
H. felis (feline strain CS2, AF506775)
H. heilmannii (feline strain ASB3, HM625818)
H. felis (canine strain DS2, AY385426)
H. felis (wild canine strain WD1, AF506785)
H. bizzozeronii (canine strain CCUG 35545, Y09404)
H. salomonis (canine strain CCUG 37845, U89351)
H. cynogastricus (canine strain JKM4, DQ004689)
H. baculiformis (feline strain M50, EF070342)
H. heilmannii (human strain HU2, AF506786)
H. heilmannii (wild feline strain BC1, AF506772)
H. felis (wild feline strain C2E, AF506774)
H. felis (wild feline strain C4E, AF506779)
H. acinonychis (wild feline strain Eaton, M88149)
H. nemestrinae (pigtailed macaque strain ATCC 49396, X67854)
H. cetorum (dolphin strain MIT 01-5202, AY143177)
H. pylori (human strain ATCC 43504, AF302106)
H. pylori (human strain ATCC 49396, AF363064)
Fig. 10.1 Phylogenetic tree based on the near-complete 16s rRNA gene sequences from all gastric
and enterohepatic Helicobacter species described so far. The alignment of the sequences and the
construction of the phylogenetic tree were performed as described before (Smet et al. 2012)
10 Non-Helicobacter pylori Helicobacter Infections in Humans and Animals 235
Instead, they thrive at the mucosal surface of the intestinal tract and/or the
liver (Sterzenbach et al. 2007). To date, Helicobacter spp. have been detected in
nearly 150 vertebrate species, including animals from every continent and all four
non-fish vertebrate taxonomic classes (Schrenzel et al. 2010). Animal-associated
helicobacters, and especially the gastric species, are characterized by their
extremely fastidious nature, which to date has resulted in a low number of
in vitro isolates available worldwide (Haesebrouck et al. 2009). Several of these
species have a pathogenic potential in different animal hosts, and some are capable
of causing disease in humans (Table 10.1). The presence and diversity of
helicobacters in the vertebrate fauna and their transfer possibilities between hosts
are critical factors on how fast the Helicobacter ecology will evolve and what their
impact is on animal and human health (Schrenzel et al. 2010). This chapter mainly
focuses on the biology and pathogenesis of animal-associated gastric Helicobacter
infections in humans and animals. An overview of the significance of enterohepatic
Helicobacter spp. in human and animal disease is summarized at the end of this
chapter.
10.2 Gastric Helicobacter Infections in Humans
In the early years of H. pylori research, pathologists examining human gastric

biopsies already reported the presence of bacteria with a long spiral-shaped mor-
phology. These microorganisms were similar to bacteria reported in the stomach
of pigs, cats, dogs and nonhuman primates and were originally referred to as
Gastrospirillum hominis (McNulty et al. 1989). Later on, they were renamed to
H. heilmannii (Heilmann and Borchard 1991). Although at that time the name
H. heilmannii had no official standing in nomenclature, it was used for many years
to refer to the group of long spiral-shaped bacteria in the human stomach, which
actually comprises several different Helicobacter species. Later on these microor-
ganisms were reclassified into H. heilmannii type 1, representing H. suis from pigs,
and H. heilmannii type 2, comprising a group of canine and feline Helicobacter spp.
(Haesebrouck et al. 2009). The valid description of H. heilmannii further added
some confusion on the nomenclature of this complex and expanding group of
microorganisms (Smet et al. 2012). Therefore, the terms H. heilmannii (sensu
lato), referring to the group of gastric non-H. pylori Helicobacter spp. (NHPH),
and H. heilmannii (sensu stricto), referring to the species, have been proposed
(Haesebrouck et al. 2011).
To date, these microorganisms have been associated with gastritis, gastric and
duodenal ulcers, and low-grade mucosa-associated lymphoid tissue (MALT) lym-
phoma in humans. Gastric NHPH have microscopically been detected in 0.2–6 % of
humans with severe gastric complaints undergoing a gastroscopy, but this is most
probably an underestimation of their true prevalence (Haesebrouck et al. 2009). It
cannot be excluded that infections with these bacteria sometimes remain
unapparent or cause mild disease signs which are often not thoroughly examined.
Table 10.1 Helicobacter spp. and their hosts

Zoonotic
Taxon Natural hosts potential
Gastric Helicobacter spp.
‘Candidatus H. bovis’ Cattle Yes
‘Candidatus H. homininae’ Chimpanzee, gorilla Unknown
H. acinonychis Cheetah, tiger, lion Unknown
H. ailurogastricus Cat Unknown
H. baculiformis Cat Unknown
H. bizzozeronii Cat, dog Yes
H. cetorum Whale, dolphin Unknown
H. cynogastricus Dog Unknown
H. felis Dog, cat, cheetah, New Guinea wild dog, rabbit Yes
H. heilmannii Dog, cat, cheetah, bobcat, tiger, lynx, leopard, Yes
puma
H. mustelae Ferret Unknown
H. pylori Human /
H. salomonis Cat, dog, rabbit Yes
H. suis Pig, mandrill monkey, rhesus macaque, Yes
crab-eating macaque
Enterohepatic Helicobacter spp.
‘Candidatus H. colifelis’ Cat Unknown
H. anseris Goose Unknown
H. aurati Hamster Unknown
H. bilis Mouse, rat, gerbil, dog, cat, sheep Yes
H. brantae Goose Unknown
H. callitrichis Marmoset Unknown
H. canadensis Bird, pig Yes
H. canis Dog, cat Yes
H. cholecystus Hamster Unknown
H. cinaedi Hamster, rat, cat, dog, rhesus monkey, baboon Yes
H. equorum Horse Unknown
H. fennelliae Dog Yes
H. ganmani Mouse Yes
H. hepaticus Mouse, gerbil Yes
H. macacae Rhesus monkey, baboon Unknown
H. marmotae Woodchuck, cat Unknown
H. magdeburgensis Mice Unknown
H. mastomyrinus Rodents Unknown
H. mesocricetorum Hamster Unknown
H. muricola Wild mouse Unknown
H. muridarum Mouse, rat Unknown
H. pamatensis Bird, pig, cat Yes
H. pullorum Poultry Yes
H. rappini Mouse, sheep, dog Yes
(continued)

Zoonotic
Taxon Natural hosts potential
H. rodentium Mouse, rat Unknown
H. suncus House musk shrew Unknown
H. trogontum Rat, pig, sheep Unknown
H. typhlonius Mouse, rat Unknown
H. valdiviensis Wild birds Unknown
H. westmeadii Human /
H. winghamensis Human, wild rodents Yes
Clinical symptoms associated with gastric NHPH infections include acute or

chronic epigastric pain, nausea, dyspepsia, reflux esophagitis, heartburn, vomiting,
hematemesis, abdominal pain, irregular defecation frequency and consistency, and
dysphagia, often accompanied by a decreased appetite (Haesebrouck et al. 2009).
In patients undergoing an endoscopy, a variety of lesions can be observed
ranging from a normal to slightly hyperemic mucosa, mucosal edema, nodular
inflammation, and the presence of ulcerations in the antrum of the stomach or in
the duodenum (Haesebrouck et al. 2009; Sykora et al. 2003). Histopathological
examination of gastric biopsies reveals infiltration with lymphocytes and plasma
cells, sometimes organized in lymphocytic aggregates (Joosten et al. 2013b). Other
lesions, such as intestinal metaplasia, have occasionally been described in patients
infected with NHPH, and some of these patients were also infected with H. pylori
(Stolte et al. 1997; Yakoob et al. 2012). Compared to an H. pylori-associated
gastritis, gastritis associated with NHPH is often less active and less severe. On
the other hand, the risk of developing MALT lymphoma is higher with NHPH than
with H. pylori (Haesebrouck et al. 2009).
Tests to rapidly diagnose infection with gastric NHPH are currently unavailable.
Urea breath tests, used to diagnose infection with H. pylori, are often negative in
patients infected with animal-associated Helicobacter spp. (Matsumoto et al. 2014).
This can be explained by the fact that infections with these bacteria are, in contrast
to H. pylori infections, more often focal and predominantly found in the antrum of
the stomach (Stolte et al. 1997). Due to the fastidious nature of gastric NHPH,
isolation of these bacteria is not an option for routine diagnostic purposes. Until
now, only H. bizzozeronii (Andersen et al. 1999; Kivisto et al. 2010) and H. felis
(Wüppenhorst et al. 2013) have been cultured from gastric biopsies. Therefore,
analysis of gastric biopsies by molecular microbiological methods and histology is
so far the only way to determine infections with these microorganisms. A German
study analyzed 89 human gastric biopsies, previously shown to be NHPH positive
(Trebesius et al. 2001). Eighty percent of these samples were positive for H. suis,
17 (19 %) samples were positive for H. heilmannii, and 5 (6 %) hybridized with a
probe specific for H. felis, H. bizzozeronii, and H. salomonis. De Groote and
co-workers (2005) screened paraffin-embedded gastric biopsies from 101 patients
with confirmed gastric NHPH infection. Fourteen samples were positive for H. suis,
whereas 49 were infected with helicobacters from cats and dogs. Another Belgian
study showed similar results. H. suis was the most prevalent species (37 %),
followed by H. salomonis (21 %), H. felis (15 %), H. heilmannii (8 %), and
H. bizzozeronii (4 %) (Van Den Bulk et al. 2005b). A Polish study evaluated the
incidence of gastric NHPH infection in dyspeptic children at the age of 4–18 years
and found a prevalence of 0.2 % (Iwanczak et al. 2012). Another study focused on
the association between coinfection with canine and feline NHPH and H. pylori and
gastric pathology in patients with dyspepsia. H. pylori was found in 67 % of the
patients, and only 6 % and 4 % of them were coinfected with H. heilmannii and
H. felis, respectively (Yakoob et al. 2012). Recently, a remarkably high prevalence
(27 %) of H. suis DNA was found in gastric biopsies from human patients with
idiopathic parkinsonism. The putative significance of this bacterium in Parkinson’s
disease requires further investigation (Blaecher et al. 2013).
Evidence is accumulating that pigs, cats, and dogs constitute reservoir hosts for
gastric Helicobacter spp. with zoonotic potential (Haesebrouck et al. 2009).
Helicobacter DNA has been detected in saliva from cats, dogs, and pigs, indicating
that the oral cavity of these animals may act as source of NHPH infection for
humans (Ekman et al. 2013; Casagrande Proieti et al. 2010; Shojaee Tabrizi
et al. 2010). Fecal-oral transmission has also been suggested as a possible route
for infection in cats (Ghil et al. 2009). Besides direct contact with animals, other
routes of transmission of NHPH should not be neglected. It has been shown that
H. felis is able to survive in water for several days, highlighting the possible role for
water in the transmission of this species (Azevedo et al. 2008). Recently, De
Cooman and co-workers (2013) demonstrated that H. suis can be present on and
survive in minced pork. This indicates that raw or undercooked pork may also
constitute a source of H. suis infection for humans. Nowadays, the prevalence of
H. pylori in humans from the Western world is decreasing from generation to
generation, leaving a niche for possible infection with these animal-associated
gastric Helicobacter spp.
For patients with severe clinical symptoms and pathology, treatment is neces-
sary. There is, however, a lack of clinical trials, and only a few reports deal with
antimicrobial susceptibility and acquired resistance of gastric NHPH (Vermoote
et al. 2011b; Van den Bulck et al. 2005a). Triple therapy using the combination of a
proton-pump inhibitor and two antimicrobial agents, like clarithromycin, metroni-
dazole, amoxicillin, or tetracycline, may be effective in most cases but not always.
A Finnish patient infected with H. bizzozeronii received a triple therapy of tetra-
cyclines, metronidazole, and lansoprazole for 1 week. The symptoms subsided, but
the infection was not cleared and the patient continued to suffer from mild nausea.
H. bizzozeronii was isolated from the stomach, and determination of its antimicro-
bial susceptibility showed resistance against tetracycline and metronidazole (Schott
et al. 2012). Furthermore, it was demonstrated that acquired resistance to metroni-
dazole in H. bizzozeronii was due to the contingency nature of an oxygen-
insensitive NAD(P)H-nitroreductase. This phenomenon was also described for
H. heilmannii (Kondadi et al. 2013).
10.3 Natural and Experimental Gastric Helicobacter

Infections in Animal Hosts
10.3.1 Gastric Non-H. pylori Helicobacter spp. Associated

with Dogs and Cats
10.3.1.1 Prevalence in Dogs and Cats
In pet animals, gastric Helicobacter spp. have been frequently described with a
prevalence of 67–86 % in clinically healthy dogs, 61–100 % in dogs presenting
chronic vomiting, and 41–100 % in healthy cats as well as cats showing chronic
vomiting (Haesebrouck et al. 2009; Shojaee Tabrizi et al. 2010). Ghil and col-
leagues (2009) reported that the prevalence of Helicobacter spp. in feral cats
was approximately twofold higher than in domestic cats. Often cats and dogs
are naturally infected with multiple gastric Helicobacter spp. (Haesebrouck
et al. 2009). The first Helicobacter species isolated from the stomach of cats
and dogs was H. felis (Lee et al. 1988). Later on, H. bizzozeronii, H. salomonis,
and H. cynogastricus were isolated from the canine gastric mucosa, whereas
H. baculiformis, H. heilmannii and H. ailurogastricus isolates were obtained from
the stomach of cats (Haesebrouck et al. 2009; Smet et al. 2012; Joosten et al. 2015).
It has been shown that H. bizzozeronii is the most predominant species in the canine
stomach, whereas H. felis and H. heilmannii are the predominant Helicobacter
spp. in cats (Priestnall et al. 2004; Svec et al. 2000; Wiinberg et al. 2005). The
prevalence of H. cynogastricus and H. baculiformis in pet animals as well as their
zoonotic potential is so far unknown. Only few data is available on the transmission
of NHPH infections in dogs and cats. Transmission of H. salomonis from a dam to
her puppies, as well as between infected and noninfected pups, has been described
(Hänninen et al. 1998). It has been suggested that in this case, transmission occurred
through oral-oral or gastric-oral contact, as nursing dogs have very close contact
with their offspring and puppies eat material vomited by the dam (Hänninen
et al. 1998).
10.3.1.2 Role of Helicobacter spp. in the Development of Gastric

Pathologies in Dogs and Cats
In general, canine and feline Helicobacter spp. have been associated with chronic
active gastritis (Haesebrouck et al. 2009). Histological changes in the lamina
propria include mild mononuclear inflammatory infiltration, the presence of lym-
phoid follicles, fibrosis, and glandular degeneration in the stomach of cats naturally
infected with H. heilmannii (Takemura et al. 2009). One study reported a correla-
tion between Helicobacter infection and the presence of feline lymphoma
(Bridgeford et al. 2008). Gastric and duodenal ulcers have been rarely reported
among cats and dogs, and an association with Helicobacter infections has not been
made (Haesebrouck et al. 2009). To study the pathogenesis of NHPH infections in

dogs and cats, several experimental infection studies have been performed. A
mononuclear infiltration throughout the gastric mucosa, with follicular organization
of the inflammatory cells, has been demonstrated in the stomach of H. felis-
infected-specific pathogen-free (SPF) cats (Scanziani et al. 2001). Another exper-
imental infection study with H. felis in young gnotobiotic dogs described lymphoid
hyperplasia in the fundus and body of the stomach (Diker et al. 2002). On the
contrary, Simpson and co-workers (1999) found a similar degree of inflammation in
both H. felis-infected SPF dogs and uninfected control dogs. These contradictory
results may be explained by differences in virulence between H. felis strains. The
review by Haesebrouck and colleagues (2009) suggested that the pathogenic sig-
nificance of gastric helicobacters in cats and dogs may be related to (1) the species
expressing its own virulence which may be increased in cases of mixed infections
due to synergistic effects, (2) differences within strains from the same species, or
(3) host differences. An Italian study investigated the localization of Helicobacter
spp. in the fundic mucosa of laboratory beagle dogs. They demonstrated that
H. bizzozeronii was present in the superficial and basal portions of the fundic
glands, while H. felis was only detected in the superficial portions of the glands.
Additionally, these helicobacters were also located free in the cytoplasm or within
lysosomes of parietal cells (Lanzoni et al. 2011). The urea breath test, widely used
for rapid and noninvasive detection of H. pylori infection in humans, has recently
been applied on laboratory beagle dogs. A sensitivity and specificity of 89 % was
reported suggesting the usefulness of this technique to monitor gastric Helicobacter
infections in dogs (Kubota et al. 2013).
10.3.1.3 Genomics, Genetics, and Experimental Studies in Rodents

on the Pathogenesis of Canine and Feline Helicobacter
Infection
Recently the genomes of H. felis, H. bizzozeronii, and H. heilmannii have been

sequenced (Arnold et al. 2011; Schott et al. 2011; Smet et al. 2013). These genomes
contain genes encoding homologues of known H. pylori virulence factors discussed
in Chaps. 3, 4, 5, 6, and 7. These genomes possess a complete comB system
conferring natural competence, but they all lack the cytotoxin-associated gene
pathogenicity island (cagPAI), a functional vacuolating cytotoxin A (VacA), and
the H. pylori adhesins identified so far. Amorim and colleagues (2014) showed that
several canine and feline helicobacters, like H. felis and H. heilmannii, are able to
adhere to the canine gastric mucosa. Which adhesins are involved is so far
unknown.
Besides SPF pets, rodent models have also been shown to be useful experimental
infection models to obtain insights into the pathogenesis of gastric NHPH infections
in animals and humans (O’Rourke et al. 2004a). H. felis infection in mice is often
used as an animal model to study H. pylori-related gastric pathology in humans.
Due to the lack of several important H. pylori virulence factors, researchers must be
aware that H. felis might not always have similar outcomes in pathogenicity
compared to H. pylori. Extrapolation of data obtained in an H. felis infection
mouse model to H. pylori infections in humans should therefore be done with
caution. To date, many reports studied the pathogenesis of canine and feline
Helicobacter infection in rodent models. One of the very first steps in the patho-
genesis of gastric infections caused by these microorganisms is colonization of the
gastric mucosa in which binding to mucins (MUC) plays an important role. MUC1,
MUC5AC, and MUC6 are the major mucins covering the gastric mucosa. Liu and
colleagues (2014) investigated the gastric mucin expression pattern in the stomach
of H. heilmannii-infected BALB/c mice. They showed a remarkable increased
expression of Muc6 and Muc13 in the first 9 weeks postinfection. Since Muc6 is
expressed by the gastric glands and, unlike H. pylori, H. heilmannii was mainly
localized in the deep glands of the gastric mucosa, the potential role of Muc6 in
H. heilmannii colonization was suggested. The MUC13 mucin is normally not
expressed in a healthy stomach and has so far only been described as a marker
for gastric cancer in humans. The increased expression already in the early stages of
infection highlights its role in H. heilmannii colonization (Liu et al. 2014). The
mucin Muc1 is constitutively expressed by the gastric mucosa and is likely the first
point of contact between the host stomach and adherent pathogens. It has been
shown that Muc1 limits H. felis binding to gastric epithelial cells. However, it does
not limit colonization and gastric pathology following infection (Every et al. 2008).
The pathological changes in the mouse stomach infected with H. salomonis,
H. bizzozeronii, or H. felis have been evaluated as a means of distinguishing
different NHPH species in terms of virulence. H. salomonis was not able to colonize
the murine stomach. H. bizzozeronii showed moderate pathological changes, while
H. felis induced the most severe inflammatory changes (De Bock et al. 2005).
Another study by the same research group demonstrated that H. felis and
H. bizzozeronii induce gastric parietal cell loss in Mongolian gerbils, highlighting
the preference of these bacteria for parietal cells as was also demonstrated in their
natural host (De Bock et al. 2006). Takaishi and co-workers (2009) studied the
effect of gastrin on H. felis-associated gastric carcinogenesis using hypergas-
trinemic, gastrin-deficient, and wild-type C57BL/6 mouse models. Gastrin is
released by G cells mainly in the antrum of the stomach in response to food intake
and stimulates the secretion of gastric acid by parietal cells. Severe corpus dysplasia
with mild gastric atrophy was noted in H. felis-infected hypergastrinemic
(INS-GAS) mice, while mild to moderate antral dysplasia was seen in the gastrin-
deficient and wild-type mice. Gastrin deficiency did not result in an alteration of
H. felis colonization, but it was shown that gastrin is an essential cofactor for the
development of gastric dysplasia in H. felis-infected C57BL/6 mice. Joosten et al.
(2013a) demonstrated that chronic H. heilmannii infection in Mongolian gerbils
was associated with decreased gastric acid secretion and increased gastrin mRNA
levels stimulated by interleukin-1 beta (IL-1β). This latter finding could be consid-
ered as a reaction to the H. heilmannii-induced hypochlorhydria. Another study
investigated the role of H. felis infection in the etiology of iron deficiency in
INS-GAS C57BL/6 mice. Decreased serum iron concentrations were associated
with a concomitant reduction in the number of parietal cells, strengthening the

association between hypochlorhydria and gastric Helicobacter-induced iron defi-
ciency. Additionally, the marked changes in gastric iron concentrations and the
reduced number of parietal cells following Helicobacter infection may be relevant
to the more rapid development of carcinogenesis in H. felis-infected INS-GAS mice
(Thomson et al. 2012). The loss of parietal cells can lead to two distinct types of
mucous metaplasia: intestinal metaplasia and spasmolytic polypeptide-expressing
metaplasia (SPEM). It has been suggested that intestinal metaplasia induced by
Helicobacter infection develops in the presence of preexisting SPEM, supporting
the role of SPEM as a neoplastic precursor in the carcinogenesis cascade (Liu
et al. 2014; El-Zaatari et al. 2013). Several reports uniformly showed that H. felis
predominantly causes a T helper (Th)1/Th17 immune response in mice, as has also
been described for H. pylori (Ding et al. 2013; Obonyo et al. 2011; Sayi et al. 2011;
Stoicov et al. 2009, Hitzler et al. 2012). Other gastric NHPH, like H. heilmannii,
have been shown to cause a predominant Th2/Th17 host immune response in
BALB/c mice which clearly differs from what is observed during H. felis infection
(Liu et al. 2014). In BALB/c mice chronically infected with H. heilmannii, MALT
lymphoma-like lesions were observed (Liu et al. 2014; O’Rourke et al. 2004a).
Similar pathological findings have also been described in BALB/c mice chronically
infected with H. felis suggesting that this mouse model can be seen as a critical
model to study gastric MALT lymphoma (Stolte et al. 2002).
10.3.2 Gastric Non-H. pylori Helicobacter spp. Associated

with Large Felines
Besides domesticated cats, Helicobacter spp. like H. felis and H. heilmannii have
occasionally been found in the stomach of wild felines, such as lynx, leopards,
pumas, bobcats, tigers, and cheetahs (Hamir et al. 2004; M€orner et al. 2008;
O’Rourke et al. 2004b; Luiz de Camargo et al. 2011). In cheetahs, gastritis caused
by these bacteria is characterized by the infiltration of lymphocytes and plasma
cells in the epithelium and lamina propria with gland destruction and parietal cell
loss. In some cases lymphoid follicles were observed, especially in captive animals
and less frequently in wild animals (Terio et al. 2005; Munson et al. 2005). Terio
and colleagues (2012) further investigated the local immune response in cheetahs
with varying degree of Helicobacter-associated gastritis. The type of cells involved
was similar among all types of gastritis with the exception that a large number of
lamina proprial activated CD79a+CD21-B cells and plasma cells were only seen in
cheetahs with severe gastritis (Terio et al. 2012). Another and more frequently
found Helicobacter species in big cat predators is H. acinonychis. This species has
been associated with severe chronic gastritis in cheetahs, tigers, and lions
(Tegtmeyer et al. 2013). Cattoli and co-workers (2000) demonstrated that eradica-
tion of this bacterium from the stomach using antimicrobial treatment resulted in
the resolution of gastric lesions in tigers. H. acinonychis has been described as the
most closely related species to H. pylori (Tegtmeyer et al. 2013). Eppinger and
colleagues (2006) sequenced the H. acinonychis genome and showed that this
species arose after a single host jump of H. pylori from humans to large felines
approximately 43,000–56,000 years ago. Comparison between the H. acinonychis
and H. pylori genomes revealed that both species share a high number of core genes
(Tegtmeyer et al. 2013). The H. acinonychis genome also possesses unique features
that confirmed the direction of the host jump from humans to large felines.
Interestingly, H. acinonychis lacks a cagPAI and a functional VacA as has been
described for other animal-associated gastric helicobacters as well. Additionally,
the H. acinonychis genome contains a high number of fragmented genes due to
frameshifts and/or stop codons which are probably caused by niche changes and
host specialization (Gressmann et al. 2005). So far, only little information is
available about the interaction of H. acinonychis with its host. Dailidiene and
co-workers (2004) showed that H. acinonychis was able to infect mice and to
coexist and recombine with H. pylori in the stomach.
10.3.3 Gastric Helicobacter Infection in Pigs
In 1990, large spiral-shaped bacteria were described for the first time in the gastric
mucus layer and on the mucosal surface of pig stomachs (Mendes et al. 1990;
Queiroz et al. 1990). Initially, Gastrospirillum suis was proposed as a name, but
subsequent characterization showed that this organism in fact belonged to the genus
Helicobacter (De Groote et al. 1999b). A new name, ‘Candidatus Helicobacter
suis’, was then proposed, and despite numerous attempts worldwide, the first
successful in vitro isolate was only obtained in 2007, by using a new biphasic
culture method, which finally led to the description of H. suis as a new species
(Baele et al. 2008). Sequence analysis of 16S rRNA, 23S rRNA, partial hsp60, and
partial ureAB gene sequences confirmed that H. suis is identical to the previously
called H. heilmannii type 1. Besides pigs and humans, this species also colonizes
the stomach of nonhuman primates, such as mandrills, macaques, and baboons
(O’Rourke et al. 2004b).
10.3.3.1 Prevalence of Helicobacter suis in Pigs
The reported prevalence of H. suis infection in pigs depends on the study. In

general, this bacterium is detected in more than 60 % of the European, Asian, and
South and North American pigs at slaughter age (Barbosa et al. 1995; Grasso
et al. 1996; Cantet et al. 1999; Park et al. 2004; Hellemans et al. 2007b; Kopta
et al. 2010). Most likely, H. suis is transmitted via the oral-oral route through saliva
or via the gastric-oral route through vomiting or regurgitation, but this remains to be
investigated. Despite a very high prevalence in adult pigs, far lower degrees of
infection have been shown in younger animals. Hellemans and co-workers (2007b)
detected a prevalence of only 2 % in suckling piglets, which however increased
rapidly after weaning.
So far, H. suis has not been detected in the stomach of wild boars. In a Polish
study, the authors did find gastric Helicobacter spp., but interestingly, these bacte-
ria were shown not to be H. suis (Fabisiak et al. 2010). More worldwide studies are,
however, required to draw firm conclusions on the presence of H. suis in these wild
ancestors of domesticated pigs.
10.3.3.2 Helicobacter suis and Its Role in the Development of Gastric

Pathology in Pigs
H. suis has been shown to cause gastritis in experimentally and naturally infected
pigs, mainly in the antrum (Mendes et al. 1991; Grasso et al. 1996; Queiroz
et al. 1996; Park et al. 2000; Hellemans et al. 2007a; De Bruyne et al. 2012).
Besides an occasional neutrophilic infiltrate, the inflammation mainly composes of
a diffuse lymphocytic/plasmacytic infiltration as well as lymphocytic aggregates
and lymphoid follicles. In experimentally infected pigs, this gastritis shows a spatial
association with the main sites of colonization: the antrum and fundus (Hellemans
et al. 2007a; De Bruyne et al. 2012). Interestingly, diffuse lymphocytic infiltration
and lymphoid follicles have been shown to be present in the stomach, and mainly in
the cardiac region, of newborn piglets without Helicobacter infection (Driessen
et al. 2002; Mazzoni et al. 2011). Most likely, germinal centers in lymphoid
follicles are formed in the presence of an antigenic stimulus, for instance, during
H. suis infection.
Besides the strong association with gastritis, H. suis infection has also been
associated with ulceration of nonglandular stratified squamous epithelium of the
pars esophagea of the stomach, although H. suis bacteria probably do not colonize
this specific stomach region (Barbosa et al. 1995; Queiroz et al. 1996; Choi
et al. 2001; Roosendaal et al. 2000; Hellemans et al. 2007b; De Bruyne
et al. 2012). Other research groups did not find this association (Grasso
et al. 1996; Cantet et al. 1999; Melnichouk et al. 1999; Park et al. 2000; Szeredi
et al. 2005), so the exact role of H. suis in the development of these lesions remains
to be elucidated. Sapierzyński and colleagues (2007) demonstrated that H. suis
infection in pigs results in an increased number of gastrin-producing cells and a
decreased number of somatostatin-producing cells in the antrum. Since gastrin
stimulates and somatostatin inhibits the secretion of hydrochloric acid by parietal
cells, this may influence gastric acid production, which may also be altered due to
the tropism of this bacterium for parietal cells (Hellemans et al. 2007a). An altered
gastric acid secretion could in turn be involved in the development of ulceration of
the pars esophagea. The discrepancies found in literature may be due to differences
in laboratory techniques, different sampling practices, or differences in virulence
between H. suis strains. In any case, hyperkeratosis and ulceration of the
nonglandular part of the stomach have been reported in many countries. Up to
80 % of the market pigs in Australia (Robertson et al. 2002) and 60 % of the sows
(Hessing et al. 1992) in the Netherlands have been described to be affected.
The development of lesions in the pars esophagea is most likely a process
involving different factors, including stress, transport, the presence in the stomach
of short-chain fatty acids, and pelleting and fine grinding of the feed (Haesebrouck
et al. 2009; Argenzio and Eisemann 1996). The latter factor may have an influence
on the fluidity of gastric contents (Elbers and Dirkzwager 1994), leading to an
increased contact of the stratified squamous epithelium of the nonglandular region
with the luminal content of the distal part with acid, bile (refluxed from the
duodenum), and pepsin.
Ulceration of the porcine gastric nonglandular mucosa may result in decreased
feed intake, a decrease in daily weight gain, and even sudden death due to fatal
hemorrhage (Ayles et al. 1996; Haesebrouck et al. 2009), thus leading to significant
economic losses. There is also little doubt that this disease can cause pain and
discomfort. Interestingly, a decrease in daily weight gain of up to 10 % has been
observed in pigs experimentally infected with H. suis, however, without a clear
association with the development of lesions in the nonglandular part (De Bruyne
et al. 2012).
Besides H. suis, other Helicobacter species have been described in the stomach
of pigs, including an H. pylori-like bacterium, responsible for ulceration of the pars
esophagea in gnotobiotic piglets (Krakowka and Ellis 2006), H. bilis and
H. trogontum (Hänninen et al. 2003, 2005). The main site of colonization of the
latter two is most probably the lower intestinal tract, and it remains to be determined
whether these bacteria are able to colonize the porcine stomach and cause gastric
pathology.
10.3.3.3 Genomics, Genetics, and Experimental Studies

on the Pathogenesis of H. suis Infection
Genome sequencing of H. suis (Vermoote et al. 2011a) revealed the presence of

homologues of H. pylori genes involved in acid acclimation, motility, chemotaxis,
oxidative stress resistance, and adhesion to gastric epithelial cells. These include
genes encoding urease A and B subunits and urease accessory proteins, genes
encoding different components of the flagellar apparatus, several che and tlp
genes, katA, sodB, genes encoding several members of the major H. pylori outer
membrane families, and hpaA. In addition, H. suis harbors a near complete comB
type IV secretion system and homologues of the H. pylori neutrophil-activating
protein and γ-glutamyl transpeptidase, but it lacks most members of the cagPAI as
well as a functional VacA.
In literature, several studies describe experimental infection of mice with
H. heilmannii. Sometimes, however, this name might be misleading, as several of
these studies have in fact used H. suis bacteria, obtained from mucus, or homog-
enized gastric tissue of infected mice, pigs, or nonhuman primates, because of the
lack of in vitro isolated strains. Infection of mice with these inocula induces
inflammation, already 7 days after inoculation (Moura et al. 1993). During long-
term infection studies of up to 2 years, infiltration of the gastric mucosa with
lymphocytes and plasma cells with subsequent development of lymphoid follicles
has been observed, both in studies using mucosal homogenates and pure in vitro
isolated H. suis strains (Park et al. 2008; Flahou et al. 2010). Conflicting reports
have been published regarding the immune response underlying H. suis-induced
gastritis. Several authors have described the inflammation and formation of gastric
lymphoid follicles to be mainly driven by a Th1 response (Cinque et al. 2006;
Mimura et al. 2011). Others have described a mixed Th1/Th2 response (Park
et al. 2008), whereas studies using pure in vitro isolated H. suis strains revealed
that experimental H. suis infection causes an upregulation of IL-17, IL-10, and IL-4
expression, in the absence of interferon (IFN)-γ upregulation (Flahou et al. 2012),
which also clearly contrasts to the immune response elicited by H. pylori in this
same animal model. Recently, however, Liang and colleagues (2015) described a
strong upregulation of IFN-γ expression in H. suis-infected Mongolian gerbils.
The resulting chronic gastritis has been shown not to depend upon the presence
of Peyer’s patches in the small intestine, in contrast to what has been described for
H. pylori (Nobutani et al. 2010). Often, this chronic gastritis evolves to more severe
histopathological lesions. In BALB/c mice experimentally infected with different
H. heilmannii bacteria, “isolated” in vivo in mice and originating both from humans
and animals, gastric MALT lymphoma has been shown to develop starting from
18 months postinfection (O’Rourke et al. 2004a). In this study, the most severe
pathological changes were seen in mice infected with in vivo “isolates” from a
human patient, a bobcat, a crab-eating macaque, and mandrill monkeys. Except for
the strain from the bobcat, these strains were in fact shown to be H. suis. Similar
lesions were observed in C57BL/6 mice infected for at least 6 months with an
in vivo “isolate” of ‘Candidatus H. heilmannii’, which in fact was shown to belong
to the species H. suis (Nakamura et al. 2007; Haesebrouck et al. 2009), as well as in
Mongolian gerbils infected with an in vitro isolated strain of H. suis (Flahou
et al. 2010). These histopathological changes resemble inflammation-related
changes in the stomach of humans infected with NHPH, including H. suis.
In addition to the involvement of several cytokines, including IL-4 and chemo-
kine (C-X-C motif) ligand 13 (CXCL-13) (Flahou et al. 2012; Yamamoto
et al. 2014), several mechanisms have been described to stimulate
lymphomagenesis in this setting. An overexpression of miR-142-5p and miR-155,
as well as increased lymphangiogenesis and angiogenesis, has been shown to be
involved in H. suis-induced gastric MALT lymphoma (Saito et al. 2012; Nakamura
et al. 2008). The latter changes are accompanied by an increased expression of
vascular endothelial growth factors (VEGF), such as VEGF-A and VEGF-C, and
some of its receptors, including Flt-1 and Flt-4 (Nishikawa et al. 2007; Nakamura
et al. 2007, 2008, 2010). In addition, the formation of peripheral lymph node
addressin (PNAd)- and mucosal addressin cell adhesion molecule 1 (MadCAM-
1)-expressing high endothelial venule-like vessels plays a role (Suzuki et al. 2010).
These alterations may cause a sustained “homing” of lymphocytes to the gastric
mucosa. Interestingly, the administration of antibodies raised against certain of the
abovementioned factors, including CXCL-13, Flt-1, and Flt-4, induces a marked

reduction of the size of the region affected by gastric MALT lymphoma, which may
have implications for the future treatment guidelines of Helicobacter-induced
gastric MALT lymphoma (Nakamura et al. 2010, 2014; Yamamoto et al. 2014).
Besides inflammation-related pathological changes, H. suis also interacts with
the mucosal epithelium. In infected humans, the majority of H. pylori bacteria
remain in the mucus layer, whereas a limited number adhere to gastric epithelial
(mucus-secreting) cells (Lindén et al. 2008). H. suis, on the other hand, is most
often observed in the vicinity of or inside the canaliculi of acid-producing parietal
cells in pigs, humans, and experimentally infected mice and Mongolian gerbils
(Hellemans et al. 2007a; Joo et al. 2007; Flahou et al. 2010). Regularly, these
parietal cells show signs of degeneration or oncosis. Many questions remain
unanswered with regard to the mechanisms underlying the epithelium-related
changes observed during H. suis infection. As described above, several important
H. pylori virulence factors, such as the cagPAI and VacA, are absent or
nonfunctional in H. suis (Vermoote et al. 2011a). So far, the H. suis γ-glutamyl
transpeptidase (GGT) is the only virulence factor from H. suis with a confirmed role
in death of gastric epithelial cells. In vitro research using a human gastric epithelial
cell line (AGS) has shown that the enzyme causes apoptosis or necrosis, depending
in part on the amount of reactive oxygen species (ROS) generated through degra-
dation of reduced glutathione (GSH), an important antioxidant (Flahou et al. 2011).
Besides its role in death of epithelial cells, the same enzyme has been shown to
impair lymphocyte function, which is in line to what has been published for
H. pylori (Zhang et al. 2013). Supplementation of GGT-treated cells with known
substrates of the enzyme was shown to modulate the observed effects: glutamine
restored normal proliferation of lymphocytes, whereas supplementation with
reduced glutathione strengthened H. suis GGT-mediated inhibition of proliferation.
Most likely, depletion of glutamine and the generation of reactive oxygen species
through degradation of GSH are involved. In this same study, H. suis outer
membrane vesicles were identified as a possible delivery route of H. suis GGT to
lymphocytes residing in the deeper mucosal layers.
Parallel to the cell death it induces, H. suis infection also causes an increased
proliferation of progenitor cells in the isthmus of gastric glands (Flahou et al. 2010).
A direct effect of the bacteria may be involved (Fast et al. 2011), or alternatively,
this may reflect a compensatory hyperproliferation due to increased epithelial cell
loss (Shirin and Moss 1998).
10.3.4 Gastric Helicobacter Infection in Nonhuman

Primates
Captive rhesus macaques are commonly infected with H. pylori (Drazek

et al. 1994). The anatomy and physiology of the stomach resemble that of humans,
which suggests that the rhesus monkey model can serve as a good experimental
model to study H. pylori infection. For instance, it has been shown that socially
housed rhesus monkeys rapidly acquire H. pylori infection, particularly during the
peripartum period (Solnick et al. 2003, 2006). Once acquired, infection is associ-
ated with chronic gastritis resembling that seen in humans.
Besides H. pylori, nonhuman primates can be naturally infected with gastric
NHPH. Although an exact species designation of the colonizing Helicobacter
species is sometimes lacking, H. suis seems to be the species involved (O’Rourke
et al. 2004b; Martin et al. 2013; Nakamura et al. 2007; Matsui et al. 2014). In the
stomachs of rhesus monkeys, gastric NHPH which were not identified to the species
level have been observed in the mucus covering the surface epithelial cells, as well
as in the lumina of gastric glands. These microorganisms were shown to be able to
invade and on occasion damage parietal cells, which was however often accompa-
nied by an apparent hyperchlorhydria. This contrasted with H. pylori infection in
this model, which appeared to cause a more pronounced gastritis, while apparently
not modifying the acid output (Dubois et al. 1991). In another study, H. suis was
shown to be present in the stomach of all rhesus monkeys that were used for
determining the effects of an experimental H. pylori infection on the gastric
microbiota. Interestingly, the populations of H. suis and H. pylori were shown to
be highly dynamic, and a potential competitive inhibition/exclusion was observed
between both species (Martin et al. 2013).
NHPH infection in baboons has been associated with the development of
gastritis by Mackie and O’Rourke (2003) but not by others (Curry et al. 1989).
NHPH have been described to be naturally present in the stomachs of cynomolgus
monkeys from different geographic regions (Reindel et al. 1999; Drevon-Gaillot
et al. 2006). Again, H. suis seems to be the species involved (O’Rourke
et al. 2004b), and these microorganisms can be found in the gastric pits, in the
superficial glands, or on the surface epithelium. In one study, no correlation was
observed between these bacteria and the infiltration of lymphoplasmacytic cells and
inflammatory lesions in these gastric tissues (Drevon-Gaillot et al. 2006).
Recently, a putative new gastric Helicobacter species was detected in
wild chimpanzees and gorillas, which was provisionally named ‘Candidatus H.
homininae’ (Flahou et al. 2014).
10.3.5 Gastric Helicobacters Associated with Ruminants

and Horses
‘Candidatus Helicobacter bovis’ has been demonstrated in the pyloric part of the
abomasum of calves and adult cattle. So far, this bacterium has not been cultivated
in vitro (De Groote et al. 1999a), and its involvement in bovine gastric disease is
unknown. Although gastric ulcers regularly occur in calves and adult cattle
(Haringsma and Mouwen 1992; Jelinski et al. 1995; Ok et al. 2001), no association
between gastric ulceration and Helicobacter colonization was observed in a recent
study, since no Helicobacter bacteria could be detected in the animals that were
sampled (Valgaeren et al. 2013). H. pylori has been demonstrated in the stomach of
sheep (Dore et al. 2001), and so far, no helicobacters have been demonstrated in the
stomach of goats (Gueneau et al. 2002; Momtaz et al. 2014). Interestingly, H. pylori
has also been detected in milk from cows, sheep, and other ruminants (Quaglia
et al. 2008; Angelidis et al. 2011; Rahimi and Kheirabadi 2012). Although Rahimi
and colleagues claim to have isolated H. pylori bacteria, most studies used poly-
merase chain reaction or fluorescence in situ hybridization to detect H. pylori.
Further studies are therefore needed to assess whether H. pylori or H. pylori-like
bacteria are involed. In addition, it needs to be confirmed whether milk consump-
tion can serve as a route of transmission for H. pylori.
Although some studies describe the absence of Helicobacter spp. in the stomach
of horses (Husted et al. 2010; Perkins et al. 2012), others describe the presence
of Helicobacter-like organisms or their DNA in this niche. So far, however,
helicobacters have not yet been cultivated from the equine stomach (Scott
et al. 2001; Contreras et al. 2007), so their possible role in the development of
gastric ulcers, which are common in horses (Haesebrouck et al. 2009), remains
speculative.
10.3.6 Gastric Helicobacters Associated with Other Animal

Species
10.3.6.1 Marine Mammals
Urease-positive helicobacters have been isolated from the main stomach or feces of
various cetaceans, including stranded wild Atlantic white-sided dolphins and cap-
tive Pacific white-sided dolphins, Atlantic bottlenose dolphins, and a beluga whale
(Harper et al. 2002, 2003a). In 2002, these bacteria were characterized and
described as H. cetorum (Harper et al. 2002). The results of a health study, in
which 20 wild Atlantic bottlenose dolphins were sampled, showed that the preva-
lence in these animals was at least 50 % (Harper et al. 2003a). In addition to the
studies described above, helicobacters with a high homology to H. cetorum have
been isolated from or detected in fecal samples from captive seals and sea lions
from Australia, South American fur seals, the stomach of an Atlantic spotted
dolphin, gastric fluids, dental plaques, saliva and gastric tissue of captive dolphins
and a killer whale from Argentina, fecal material from wild and captive Yangtze
finless porpoises, the stomach of common dolphins, an Atlantic white-sided dolphin
and a striped dolphin from European waters, and the aquatic environment of captive
dolphins (Oxley and McKay 2005; Goldman et al. 2009a, b, 2011; Suárez
et al. 2010; McLaughlin et al. 2011; Davison et al. 2014).
Some of the captive animals from which H. cetorum was recovered showed
clinical signs, such as intermittent inappetence, lethargy, or chronic regurgitation.
Endoscopic or gross examination revealed the presence of esophageal and
forestomach ulcers, as well as gastric mucosal hemorrhages (Harper et al. 2002;

Davison et al. 2014). Cytological examination of gastric fluid of some animals
indicated the presence of inflammation in the stomach (Harper et al. 2002). This
was confirmed by histopathological analysis, revealing Helicobacter colonization
of the epithelial surface accompanied by a diffuse lymphoplasmacytic gastritis in
the main stomach and to a lesser extent in the pyloric stomach, and a mild distortion
of the adjacent glands (Harper et al. 2002; Suárez et al. 2010).
In a recent study, two genomes of H. cetorum were sequenced: one strain
originated from a dolphin and one strain isolated from a captive Beluga whale
(Kersulyte et al. 2013). Although these genomes, differing markedly from one
another in gene content, appeared to be larger than H. pylori genomes, the strains
were shown to be more closely related to H. pylori and H. acinonychis than to other
known species. They lack the cagPAI but do possess novel alleles of the vacA gene.
In addition, they reveal an extra triplet of vacA genes, metabolic genes distinct from
H. pylori, as well as genes encoding both an iron- and nickel-cofactored urease.
Besides H. cetorum, other putative novel Helicobacter spp., distinct from
H. cetorum, have been detected in the gastric fluids, gastric mucosa, or dental
plaque from dolphins, harp seals, and a sea lion with chronic gastritis (Harper
et al. 2003b; Oxley et al. 2004, 2005; Goldman et al. 2011).
10.3.6.2 Ferrets
Not so long after the discovery and description of H. pylori in humans, spiral
organisms were isolated from a gastric ulcer of a ferret and from the gastric mucosa
of two healthy ferrets (Fox et al. 1986). In 1989, these organisms were named
Helicobacter mustelae (Goodwin et al. 1989). Only a minority of ferrets younger
than 6 weeks are colonized by this bacterium, in contrast to the vast majority of
adult ferrets (Fox et al. 1988), indicating that widespread colonization and persis-
tence occur after weaning (Fox et al. 1991a; Forester et al. 2000). Keeping in mind
the ease by which ferrets vomit, oral-oral and gastric-oral contact most likely play a
role in transmission of H. mustelae (Fox et al. 1991a). Fecal-oral transmission has,
however, also been suggested. H. mustelae has indeed been isolated successfully
from feces of ferrets, and successful isolation correlated with periods of transient
hypochlorhydria, which may allow larger numbers of bacteria to exit the stomach
(Fox et al. 1992).
H. mustelae colonizes the mucosal surface in the corpus region, which often
induces only a superficial gastritis (Marini and Fox 1999). In the antrum, however,
H. mustelae colonizes the surface, gastric pits, and superficial portion of the glands,
leading to the development of a diffuse mononuclear gastritis with inflammatory
cells often occupying the full thickness of the mucosa (Fox et al. 1991b).
The incidence of gastric ulceration in ferrets varies between 1.4 and 35 %
(Andrews et al. 1979). Both gastric and duodenal ulcerations have been reported
in ferrets infected with H. mustelae (Fox et al. 1986, 1990). Given the high
prevalence of H. mustelae in adult ferrets, long-term observations of experimentally
infected pathogen-free ferrets are needed to elucidate the exact role of H. mustelae
infection in the development of peptic ulcer disease. An increased epithelial cell
proliferation has been detected in the gastric mucosa of ferrets infected with
H. mustelae, which may play a role in the development of gastric tumors
(Yu et al. 1995). Indeed, gastric adenocarcinoma has been described in the pyloric
mucosa of two ferrets infected with H. mustelae (Fox et al. 1997). In both cases, the
invasion of neoplastic tubules into the deep submucosa was described. Gastric
MALT lymphoma, accompanied by destruction of the gastric glands, has also
been described in the antrum of ferrets infected with H. mustelae (Erdman
et al. 1997). For both tumor types, however, evidence remains so far circumstantial
(Solnick and Schauer 2001).
H. mustelae adheres firmly to the gastric epithelium, and only a few bacteria are
seen lying in the mucus (O’Rourke et al. 1992). In H. mustelae infected ferrets, the
gastric mucosal hydrophobicity is reduced, which correlates with the degree of
mucosal inflammation (Gold et al. 1996). This may promote the attachment of
H. mustelae, which is thought to be mainly hydrophilic. H. mustelae binds to the
same receptor lipids as H. pylori, in particular phosphatidylethanolamine (Gold
et al. 1995). Like other gastric helicobacters, H. mustelae possesses a urease
enzyme and flagella, consisting of a body, hook, and flagellar filament composed
of FlaA and FlaB subunits. Clyne and coworkers (2000) showed that these flagella
do not play a direct role in promoting adherence of H. mustelae to gastric epithelial
cells. Double mutants of H. mustelae in flaA and flaB genes were shown to be
completely nonmotile and unable to colonize the ferret, whereas single-gene flaA
and flaB mutants have a decreased motility but are still able to colonize the ferret’s
stomach (Andrutis et al. 1997; Josenhans et al. 1995). An isogenic urease-negative
mutant of H. mustelae was shown to fail to colonize the ferret stomach (Andrutis
et al. 1995; Solnick et al. 1995). In addition to the standard nickel-dependent
UreAB, H. mustelae has been shown to possess a second, nickel-independent urease
(Stoof et al. 2008). Instead, this UreA2B2 is activated with ferrous ions in the
absence of auxiliary proteins. This unique metalloprotein is sufficient for the
bacteria to survive an acid shock in the presence of urea, and it is thought to play
a role in survival of the bacteria in the stomach of carnivores, with a diet rich in iron
and low in nickel (Stoof et al. 2008; Carter et al. 2011, 2012).
H. mustelae produces an array of surface rings, which seem to be unique to this
Helicobacter species. They are composed of the Helicobacter surface ring (Hsr)
protein, comprising approximately 25 % of the total envelope protein of H. mustelae
(O’Toole et al. 1994). These surface rings have been shown to play a role in
bacterial colonization and the pathogenesis of H. mustelae infection, as shown by
reduced numbers of bacteria recovered from mutant-dosed ferrets (Patterson
et al. 2003). Moreover, animals inoculated with the Hsr-negative strain show less
inflammation compared to ferrets infected with the wild-type strain. Like other
animal-associated gastric Helicobacter species, H. mustelae lacks a cagPAI and
VacA.
10.3.6.3 Hamsters and Mice
H. aurati has been isolated from the inflamed stomachs and ceca of adult
Syrian hamsters (Patterson et al. 2000a). Certain features, including the fusiform
shape and the presence of periplasmic fibrils, distinguish it from other enterohepa-
tic Helicobacter species detected in hamsters, such as H. cholecystus,
H. mesocricetorum, and H. cinaedi (Solnick and Schauer 2001; Whary and Fox
2004; Ceelen et al. 2007a). Although H. aurati possesses urease activity, the fact
that bacteria were recovered from cecal samples more often than from antral
samples indicates that the preferential colonization site of H. aurati in hamsters is
probably the intestinal tract (cecum) with subsequent spreading of this bacterial
agent to the stomach in some animals, possibly due to the coprophagic behavior of
hamsters (Patterson et al. 2000b). The precise role of H. aurati in gastric diseases
in hamsters has not yet been fully elucidated, although the organism has been
identified in hamsters suffering from chronic gastritis and intestinal metaplasia
(Patterson et al. 2000a, b). In the stomach of these same hamsters, the authors
reported the presence of another helical, urease-negative Helicobacter species, as
well as a smaller, urease-negative Campylobacter species. Likewise, natural infec-
tion with different Helicobacter species, including H. aurati, was reported in a
Syrian hamster with gastritis-associated adenocarcinoma (Nambiar et al. 2005).
There are no indications that H. aurati is of zoonotic significance.
Also in mice, urease-positive helicobacters have been described in the stomach.
In line with what has been described for H. aurati infection in Syrian hamsters,
H. muridarum has occasionally been detected in the stomach, with or without
concurrent inflammation, although these bacteria are found more frequently in the
ileal and cecal mucosa of the animals (Lee et al. 1992). H. suncus has been isolated
from the stomach of house musk shrews with chronic gastritis (Goto et al. 1998).
10.3.6.4 Rabbits
Only two reports describe the presence of H. felis and H. salomonis DNA in the
stomach of rabbits. So far these bacteria have not been cultivated from rabbits, and
their pathogenicity toward these animals is unknown (Haesebrouck et al. 2009; Van
den Bulck et al. 2006).
10.4 Enterohepatic Helicobacter Species
The NHPH species described above are mainly found colonizing the stomach of
their hosts. A large number of Helicobacter species, however, prefer to settle in
the lower intestinal tract or the liver of a wide variety of mammalian, reptilian,
avian, or amphibian host species as well as humans (Schrenzel et al. 2010; Hansen
et al. 2011).
Several enterohepatic Helicobacter species are found in rodents with or

without signs of intestinal or hepatic disease, including H. bilis, H. hepaticus,
H. cholecystus, H. muridarum, H. mastomyrinus, H. typhlonius, H. rodentium,
H. mesocricetorum, H. magdeburgensis, H. aurati, H. cinaedi, H. ganmani,
H. trogontum, and H. muricola (Lee et al. 1992; Fox et al. 1994, 1995; Patterson
et al. 2000a; Vandamme et al. 2000; Robertson et al. 2001; Solnick and Schauer
2001; Won et al. 2002; Shen et al. 2005; Traverso et al. 2010). Several models of
experimental enterohepatic Helicobacter infection, including H. hepaticus infec-
tion in immunodeficient IL-10-/- knockout mice, are used to model the development
of colitis/inflammatory bowel disease in humans (Solnick and Schauer 2001;
Hansen et al. 2011).
H. canis has been detected in or isolated from healthy dogs and cats as well as
dogs suffering from endemic diarrhea or necrotic hepatitis (Solnick and Schauer
2001; Shen et al. 2001). Several other enterohepatic Helicobacter species have
been demonstrated in dogs and cats, including H. bilis, H. cinaedi, ‘Candidatus
H. colifelis’, H. marmotae, and H. fennelliae (Solnick and Schauer, 2001; Fox
et al. 2002; Misawa et al. 2002; Hänninen et al. 2005; Rossi et al. 2008; Otte
et al. 2012; Castiglioni et al. 2012). The pathogenic significance of these microor-
ganisms for dogs and cats remains uncertain.
H. equorum has been isolated from the feces of healthy horses (Moyaert
et al. 2007a, b). The prevalence of this microorganism in different adult horse
populations varies between 0.8 % and 7.9 % but is much higher (66 %) in 1- to
6-month-old foals (Moyaert et al. 2009). Experimental infections revealed that this
microorganism colonizes the cecum, colon, and rectum of adult horses without
causing apparent pathological changes (Moyaert et al. 2007c).
Several Helicobacter spp., including H. trogontum, H. bilis, and H. canis, have
been identified or isolated from sheep, and infection with the first two species has
been associated with abortion and the presence of hepatic necrosis in aborted lambs
(Dewhirst et al. 2000; Solnick and Schauer 2001; Hänninen et al. 2003, 2005;
Swennes et al. 2014). Enterohepatic Helicobacter species have not been described
in other ruminants, including cattle and goats.
H. pamatensis (Seymour et al. 1994), H. trogontum (Hänninen et al. 2003),
H. bilis (Hänninen et al. 2005), and atypical H. canadensis strains (Inglis
et al. 2006) have been isolated from the gastrointestinal tract or feces of pigs, but
their pathogenic significance for this animal host is not known.
H. pullorum has been isolated from the ceca and feces of apparently healthy
chickens, as well as from laying hens with vibrionic hepatitis (Stanley et al. 1994).
In two in vivo studies, experimentally infected chickens remained clinically healthy
although mild lesions were observed in the ceca (Neubauer and Hess 2006; Ceelen
et al. 2007b). H. pullorum or its DNA have also been detected in other bird species,
including turkeys (Zanoni et al. 2011) and a parakeet suffering from diarrhea
(Ceelen et al. 2006b). Other Helicobacter species detected in birds include
H. canadensis, which has mainly been associated with geese but also with chickens,
guinea fowl, and pheasants (Fox et al. 2000; Nebbia et al. 2007; Robino et al. 2010).
H. anseris and H. brantae have been isolated from the feces of resident Canada
geese (Fox et al. 2006), and still now, new species are being discovered, including
H. valdiviensis, isolated from wild bird fecal samples (Collado et al. 2014).
Also in monkeys, enterohepatic helicobacters have been detected, including
H. callitrichis in common marmosets, H. cinaedi and H. macacae in rhesus
macaques and baboons, and several enterohepatic Helicobacter spp. in gorillas
and chimpanzees (Fernandez et al. 2002; Garcı́a et al. 2006; Fox et al. 2007;
Won et al. 2007; Flahou et al. 2014). It has been suggested that infection with
H. macacae plays a role in the development of chronic idiopathic colitis and
intestinal adenocarcinoma in rhesus macaques (Lertpiriyapong et al. 2014).
Several enterohepatic Helicobacter species have been associated with disease in
humans. These reports include associations between H. canis/H. winghamensis
and gastroenteritis; H. hepaticus and cholecystitis, liver carcinogenesis, or chronic
pancreatitis; H. bilis and chronic cholecystitis, biliary duct, and gallbladder
cancer; H. ganmani and liver disorders; H. pullorum and enteritis or diarrhea;
H. canadensis and enteritis; and H. cinaedi/H. fennelliae and chronic diarrhea,
enteritis, proctitis, or proctocolitis in homosexual men (Totten et al. 1985; Stanley
et al. 1994; Steinbrueckner et al. 1997; Fox et al. 2000; Melito et al. 2001; Solnick
and Schauer 2001; Matsukura et al. 2002; Murata et al. 2004; Tolia et al. 2004;
Kobayashi et al. 2005; Apostolov et al. 2005; Nilsson et al. 2006; Zhang et al. 2006;
Hamada et al., 2009). Enterohepatic Helicobacter species have also been associated
with the various forms of inflammatory bowel disease (Hansen et al. 2011), and
several species, including H. cinaedi, H. fennelliae, H. canadensis, H. canis,
H. westmaedii, and H. rappini, have been isolated from (immunosuppressed)
patients with bacteremia (Solnick and Schauer 2001; Tee et al. 2001, Matsumoto
et al. 2007; Prag et al. 2007; Abidi et al. 2013; Rimbara et al. 2013).
Most enterohepatic Helicobacter species do not contain an urease enzyme,
although there are exceptions, including H. hepaticus and H. bilis. A large number
of enterohepatic Helicobacter species contain a bacterial toxin called the cytolethal
distending toxin (CDT). The toxic effects of this major virulence factor involve
cellular distension, actin cytoskeleton remodeling, G2/M cell cycle arrest, and
cytolethality (Ceelen et al. 2006a; Varon et al. 2014). CDT is typically composed
of three subunits: CdtA, CdtB, and CdtC, which are all required for a maximal
cytotoxic activity (Liyanage et al. 2013; Varon et al. 2014). Other factors involved
in host-bacteria interactions include a type VI secretion system, which is expressed
by several enterohepatic helicobacters, including H. hepaticus, H. pullorum,
H. cinaedi, and H. trogontum (Chow and Mazmanian 2010; Goto et al. 2012;
Kaakoush et al. 2013; Sirianni et al. 2013).
There are clear indications that gastric NHPH species can cause disease in humans.
Some distinct features, such as the association with gastric MALT lymphoma,
indicate that these zoonotic bacteria should not just be considered as a “light”
version of H. pylori. There are clear indications that domestic animals constitute
reservoir hosts for these gastric Helicobacter species with zoonotic potential. A
correct diagnosis to the species level remains sometimes problematic. Therefore,
diagnostic methods enabling the correct identification of these bacteria are needed
to help clarify the epidemiology and pathology of these infections in humans. The
successful in vitro isolation of several of these species has opened new perspectives
for understanding the pathogenesis of non-H. pylori Helicobacter associated gastric
pathology in their natural hosts as well as humans. An increased knowledge may in
the end contribute to the development of new treatment and prevention measures.
References
Abidi MZ, Wilhelm MP, Neff JL, Hughes JG, Cunningham SA, Patel R (2013) Helicobacter canis
bacteremia in a patient with fever of unknown origin. J Clin Microbiol 51:1046–1048
Amorim and colleagues I, Freitas DP, Magalhaes A, Faria F, Lopes C, Faustino AM, Smet A,
Haesebrouck F, Reis CA, Gärtner F (2014) A comparison of Helicobacter pylori and
non-Helicobacter pylori Helicobacter spp. binding to canine gastric mucosa with defined
gastric glycophenotype. Helicobacter 19:249–259. doi:10.1111/hel.12125
Andersen LP, Boye K, Blom J, Holck S, Norgaard A, Elsborg L (1999) Characterization of a
culturable “Gastrospirillum hominis” (Helicobacter heilmannii) strain isolated from human
gastric mucosa. J Clin Microbiol 37:1069–1076
Andrews PL, Illman O, Mellersh A (1979) Some observations of anatomical abnormalities and
disease states in a population of 350 ferrets (Mustela furo L.). Z Verstierkd 21:346–353
Andrutis KA, Fox JG, Schauer DB, Marini RP, Murphy JC, Yan L, Solnick JV (1995) Inability of
an isogenic urease-negative mutant strain of Helicobacter mustelae to colonize the ferret
stomach. Infect Immun 63:3722–3725
Andrutis KA, Fox JG, Schauer DB, Marini RP, Li X, Yan L, Josenhans C, Suerbaum S (1997)
Infection of the ferret stomach by isogenic flagellar mutant strains of Helicobacter mustelae.
Angelidis AS, Tirodimos I, Bobos M, Kalamaki MS, Papageorgiou DK, Arvanitidou M (2011)
Detection of Helicobacter pylori in raw bovine milk by fluorescence in situ hybridization
(FISH). Int J Food Microbiol 151:252–256
Apostolov E, Al-Soud WA, Nilsson I, Kornilovska I, Usenko V, Lyzogubov V, Gaydar Y,
Wadstr€ om T, Ljungh A (2005) Helicobacter pylori and other Helicobacter species in gallblad-
der and liver of patients with chronic cholecystitis detected by immunological and molecular
methods. Scand J Gastroenterol 40:96–102
Argenzio RA, Eisemann J (1996) Mechanisms of acid injury in porcine gastroesophageal mucosa.
Am J Vet Res 57:564–573
Arnold I, Zigova Z, Holden M, Lawley T, Rad R, Dougan G, Falkow S, Bentley SD, Müller A
(2011) Comparative whole genome sequence analysis of the carcinogenic model pathogen
Helicobacter felis. Genome Biol Evol 3:302–308
Ayles HL, Friendship RM, Ball EO (1996) Effect of dietary particle size on gastric ulcers, assessed
by endoscopic examination, and relationship between ulcer severity and growth performance
of individually fed pigs. Swine Health Prod 4:211–216
Azevedo NF, Almeida C, Fernendes I, Cerqueira L, Dias S, Keevil CW, Vieira MJ (2008) Survival
of gastric and enterohepatic Helicobacter spp. in water: implications for transmission. Appl
Environ Microbiol 74:1805–1811
Baele M, Decostere A, Vandamme P, Ceelen L, Hellemans A, Chiers K, Ducatelle R, Haesebrouck

F (2008) Isolation and characterization of Helicobacter suis sp. nov. from pig stomachs. Int J
Syst Evol Microbiol 58:1350–1358
Barbosa AJA, Silva JCP, Nogueira AMMF, Paulino E, Miranda CR (1995) Higher incidence of
Gastrospirillum sp. in swine with gastric ulcer of the pars oesophagea. Vet Pathol 32:134–139
Blaecher C, Smet A, Flahou B, Pasmans F, Ducatelle R, Taylor D, Weller C, Bjarnason I,
Charlett A, Lawson AJ, Dobbs RJ, Dobbs SM, Haesebrouck F (2013) Significantly higher
frequency of Helicobacter suis in patients with idiopathic parkinsonism than in control
patients. Aliment Pharmacol Ther 38:1347–1353
Bridgeford EC, Marini RP, Feng Y, Parry NM, Rickman B, Fox JG (2008) Gastric Helicobacter
species as a cause of feline gastric lymphoma: a viable hypothesis. Vet Immunol
Immunopathol 123:106–113
Cantet F, Magras C, Marais A, Federighi M, Mégraud F (1999) Helicobacter species colonizing
pig stomach: molecular characterization and determination of prevalence. Appl Environ
Carter EL, Tronrud DE, Taber SR, Karplus PA, Hausinger RP (2011) Iron-containing urease in a
pathogenic bacterium. Proc Natl Acad Sci U S A 108:13095–13099
Carter EL, Proshlyakov DA, Hausinger RP (2012) Apoprotein isolation and activation, and
vibrational structure of the Helicobacter mustelae iron urease. J Inorg Biochem 111:195–202
Casagrande Proietti P, Bietta A, Brachelente C, Lepri E, Davidson I, Franciosini MP (2010)
Detection of Helicobacter spp. in gastric fecal and saliva samples from swine affected by
gastric ulceration. J Vet Sci 11:221–225
Castiglioni V, Vailati Facchini R, Mattiello S, Luini M, Gualdi V, Scanziani E, Recordati C (2012)
Enterohepatic Helicobacter spp. in colonic biopsies of dogs: molecular, histopathological and
immunohistochemical investigations. Vet Microbiol 159:107–114
Cattoli G, Bart A, Klaver PS, Robijn RJ, Beumer HJ, van Vugt R, Pot RG, van der Gaag I,
Vandenbroucke-Grauls CM, Kuipers EJ, Kusters JG (2000) Helicobacter acinonychis eradi-
cation leading to the resolution of gastric lesions in tigers. Vet Rec 147:164–165
Ceelen L, Decostere A, Ducatelle R, Haesebrouck F (2006a) Cytolethal distending toxin generates
cell death by inducing a bottleneck in the cell cycle. Microbiol Res 161:109–120
Ceelen L, Decostere A, Martel A, Pasmans F, Haesebrouck F (2006b) First report of Helicobacter
pullorum in the faeces of a diarrhoeic psittacine bird (Psephotus haematogaster). Vet Rec
159:389–390
Ceelen L, Haesebrouck F, Ducatelle R, Decostere A (2007a) The occurrence and clinical signif-
icance of enterohepatic Helicobacter species in laboratory rodents. Vlaams Diergen Tijds
76:103–116
Ceelen L, Decostere A, Chiers K, Ducatelle R, Maes D, Haesebrouck F (2007b) Pathogenesis of
Helicobacter pullorum infections in broilers. Int J Food Microbiol 116:207–213
Choi YK, Han JH, Joo HS (2001) Identification of novel Helicobacter species in pig stomachs by
PCR and partial sequencing. J Clin Microbiol 39:3311–3315
Chow J, Mazmanian SK (2010) A pathobiont of the microbiota balances host colonization and
intestinal inflammation. Cell Host Microbe 7:265–276
Cinque SMS, Rocha GA, Correa-Oliveira R, Soares TF, Moura SB, Rocha AMC, Nogueira AM,
Cabral MM, Vieira LQ, Martins-Filho OA, Queiroz DM (2006) The role of IFN-γ and IL-4 in
gastric mucosa inflammation associated with Helicobacter heilmannii type 1 infection. Braz J
Med Biol Res 39:253–261
Clyne M, Cr oinı́n TO, Suerbaum S, Josenhans C, Drumm B (2000) Adherence of isogenic
flagellum-negative mutants of Helicobacter pylori and Helicobacter mustelae to human and
ferret gastric epithelial cells. Infect Immun 68:4335–4339
Collado L, Jara R, Gonzalez S (2014) Description of Helicobacter valdiviensis sp. nov., a novel
Epsilonproteobacteria isolated from wild bird faecal samples in Southern Chile. Int J Syst Evol
Microbiol 64:1913–1919. doi:10.1099/ijs.0.057141-0
Contreras M, Morales A, Garcia-Amado MA, De Vera M, Bermúdez V, Gueneau P (2007)

Detection of Helicobacter-like DNA in the gastric mucosa of thoroughbred horses. Lett Appl
Curry A, Jones DM, Skelton-Stroud P (1989) Novel ultrastructural findings in a helical bacterium
found in the baboon (Papio anubis) stomach. J Gen Microbiol 135:2223–2231
Daidiliene D, Dailide G, Ogura K, Zhang M, Mukhopadhyay AK, Eaton KA, Cattoli G, Kusters
JG, Berg DE (2004) Helicobacter acinonychis: genetic and rodent infection studies of a
Helicobacter pylori-like gastric pathogen of cheetahs and other big cats. J Bacteriol
186:356–365
Davison NJ, Barnett JE, Koylass M, Whatmore AM, Perkins MW, Deaville RC, Jepson PD (2014)
Helicobacter cetorum infection in striped dolphin (Stenella coeruleoalba), Atlantic white-
sided dolphin (Lagenorhynchus acutus), and short-beaked common dolphin (Delphinus
delphus) from the Southwest Coast of England. J Wildl Dis. doi:10.7589/2013-02-047
De Bock M, Decostere A, Van den Bulck K, Baele M, Duchateau L, Haesebrouck F, Ducatelle R
(2005) The inflammatory response in the mouse stomach to Helicobacter bizzozeronii,
Helicobacter salomonis and two Helicobacter felis strains. J Comp Pathol 133:83–91
De Bock M, Decostere A, Hellemans A, Haesebrouck F, Ducatelle R (2006) Helicobacter felis and
Helicobacter bizzozeronii induce gastric parietal cell loss in Mongolian gerbils. Microbes
Infect 8:503–510
De Bruyne E, Flahou B, Chiers K, Meyns T, Kumar S, Vermoote M, Pasmans F, Millet S,
Dewulf J, Haesebrouck F, Ducatelle R (2012) An experimental Helicobacter suis infection
causes gastritis and reduced daily weight gain in pigs. Vet Microbiol 160:449–454
De Cooman L, Fahou B, Houf K, Smet A, Ducatelle R, Pasmans F, Haesebrouck F (2013) Survival
of Helicobacter suis bacteria in retail pig meat. Int J Food Microbiol 166:164–167
De Groote D, Van Doorn LJ, Ducatelle R, Verschuuren A, Tilmant K, Quint WGV,
Haesebrouck F, Vandamme P (1999a) Phylogenetic characterization of ‘Candidatus
Helicobacter bovis’, a new gastric Helicobacter in cattle. Int J Syst Bacteriol 49:1707–1715
De Groote D, van Doorn LJ, Ducatelle R, Verschuuren A, Haesebrouck F, Quint WGV, Jalava K,
Vandamme P (1999b) ‘Candidatus Helicobacter suis’, a gastric Helicobacter from pigs, and its
phylogenetic relatedness to other gastrospirilla. Int J Syst Bacteriol 49:1769–1777
De Groote D, Van Doorn LJ, Van den Bulck K, Vandamme P, Vieth M, Stolte M, Debongnie JC,
Burette A, Haesebrouck F, Ducatelle R (2005) Detection of non-pylori Helicobacter species in
“Helicobacter heilmannii”-infected humans. Helicobacter 10:398–406
Dewhirst FE, Fox JG, Mendes EN, Paster BJ, Gates CE, Kirkbride CA, Eaton KA (2000)
‘Flexispira rappini’ strains represent at least 10 Helicobacter taxa. Int J Syst Evol Microbiol
50:1781–1787
Diker KS, Haziroglu R, Akan M, Celik S, Kabakci N (2002) The prevalence colonization sites and
pathological effects of gastric helicobacters in dogs. Turk J Vet Anim Sci 26:345–351
Ding H, Nedrud JG, Blanchard TG, Zagorski BM, Li G, Shiu J, Xu J, Czinn SJ (2013)
Th1-mediated immunity against Helicobacter pylori can compensate for lack of Th17 cells
and can protect mice in the absence of immunization. PLoS ONE 8:e69384
Dore MP, Sepulveda AR, El-Zimaity H, Yamaoka Y, Osato MS, Mototsugu K, Nieddu AM,
Realdi G, Graham DY (2001) Isolation of Helicobacter pylori from sheep-implications for
transmission to humans. Am J Gastroenterol 96:1396–1401
Drazek ES, Dubois A, Holmes RK (1994) Characterization and presumptive identification of
Helicobacter pylori isolates from rhesus monkeys. J Clin Microbiol 32:1799–1804
Drevon-Gaillot E, Perron-Lepage MF, Clement C, Burnett R (2006) A review of background
findings in cynomolgus monkeys (Macaca fascicularis) from three different geographical
origins. Exp Toxicol Pathol 58:77–88
Driessen A, Van Ginneken C, Creemers J, Lambrichts I, Weyns A, Geboes K, Ectors N (2002)
Histological and immunohistochemical study of the lymphoid tissue in the normal stomach of
the gnotobiotic pig. Virchows Arch 441:589–598
Dubois A, Tarnawski A, Newell DG, Fiala N, Dabros W, Stachura J, Krivan H, Heman-Ackah LM

(1991) Gastric injury and invasion of parietal cells by spiral bacteria in rhesus monkeys. Are
gastritis and hyperchlorhydria infectious diseases? Gastroenterology 100:884–891
Ekman E, Fredriksson M, Trowald-Wigh G (2013) Helicobacter spp. in the saliva, stomach,
duodenum and faeces of colony dogs. Vet J 195:127–129
Elbers ARW, Dirkzwager A (1994) Changes in stomach mucosa in swine: a literature review.
Tijdschr Diergeneeskd 119:669–674
El-Zaatari M, Kao JY, Tessier A, Bai L, Hayes MM, Fontaine C, Eaton KA, Merchant JL (2013)
Gli1 deletion prevents Helicobacter-induced gastric metaplasia and expansion of myeloid cell
subsets. PLoS ONE 8:e58935
Eppinger M, Baar C, Linz B, Raddatz G, Lanz C, Keller H, Morelli G, Gressmann H, Achtman M,
Schuster SC (2006) Who ate whom? Adaptive Helicobacter genomic changes that accompa-
nied a host jump from early humans to large felines. PLoS Genet 7:e120
Erdman SE, Correa P, Coleman LA, Schrenzel MD, Li X, Fox JG (1997) Helicobacter mustelae-
associated gastric MALT lymphoma in ferrets. Am J Pathol 151:273–280
Every AL, Chionh YT, Skene CD, McGuckin MA, Sutton P (2008) Muc1 limits Helicobacter felis
binding to gastric epithelial cells but does not limit colonization and gastric pathology
following infection. Helicobacter 13:489–493
Fabisiak M, Sapierzyński R, Salamaszyńska-Guz A, Kizerwetter-Swida M (2010) The first
description of gastric Helicobacter in free-ranging wild boar (Sus scrofa) from Poland. Pol J
Vet Sci 13:171–174
Fast EM, Toomey ME, Panaram K, Desjardins D, Kolaczyk ED, Frydman HM (2011) Wolbachia
enhance Drosophila stem cell proliferation and target the germline stem cell niche. Science
334:990–992
Fernandez KR, Hansen LM, Vandamme P, Beaman BL, Solnick JV (2002) Captive rhesus
monkeys (Macaca mulatta) are commonly infected with Helicobacter cinaedi. J Clin
Flahou B, Haesebrouck F, Pasmans F, D’Herde K, Driessen A, Van Deun K, Smet A, Duchateau L,
Chiers K, Ducatelle R (2010) Helicobacter suis causes severe gastric pathology in mouse and
mongolian gerbil models of human gastric disease. PLoS ONE 5(11):e14083. doi:10.1371/
journal.pone.0014083
Flahou B, Haesebrouck F, Chiers K, Van Deun K, De Smet L, Devreese B, Vandenberghe I,
Favoreel H, Smet A, Pasmans F, D’Herde K, Ducatelle R (2011) Gastric epithelial cell death
caused by Helicobacter suis and Helicobacter pylori γ-glutamyl transpeptidase is mainly
glutathione degradation-dependent. Cell Microbiol 13:1933–1955
Flahou B, Van Deun K, Pasmans F, Smet A, Volf J, Rychlik I, Ducatelle R, Haesebrouck F (2012)
The local immune response of mice after Helicobacter suis infection: strain differences and
distinction with Helicobacter pylori. Vet Res 43:75
Flahou B, Modrý D, Pomajbı́ková K, Petrželková KJ, Smet A, Ducatelle R, Pasmans F, Sá RM,
Todd A, Hashimoto C, Mulama M, Kiang J, Rossi M, Haesebrouck F (2014) Diversity of
zoonotic enterohepatic Helicobacter species and detection of a putative novel gastric
Helicobacter species in wild and wild-born captive chimpanzees and western lowland gorillas.
Vet Microbiol 174:186–194
Forester NT, Parton K, Lumsden JS, O’Toole PW (2000) Isolation of Helicobacter mustelae from
ferrets in New Zealand. N Z Vet J 48:65–69
Fox JG, Edrise BM, Cabot EB, Beaucage C, Murphy JC, Prostak KS (1986) Campylobacter-like
organisms isolated from gastric mucosa of ferrets. Am J Vet Res 47:236–239
Fox JG, Cabot EB, Taylor NS, Laraway R (1988) Gastric colonization by Campylobacter pylori
subsp. mustelae in ferrets. Infect Immun 56:2994–2996
Fox JG, Correa P, Taylor NS, Lee A, Otto G, Murphy JC, Rose R (1990) Helicobacter mustelae-
associated gastritis in ferrets. Gastroenterology 99:352–361
Fox JG, Otto G, Murphy JC, Taylor NS, Lee A (1991a) Gastric colonization of the ferret with
Helicobacter species: natural and experimental infections. Rev Infect Dis 13(Suppl 8):S671–
S680
Fox JG, Otto G, Taylor NS, Rosenblad W, Murphy JC (1991b) Helicobacter mustelae-induced
gastritis and elevated gastric pH in the ferret (Mustela putorius furo). Infect Immun
59:1875–1880
Fox JG, Paster BJ, Dewhirst FE, Taylor NS, Yan LL, Macuch PJ, Chmura LM (1992) Helicobacter
mustelae isolation from feces of ferrets: evidence to support fecal-oral transmission of a gastric
Helicobacter. Infect Immun 60:606–611
Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor NS, Collins MJ Jr, Gorelick PL, Ward
JM (1994) Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers
and intestinal mucosal scrapings from mice. J Clin Microbiol 32(5):1238–1245
Fox JG, Yan LL, Dewhirst FE, Paster BJ, Shames B, Murphy JC, Hayward A, Belcher JC, Mendes
EN (1995) Helicobacter bilis sp. nov., a novel Helicobacter species isolated from bile, livers,
and intestines of aged, inbred mice. J Clin Microbiol 33(2):445–454
Fox JG, Dangler CA, Sager W, Borkowski R, Gliatto JM (1997) Helicobacter mustelae-associated
gastric adenocarcinoma in ferrets (Mustela putorius furo). Vet Pathol 34:225–229
Fox JG, Chien CC, Dewhirst FE, Paster BJ, Shen Z, Melito PL, Woodward DL, Rodgers FG
(2000) Helicobacter canadensis sp. nov. isolated from humans with diarrhea as an example of
an emerging pathogen. J Clin Microbiol 38:2546–2549
Fox JG, Shen Z, Xu S, Feng Y, Dangler CA, Dewhirst FE, Paster BJ, Cullen JM (2002)
Helicobacter marmotae sp. nov. isolated from livers of woodchucks and intestines of cats. J
Fox JG, Taylor NS, Howe S, Tidd M, Xu S, Paster BJ, Dewhirst FE (2006) Helicobacter anseris
sp. nov. and Helicobacter brantae sp. nov., isolated from feces of resident Canada geese in the
greater Boston area. Appl Environ Microbiol 72:4633–4637
Fox JG, Boutin SR, Handt LK, Taylor NS, Xu S, Rickman B, Marini RP, Dewhirst FE, Paster BJ,
Motzel S, Klein HJ (2007) Isolation and characterization of a novel Helicobacter species,
“Helicobacter macacae,” from rhesus monkeys with and without chronic idiopathic colitis. J
Garcı́a A, Xu S, Dewhirst FE, Nambiar PR, Fox JG (2006) Enterohepatic Helicobacter species
isolated from the ileum, liver and colon of a baboon with pancreatic islet amyloidosis. J Med
Ghil HM, Yoo JH, Jung WS, Chung TH, Youn HY, Hwang CY (2009) Survey of Helicobacter
infection in domestic and feral cats in Korea. J Vet Sci 10:67–72
Gold BD, Dytoc M, Huesca M, Philpott D, Kuksis A, Czinn S, Lingwood CA, Sherman PM (1995)
Comparison of Helicobacter mustelae and Helicobacter pylori adhesion to eukaryotic cells
in vitro. Gastroenterology 109:692–700
Gold BD, Islur P, Policova Z, Czinn S, Neumann AW, Sherman PM (1996) Surface properties of
Helicobacter mustelae and ferret gastrointestinal mucosa. Clin Investig Med 19:92–100
Goldman CG, Loureiro JD, Matteo MJ, Catalano M, Gonzalez AB, Heredia SR, Zubillaga MB,
Solnick JV, Cremaschi GA (2009a) Helicobacter spp. from gastric biopsies of stranded South
American fur seals (Arctocephalus australis). Res Vet Sci 86:18–21
Goldman CG, Matteo MJ, Loureiro JD, Degrossi J, Teves S, Heredia SR, Alvarez K, González
AB, Catalano M, Boccio J, Cremaschi G, Solnick JV, Zubillaga MB (2009b) Detection of
Helicobacter and Campylobacter spp. from the aquatic environment of marine mammals. Vet
Goldman CG, Matteo MJ, Loureiro JD, Almuzara M, Barberis C, Vay C, Catalano M, Heredia SR,
Mantero P, Boccio JR, Zubillaga MB, Cremaschi GA, Solnick JV, Perez-Perez GI, Blaser MJ
(2011) Novel gastric helicobacters and oral campylobacters are present in captive and wild
cetaceans. Vet Microbiol 152:138–145
Goodwin CS, Armstrong JA, Chilvers T, Peters M, Colins MD, Sly L, Mcconnell W, Harper WES
(1989) Transfer of Campylobacter pylori and Campylobacter mustelae to Helicobacter gen.
nov. as Helicobacter pylori comb. nov. and Helicobacter mustelae comb. nov., respectively.
Int J Syst Bacteriol 39:397–405
Goto K, Ohashi H, Ebukuro S, Itoh K, Tohma Y, Takakura A, Wakana S, Ito M, Itoh T (1998)
Isolation and characterization of Helicobacter species from the stomach of the house musk
shrew (Suncus murinus) with chronic gastritis. Curr Microbiol 37:44–51
Goto T, Ogura Y, Hirakawa H, Tomida J, Morita Y, Akaike T, Hayashi T, Kawamura Y (2012)
Complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of
human bacteremia. J Bacteriol 194:3744–3745
Grasso GM, Ripabelli G, Sammarco ML, Ruberto A, Iannitto G (1996) Prevalence of
Helicobacter-like organisms in porcine gastric mucosa: a study of swine slaughtered in Italy.
Comp Immunol Microbiol Infect Dis 19:213–217
Gressmann H, Linz B, Ghai R, Pleissner KP, Schlapbach R, Yamaoka Y, Kraft C, Suerbaum S,
Meyer TF, Achtman M (2005) Gain and loss of multiple genes during the evolution of
Helicobacter pylori. PLoS Genet 4:e43
Gueneau P, Fuenmayor J, Aristimuno OC, Cedeno S, Baez E, Reyes N, Michelangeli F,
Domı́nguez-Bello MG (2002) Are goats naturally resistant to gastric Helicobacter infection?
Vet Microbiol 84:115–121
Haesebrouck F, Pasmans F, Flahou B, Chiers K, Baele M, Meyns T, Decostere A, Ducatelle R
(2009) Gastric helicobacters in domestic animals and nonhuman primates and their signifi-
cance for human health. Clin Microbiol Rev 22:202–223
Haesebrouck F, Pasmans F, Flahou B, Smet A, Vandamme P, Ducatelle R (2011)
Non-Helicobacter pylori Helicobacter species in the human gastric mucosa: a proposal to
introduce the terms H. heilmannii sensu lato and sensu stricto. Helicobacter 16:339–340
Hamada T, Yokota K, Ayada K, Hirai K, Kamada T, Haruma K, Chayama K, Oguma K (2009)
Detection of Helicobacter hepaticus in human bile samples of patients with biliary disease.
Hamir AN, Stasko J, Rupprecht CE (2004) Observation of Helicobacter-like organisms in gastric
mucosa of grey foxes (Urocyon cinereoargenteus) and bobcats (Lynx rufus). Can J Vet Res
68:154–156
Hänninen ML, Happonen I, Jalava K (1998) Transmission of canine gastric Helicobacter
salomonis infection from dam to offspring and between puppies. Vet Microbiol 62:47–58
Hänninen ML, Utriainen M, Happonen I, Dewhirst FE (2003) Helicobacter sp. flexispira 16S
rDNA taxa 1, 4 and 5 and Finnish porcine Helicobacter isolates are members of the species
Helicobacter trogontum (taxon 6). Int J Syst Evol Microbiol 53:425–433
Hänninen ML, Kärenlampi RI, Koort JM, Mikkonen T, Bj€ orkroth KJ (2005) Extension of the
species Helicobacter bilis to include the reference strains of Helicobacter sp. flexispira taxa
2, 3 and 8 and Finnish canine and feline flexispira strains. Int J Syst Evol Microbiol 55:891–898
Hansen R, Thomson JM, Fox JG, El-Omar EM, Hold GL (2011) Could Helicobacter organisms
cause inflammatory bowel disease? FEMS Immunol Med Microbiol 61(1):1–14
Haringsma PC, Mouwen JMVM (1992) Mogelijke betekenis van spirilvormige bacteriën bij het
ontstaan van lebmaagzweren bij het volwassen rund. Tijdschr Diergeneeskd 117:485–486
Harper CG, Feng Y, Xu S, Taylor NS, Kinsel M, Dewhirst FE, Paster BJ, Greenwell M, Levine G,
Rogers A, Fox JG (2002) Helicobacter cetorum sp. nov., a urease-positive Helicobacter
species isolated from dolphins and whales. J Clin Microbiol 40:4536–4543
Harper CG, Whary MT, Feng Y, Rhinehart HL, Wells RS, Xu S, Taylor NS, Fox JG (2003a)
Comparison of diagnostic techniques for Helicobacter cetorum infection in wild Atlantic
bottlenose dolphins (Tursiops truncatus). J Clin Microbiol 41:2842–2848
Harper CG, Xu S, Rogers AB, Feng Y, Shen Z, Taylor NS, Dewhirst FE, Paster BJ, Miller M,
Hurley J, Fox JG (2003b) Isolation and characterization of novel Helicobacter spp. from the
gastric mucosa of harp seals Phoca groenlandica. Dis Aquat Organ 57:1–9
Heilmann KL, Borchard F (1991) Gastritis due to spiral shaped bacteria other than Helicobacter
pylori: clinical, histological, and ultrastructural findings. Gut 32:137–140
Hellemans A, Chiers K, Decostere A, De Bock M, Haesebrouck F, Ducatelle R (2007a) Exper-

imental infection of pigs with “Candidatus Helicobacter suis”. Vet Res Commun 31:385–395
Hellemans A, Chiers K, Maes D, De Bock M, Decostere A, Haesebrouck F, Ducatelle R (2007b)
Prevalence of “Candidatus Helicobacter suis” in pigs of different ages. Vet Rec 161:189–192
Hessing MJC, Geudeke MJ, Scheepens CJM, Tielen MJM, Schouten WGP, Wiepkema PR (1992)
Mucosal lesions in the pars oesophagea in pigs: prevalence and influence of stress. Tijdschr
Diergeneeskd 117:445–450
Hitzler I, Kohler E, Engler DB, Yazgan AS, Müller A (2012) The role of Th cell subsets in the
control of Helicobacter infections and in T cell-driven gastric immunopathology. Front
Immunol 3:142
Husted L, Jensen TK, Olsen SN, Mølbak L (2010) Examination of equine glandular stomach
lesions for bacteria, including Helicobacter spp. by fluorescence in situ hybridisation. BMC
Microbiol 10:84
Inglis GD, McConville M, de Jong A (2006) Atypical Helicobacter canadensis strains associated
with swine. Appl Environ Microbiol 72:4464–4471
Iwanczak B, Biernat M, Iwanczak F, Grabinska J, Matusiewicz K, Gosciniak G (2012) The clinical
aspects of Helicobacter heilmannii infection in children with dyspeptic symptoms. J Physiol
Pharmacol 63:133–136
Jelinski MD, Ribble CS, Chirino-Trejo M, Clark EG, Janzen ED (1995) The relationship between
the presence of Helicobacter pylori, Clostridium perfringens type A, Campylobacter spp, or
fungi and fatal abomasal ulcers in unweaned beef calves. Can Vet J 36:379–382
Joo M, Kwak JE, Chang SH, Kim H, Chi JG, Kim KA, Yang JH, Lee JS, Moon YS, Kim KM
(2007) Helicobacter heilmannii-associated gastritis: clinicopathologic findings and comparison
with Helicobacter pylori-associated gastritis. J Korean Med Sci 22:63–69
Joosten M, Blaecher C, Flahou B, Ducatelle R, Haesebrouck F, Smet A (2013a) Diversity in
bacterium-host interactions within the species Helicobacter heilmannii sensu stricto. Vet Res
44:65
Joosten M, Flahou B, Meyns T, Smet A, Arts J, De Cooman L, Pasmans F, Ducatelle R,
Haesebrouck F (2013b) Case report: Helicobacter suis infection in a pig veterinarian.
Joosten M, Lindén S, Rossi M, Tay A, Skoog E, Padra M, Thirriot F, Perkins T, Vandamme P, Van
Nieuwerburg F, Flahou B, Deforce D, Ducatelle R, Haesebrouck F, Smet A (2015) Genomic
divergence and differences in adhesion to the gastric mucosa between Helicobacter heilmannii
and its closest relative Helicobacter ailurogastricus sp. nov. Infect Immun 84:293–306
Josenhans C, Labigne A, Suerbaum S (1995) Comparative ultrastructural and functional studies of
Helicobacter pylori and Helicobacter mustelae flagellin mutants: both flagellin subunits, FlaA
and FlaB, are necessary for full motility in Helicobacter species. J Bacteriol 177:3010–3020
Kaakoush NO, Sirianni A, Raftery MJ, Mitchell HM (2013) The secretome of Helicobacter
trogontum. Helicobacter 18:316–320
Kersulyte D, Rossi M, Berg DE (2013) Sequence divergence and conservation in genomes of
Helicobacter cetorum strains from a dolphin and a whale. PLoS ONE 8:e83177
Kivist€o R, Linros J, Rossi M, Rautelin H, Hänninen ML (2010) Characterization of multiple
Helicobacter bizzeronii isolates from a Finish patient with severe dyspeptic symptoms and
chronic active gastritis. Helicobacter 15:58–66
Kobayashi T, Harada K, Miwa K, Nakanuma Y (2005) Helicobacter genus DNA fragments are
commonly detectable in bile from patients with extrahepatic biliary diseases and associated
with their pathogenesis. Dig Dis Sci 50:862–867
Kondadi PK, Pacini C, Revez J, Hänninen ML, Rossi M (2013) Contingency nature of
Helicobacter bizzozeronii oxygen-insensitive NAD(P)H-nitroreductase (HBZC1 00960) and
its role in metronidazole resistance. Vet Res 44:56
Kopta LA, Paquette JA, Bowersock TL, Choromanski LJ, Godbee TK, Galvin JE, Foss DL (2010)
Information of Helicobacter suis in pig-producing regions of North America. Conference of
Research Workers in Animal Diseases, Chicago, 5–7 December 1998
Krakowka S, Ellis J (2006) Reproduction of severe gastroesophageal ulcers (GEU) in gnotobiotic

swine infected with porcine Helicobacter pylori-like bacteria. Vet Pathol 43:956–962
Kubota S, Ohno K, Tsukamoto A, Maeda S, Murata Y, Nakashima K, Fukushima K, Uchida K,
Fujino Y, Tsujimoto H (2013) Value of the 13C urea breath test for the detection of gastric
Helicobacter spp. infection in dogs undergoing endoscopic examination. J Vet Med Sci
75:1049–1054
Lanzoni A, Faustinelli I, Cristofori P, Luini M, Simpson KW, Scanziani E, Recordati C (2011)
Localization of Helicobacter spp. in the fundic mucosa of laboratory beagle dogs: an ultra-
structural study. Vet Res 42:42
Lee A, Hazell SL, O’Rourke J, Kouprach S (1988) Isolation of a spiral-shaped bacterium from the
cat stomach. Infect Immun 56:2843–2850
Lee A, Phillips MW, O’Rourke JL, Paster BJ, Dewhirst FE, Fraser GJ, Fox JG, Sly LI, Romaniuk
PJ, Trust TJ, Kouprach S (1992) Helicobacter muridarum sp. nov., a microaerophilic helical
bacterium with a novel ultrastructure isolated from the intestinal mucosa of rodents. Int J Syst
Bacteriol 42:27–36
Lertpiriyapong K, Handt L, Feng Y, Mitchell TW, Lodge KE, Shen Z, Dewhirst FE,
Muthupalani S, Fox JG (2014) Pathogenic properties of enterohepatic Helicobacter spp.
isolated from Rhesus macaques with intestinal adenocarcinoma. J Med Microbiol
63:1004–1016
Liang J, De Bruyne E, Ducatelle R, Smet A, Haesebrouck F, Flahou B (2015) Purification of
Helicobacter suis strains from biphasic cultures by single colony isolation: influence on strain
characteristics. Helicobacter 20:206–216. doi:10.1111/hel.12192
Lindén SK, Wickstr€ om C, Lindell G, Gilshenan K, Carlstedt I (2008) Four modes of adhesion are
used during Helicobacter pylori binding to human mucins in the oral and gastric niches.
Liu C, Smet A, Blaecher C, Flahou B, Ducatelle R, Linden S, Haesebrouck F (2014) Gastric de
novo Muc13 expression and spasmolytic polypeptide-expressing metaplasia during
Helicobacter heilmannii infection. Infect Immun 82:3227–3239
Liyanage NP, Dassanayake RP, Kuszynski CA, Duhamel GE (2013) Contribution of Helicobacter
hepaticus cytolethal distending toxin subunits to human epithelial cell cycle arrest and apo-
ptotic death in vitro. Helicobacter 18:433–443
Luiz de Camargo P, Akemi Uenaka S, Bette Motta M, Harumi Adania C, Yamasaki L, Alfieri AA,
Bracarense AP (2011) Gastric helicobacter spp. Infection in captive neotropical brazilian
feline. Braz J Microbiol 42:290–297
Mackie JT, O’Rourke JL (2003) Gastritis associated with Helicobacter-like organisms in baboons.
Vet Pathol 40:563–566
Marini RP, Fox JG (1999) Animal models of Helicobacter (ferrets). In: Zak O, Sande MA (eds)
Handbook of animal models of infection. Academic, London, pp 273–284
Martin ME, Bhatnagar S, George MD, Paster BJ, Canfield DR, Eisen JA, Solnick JV (2013) The
impact of Helicobacter pylori infection on the gastric microbiota of the rhesus macaque. PLoS
ONE 8:e76375
Matsui H, Takahashi T, Murayama SY, Uchiyama I, Yamaguchi K, Shigenobu S, Matsumoto T,
Kawakubo M, Horiuchi K, Ota H, Osaki T, Kamiya S, Smet A, Flahou B, Ducatelle R,
Haesebrouck F, Takahashi S, Nakamura S, Nakamura M (2014) Development of new PCR
primers by comparative genomics for the detection of Helicobacter suis in gastric biopsy
specimens. Helicobacter 19:260–271. doi:10.1111/hel.12127
Matsukura N, Yokomuro S, Yamada S, Tajiri T, Sundo T, Hadama T, Kamiya S, Naito Z, Fox JG
(2002) Association between Helicobacter bilis in bile and biliary tract malignancies: H. bilis in
bile from Japanese and Thai patients with benign and malignant diseases in the biliary tract.
Jpn J Vet Res 93:842–847
Matsumoto T, Goto M, Murakami H, Tanaka T, Nishiyama H, Ono E, Okada C, Sawabe E,
Yagoshi M, Yoneyama A, Okuzumi K, Tateda K, Misawa N, Yamaguchi K (2007) Multicenter
study to evaluate bloodstream infection by Helicobacter cinaedi in Japan. J Clin Microbiol

45:2853–2857
Matsumoto T, Masatomo K, Akamatsu T, Koide N, Ogiwara N, Kubota S, Sugano M,
Kawakami Y, Katsuyama T, Ota H (2014) Helicobacter heilmannii sensu stricto-related gastric
ulcers: a case report. World J Gastroenterol 20:3376–3382
Mazzoni M, Bosi P, De Sordi N, Lalatta-Costerbosa G (2011) Distribution, organization and
innervation of gastric MALT in conventional piglet. J Anat 219:611–621
McLaughlin RW, Zheng JS, Chen MM, Zhao QZ, Wang D (2011) Detection of Helicobacter in the
fecal material of the endangered Yangtze finless porpoise Neophocaena phocaenoides
asiaeorientalis. Dis Aquat Organ 95:241–245
McNulty CA, Dent JC, Curry A, Uff JS, Ford GA, Gear MW, Wilkinson SP (1989) New spiral
bacterium in gastric mucosa. J Clin Pathol 42:585–591
Melito PL, Munro C, Chipman PR, Woodward DL, Booth TF, Rodgers FG (2001) Helicobacter
winghamensis sp. nov., a novel Helicobacter sp. isolated from patients with gastroenteritis. J
Melnichouk SI, Friendship RM, Dewey CE, Bildfell RJ, Smart NL (1999) Helicobacter-like
organisms in the stomach of pigs with and without gastric ulceration. Swine Health Prod
7:201–205
Mendes EN, Queiroz DMM, Rocha GA, Moura SB, Leite VHR, Fonseca MEF (1990) Ultrastruc-
ture of a spiral micro-organism from pig gastric mucosa (“Gastrospirillum suis”). J Med
Mendes EN, Queiroz DMM, Rocha GA, Nogueira AMMF, Carvalho ACT, Lage AP, Barbosa AJ
(1991) Histopathological study of porcine gastric mucosa with and without a spiral bacterium
(“Gastrospirillum suis”). J Med Microbiol 35:345–348
Mimura T, Yoshida M, Nishiumi S, Tanaka H, Nobutani K, Takenaka M, Suleiman YB,
Yamamoto K, Ota H, Takahashi S, Matsui H, Nakamura M, Miki I, Azuma T (2011) IFN-γ
plays an essential role in the pathogenesis of gastric lymphoid follicles formation caused by
Helicobacter suis infection. FEMS Immunol Med Microbiol 63(1):25–34
Misawa N, Kawashima K, Kondo F, Kushima E, Kushima K, Vandamme P (2002) Isolation and
characterization of Campylobacter, Helicobacter and Anaerobiospirillum strains from a puppy
with bloody diarrhea. Vet Microbiol 87:353–364
Momtaz H, Dabiri H, Souod N, Gholami M (2014) Study of Helicobacter pylori genotype status in
cows, sheep, goats and human beings. BMC Gastroenterol 14:61
M€
orner T, Br€ ojer C, Ryser-Degiorgis MP, Gavier-Widén D, Nilsson HO, Wadstr€ om T (2008)
Detection of gastric Helicobacter species in free-ranging lynx (Lynx lynx) and red foxes
(Vulpes vulpes) in Sweden. J Wildl Dis 44:697–700
Moura SB, Queiroz DMM, Mendes EN, Nogueira AMMF, Rocha GA (1993) The inflammatory
response of the gastric mucosa of mice experimentally infected with “Gastrospirillum suis”. J
Moyaert H, Decostere A, Vandamme P, Debruyne L, Mast J, Baele M, Ceelen L, Ducatelle R,
Haesebrouck F (2007a) Helicobacter equorum sp. nov., a urease-negative Helicobacter species
isolated from horse faeces. Int J Syst Evol Microbiol 57:213–218
Moyaert H, Haesebrouck F, Baele M, Picavet T, Ducatelle R, Chiers K, Ceelen L, Decostere A
(2007b) Prevalence of Helicobacter equorum in faecal samples from horses and humans. Vet
Moyaert H, Decostere A, Pasmans F, Baele M, Ceelen L, Smits K, Ducatelle R, Haesebrouck F
(2007c) Acute in vivo interactions of Helicobacter equorum with its equine host. Equine Vet J
39:370–372
Moyaert H, Haesebrouck F, Ducatelle R, Pasmans F (2009) Helicobacter equorum is highly
prevalent in foals. Vet Microbiol 133:190–192
Munson L, Terio KA, Worley M, Jago M, Bagot-Smith A, Marker L (2005) Extrinsic factors
significantly affect patterns of disease in free-ranging and captive cheetah (Acinonyx jubatus)
populations. J Wildl Dis 41:542–548
Murata H, Tsuji S, Tjusii M, Fu HY, Tanimura H, Tjujimoto M, Matsuura N, Kawano S, Hori M

(2004) Helicobacter bilis infection in biliary tract cancer. Aliment Pharmacol Ther 20:90–94
Nakamura M, Murayama SY, Serizawa H, Sekiya Y, Eguchi M, Takahashi S, Nishikawa K,
Takahashi T, Matsumoto T, Yamada H, Hibi T, Tsuchimoto K, Matsui H (2007) “Candidatus
Helicobacter heilmannii” from a cynomolgus monkey induces gastric mucosa-associated
lymphoid tissue lymphomas in C57BL/6 mice. Infect Immun 75:1214–1222
Nakamura M, Takahashi S, Matsui H, Murayama SY, Aikawa C, Sekiya Y, Nishikawa K,
Matsumoto T, Yamada H, Tsuchimoto K (2008) Microcirculatory alteration in low-grade
gastric mucosa-associated lymphoma by Helicobacter heilmannii infection: its relation to
vascular endothelial growth factor and cyclooxygenase-2. J Gastroenterol Hepatol 23(Suppl
2):S157–S160
Nakamura M, Matsui H, Takahashi T, Ogawa S, Tamura R, Murayama SY, Takahashi S,
Tsuchimoto K (2010) Suppression of lymphangiogenesis induced by Flt-4 antibody in gastric
low-grade mucosa-associated lymphoid tissue lymphoma by Helicobacter heilmannii infec-
tion. J Gastroenterol Hepatol 25(Suppl 1):S1–S6
Nakamura M, Takahashi T, Matsui H, Takahashi S, Murayama SY, Suzuki H, Tsuchimoto K
(2014) New pharmaceutical treatment of gastric MALT lymphoma: anti-angiogenesis treat-
ment using VEGF receptor antibodies and celecoxib. Curr Pharm Des 20:1097–1103
Nambiar PR, Kirchain S, Fox JG (2005) Gastritis-associated adenocarcinoma and intestinal
metaplasia in a Syrian hamster naturally infected with Helicobacter species. Vet Pathol
42:386–390
Nebbia P, Tramuta C, Ortoffi M, Bert E, Cerruti Sola S, Robino P (2007) Identification of enteric
Helicobacter in avian species. Schweiz Arch Tierheilkd 149:403–407
Neubauer C, Hess M (2006) Tissue tropism of Helicobacter pullorum in specific pathogen-free
chickens determined by culture and nucleic acid detection. Avian Dis 50:620–623
Nilsson HO, Stenram U, Ihse I, Wadstr€om T (2006) Helicobacter species ribosomal DNA in the
pancreas, stomach and duodenum of pancreatic cancer patients. J Gastroenterol 12:3038–3043
Nishikawa K, Nakamura M, Takahashi S, Matsui H, Murayama SY, Matsumoto T, Yamada H,
Tsuchimoto K (2007) Increased apoptosis and angiogenesis in gastric low-grade mucosa-
associated lymphoid tissue-type lymphoma by Helicobacter heilmannii infection in C57BL/6
mice. FEMS Immunol Med Microbiol 50:268–272
Nobutani K, Yoshida M, Nishiumi S, Nishitani Y, Takagawa T, Tanaka H, Yamamoto K,
Mimura T, Bensuleiman Y, Ota H, Takahashi S, Matsui H, Nakamura M, Azuma T (2010)
Helicobacter heilmannii can induce gastric lymphoid follicles in mice via a Peyer’s patch-
independent pathway. FEMS Immunol Med Microbiol 60:156–164
O’Rourke J, Lee A, Fox JG (1992) An ultrastructural study of Helicobacter mustelae and evidence
of a specific association with gastric mucosa. J Med Microbiol 36:420–427
O’Rourke JL, Dixon MF, Jack A, Enno A, Lee A (2004a) Gastric B-cell mucosa-associated
lymphoid tissue (MALT) lymphoma in an animal model of ‘Helicobacter heilmannii’ infec-
tion. J Pathol 203:896–903
O’Rourke JL, Solnick JV, Neilan BA, Seidel K, Hayter R, Hansen LM, Lee A (2004b) Description
of “Candidatus Helicobacter heilmannii” based on DNA sequence analysis of 16S rRNA and
urease genes. Int J Syst Evol Microbiol 54:2203–2211
O’Toole PW, Austin JW, Trust TJ (1994) Identification and molecular characterization of a major
ring-forming surface protein from the gastric pathogen Helicobacter mustelae. Mol Microbiol
11:349–361
Obonyo M, Rickman B, Guiney DG (2011) Effects of myeloid differentiation primary response
gene 88 (MyD88) activation on Helicobacter infection in vivo and induction of a Th17
response. Helicobacter 16:398–404
Ok M, Sen I, Turgut K, Irmak K (2001) Plasma gastrin activity and the diagnosis of bleeding
abomasal ulcers in cattle. J Vet Med A 48:563–568
Otte CM, Gutiérrez OP, Favier RP, Rothuizen J, Penning LC (2012) Detection of bacterial DNA in
bile of cats with lymphocytic cholangitis. Vet Microbiol 156:217–221
Oxley AP, McKay DB (2005) Comparison of Helicobacter spp. genetic sequences in wild and
captive seals, and gulls. Dis Aquat Organ 65:99–105
Oxley AP, Powell M, McKay DB (2004) Species of the family Helicobacteraceae detected in an
Australian sea lion (Neophoca cinerea) with chronic gastritis. J Clin Microbiol 42:3505–3512
Oxley AP, Argo JA, McKay DB (2005) Helicobacter spp. from captive bottlenose dolphins
(Tursiops spp.) and polar bears (Ursus maritimus). Vet J 170:377–380
Park JH, Lee BJ, Lee YS, Park JH (2000) Association of tightly spiraled bacterial infection and
gastritis in pigs. J Vet Med Sci 62:725–729
Park JH, Seok SH, Cho SA, Baek MW, Lee HY, Kim DJ, Park JH (2004) The high prevalence of
Helicobacter sp. in porcine pyloric mucosa and its histopathological and molecular character-
istics. Vet Microbiol 104:219–225
Park JH, Seok SH, Baek MW, Lee HY, Kim DJ, Park JH (2008) Gastric lesions and immune
responses caused by long-term infection with Helicobacter heilmannii in C57BL/6 mice. J
Comp Pathol 139:208–217
Patterson MM, Schrenzel MD, Feng Y, Fox JG (2000a) Gastritis and intestinal metaplasia in
Syrian hamsters infected with Helicobacter aurati and two other microaerobes. Vet Pathol
37:589–596
Patterson MM, Schrenzel MD, Feng Y, Xu S, Dewhirst FE, Paster BJ, Thibodeau SA,
Versalovic J, Fox JG (2000b) Helicobacter aurati sp. nov., a urease-positive Helicobacter
species cultured from gastrointestinal tissues of Syrian hamsters. J Clin Microbiol
38:3722–3728
Patterson MM, O’Toole PW, Forester NT, Noonan B, Trust TJ, Xu S, Taylor NS, Marini RP, Ihrig
MM, Fox JG (2003) Failure of surface ring mutant strains of Helicobacter mustalae to
persistently infect the ferret stomach. Infect Immun 71:2350–2355
Perkins GA, den Bakker HC, Burton AJ, Erb HN, McDonough SP, McDonough PL, Parker J,
Rosenthal RL, Wiedmann M, Dowd SE, Simpson KW (2012) Equine stomachs harbor an
abundant and diverse mucosal microbiota. Appl Environ Microbiol 78:2522–2532
Pot RGJ, Stoof J, Nuijten PJM, De Haan LAM, Loeffen P, Kuipers EJ, van Vliet AH, Kusters JG
(2007) UreA2B2: a second urease system in the gastric pathogen Helicobacter felis. FEMS
Immunol Med Microbiol 50:273–279
Prag J, Blom J, Krogfelt K (2007) Helicobacter canis bacteraemia in a 7-month-old child. FEMS
Immunol Med Microbiol 50:264–267
Priestnall SL, Wiinberg B, Spohr A, Neuhaus B, Kuffer M, Wiedmann M, Simpson KW (2004)
Evaluation of Helicobacter heilmannii subtypes in the gastric mucosa of cats and dogs. J Clin
Quaglia NC, Dambrosio A, Normanno G, Parisi A, Patrono R, Ranieri G, Rella A, Celano GV
(2008) High occurrence of Helicobacter pylori in raw goat, sheep and cow milk inferred by
glmM gene: a risk of food-borne infection? Int J Food Microbiol 124:43–47
Queiroz DMM, Rocha GA, Mendes EN, Lage AP, Carvalho ACT, Barbosa AJA (1990) A spiral
microorganism in the stomach of pigs. Vet Microbiol 24:199–204
Queiroz DMM, Rocha GA, Mendes EN, Moura SB, Rocha De Oliveira AM, Miranda D (1996)
Association between Helicobacter and gastric ulcer disease of the pars oesophagea in swine.
Rahimi E, Kheirabadi EK (2012) Detection of Helicobacter pylori in bovine, buffalo, camel,
ovine, and caprine milk in Iran. Foodborne Pathog Dis 9:453–456
Reindel JF, Fitzgerald AL, Breider MA, Gough AW, Yan C, Mysore JV, Dubois A (1999) An
epizootic of lymphoplasmacytic gastritis attributed to Helicobacter pylori infection in
Cynomolgus Monkeys (Macaca fascicularis). Vet Pathol 36:1–13
Rimbara E, Mori S, Kim H, Matsui M, Suzuki S, Takahashi S, Yamamoto S, Mukai M, Shibayama
K (2013) Helicobacter cinaedi and Helicobacter fennelliae transmission in a hospital from
2008 to 2012. J Clin Microbiol 51:2439–2442
Robertson BR, O’Rourke JL, Vandamme P, On SL, Lee A (2001) Helicobacter ganmani sp. nov., a
urease-negative anaerobe isolated from the intestines of laboratory mice. Int J Syst Evol
Microbiol 51(Pt 5):1881–1889
Robertson LD, Accioly JM, Moore KM, Driesen SJ, Pethick DW, Hampson DJ (2002) Risk factors
for gastric ulcers in Australian pigs at slaughter. Prev Vet Med 53:293–303
Robino P, Tomassone L, Tramuta C, Rodo M, Giammarino M, Vaschetti G, Nebbia P (2010)
Prevalence of Campylobacter jejuni, Campylobacter coli and enteric Helicobacter in domestic
and free living birds in North-Western Italy. Schweiz Arch Tierheilkd 152:425–431
Roosendaal R, Vos JH, Roumen T, van Vugt R, Cattoli G, Bart A, Klaasen HL, Kuipers EJ,
Vandenbroucke-Grauls CM, Kusters JG (2000) Slaughter pigs are commonly infected by
closely related but distinct gastric ulcerative lesion-inducing gastrospirilla. J Clin Microbiol
38:2661–2664
Rossi M, Hänninen ML, Revez J, Hannula M, Zanoni RG (2008) Occurrence and species level
diagnostics of Campylobacter spp., enteric Helicobacter spp. and Anaerobiospirillum spp. in
healthy and diarrheic dogs and cats. Vet Microbiol 129:304–314
Saito Y, Suzuki H, Tsugawa H, Imaeda H, Matsuzaki J, Hirata K, Hosoe N, Nakamura M,
Mukai M, Saito H, Hibi T (2012) Overexpression of miR-142-5p and miR-155 in gastric
mucosa-associated lymphoid tissue (MALT) lymphoma resistant to Helicobacter pylori erad-
ication. PLoS ONE 7:e47396
Sapierzyński R, Fabisiak M, Kizerwetter-Swida M, Cywińska A (2007) Effect of Helicobacter
sp. infection on the number of antral gastric endocrine cells in swine. Pol J Vet Sci 10:65–70
Sayi A, Kohler E, Toller IM, Flavell RA, Müller W, Roers A, Müller A (2011) TLR-2-activated B
cells suppress Helicobacter-induced preneoplastic gastric immunopathology by inducing T
regulatory-1 cells. J Immunol 186:878–890
Scanziani E, Simpson KW, Monestiroli S, Soldati S, Strauss-Ayali D, Del Pierro F (2001)
Histological and immunohistochemical detection of different Helicobacter species in the
gastric mucossa of cats. J Vet Diagn Invest 13:3–12
Schott T, Rossi M, Hänninen M (2011) Genome sequence of Helicobacter bizzozeronii strain CIII-
1, an isolate from human gastric mucosa. J Bacteriol 193:4565–4566
Schott T, Kondadi PK, Hänninen ML, Rossi M (2012) Microevolution of a zoonotic Helicobacter
population colonizing the stomach of a human host before and after failed treatment. Genome
Biol Evol 4:1310–1315
Schrenzel MD, Witte CL, Bahl J, Tucker TA, Fabian N, Greger H, Hollis C, Hsia G, Siltamaki E,
Rideout BA (2010) Genetic characterization and epidemiology of helicobacters in
non-domestic animals. Helicobacter 15:126–142
Scott DR, Marcus EA, Shirazi-Beechey SSP (2001) Evidence of Helicobacter infection in the
horse. Proceedings of the American Society for Microbiology, Washington, DC, p 287
Seymour C, Lewis RG, Kim M, Gagnon DF, Fox JG, Dewhirst FE, Paster BJ (1994) Isolation of
Helicobacter strains from wild bird and swine feces. Appl Environ Microbiol 60:1025–1028
Shen Z, Feng Y, Dewhirst FE, Fox JG (2001) Coinfection of enteric Helicobacter spp. and
Campylobacter spp. in cats. J Clin Microbiol 39:2166–2172
Shen Z, Xu S, Dewhirst FE, Paster BJ, Pena JA, Modlin IM, Kidd M, Fox JG (2005) A novel
enterohepatic Helicobacter species ‘Helicobacter mastomyrinus’ isolated from the liver and
intestine of rodents. Helicobacter 10:59–70
Shirin H, Moss SF (1998) Helicobacter pylori induced apoptosis. Gut 43:592–594
Shojaee Tabrizi A, Jamshidi S, Oghalaei A, Zahraei Salehi T, Bayati Eshkaftaki A, Mohammadi M
(2010) Identification of Helicobacter spp. in oral secretions vs gastric mucosa of stray cats. Vet
Simpson KW, McDonough PL, Strauss-Ayali D, Chang YF, Harpending P, Valentina BA (1999)
Helicobacter felis infection in dogs: effect on gastric structure and function. Vet Pathol
36:237–248
Sirianni A, Kaakoush NO, Raftery MJ, Mitchell HM (2013) The pathogenic potential of
Helicobacter pullorum: possible role for the type VI secretion system. Helicobacter
18:102–111
Smet A, Flahou B, D’Herde K, Vandamme P, Cleenwerck I, Ducatelle R, Pasmans F, Haesebrouck
F (2012) Helicobacter heilmannii sp. nov., isolated from the feline gastric mucosa. Int J Syst
Evol Microbiol 62:299–306
Smet A, Van Nieuwerburgh F, Ledesma J, Flahou B, Deforce D, Ducatelle R, Haesebrouck F
(2013) Genome sequence of helicobacter heilmannii Sensu Stricto ASB1 isolated from the
gastric mucosa of a kitten with severe gastritis. Genome Announc 1 pii:e00033-12
Solnick JV, Schauer DB (2001) Emergence of diverse Helicobacter species in the pathogenesis of
gastric en enterohepatic diseases. Clin Microbiol Rev 14:59–97
Solnick JV, Josenhans C, Suerbaum S, Tompkins LS, Labigne A (1995) Construction and
characterization of an isogenic urease-negative mutant of Helicobacter mustelae. Infect
Immun 63:3718–3721
Solnick JV, Chang K, Canfield DR, Parsonnet J (2003) Natural acquisition of Helicobacter pylori
infection in newborn rhesus macaques. J Clin Microbiol 41:5511–5516
Solnick JV, Fong J, Hansen LM, Chang K, Canfield DR, Parsonnet J (2006) Acquisition of
Helicobacter pylori infection in Rhesus Macaques is most consistent with oral-oral transmis-
sion. J Clin Microbiol 44:3799–3803
Stanley J, Linton D, Burnens A, Dewhirst FE, On SLW, Porter A, Owen RJ, Costas M (1994)
Helicobacter pullorum sp. nov. – genotype and phenotype of new species isolated from poultry
and from humans patients with gastroenteritis. Microbiology 140:3441–3449
Steinbrueckner B, Haerter G, Pelz K, Weiner S, Rump JA, Deissler W, Bereswill S, Kist M (1997)
Isolation of Helicobacter pullorum from patients with enteritis. Scand J Infect Dis 29:315–318
Sterzenbach T, Lee SK, Brenneke B, von Goetz F, Schauer DB, Fox JG, Suerbaum S, Josenhans C
(2007) Inhibitory effect of enterohepatic Helicobacter hepaticus on innate immune responses
of mouse intestinal epithelial cells. Infect Immun 75:2717–2728
Stoicov C, Fan X, Liu JH, Bowen G, Whary M, Kurt-Jones E, Houghton J (2009) T-bet knockout
prevents Helicobacter felis-induced gastric cancer. J Immunol 183:642–649
Stolte M, Kroger G, Meining A, Morgner A, Bayerd€orffer E, Bethke B (1997) A comparison of
Helicobacter pylori and H. heilmannii gastritis. A matched control study involving
404 patients. Scand J Gastroenterol 32:28–33
Stolte M, Bayerd€ orffer E, Morgner A, Alpen B, Wündisch T, Thiede C, Neubauer A (2002)
Helicobacter and gastric MALT lymphoma. Gut 50:iii19–iii24
Stoof J, Breijer S, Pot RG, van der Neut D, Kuipers EJ, Kusters JG, van Vliet AH (2008) Inverse
nickel-responsive regulation of two urease enzymes in the gastric pathogen Helicobacter
mustelae. Environ Microbiol 10:2586–2597
Suárez P, Contreras M, Fernández-Delgado M, Salazar V, Pe~ na R, Michelangeli F, Garcı́a-Amado
MA (2010) Detection of Helicobacter in the digestive tract of an Atlantic spotted dolphin
(Stenella frontalis). J Wildl Dis 46:622–626
Suzuki A, Kobayashi M, Matsuda K, Matsumoto T, Kawakubo M, Kumazawa S, Koide N,
Miyagawa S, Ota H (2010) Induction of high endothelial venule-like vessels expressing
GlcNAc6ST-1-mediated L-selectin ligand carbohydrate and mucosal addressin cell adhesion
molecule 1 (MAdCAM-1) in a mouse model of “Candidatus Helicobacter heilmannii”-induced
gastritis and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Helicobacter
15:538–548
Svec A, Kordas P, Pavliz Z, Novotný J (2000) High prevalence of Helicobacter heilmannii-
associated gastritis in a small, predominantly rural area: further evidence in support of a
zoonosis? Scand J Gastroenterol 35:925–928
Swennes AG, Turk ML, Trowel EM, Cullin C, Shen Z, Pang J, Petersson KH, Dewhirst FE, Fox
JG (2014) Helicobacter canis colonization in sheep: a zoonotic link. Helicobacter 19:65–68
Sykora J, Hejda V, Varvarovska J, Stozicky F, Gottrand F, Siala K (2003) Helicobacter heilmannii
related gastric ulcer in childhood. J Pediatr Gastroenterol Nutr 36:410–413
Szeredi L, Palkovics G, Solymosi N, Tekes L, Méhesfalvi J (2005) Study on the role of gastric
Helicobacter infection in gross pathological and histological lesions of the stomach in finishing
pigs. Acta Vet Hung 53:371–383
Takaishi S, Tu S, Dubeykovskaya ZA, Whary MT, Muthupalani S, Rickman BH, Rogers AB,
Lertkowit N, Varro A, Fox JG, Wang TC (2009) Gastrin is an essential cofactor for
helicobacter-associated gastric corpus carcinogenesis in C57BL/6 mice. Am J Pathol
175:365–375
Takemura LS, Camargo PL, Alfieri AA, Bracarense AP (2009) Helicobacter spp. In cats:
association between infecting species and epithelial proliferation within the gastric lamina
propria. J Comp Pathol 141:127–134
Tee W, Montgomery J, Dyall-Smith M (2001) Bacteremia caused by a Helicobacter pullorum-like
organism. J Infect Dis 188:1893–1898
Tegtmeyer N, Rivas Traverso F, Rohde M, Oyarzabal OA, Lehn N, Schneider-Brachert W, Ferrero
RL, Fox JG, Berg DE, Backert S (2013) Electron microscopic, genetic and protein expression
analyses of Helicobacter acinonychis strains from a Bengal tiger. PLoS ONE 8:e71220
Terio KA, Munson L, Marker L, Aldridge BM, Solnick JV (2005) Comparison of Helicobacter
spp. in Cheetahs (Acinonyx jubatus) with and without gastritis. J Clin Microbiol 43:229–234
Terio KA, Munson L, Moore PF (2012) Characterization of the gastric immune response in
cheetahs (Acinonyx jubatus) with Helicobacter-associated gastritis. Vet Pathol 49:824–833
Thomson MJ, Pritchard DM, Boxal SA, Abuderman AA, Williams JM, Varro A, Crabtree JE
(2012) Gastric Helicobacter infection induces iron deficiency in the INS-GAS mouse. PLoS
ONE 7:e50194
Tolia V, Nilsson HO, Boyer K, Wuerth A, AL-Soud WA, Rabah R, Wadstr€ om T (2004) Detection
of Helicobacter ganmani-like 16S rDNA in pediatric liver tissue. Helicobacter 9:460–468
Totten PA, Fennell CL, Tenover FC, Wezenberg JM, Perine PL, Stamm WE, Holmes KK (1985)
Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp. nov.): two new Campylo-
bacter species associated with enteric disease in homosexual men. J Infect Dis 151:131–139
Traverso FR, Bohr UR, Oyarzabal OA, Rohde M, Clarici A, Wex T, Kuester D, Malfertheiner P,
Fox JG, Backert S (2010) Morphologic, genetic, and biochemical characterization of
Helicobacter magdeburgensis, a novel species isolated from the intestine of laboratory mice.
Trebesius K, Adler K, Vieth M, Stolte M, Haas R (2001) Specific detection and prevalence of
Helicobacter heilmannii-like organisms in the human gastric mucosa by fluorescent in situ
hybridization and partial 16S ribosomal DNA sequencing. J Clin Microbiol 39:1510–1516
Valgaeren BR, Pardon B, Flahou B, Verherstraeten S, Goossens E, Timbermont L, Haesebrouck F,
Ducatelle R, Van Immerseel F, Deprez PR (2013) Prevalence and bacterial colonisation of
fundic ulcerations in veal calves. Vet Rec 172:269
Van den Bulck K, Decostere A, Gruntar I, Baele M, Krt B, Ducatelle R, Haesbrouck F (2005a) In
vitro susceptibility testing of Helicobacter felis, H. bizzozeronii and H. salomonis. Antimicrob
Agents Chemother 49:2997–3000
Van den Bulck K, Decostere A, Baele M, Driessen A, Debongnie JC, Burette A, Stolte M,
Ducatelle R, Haesebrouck F (2005b) Identification of non-Helicobacter pylori spiral organisms
in gastric samples from humans, dogs and cats. J Clin Microbiol 43:2256–2260
Van den Bulck K, Decostere A, Baele M, Marechal M, Ducatelle R, Haesebrouck (2006) Low
frequency of Helicobacter species in the stomachs of experimental rabbits. Lab Anim
40:282–287
Vandamme P, Harrington CS, Jalava K, On SL (2000) Misidentifying helicobacters: the
Helicobacter cinaedi example. J Clin Microbiol 38:2261–2266
Varon C, Mocan I, Mihi B, Péré-Védrenne C, Aboubacar A, Moraté C, Oleastro M, Doignon F,
Laharie D, Mégraud F, Ménard A (2014) Helicobacter pullorum cytolethal distending toxin
targets vinculin and cortactin and triggers formation of lamellipodia in intestinal epithelial
cells. J Infect Dis 209:588–599
Vermoote M, Vandekerckhove TTM, Flahou B, Pasmans F, Smet A, De Groote D, Van

Criekinge W, Ducatelle R, Haesebrouck F (2011a) Genome sequence of Helicobacter suis
supports its role in gastric pathology. Vet Res 42:51
Vermoote M, Pasmans F, Flahou B, Van Deun K, Ducatelle R, Haesebrouck F (2011b) Antimi-
crobial susceptibility pattern of Helicobacter suis strains. Vet Microbiol 153:339–342
Warren JR, Marshal BJ (1983) Unidentified curved bacilli on gastric epithelium in active chronic
gastritis. Lancet 1:1273–1275
Whary MT, Fox JG (2004) Natural and experimental Helicobacter infections. Comp Med
54:128–158
Wiinberg B, Spohr A, Dietz HH, Egelund T, Greiter-Wilke A, McDonough SP, Olsen J,
Priestnall S, Chang YF, Simpson KW (2005) Quantitative analysis of inflammatory and
immune responses in dogs with gastritis and their relationship to Helicobacter spp. infection.
J Vet Intern Med 19:4–14
Won YS, Yoon JH, Lee CH, Kim BH, Hyun BH, Choi YK (2002) Helicobacter muricola sp. nov.,
a novel Helicobacter species isolated from the ceca and feces of Korean wild mouse (Mus
musculus molossinus). FEMS Microbiol Lett 209:45–51
Won YS, Vandamme P, Yoon JH, Park YH, Hyun BH, Kim HC, Itoh T, Tanioka Y, Choi YK
(2007) Helicobacter callitrichis sp. nov., a novel Helicobacter species isolated from the feces
of the common marmoset (Callithrix jacchus). FEMS Microbiol Lett 271:239–244
Wüppenhorst N, von Loewenich F, Hobmaier B, Vetter-Knoll M, Mohadjer S, Kist M (2013)
Culture of a gastric non-Helicobacter pylori Helicobacter from the stomach of a 14-year old
girl. Helicobacter 18:1–5
Yakoob J, Abbas Z, Khan R, Ahmad Z, Islam M, Awan S, Jafri F, Jafri W (2012) Prevalence of
non-Helicobacter pylori species in patients presenting with dyspepsia. BMC Gastroenterol
12:3
Yamamoto K, Nishiumi S, Yang L, Klimatcheva E, Pandina T, Takahashi S, Matsui H,
Nakamura M, Zauderer M, Yoshida M, Azuma T (2014) Anti-CXCL13 antibody can inhibit
the formation of gastric lymphoid follicles induced by Helicobacter infection. Mucosal
Immunol. doi:10.1038/mi.2014.14
Yu J, Russell RM, Salomon RN, Murphy JC, Palley LS, Fox JG (1995) Effect of Helicobacter
mustelae infection on ferret gastric epithelial cell proliferation. Carcinogenesis 16:1927–1931
Zanoni RG, Piva S, Rossi M, Pasquali F, Lucchi A, De Cesare A, Manfreda G (2011) Occurrence
of Helicobacter pullorum in turkeys. Vet Microbiol 149:492–496
Zhang L, Day A, McKenzie G, Mitchell H (2006) Nongastric Helicobacter species detected in the
intestinal tract of children. J Clin Microbiol 44:2276–2279
Zhang G, Ducatelle R, Pasmans F, D’Herde K, Huang L, Smet A, Haesebrouck F, Flahou B (2013)
Effects of Helicobacter suis γ-glutamyl transpeptidase on lymphocytes: modulation by gluta-
mine and glutathione supplementation and outer membrane vesicles as a putative delivery
route of the enzyme. PLoS ONE 8:e77966
Part II
Immune Responses and Host Factors
Chapter 11
Animal Models of Helicobacter pylori
Infection
Jay V. Solnick, Kathryn A. Eaton, and Richard M. Peek Jr.
Abstract Experimentation in animal models is essential to fully understand the

biology of H. pylori. Although a variety of animals have been successfully infected
with H. pylori, current studies most commonly use either mice, Mongolian gerbils,
or nonhuman primates, each of which has strengths and weaknesses. The mouse is
inexpensive and convenient, and the elegant genetics permits dissection of the host
response to infection. However, the function of the H. pylori type IV secretion
system—the best-known virulence factor—is commonly lost during colonization of
mice. This occurs less frequently in the gerbil, which also has the major advantage
that some H. pylori strains cause gastric adenocarcinoma during relatively short-
term colonization. But there is no genetics available in the gerbil and there are
fewer reagents available than for the mouse. Nonhuman primates are physiologi-
cally most similar to humans and are naturally infected with H. pylori, but are
limited by high cost and availability. Choice of the most appropriate model depends
on the question, resources, and the availability of local expertise.
Keywords Helicobacter pylori • Animal model • Mouse • Gerbil • Nonhuman

primate
J.V. Solnick, M.D., Ph.D. (*)

Departments of Medicine and Microbiology & Immunology, Center for Comparative
Medicine, University of California, Davis School of Medicine, Davis, CA 95616, USA
K.A. Eaton, D.V.M., Ph.D.
Department of Microbiology & Immunology, Laboratory Animal Medicine Unit, University of
Michigan School of Medicine, Ann Arbor, MI 48109, USA
R.M. Peek Jr., M.D.
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232,
USA

DOI 10.1007/978-4-431-55936-8_11
274 J.V. Solnick et al.
11.1 Introduction
Despite recent controversy over the relatively few animal models that dominate
biomedical research, and the extent to which they recapitulate disease processes in
humans (Bolker 2012; Seok et al. 2013; Couzin-Frankel 2013), animal experimen-
tation remains fundamental to our understanding of human health. Because of cost,
convenience, and particularly the availability of reagents and genetic tools, the
laboratory mouse is most often the model of choice. But for many infectious
diseases, mice cannot be readily infected with any given human pathogen, so
investigators must use either a different animal or a surrogate pathogen that is
similar to that which infects humans. Such was the case soon after the discovery of
Helicobacter pylori, when early attempts to infect mice and rats were unsuccessful,
even if they were germ-free (Cantorna and Balish 1990; Ehlers et al. 1988). The first
animal experimental infection was by Krakowka and coworkers, who demonstrated
that neonatal gnotobiotic piglets were susceptible to infection with H. pylori (then
Campylobacter pylori), which reproduced many of the histologic features found in
chronically infected humans (Krakowka et al. 1987). At about the same time,
natural H. pylori infection was identified in captive rhesus monkeys (Baskerville
and Newell 1988), and closely related species were cultured from domestic and
laboratory animals, including H. felis in cats (Lee et al. 1990) and H. mustelae in
ferrets (Fox et al. 1990). For the first 10 years after the discovery of H. pylori,
animal experimentation was predominantly performed using these surrogate
models—H. felis in mice and H. mustelae in ferrets—with a few studies of
H. pylori in pigs and nonhuman primates.
Although the first experimental rodent infection with H. pylori was described by
Karita and colleagues in 1991 (Karita et al. 1991), the mouse model was largely
ignored until 1995 when a seminal paper was published in Science (Marchetti
et al. 1995). Initially, many investigators had difficulty reproducing these findings.
The trick appeared to be that only some strains would robustly colonize mice and
that they needed to be fresh human clinical isolates, ideally adapted to the mouse by
repeated in vivo passage. This was validated in 1997 with the introduction of
Sydney Strain 1 (SS1), a mouse adapted strain that soon became the international
standard for animal experimentation with H. pylori (Lee et al. 1997). In addition to
the mouse, some investigators use the Mongolian gerbil because it is also a model
of gastric adenocarcinoma, or the nonhuman primate because it physiologically and
anatomically most closely resembles humans. Here, we critically review these three
model systems—mouse, gerbil, and nonhuman primate—and briefly consider the
human challenge model that has been developed for studies of H. pylori immuni-
zation. Other models such as dogs (Rossi et al. 1999), cats (Fox et al. 1995), pigs
(Kronsteiner et al. 2013), guinea pigs (Shomer et al. 1998), rats (Elseweidy
et al. 2010), and even Drosophila (Wandler and Guillemin 2012) are sometimes
used, but they are relatively uncommon and are not considered further.
11 Animal Models of Helicobacter pylori Infection 275
11.2 Mouse Model
11.2.1 Early Studies
Because mouse colonization was initially unsuccessful, the gnotobiotic piglet

model was developed (Krakowka et al. 1987) and used to investigate H. pylori
virulence factors (Eaton et al. 1991, 1997), vaccination strategies (Eaton and
Krakowka 1992; Eaton et al. 1998), and host immune responses. But the piglet
model was complex and expensive, so other helicobacter species were examined as
surrogates for H. pylori. In 1989, Adrian Lee’s group isolated Helicobacter felis,
which caused severe gastritis and preneoplastic proliferation (Lee et al. 1990).
H. felis was the first model used in genetically engineered mice to investigate the
role of host and bacterial factors in gastritis and cancer, and it provided a major
advance in pathogenesis studies.
11.2.2 H. felis in Mice
H. felis in mice has been used principally to study the host immune response (Sayi
et al. 2009, 2011; Lee et al. 1990; Mohammadi et al. 1997; Roth et al. 1999) and
carcinogenesis (Fox et al. 1996; Wang et al. 2000; Houghton et al. 2004; Stoicov
et al. 2009). H. felis was also the first helicobacter species used in the adoptive
transfer mouse model to demonstrate the importance of helper T cells (Th1) in
disease (Mohammadi et al. 1996). H. felis lacks some H. pylori virulence factors
such as the cag pathogenicity island (cagPAI) and the vacuolating cytotoxin
(VacA), but the model has the advantage of robust colonization and induction of
a severe local inflammatory response that leads to gastric epithelial damage and
preneoplastic lesions. For this reason, H. felis continues to be an excellent model
organism to study the host immune response to gastric helicobacter, as well as
examination of host and environmental factors that promote gastric neoplasia.
11.2.3 H. pylori in Mice
The mouse model was not commonly used until 1997 when strain SS1 was
introduced (Lee et al. 1997). SS1 is an H. pylori isolate from a human patient that
was adapted to mouse colonization by serial in vivo passage. It colonizes mice well,
persists indefinitely in most mouse strains, causes histological gastritis, and is
genetically tractable, allowing mutational analysis of bacterial colonization and
virulence factors. SS1 rapidly became the standard for use in mouse models. It has
been used to confirm the roles of urease, flagella, chemoreceptors, and other
virulence factors in gastric colonization (Eaton et al. 2002; Kim et al. 1999;
Andermann et al. 2002) and is commonly used for vaccine development (Pappo
et al. 1999; Garhart et al. 2003; Akhiani et al. 2002; Moss et al. 2011) and studies of
host immune responses (Gray et al. 2013; Eaton et al. 2001, 2006). Since the
isolation of SS1, several laboratories have isolated other mouse-colonizing strains
or have adapted strains by serial passage in mice, but SS1 remains a commonly used
strain for in vivo studies.
In 2002, a controversy arose regarding the use of SS1 in a mouse disease model.
This was based on the finding that while SS1 contained the cagPAI, it did not
appear to have a functional type IV secretion system (T4SS) as determined in cell
culture (Crabtree et al. 2002; Philpott et al. 2002). To address this limitation,
investigators have used pre-mouse SS1 (PMSS1), the original human isolate that
has a functional T4SS and gave rise to SS1 after mouse passage (Arnold
et al. 2010). Infection of mice with PMSS1 can produce T4SS-dependent gastritis,
gastric atrophy, epithelial hyperplasia, and metaplasia. Some have interpreted this
to suggest that PMSS1 is unique and does not undergo loss of T4SS function during
mouse passage. However, recent experiments show that loss of T4SS function in
H. pylori PMSS1 is not unusual following gastric colonization, both in mouse
and nonhuman primate models. The mechanism appears to be in-frame recombi-
nation in cagY, which encodes an essential protein in the H. pylori T4SS (Barrozo
et al. 2013). This is perhaps not surprising, since H. pylori is known to be
genetically polymorphic and subject to adaptive changes in vivo (Linz
et al. 2014). There are likely also other genomic changes in the cagPAI or elsewhere
in the genome during in vivo mouse passage. Thus, SS1 remains a useful strain for
examination of host immune response and gastritis due to H. pylori, but not for the
effects of the T4SS, which can only be examined in the mouse model during short-
term infection (typically less than 8 weeks) before the loss of T4SS function.
11.2.4 Gastritis in Helicobacter Mouse Models
Gastritis due to helicobacter infection in mice is similar but not identical to gastritis
in humans. In both host species, gastritis is mild and develops slowly in most
individuals, although individual human patients and immune-modulated mouse
strains may develop more severe manifestations of disease. The inflammatory
infiltrate in both mice and humans consists of a mixture of neutrophils (referred
to as “active” gastritis) and mononuclear inflammatory cells that are rarely specif-
ically identified, but are likely a mixture of lymphocytes, plasma cells, and macro-
phages or dendritic cells. In humans, chronic infection is sometimes associated with
loss of gastric glands and fibrosis (atrophic gastritis) and replacement of gastric-
type glandular epithelium with intestinal type epithelium, complete with absorptive
and goblet cells (intestinal metaplasia). Gastric cancer is thought to develop as a
result of intestinal metaplasia progressing to dysplasia and neoplasia (Correa and
Houghton 2007).
Disease due to gastric helicobacter in mice is somewhat bacterial and host strain
dependent. Most commonly, H. pylori causes mild slowly progressive chronic
gastritis and lymphofollicular hyperplasia. Mice do not develop atrophy or intesti-
nal metaplasia. Instead, chronic colonization leads to epithelial hyperplasia and a
type of metaplasia known as spasmolytic polypeptide-expressing metaplasia
(SPEM) (Weis and Goldenring 2009). In chronically infected C57BL/6 mice,
chronic active gastritis begins at the junction of pyloric and fundic epithelium
and spreads into the fundus. SPEM develops late in disease and eventually pro-
gresses to grossly apparent gastric hypertrophy. Proliferative lesions may become
extensive and severe, eventually involving most of the glandular gastric mucosa
and sometimes herniating through the gastric muscularis mucosae. SPEM is rarely
described in humans, but it is the most common type of gastric metaplasia in mice
where it is thought to be a preneoplastic lesion (Nomura et al. 2004; Weis and
Goldenring 2009). H. felis causes lesions that are similar to those caused by
H. pylori, but are more severe and rapidly progressive (Court et al. 2002). Because
it causes extensive gastric epithelial proliferation in mice, H. felis is commonly
used in conjunction with cocarcinogens and/or genetically engineered mice to
investigate the progression to neoplasia.
Progression and severity of gastric lesions in helicobacter-infected mice are
enhanced by adoptive transfer of CD4+ T cells or by absence or suppression of
T-cell regulatory cytokines such as IL-10 (Eaton et al. 2001) or T-regulatory cell
subsets (Gray et al. 2013). Gastritis and secondary epithelial lesions are enhanced
by Th1 and Th17 cytokines, particularly interferon gamma IFN-γ (Sayi et al. 2009),
which appears to be a principal inducer of gastritis and metaplasia in mouse models
(Syu et al. 2012). Other pro-inflammatory cytokines, such as tumor necrosis factor
alpha (TNF-α) (Thalmaier et al. 2002) and IL-1β (Huang et al. 2013), have also
been associated with disease in mouse models.
One experimental strategy that has proven useful in investigation of both
bacterial and host immune contributions to disease is the adoptive transfer of
CD4+ T cells from C57BL/6 mice to helicobacter-infected congenic T- and B-
cell-deficient (SCID or RAG/) mice, which results in severe, rapidly progressive
proliferative gastritis (Gray and Eaton 2012; Eaton et al. 1999). Use of specific
T-cell subsets or T cells from cytokine-deficient mice can be used to isolate the role
of specific host factors in disease (Eaton et al. 2001, 2006; Raghavan et al. 2003;
Roth et al. 1999; Lucas et al. 2001; Sayi et al. 2009). The importance of Th1 T cells,
IFN-γ, TNF-α, and IL-17 in gastritis and the role of T-regs in regulating host
responses and gastritis were all discovered using the adoptive transfer H. pylori
model.
11.2.5 Cancer Models
Long-term infection by either H. pylori or H. felis causes gastric epithelial hyper-

plasia, SPEM-type metaplasia, and dysplasia, but H. pylori infection alone does not
appear to cause neoplastic transformation in mice. Proliferative and dysplastic

lesions are more pronounced in mice infected with H. felis, and several laboratories
have interpreted advanced lesions as neoplastic (Cai et al. 2005; Houghton
et al. 2004), but functional characteristics of neoplastic transformation are rarely
examined, precluding definitive diagnosis. In one study, neoplastic cells isolated
from proliferative lesions in gastrin-deficient mice demonstrated anchorage-
independent growth (Zavros et al. 2005), but this has not been demonstrated in
helicobacter-associated proliferative lesions in mice. Regardless of differences in
interpretation of morphologic lesions, SPEM and dysplasia are generally consid-
ered preneoplastic in mice (Weis and Goldenring 2009), and for this reason,
infection with H. felis (and to a lesser extent H. pylori) in mice has been used to
model cancer progression, cocarcinogenic agents, and the role of genetic mutations
in tumor development.
The mouse glandular gastric mucosa is relatively resistant to agents such as
N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and N-Nitroso-N-methylurea
(MNU) that cause gastric adenocarcinoma in rats, though several studies have
used these agents as potential cocarcinogens in helicobacter-infected mice. Because
of the increasing availability of genetically engineered strains, mice offer the
best opportunity to evaluate the role of specific genes in cancer progression.
Several models have been used for this purpose, including p53 hemizygous trans-
genic mice (Fox et al. 1996), adenomatous polyposis coli (APC)-gene mutant
mice (Fox et al. 1997), and knockout mice with deletions in trefoil factor 2 (Fox
et al. 2007), p27 (cyclin-dependent kinase inhibitor) (Kuzushita et al. 2005), iNOS
(Nam et al. 2004), p53 (Fox et al. 2002), and MyD88 (Banerjee et al. 2014), among
others. Insulin-gastrin (INS-GAS) transgenic mice, which express gastrin under
control of the insulin promoter, are predisposed to development of gastric prolifer-
ative lesions when colonized with H. felis (Wang et al. 2000) and H. pylori (Lofgren
et al. 2011). Gastric microbiota other than helicobacter species may also contribute
to the development of neoplasia, since INS-GAS mice with a complex gut
microbiota progress more rapidly to neoplasia following H. pylori infection than
germ-free mice colonized with H. pylori alone (Lofgren et al. 2011). Even coloni-
zation with three members of the altered Schaedler flora (ASF) is sufficient to
recapitulate the more aggressive pathology found in conventional INS-GAS mice
(Lertpiriyapong et al. 2014), which suggests that there is not a single or even a few
unique species that, together with H. pylori, promote neoplasia. Nevertheless, if a
cancer-promoting microbial fingerprint could be identified by bacterial phylogeny
or metagenomics, this might provide another biomarker that could be used to
identify H. pylori-infected patients who are at greatest risk of gastric cancer
(Cooke et al. 2013).
11.3 Mongolian Gerbil Model
11.3.1 Background
The first published description of the gerbil (Meriones unguiculatus) model

appeared in 1991, when Yokota and coworkers reported that gerbils developed
mild gastritis 2 months after H. pylori oral gavage (Yokota et al. 1991). H. pylori
colonizes deep within the gastric mucus gel layer near the epithelia, similar in
topography to where the bacteria are found in human gastric specimens (Schreiber
et al. 2004). Gerbils infected with H. pylori can develop gastric ulcer and intestinal
metaplasia in the stomach (Hirayama et al. 1996), as well as gastric carcinoma
following the administration of chemical carcinogens such as MNU or MNNG
(Tokieda et al. 1999). In 1998, a groundbreaking article demonstrated that long-
term (>1 year) infection of Mongolian gerbils with H. pylori led to gastric adeno-
carcinoma in approximately one-third of infected animals, without the
coadministration of carcinogens, which was subsequently confirmed by others
(Watanabe et al. 1998; Ogura et al. 2000). Carcinomas that developed in
H. pylori-infected gerbils typically occurred in the distal stomach and the pyloric
region, and contained well-differentiated intestinal-type epithelium, reflecting
many of the features of intestinal-type gastric adenocarcinoma in humans.
One caveat of using rodent models of gastric cancer such as Mongolian gerbils is
that there has been some difficulty in distinguishing herniation of nonneoplastic
mucosa from invasive carcinoma, particularly gastrointestinal neoplasia that arises
in the setting of inflammation. To address this, guidelines for interpretation of these
lesions have been established by a multidisciplinary working group (Boivin
et al. 2003). Originally formulated for evaluation of intestinal tumors, these guide-
lines have also been applied to gastric lesions (Hagiwara et al. 2011; Houghton
et al. 2004; Rogers et al. 2005). In many studies on H. pylori-induced cancer in
gerbils, several features are used to distinguish invasive carcinoma from mucosal
herniation (Table 11.1).
11.3.2 Host Factors
Although somewhat limited due to their outbred nature, Mongolian gerbils have
been used to study the effects of some host constituents on the development of
gastric cancer. IL-1β is a Th1 cytokine that inhibits acid secretion and is increased
within gastric mucosa of H. pylori-infected persons (Noach et al. 1994). In humans,
polymorphisms in the IL-1β gene cluster (IL-1β31 and IL-1β511) are associated
with increased IL-1β production and a significantly increased risk for
hypochlorhydria, gastric atrophy, and distal gastric adenocarcinoma, but only
among those infected with H. pylori (El-Omar et al. 2000; Figueiredo
et al. 2002). Takashima and colleagues examined these relationships in greater
Table 11.1 Features of invasive carcinoma in the Mongolian gerbils

Higher grades of cytologic dysplasia than the overlying mucosa
Desmoplasia unassociated with a prominent inflammatory infiltrate
Irregular, sharp, or angulated, rather than rounded glands
Invading glands spread laterally to the surface component
Invading glands lack circumferential lining by epithelial cells
Invading glands lack full investment of basement membrane
More than two glands in the submucosa or deeper gastric wall
depth in gerbils infected with H. pylori by quantifying changes in gastric acidity and
then defining the role that IL-1β played in this response (Takashima et al. 2001).
Compared to uninfected animals, gerbils infected for 6 or 12 weeks with H. pylori
developed significantly increased levels of inflammation and IL-1β expression
within the gastric mucosa, which was accompanied by a loss of acid secretion.
More definitive experiments demonstrated that treatment of H. pylori-infected
gerbils with an IL-1β antagonist abolished the loss of acid secretion, implicating
this cytokine in the development of achlorhydria within an H. pylori-infected
stomach (Takashima et al. 2001).
Other groups have also developed innovative reagents specific for Mongolian
gerbils. Primers targeting pro-inflammatory cytokines including IL-1β, IL-4, IL-6,
IL-10, IL-12, KC, IFN-γ, TNF-α, and TGF as well as somatostatin, COX-2, iNOS,
and H+K+ATPase have been developed based on species-specific gerbil cDNA.
Crabtree et al. used such reagents to demonstrate that long-term H. pylori infection
increases the levels of IL-12 and IFN-γ expression within gerbil gastric mucosa
(Crabtree et al. 2004). H. pylori infection of gerbils has also been shown to increase
serum levels of gastrin, which can promote cell growth, and increased gastrin levels
are directly related to heightened gastric epithelial cell proliferation (Peek
et al. 2000). Nozaki and coworkers developed a gastric cell line from a gerbil
gastric cancer specimen, which was used to demonstrate that H. pylori can activate
transcription factor NF-κB signaling in a cagPAI-dependent manner (Nozaki
et al. 2005).
11.3.3 Bacterial Factors
Compared to gerbils infected with wild-type H. pylori, gerbils colonized with

cagPAI mutant strains develop significantly less severe gastritis (Ogura
et al. 2000; Akanuma et al. 2002; Israel et al. 2001; Saito et al. 2005). Rieder and
coworkers investigated alterations not only in the intensity but also the topography
of inflammation in gerbils infected with wild-type H. pylori or isogenic cagA or
cagY mutants (Rieder et al. 2005). Inactivation of cagA or cagY resulted in an
inflammatory response that was primarily restricted to the gastric antrum, largely
sparing the acid-secreting corpus. Consistent with these histologic changes,
intragastric pH values were increased only in gerbils challenged with the wild-type
H. pylori strain (Rieder et al. 2005). These results indicate that a functional cagPAI
T4SS is required to induce corpus-predominant gastritis, a precursor lesion in the
progression to intestinal-type gastric adenocarcinoma. H. pylori possesses polar
flagella consisting of two major subunits, FlaA and FlaB (O’Toole et al. 2000), and
deletion of flaA results in flagellar truncation and decreased motility (Josenhans
et al. 1995). Using signature-tagged mutagenesis, Kavermann and coworkers iden-
tified flaA as an essential gene for colonization of gerbils by H. pylori, along with
several other genes involved in either flagellar assembly or that encode structural
components of flagella (Kavermann et al. 2003). Other essential colonization genes
that were identified using this technique include those encoding outer membrane
proteins, secretion and transport systems, and mediators of chemotaxis, acid sur-
vival, and stress responses (Kavermann et al. 2003).
Although gerbils can develop gastric cancer in response to H. pylori alone, the
prolonged time course required for transformation in earlier studies precluded
large-scale analyses that comprehensively evaluate mediators that are critical in
the carcinogenic cascade (Watanabe et al. 1998). Since serial passage of H. pylori
in rodents increases colonization efficiency, the Peek lab investigated whether
in vivo adaptation of a human H. pylori strain (B128) would enhance its carcino-
genic potential. A gerbil infected with H. pylori strain B128 was sacrificed 3 weeks
post-challenge, and a single-colony output derivative (strain 7.13) was used to
infect an independent population of gerbils (Franco et al. 2005). The kinetics and
intensity of inflammation induced by strain 7.13 were similar to those induced by
parental strain B128. However, after 8 weeks of infection with strain 7.13, gastric
dysplasia and adenocarcinoma developed in approximately 75 % and 60 %, respec-
tively, while these lesions were not present in any gerbils infected with the
progenitor H. pylori strain B128 (Franco et al. 2005). H. pylori strain 7.13 also
activated β-catenin to a significantly higher level in gastric epithelial cells than
B128, and isogenic inactivation of cagA in strain 7.13 revealed that β-catenin
activation was mediated by CagA. Mimicking the outcome of human infection,
gerbils infected with H. pylori wild-type strain 7.13 developed gastric cancer to a
greater degree than those infected with a 7.13 cagA deletion mutant (Franco
et al. 2008).
To identify additional proteins that may mediate the development of H. pylori-
induced gastric cancer, these investigators then combined two-dimensional differ-
ence gel electrophoresis (2D-DIGE) with mass spectrometry to identify differen-
tially expressed membrane and cytosolic proteins from the non-carcinogenic
H. pylori strain B128 and its carcinogenic derivative, strain 7.13. Twenty-eight
significant expression changes in proteins were detected. A novel modification in
FlaA was also detected, which was due to an amino acid substitution that resulted in
altered motility (Franco et al. 2009). Another virulence determinant of H. pylori
that is important for adherence is OipA. Expression of OipA has been associated
with increased secretion of the pro-inflammatory cytokine IL-8 in vitro, and
infection of gerbils with H. pylori oipA mutants has revealed a role for this outer
membrane protein in inflammation and gastric cancer (Franco et al. 2008).
11.3.4 Dietary Factors
Diet is likely an important risk factor for gastric cancer, particularly diets high in
salt (Tsugane and Sasazuki 2007). However, epidemiologic studies of diet in
humans are subject to many limitations, and it is difficult to determine whether
dietary parameters are causally linked to gastric cancer or merely represent markers
for other factors that are important in gastric cancer pathogenesis. To further
investigate potential relationships between diet and gastric cancer risk, several
studies have examined the role of diet in the gerbil model of H. pylori-induced
gastric cancer. H. pylori infection and a high-salt diet can independently induce
gastric atrophy and intestinal metaplasia in Mongolian gerbils (Bergin et al. 2003),
but they appear to be synergistic when animals also receive a chemical carcinogen
(Kato et al. 2006; Nozaki et al. 2002). CagA-dependent gastric adenocarcinoma
was detected in a significantly higher proportion of H. pylori-infected gerbils
maintained on a high-salt diet compared to infected animals on a regular diet
(Gaddy et al. 2013). Increased cagA transcription within the gastric mucosa was
also detected in H. pylori-infected gerbils fed a high-salt diet compared to those on
a regular diet (Gaddy et al. 2013), suggesting that high-salt diets potentiate the
carcinogenic effects of cagA+ H. pylori strains.
Dietary iron depletion may also promote H. pylori-induced gastritis and gastric
cancer in gerbils infected with cagA-positive H. pylori (Noto et al. 2013). A
low-iron diet did not produce these effects in animals infected with an isogenic
cagA mutant strain, and these mutants exhibited a significant decrease in coloniza-
tion density compared to wild-type H. pylori (Tan et al. 2011). To define mecha-
nisms through which low iron concentrations may augment virulence, H. pylori
strains cultured from gerbils fed a low-iron diet or a regular diet were compared
using 2D-DIGE and mass spectrometry. Multiple proteins differed in abundance
when comparing H. pylori strains isolated from iron-deplete versus iron-replete
gerbils, including proteins that mediate survival, microbial adherence, and function
of the cag T4SS (Noto et al. 2013). H. pylori FlaA and FlaB, the major flagellin
subunits, were also significantly upregulated in bacteria cultured from iron-deplete
animals. H. pylori strains isolated from iron-depleted gerbils also expressed signif-
icantly higher levels of CagA (Noto et al. 2013). Strains isolated from iron-depleted
gerbils showed more T4SS pili, translocated more CagA, and induced higher levels
of IL-8 when compared to strains isolated from iron-replete gerbils (Noto
et al. 2013), effects that reversed during passage in vitro (Noto et al. 2015). These
experiments indicate that exposure of H. pylori to low-iron conditions enhances the
ability of the bacteria to induce responses with carcinogenic potential in gastric
epithelial cells.
11.3.5 Role of the Gastric Microbiota
Although H. pylori infection is the strongest identified risk factor for gastric cancer,
recent studies in humans have indicated that other residents of the gastric
microbiome may influence malignant transformation (Abreu and Peek 2014). One
clinical trial reported that treatment of H. pylori was associated with a significant
reduction in gastric cancer incidence rates, yet only 47 % of treated persons
remained free of H. pylori at the time of evaluation (Ma et al. 2012). This suggests
that antibiotic therapy alters other microbial species that could affect the develop-
ment of gastric cancer.
Few studies have examined the gastric microbiota in gerbils. The most com-
monly represented phyla of the gerbil gastric microbiome include Firmicutes,
Actinobacteria, Proteobacteria, and Bacteroidetes (Fox and Sheh 2013; Yang
et al. 2013). Similar to mice, the genus Lactobacillus dominates the gastric
microbiota of uninfected gerbils (Fox and Sheh 2013; Yang et al. 2013). One
study that used molecular techniques to compare differences in the gerbil gastric
microbiota before and after H. pylori infection demonstrated a reduction in the
diversity of Lactobacillus following H. pylori infection (Sun et al. 2003). Another
group utilized quantitative PCR to follow the relative abundance of 15 microbial
species in the gerbil stomach (Osaki et al. 2012). In uninfected gerbils, the most
abundant genera were Lactobacillus and Enterococcus, followed by equal levels of
Atopobium and Clostridium. In gerbils that were successfully infected with
H. pylori, the relative abundance of Clostridium coccoides increased, compared
to uninfected gerbils. In gerbils that were challenged with H. pylori but were not
successfully colonized, the proportion of C. coccoides, C. leptum, and
Bifidobacterium was reduced. Gerbils that were challenged with H. pylori but not
colonized represented the only group that harbored members of the Eubacterium
cylindroides and Prevotella species (Osaki et al. 2012). However, the importance of
differences in microbial composition to the development of gastric cancer in the
gerbil model has yet to be determined.
11.4 Nonhuman Primate Model
11.4.1 Background
Spiral bacteria were first observed in the stomach of rhesus monkeys early in the
twentieth century (Doenges 1938, 1939) and described again in 1982 (Sato and
Takeuchi 1982). However, the bacteria found in rhesus monkeys at that time were
almost certainly not H. pylori, but rather the large tightly coiled spiral organisms
that morphologically resemble those seen in a variety of animal hosts (Haesebrouck
et al. 2009), and which today would probably be identified as either Helicobacter
suis or Helicobacter heilmannii. Subsequently, several groups reported cultivation
of H. pylori from naturally infected rhesus (Macaca mulatta), cynomolgus

(M. fasicularis), and pigtailed (M. nemestrinae) macaques (Brondson and
Schoenknecht 1988; Baskerville and Newell 1988; Newell et al. 1988; Reindel
et al. 1999; Perry et al. 2010). The isolate from pigtailed macaques was originally
designated a novel species (Brondson et al. 1991), but was later found to be a
heterotypic synonym of H. pylori (Suerbaum et al. 2001). More extensive pheno-
typic testing and sequencing of the 16S rRNA gene confirmed that H. pylori
isolated from naturally infected macaques was indistinguishable from strains that
infect humans (Drazek et al. 1994; Doi et al. 2005). Comparative genomic hybrid-
ization also suggests that H. pylori isolates from naturally infected rhesus monkeys
are not distinguishable from human strains, though they cluster together, probably
because they were collected from the same primate center (Joyce et al. 2002). Most
H. pylori isolates from naturally infected cynomolgus (Doi et al. 2005) and rhesus
(Solnick unpublished observations) monkeys appear to contain the cagPAI.
Whether H. pylori from naturally infected macaques is autochthonous is unknown,
because samples from feral primates without human contact are difficult to obtain.
To address this, we analyzed the DNA sequences of seven housekeeping genes
from five H. pylori strains cultured from rhesus macaques at the California National
Primate Research Center. Comparison to a large sequence database used to char-
acterize strains from human populations worldwide (Falush et al. 2003; Linz
et al. 2007) showed that the rhesus H. pylori strains belong to the hpEurope
clade, strongly suggesting that they were acquired from humans (Solnick and
Achtman unpublished observations).
11.4.2 Strengths and Limitations of the Nonhuman Primate

Model
Nonhuman primates provide the opportunity to study H. pylori in an animal that is

anatomically and physiologically most similar to humans. For example, nonhuman
primates have a gastric anatomy and pH similar to humans, while the rodent
stomach has a higher pH and is divided into two anatomical regions, the large
forestomach lined by squamous epithelium and the more acidic glandular portion
(Kararli 1995). Other than one report in a single colony of experimental cats (Handt
et al. 1995a), natural H. pylori infection has been described only in primates. While
H. pylori is probably not autochthonous in feral nonhuman primates, socially
housed captive macaques are often naturally infected, typically in the first year of
life (Solnick et al. 2003), a pattern that resembles the epidemiology of human
infection in countries with a high prevalence (Bardhan 1997). Natural transmission
can be readily demonstrated experimentally among cohoused animals (Solnick
et al. 2006). There are also extensive resources available for studies of nonhuman
primates. For example, multiple genomes have been sequenced, including the
rhesus macaque; transcriptome analyses of rhesus and other nonhuman primate
species are in progress. Many reagents for studies in nonhuman primates are also
readily available and can be found at the National Primate Reagent Resource
maintained by the National Institutes of Health (http://www.nhpreagents.org).
From a practical perspective, gastric samples can be obtained repeatedly by endo-
scopic biopsy to follow the course of infection over time.
But the nonhuman primate model also has disadvantages, particularly cost and
availability. The US national primate research centers (NPRCs) funded by the
National Institutes of Health are intended to support investigators who receive
their primary research project funding from NIH either at the home institution or
across the country, but they may also be used by investigators funded by other
agencies, foundations, and the private sector. Together, the NPRCs have more than
26,000 nonhuman primates representing more than 20 species, predominantly
macaques. Primate research is also conducted on a smaller scale at many other
universities and private centers. In the European Union, the European Primate
Network (EUPRIM-Net) brings together nine European primate centers that com-
bine research and breeding to form a virtual European Primate Center. Costs vary
across centers, but at the California NPRC, some representative charges in 2016 are
approximately $7,000 for animal purchase, $3,500 use fee for non-terminal exper-
iments, and $8.00 for indoor housing per diem, though this can range from $4.00 for
outdoor housing in large field cages to $44.00 in the newborn nursery. Another
limitation of the nonhuman primate model of H. pylori is that it is typically not a
disease model. Gastric diseases associated with H. pylori in humans—peptic ulcers,
gastric adenocarcinoma, and MALT lymphoma—have been seen in rhesus mon-
keys naturally colonized with H. pylori (D.R. Canfield, personal communication;
Kimbrough 1966; Parker et al. 1981). However, in typical short-term experiments
lasting a few weeks or months, one typically sees only histologic gastritis. Ethical
and safety issues unique to research with nonhuman primates should also be
considered.
11.4.3 Experimental Infection
The rhesus macaque is the most common nonhuman primate used in experimental
research in the USA (Conlee et al. 2004), and it is typically the species of choice for
studies of H. pylori. Identification of animals without prior H. pylori infection is
challenging, largely because infection is so common (Dubois et al. 1994; Solnick
et al. 1999). Noninvasive testing by enzyme-linked immunosorbent assay (ELISA)
with human or rhesus-derived strains has been variably successful (Handt
et al. 1995b, 1997); our experience is that it is sensitive but not specific. In a
prospective study of socially housed rhesus macaques, 8 of 20 newborn monkeys
(40 %) were culture positive for H. pylori by 12 weeks of age, reaching 90 % by
1 year (Solnick et al. 2003). In older animals, the frequency of positive cultures
declines, though they almost always remain seropositive (Solnick et al. 2003). This
discordance might reflect the limited sensitivity of culture (typically about 102
CFU/g of tissue) or that infection is multifocal. It could also be that animals

sometimes clear the infection yet remain serofast, perhaps because of frequent
encounters that fail to produce a persistent infection. Concomitant infection with
H. suis or H. heilmannii might also confound the results of H. pylori serology, as
well as the urea breath test (Solnick et al. 1999, 2002), since these large gastric
spiral organisms are very common and are urease positive (Solnick et al. 1994).
Specific pathogen-free (SPF) rhesus monkeys lacking H. pylori can be derived by
removing them from the dam within a few hours of birth and raising them in a
nursery (Solnick et al. 1999), though this is obviously expensive. This approach is
also limited by the difficulty of performing endoscopy in newborn macaques, which
typically cannot be done easily before about 4–6 months of age, and requires a
pediatric bronchoscope with limited biopsy capability. SPF monkeys derived at the
NIH-sponsored NPRCs are unlikely to be free of H. pylori because they are
screened predominantly for viruses, most notably herpes B virus, but not
H. pylori (Kanthaswamy et al. 2010). Fortunately, depending on the question
being addressed, it may be unnecessary to use monkeys that have never been
infected with H. pylori, since the infection can be treated with antibiotics and the
animals reinfected (Dubois et al. 1999), albeit with a lower bacterial load (Dubois
et al. 1999; Solnick et al. 2001). Experimental infection of SPF rhesus macaques
with H. pylori closely recapitulates the gastritis seen in human infection (Fig. 11.1).
Many different H. pylori strains have been used successfully for experimental
challenge of nonhuman primates, which is typically performed by gastric gavage.
Early studies suggested that H. pylori strains derived from monkeys might colonize
better than those from humans (Euler et al. 1990). While this may be true, there is
no doubt that many human strains will colonize as well, though mixed infections
suggest strongly that some strains are better suited to monkeys than others (Dubois
et al. 1999; Solnick et al. 2001). Like in mouse and gerbil models, it is important
that the strains be freshly isolated from their host with minimal laboratory passage.
Many investigators have attempted to increase the likelihood of experimental
infection by suppressing gastric acid and using a large inoculum (e.g., 109
colony-forming units (CFU)), though neither is necessary if the strain is well suited
to monkeys (Solnick et al. 2001). Inoculation of as few as 104 CFU has been
successful (Solnick et al. 2001). Both cagPAI-positive and cagPAI-negative strains
will colonize, the latter producing less inflammation and higher colonization
density (Hornsby et al. 2008). As in the mouse model, strains isolated from
experimentally infected monkeys lose function of the T4SS, which is mediated
by recombination in the highly repetitive region of cagY (Barrozo et al. 2013).
Interestingly, cagY-mediated gain of function in the T4SS is also seen in macaques
(Barrozo et al. 2013). Since cagY codons are under strong positive selection, and
recombination can both up- and downmodulate T4SS function in a graded fashion,
it seems likely that this is a strategy to “tune” or optimize the host inflammatory
response to achieve a homeostatic balance. Colonization in rhesus macaques also
results in loss of expression of the babA adhesin, either by phase variation or by
gene conversion with the babB paralog (Solnick et al. 2004; Styer et al. 2010). Loss
of T4SS function and expression of babA typically occur during the first 4–8 weeks
Fig. 11.1 Helicobacter sp. infection in rhesus macaques. (a) Normal uninfected pyloric antrum
with only a few scattered inflammatory cells in the lamina propria (H&E stain, scale bar ¼ 50 μ).
(b) Pyloric antrum illustrating characteristic gastritis secondary to H. pylori infection. The
superficial lamina propria is expanded by inflammation comprising mainly plasma cells with
lesser numbers of lymphocytes and scattered neutrophils (H&E stain). (c) Higher magnification of
H. pylori organisms characteristically located on the luminal surface of epithelial cells lining
gastric pits (modified Steiner silver stain, scale bar ¼ 20 μ); inset shows higher power magnifica-
tion of H. pylori. (d) In contrast to H. pylori, H. heilmannii inhabits the gastric corpus, generally
extending deeper into glands and often residing inside parietal cells, and elicits little or no
inflammation. Inset illustrates the corkscrew spiral shape of the larger organisms, which are likely
either H. heilmannii or H. suis. Photomicrograph courtesy of D.R. Canfield, DVM
after challenge, which appears to be a period of rapid change in the H. pylori

genome during acute infection in both rhesus macaques and humans (Linz
et al. 2014).
Once established, infection in a well-adapted strain is typically persistent,
though some strains will show transient infections and others will not colonize at
all. The outcome can be readily monitored by serial endoscopic biopsies, which can
be processed for quantitative CFU, histopathology, host (Huff et al. 2004) and
bacterial (Boonjakuakul et al. 2005) gene expression, bacterial genomic evolution
(Linz et al. 2007; Solnick et al. 2004), and other assays. Colonization density is
typically 105–106 CFU/g of gastric tissue, but may decline somewhat after the
immune response develops in the first few weeks after infection.
11.4.4 How Has the Nonhuman Primate Model Been Used?
The nonhuman primate model has been used to address a number of important
questions in fundamental and clinical aspects of H. pylori biology. Here, we give a
few illustrative examples. Studies in socially housed macaques suggest that natural
H. pylori transmission occurs very early in life, probably by the oral-oral route
(Solnick et al. 2003, 2006). An early study found that immunization with urease and
Escherichia coli LT appeared to protect rhesus macaques from natural infection
(Dubois et al. 1998), but the infection status of the animals before immunization
was only examined by serology. Subsequent studies under more controlled condi-
tions were disappointing (Lee et al. 1999; Solnick et al. 2000). The macaque model
has also provided an opportunity to examine host and bacterial gene expression
(Boonjakuakul et al. 2005; Huff et al. 2004; Suerbaum and Josenhans 2007), and
the development of H. pylori genomic diversity, which is perhaps greater than
that in any other bacterial species known (Suerbaum and Josenhans 2007). The
results suggest that there is a mutation burst early during acute infection (Linz
et al. 2014), with rapid genomic changes that alter expression of outer membrane
proteins (OMPs) and the T4SS (Barrozo et al. 2013; Solnick et al. 2004; Styer
et al. 2010). Alterations in H. pylori OMPs likely reflect a complex cross talk
between expression of bacterial lectins and changes in host glycosylation (Cooke
et al. 2009; Linden et al. 2008; Mahdavi et al. 2002; Wirth et al. 2006). Although
not typically a disease model, the rhesus macaque has also been used to study the
effects of H. pylori and diet on the gastric cancer cascade. Macaques experimen-
tally infected with cagPAI-positive H. pylori and chronically fed N-ethyl-N-
nitrosoguanidine (ENNG) developed gastric intestinal metaplasia starting at
2 years (Liu et al. 2009). Untreated control monkeys and monkeys treated singly
with either H. pylori or ENNG showed no neoplastic changes. These data demon-
strate the synergistic effects of H. pylori infection with a dietary carcinogen that is
present in East Asian diets.
11.4.5 Human Challenge Model
Two clinical studies have been published describing the results of experimental
H. pylori infection in H. pylori-naive humans, which demonstrated successful
colonization in most individuals (Aebischer et al. 2008; Graham et al. 2004).
Both studies used the cagPAI-negative, antibiotic-susceptible Baylor strain
(BCS100; ATCC BAA-945) isolated from a patient with mild gastritis. At least
two other human trials have been performed but remain unpublished (David
Graham, personal communication). Although potentially useful in vaccine studies
for which the human challenge model was developed, safety and regulatory issues
will likely preclude its widespread use.
Animal models are essential for a full understanding of H. pylori pathogenesis and
for vaccine development. Cost, convenience, and the ever-increasing availability of
genetically engineered mice will ensure that the mouse remains the dominant model
for in vivo studies of H. pylori, using either H. felis to examine inflammatory and
carcinogenic mechanisms, or H. pylori to examine vaccines and specific bacterial
virulence factors. The gerbil is arguably the best disease model, though it is more
expensive than mice, and there are no genetically engineered animals and relatively
few reagents. The nonhuman primate remains the model most closely resembling
humans, but cost and availability limit its use to specialized centers.
References
Abreu MT, Peek RM Jr (2014) Gastrointestinal malignancy and the microbiome. Gastroenterology
146:1534–1546
Aebischer T, Bumann D, Epple HJ, Metzger W, Schneider T, Cherepnev G, Walduck AK,
Kunkel D, Moos V, Loddenkemper C, Jiadze I, Panasyuk M, Stolte M, Graham DY,
Zeitz M, Meyer TF (2008) Correlation of T cell response and bacterial clearance in human
volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination
with Ty21a-based Salmonella vaccines. Gut 57:1065–1072
Akanuma M, Maeda S, Ogura K, Mitsuno Y, Hirata Y, Ikenoue T, Otsuka M, Watanabe T,
Yamaji Y, Yoshida H, Kawabe T, Shiratori Y, Omata M (2002) The evaluation of putative
virulence factors of Helicobacter pylori for gastroduodenal disease by use of a short-term
Mongolian gerbil infection model. J Infect Dis 185:341–347
Akhiani AA, Pappo J, Kabok Z, Sch€on K, Gao W, Franzén LE, Lycke N (2002) Protection against
Helicobacter pylori infection following immunization is IL-12-dependent and mediated by
Th1 cells. J Immunol 169:6977–6984
Andermann TM, Chen YT, Ottemann KM (2002) Two predicted chemoreceptors of Helicobacter
pylori promote stomach infection. Infect Immun 70:5877–5881
Arnold IC, Lee JY, Amieva MR, Roers A, Flavell RA, Sparwasser T, Muller A (2010) Tolerance
Banerjee A, Thamphiwatana S, Carmona EM, Rickman B, Doran KS, Obonyo M (2014) Defi-
ciency of the myeloid differentiation primary response molecule MyD88 leads to an early and
rapid development of Helicobacter-induced gastric malignancy. Infect Immun 82:356–363
Bardhan PK (1997) Epidemiological features of Helicobacter pylori in developing countries. Clin
Infect Dis 25:973–978
Barrozo RM, Cooke CL, Hansen LM, Lam AM, Gaddy JA, Johnson EM, Cariaga TA, Suarez G,
Peek RM Jr, Cover TL, Solnick JV (2013) Functional plasticity in the type IV secretion system
of Helicobacter pylori. PLoS Pathog 9:e1003189
Baskerville A, Newell DG (1988) Naturally occurring chronic gastritis and C pylori infection in
the rhesus monkey: a potential model for gastritis in man. Gut 29:465–472
Bergin IL, Sheppard BJ, Fox JG (2003) Helicobacter pylori infection and high dietary salt
independently induce atrophic gastritis and intestinal metaplasia in commercially available
outbred Mongolian gerbils. Dig Dis Sci 48:475–485
Boivin GP, Washington K, Yang K, Ward JM, Pretlow TP, Russell R, Besselsen DG, Godfrey VL,
Doetschman T, Dove WF, Pitot HC, Halberg RB, Itzkowitz SH, Groden J, Coffey RJ (2003)
Pathology of mouse models of intestinal cancer: consensus report and recommendations.

Bolker J (2012) Model organisms: there’s more to life than rats and flies. Nature 491:31–33
Boonjakuakul JK, Canfield DR, Solnick JV (2005) Comparison of Helicobacter pylori virulence
gene expression in vitro and in the rhesus macaque. Infect Immun 73:4895–4904
Brondson MA, Schoenknecht FD (1988) Campylobacter pylori isolated from the stomach of the
monkey, Macaca nemestrina. J Clin Microbiol 26:1725–1728
Brondson M, Goodwin C, Sly L, Chilvers T, Schoenknecht F (1991) Helicobacter nemestrinae
sp. nov., a spiral bacterium found in the stomach of a pigtailed macaque (Macaca nemestrina).
Int J Syst Bacteriol 41:148–153
Cai X, Stoicov C, Li H, Carlson J, Whary M, Fox JG, Houghton J (2005) Overcoming
Fas-mediated apoptosis accelerates Helicobacter-induced gastric cancer in mice. Cancer Res
65:10912–10920
Cantorna MT, Balish E (1990) Inability of human clinical strains of Helicobacter pylori to
colonize the alimentary tract of germfree rodents. Can J Microbiol 36:237–241
Conlee KM, Hoffeld EH, Stephens ML (2004) A demographic analysis of primate research in the
United States. Altern Lab Anim ATLA 32(Suppl 1A):315–322
Cooke CL, An HJ, Kim J, Canfield DR, Torres J, Lebrilla CB, Solnick JV (2009) Modification of
gastric mucin oligosaccharide expression in rhesus macaques after infection with Helicobacter
pylori. Gastroenterology 137:1061–1071, 1071 e1061–1068
Cooke CL, Torres J, Solnick JV (2013) Biomarkers of Helicobacter pylori-associated gastric
cancer. Gut Microbes 4(6):532–540. doi:10.4161/gmic.25720, Epub 2013 Jul 12. Review
Correa P, Houghton J (2007) Carcinogenesis of Helicobacter pylori. Gastroenterology
133:659–672
Court M, Robinson PA, Dixon MF, Crabtree JE (2002) Gastric Helicobacter species infection in
murine and gerbil models: comparative analysis of effects of H. pylori and H. felis on gastric
epithelial cell proliferation. J Infect Dis 186:1348–1352
Couzin-Frankel J (2013) When mice mislead. Science 342:922–923, 925
Crabtree JE, Ferrero RL, Kusters JG (2002) The mouse colonizing Helicobacter pylori strain SS1
may lack a functional cag pathogenicity island. Helicobacter 7:139–140
Crabtree JE, Court M, Aboshkiwa MA, Jeremy AH, Dixon MF, Robinson PA (2004) Gastric
mucosal cytokine and epithelial cell responses to Helicobacter pylori infection in Mongolian
gerbils. J Pathol 202:197–207
Doenges J (1938) Sprochetes in the gastric glands of macacus rhesus and humans without definite
history of related disease. Proc Soc Exp Biol Med 38:535–538
Doenges JL (1939) Spirochetes in the gastric glands of cacacus rhesus and of man without related
disease. Arch Pathol 25:469–477
Doi SQ, Kimbason T, Reindel J, Dubois A (2005) Molecular characterization of Helicobacter
pylori strains isolated from cynomolgus monkeys (M. fascicularis). Vet Microbiol
108:133–139
Drazek ES, Dubois A, Holmes RK (1994) Characterization and presumptive identification of
Helicobacter pylori isolates from rhesus monkeys. J Clin Microbiol 32:1799–1804
Dubois A, Fiala N, Heman-Ackah LM, Drazek ES, Tarnawski A, Fishbein WN, Perez-Perez GI,
Blaser MJ (1994) Natural gastric infection with Helicobacter pylori in monkeys: a model for
spiral bacteria infection in humans. Gastroenterology 106:1405–1417
Dubois A, Lee CK, Fiala N, Kleanthous H, Mehlman PT, Monath T (1998) Immunization against
natural Helicobacter pylori infection in nonhuman primates. Infect Immun 66:4340–4346
Dubois A, Berg DE, Incecik ET, Fiala N, Heman-Ackah LM, Del Valle J, Yang M, Wirth HP,
Perez-Perez GI, Blaser MJ (1999) Host specificity of Helicobacter pylori strains and host
responses in experimentally challenged nonhuman primates. Gastroenterology 116:90–96
Eaton KA, Krakowka S (1992) Chronic active gastritis due to Helicobacter pylori in immunized
gnotobiotic piglets. Gastroenterology 103:1580–1586
Eaton KA, Brooks CL, Morgan DR, Krakowka S (1991) Essential role of urease in pathogenesis of
gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun 59:2470–2475
Eaton KA, Cover TL, Tummuru MK, Blaser MJ, Krakowka S (1997) Role of vacuolating
cytotoxin in gastritis due to Helicobacter pylori in gnotobiotic piglets. Infect Immun
65:3462–3464
Eaton KA, Ringler SS, Krakowka S (1998) Vaccination of gnotobiotic piglets against
Helicobacter pylori. J Infect Dis 178:1399–1405
Eaton KA, Ringler SR, Danon SJ (1999) Murine splenocytes induce severe gastritis and delayed-
type hypersensitivity and suppress bacterial colonization in Helicobacter pylori-infected SCID
mice. Infect Immun 67:4594–4602
Eaton KA, Mefford M, Thevenot T (2001) The role of T cell subsets and cytokines in the
pathogenesis of Helicobacter pylori gastritis in mice. J Immunol 166:7456–7461
Eaton KA, Gilbert JV, Joyce EA, Wanken AE, Thevenot T, Baker P, Plaut A, Wright A (2002) In
vivo complementation of ureB restores the ability of Helicobacter pylori to colonize. Infect
Immun 70:771–778
Eaton KA, Benson LH, Haeger J, Gray BM (2006) Role of transcription factor T-bet expression by
CD4+ cells in gastritis due to Helicobacter pylori in mice. Infect Immun 74:4673–4684
Ehlers S, Warrelmann M, Hahn H (1988) In search of an animal model for experimental
Campylobacter pylori infection: administration of Campylobacter pylori to rodents. Zbl
Bakteriologie Mikrobiol Hyg Ser A Med Microbiol Infect Dis Virol Parasitol 268:341–346
El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, Herrera J,
Lissowska J, Yuan CC, Rothman N, Lanyon G, Martin M, Fraumeni JF Jr, Rabkin CS
(2000) Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature
404:398–402
Elseweidy MM, Taha MM, Younis NN, Ibrahim KS, Hamouda HA, Eldosouky MA, Soliman H
(2010) Gastritis induced by Helicobacter pylori infection in experimental rats. Dig Dis Sci
55:2770–2777
Euler AR, Zurenko GE, Moe JB, Ulrich RG, Yagi Y (1990) Evaluation of two monkey species
(Macaca mulatta and Macaca fascicularis) as possible models for human Helicobacter pylori
disease. J Clin Microbiol 28:2285–2290
Falush D, Wirth T, Linz B, Pritchard JK, Stephens M, Kidd M, Blaser MJ, Graham DY, Vacher S,
Perez-Perez GI, Yamaoka Y, Megraud F, Otto K, Reichard U, Katzowitsch E, Wang X,
Achtman M, Suerbaum S (2003) Traces of human migrations in Helicobacter pylori
populations. Science 299:1582–1585
Figueiredo C, Machado JC, Pharoah P, Seruca R, Sousa S, Carvalho R, Capelinha AF, Quint W,
Caldas C, van Doorn LJ, Carneiro F, Sobrinho-Simoes M (2002) Helicobacter pylori and
interleukin 1 genotyping: an opportunity to identify high-risk individuals for gastric carcinoma.
J Natl Cancer Inst 94:1680–1687
Fox J, Sheh A (2013) The role of the gastrointestinal microbiome in Helicobacter pylori patho-
genesis. Gut Microbes 4:505–531
Fox JG, Correa P, Taylor NS, Lee A, Otto G, Murphy JC, Rose R (1990) Helicobacter mustelae-
associated gastritis in ferrets: an animal model of Helicobacter pylori gastritis in humans.
Fox JG, Batchelder M, Marini R, Yan L, Handt L, Li X, Shames B, Hayward A, Campbell J,
Murphy JC (1995) Helicobacter-pylori induced gastritis in the domestic cat. Infect Immun
63:2674–2681
Fox J, Li X, Cahill R, Andrutis K, Rustgi A, Odze R, Wang T (1996) Hypertrophic gastropathy in
Helicobacter felis-infected wild-type C57BL/6 mice and p53 hemizygous transgenic mice.
Fox JG, Dangler CA, Whary MT, Edelman W, Kucherlapati R, Wang TC (1997) Mice carrying a
truncated Apc gene have diminished gastric epithelial proliferation, gastric inflammation, and
humoral immunity in response to Helicobacter felis infection. Cancer Res 57:3972–3978
Fox JG, Sheppard BJ, Dangler CA, Whary MT, Ihrig M, Wang TC (2002) Germ-line p53-targeted
disruption inhibits Helicobacter-induced premalignant lesions and invasive gastric carcinoma
through down-regulation of Th1 proinflammatory responses. Cancer Res 62:696–702
Fox JG, Rogers AB, Whary MT, Ge Z, Ohtani M, Jones EK, Wang TC (2007) Accelerated
progression of gastritis to dysplasia in the pyloric antrum of TFF2-/- C57BL6 x Sv129
Helicobacter pylori-infected mice. Am J Pathol 171:1520–1528
Franco AT, Israel DA, Washington MK, Krishna U, Fox JG, Rogers AB, Neish AS, Collier-
Hyams L, Perez-Perez GI, Hatakeyama M, Whitehead R, Gaus K, O’Brien DP, Romero-
Gallo J, Peek RM Jr (2005) Activation of b-catenin by carcinogenic Helicobacter pylori. Proc
virulence factors. Cancer Res 68:379–387
Franco AT, Friedman DB, Nagy TA, Romero-Gallo J, Krishna U, Kendall A, Israel DA,
Tegtmeyer N, Washington MK, Peek RM Jr (2009) Delineation of a carcinogenic Helicobacter
pylori proteome. Mol Cell Proteomics 8:1947–1958
Gaddy JA, Radin JN, Loh JT, Zhang F, Washington MK, Peek RM Jr, Algood HM, Cover TL
(2013) High dietary salt intake exacerbates Helicobacter pylori-induced gastric carcinogene-
sis. Infect Immun 81:2258–2267
Garhart CA, Nedrud JG, Heinzel FP, Sigmund NE, Czinn SJ (2003) Vaccine-induced protection
against Helicobacter pylori in mice lacking both antibodies and interleukin-4. Infect Immun
71:3628–3633
Graham DY, Opekun AR, Osato MS, El-Zimaity HM, Lee CK, Yamaoka Y, Qureshi WA,
Cadoz M, Monath TP (2004) Challenge model for Helicobacter pylori infection in human
volunteers. Gut 53:1235–1243
Gray BM, Eaton KA (2012) Adoptive transfer of splenocytes to immunocompromised mice.
Methods Mol Biol 921:109–112
Gray BM, Fontaine CA, Poe SA, Eaton KA (2013) Complex T cell interactions contribute to
Helicobacter pylori gastritis in mice. Infect Immun 81:740–752
Haesebrouck F, Pasmans F, Flahou B, Chiers K, Baele M, Meyns T, Decostere A, Ducatelle R
(2009) Gastric helicobacters in domestic animals and nonhuman primates and their signifi-
cance for human health. Clin Microbiol Rev 22:202–223
Hagiwara T, Mukaisho K, Nakayama T, Sugihara H, Hattori T (2011) Long-term proton pump
inhibitor administration worsens atrophic corpus gastritis and promotes adenocarcinoma
development in Mongolian gerbils infected with Helicobacter pylori. Gut 60:624–630
Handt LK, Fox JG, Stalis IH, Rosemarie R, Lee G, Linn J, Li X, Kleanthous H (1995a)
Characterization of feline Helicobacter pylori strains and associated gastritis in a colony of
domestic cats. J Clin Microbiol 33:2280–2289
Handt LK, Klein HJ, Rozmiarek H, Fox JG, Pouch WJ, Motzel SL (1995b) Evaluation of two
commercial serologic tests for the diagnosis of Helicobacter pylori infection in the rhesus
monkey. Lab Anim Sci 45:613–616
Handt LK, Fox JG, Yan LL, Shen Z, Pouch WJ, Ngai D, Motzel SL, Nolan TE, Klein HJ (1997)
Diagnosis of Helicobacter pylori infection in a colony of rhesus monkeys (Macaca mulatta). J
Hirayama F, Takagi S, Kusuhara H, Iwao E, Yokoyama Y, Ikeda Y (1996) Induction of gastric
ulcer and intestinal metaplasia in Mongolian gerbils infected with Helicobacter pylori. J
Hornsby M, Huff JL, Kays R, Canfield D, Bevins C, Solnick J (2008) Helicobacter pylori induces
an antimicrobial response in rhesus macaques in a Cag Pathogenicity island-dependent man-
ner. Gastroenterology 134:1049–1057
Houghton J, Stoicov C, Nomura S, Rogers AB, Carlson J, Li H, Cai X, Fox JG, Goldenring JR,
Wang TC (2004) Gastric cancer originating from bone marrow-derived cells. Science
306:1568–1571
Huang FY, Chan AO, Lo RC, Rashid A, Wong DK, Cho CH, Lai CL, Yuen MF (2013)
Characterization of interleukin-1beta in Helicobacter pylori-induced gastric inflammation
and DNA methylation in interleukin-1 receptor type 1 knockout (IL-1R1-/-) mice. Eur J Cancer
49:2760–2770
Huff J, Hansen LM, Solnick JV (2004) A gastric transcription profile of Helicobacter pylori
infection in the rhesus macaque. Infect Immun 72:5216–5226
Israel DA, Salama N, Arnold CN, Moss SF, Ando T, Wirth HP, Tham KT, Camorlinga M, Blaser
MJ, Falkow S, Peek RM Jr (2001) Helicobacter pylori strain-specific differences in genetic
content, identified by microarray, influence host inflammatory responses. J Clin Invest
107:611–620
Josenhans C, Labigne A, Suerbaum S (1995) Comparative ultrastructural and functional studies of
Helicobacter pylori and Helicobacter mustelae flagellin mutants: both flagellin subunits, FlaA
and FlaB, are necessary for full motility in Helicobacter species. J Bacteriol 177:3010–3020
Joyce EA, Chan K, Salama NR, Falkow S (2002) Redefining bacterial populations: a post-genomic
reformation. Nat Rev Genet 3:462–473
Kanthaswamy S, Kou A, Satkoski J, Penedo MC, Ward T, Ng J, Gill L, Lerche NW, Erickson BJ,
Smith DG (2010) Genetic characterization of specific pathogen-free rhesus macaque (Macaca
mulatta) populations at the California National Primate Research Center (CNPRC). Am J
Primatol 72:587–599
Kararli TT (1995) Comparison of the gastrointestinal anatomy, physiology, and biochemistry of
humans and commonly used laboratory animals. Biopharm Drug Dispos 16:351–380
Karita M, Kouchiyama T, Okita K, Nakazawa T (1991) New small animal model for human
gastric Helicobacter pylori infection: success in both nude and euthymic mice. Am J
Kato S, Tsukamoto T, Mizoshita T, Tanaka H, Kumagai T, Ota H, Katsuyama T, Asaka M,
Tatematsu M (2006) High salt diets dose-dependently promote gastric chemical carcinogenesis
in Helicobacter pylori-infected Mongolian gerbils associated with a shift in mucin production
from glandular to surface mucous cells. Int J Cancer 119:1558–1566
Kavermann H, Burns BP, Angermuller K, Odenbreit S, Fischer W, Melchers K, Haas R (2003)
Identification and characterization of Helicobacter pylori genes essential for gastric coloniza-
tion. J Exp Med 197:813–822
Kim JS, Chang JH, Chung S, Yum JS (1999) Molecular cloning and characterization of the
Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility. J Bacteriol
181:6969–6976
Kimbrough R (1966) Spontaneous malignant gastric tumor in a Rhesus monkey (Macaca mulatta).
Arch Pathol 81:343–351
Krakowka S, Morgan DR, Kraft WG, Leunk R (1987) Establishment of gastric Campylobacter
pylori infection in the neonatal gnotobiotic piglet. Infect Immun 55:2789–2796
Kronsteiner B, Bassaganya-Riera J, Philipson C, Viladomiu M, Carbo A, Pedragosa M, Vento S,
Hontecillas R (2013) Helicobacter pylori infection in a pig model is dominated by Th1 and
cytotoxic CD8+ T cell responses. Infect Immun 81:3803–3813
Kuzushita N, Rogers AB, Monti NA, Whary MT, Park MJ, Aswad BI, Shirin H, Koff A, Eguchi H,
Moss SF (2005) p27(kip1) deficiency confers susceptibility to gastric carcinogenesis in
Helicobacter pylori-infected mice. Gastroenterology 129:1544–1556
Lee A, Fox JG, Otto G, Murphy J (1990) A small animal model of human Helicobacter pylori
active chronic gastritis. Gastroenterology 99:1315–1323
Lee A, O’Rourke J, De Ungria MC, Robertson B, Daskalopoulos G, Dixon MF (1997) A
standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain.
Lee CK, Soike K, Giannasca P, Hill J, Weltzin R, Kleanthous H, Blanchard J, Monath TP (1999)
Immunization of rhesus monkeys with a mucosal prime, parenteral boost strategy protects
against infection with Helicobacter pylori. Vaccine 17:3072–3082
Lertpiriyapong K, Whary MT, Muthupalani S, Lofgren JL, Gamazon ER, Feng Y, Ge Z, Wang
TC, Fox JG (2014) Gastric colonisation with a restricted commensal microbiota replicates the
promotion of neoplastic lesions by diverse intestinal microbiota in the Helicobacter pylori
INS-GAS mouse model of gastric carcinogenesis. Gut 63:54–63
Linden S, Mahdavi J, Semino-Mora C, Olsen C, Carlstedt I, Boren T, Dubois A (2008) Role of
ABO secretor status in mucosal innate immunity and H. pylori infection. PLoS Pathog 4:e2
Linz B, Balloux F, Moodley Y, Manica A, Liu H, Roumagnac P, Falush D, Stamer C, Prugnolle F,
van der Merwe SW, Yamaoka Y, Graham DY, Perez-Trallero E, Wadstrom T, Suerbaum S,
Achtman M (2007) An African origin for the intimate association between humans and
Helicobacter pylori. Nature 445:915–918
Linz B, Windsor HM, McGraw JJ, Hansen LM, Gajewski JP, Tomsho LP, Hake CM, Solnick JV,
Schuster SC, Marshall BJ (2014) A mutation burst during the acute phase of Helicobacter
pylori infection in humans and rhesus macaques. Nat Commun 5:4165–4172
Liu H, Merrell DS, Semino-Mora C, Goldman M, Rahman A, Mog S, Dubois A (2009) Diet
synergistically affects Helicobacter pylori-induced gastric carcinogenesis in nonhuman pri-
mates. Gastroenterology 137:1367–1379
Lofgren JL, Whary MT, Ge Z, Muthupalani S, Taylor NS, Mobley M, Potter A, Varro A,
Eibach D, Suerbaum S, Wang TC, Fox JG (2011) Lack of commensal flora in Helicobacter
pylori-infected INS-GAS mice reduces gastritis and delays intraepithelial neoplasia. Gastro-
Lucas B, Bumann D, Walduck A, Koesling J, Develioglu L, Meyer TF, Aebischer T (2001)
Adoptive transfer of CD4+ T cells specific for subunit A of Helicobacter pylori urease reduces
H. pylori stomach colonization in mice in the absence of interleukin-4 (IL-4)/IL-13 receptor
signaling. Infect Immun 69:1714–1721
Ma JL, Zhang L, Brown LM, Li JY, Shen L, Pan KF, Liu WD, Hu Y, Han ZX, Crystal-Mansour S,
Pee D, Blot WJ, Fraumeni JF Jr, You WC, Gail MH (2012) Fifteen-year effects of Helicobacter
pylori, garlic, and vitamin treatments on gastric cancer incidence and mortality. J Nat Cancer
Inst 104:488–492
Teneberg S, Karlsson KA, Altraja S, Wadstrom T, Kersulyte D, Berg DE, Dubois A,
Hammarstrom L, Boren T (2002) Helicobacter pylori SabA adhesin in persistent infection
Marchetti M, Arico B, Burroni D, Figura N, Rappuoli R, Ghiara P (1995) Development of a mouse
model of Helicobacter pylori infection that mimics human disease. Science 267:1655–1658
Mohammadi M, Czinn S, Redline R, Nedrud J (1996) Helicobacter-specific cell-mediated
immune responses display a predominant Th1 phenotype and promote a delayed-type hyper-
sensitivity response in the stomachs of mice. J Immunol 156:4729–4738
Mohammadi M, Nedrud J, Redline R, Lycke N, Czinn SJ (1997) Murine CD4 T-cell response to
Helicobacter infection: TH1 cells enhance gastritis and TH2 cells reduce bacterial load.
Moss SF, Moise L, Lee DS, Kim W, Zhang S, Lee J, Rogers AB, Martin W, De Groot AS (2011)
HelicoVax: epitope-based therapeutic Helicobacter pylori vaccination in a mouse model.
Vaccine 29:2085–2091
Nam KT, Oh SY, Ahn B, Kim YB, Jang DD, Yang KH, Hahm KB, Kim DY (2004) Decreased
Helicobacter pylori associated gastric carcinogenesis in mice lacking inducible nitric oxide
synthase. Gut 53:1250–1255
Newell DG, Hudson MJ, Baskerville A (1988) Isolation of a gastric campylobacter-like organism
from the stomach of four rhesus monkeys, and identification as Campylobacter pylori. J Med
Noach LA, Bosma NB, Jansen J, Hoek FJ, van Deventer SJ, Tytgat GN (1994) Mucosal tumor
necrosis factor-alpha, interleukin-1 beta, and interleukin-8 production in patients with
Helicobacter pylori infection. Scand J Gastroenterol 29:425–429
Nomura S, Baxter T, Yamaguchi H, Leys C, Vartapetian AB, Fox JG, Lee JR, Wang TC,
Goldenring JR (2004) Spasmolytic polypeptide expressing metaplasia to preneoplasia in
H. felis-infected mice. Gastroenterology 127:582–594
Noto JM, Gaddy JA, Lee JY, Piazuelo MB, Friedman DB, Colvin DC, Romero-Gallo J, Suarez G,
Loh J, Slaughter JC, Tan S, Morgan DR, Wilson KT, Bravo LE, Correa P, Cover TL, Amieva
MR, Peek RM Jr (2013) Iron deficiency accelerates Helicobacter pylori-induced carcinogen-
esis in rodents and humans. J Clin Invest 123:479–492
Noto JM, Lee JY, Gaddy JA, Cover TL, Amieva MR, Peek RM, Jr. (2015) Regulation of
Helicobacter pylori virulence within the context of iron deficiency. J Infect Dis 211:1790–1794
Nozaki K, Shimizu N, Inada K, Tsukamoto T, Inoue M, Kumagai T, Sugiyama A, Mizoshita T,
Kaminishi M, Tatematsu M (2002) Synergistic promoting effects of Helicobacter pylori
infection and high-salt diet on gastric carcinogenesis in Mongolian gerbils. Jpn J Cancer Res
93:1083–1089
Nozaki K, Tanaka H, Ikehara Y, Cao X, Nakanishi H, Azuma T, Yamazaki S, Yamaoka Y,
Shimizu N, Mafune K, Kaminishi M, Tatematsu M (2005) Helicobacter pylori-dependent
NF-kappa B activation in newly established Mongolian gerbil gastric cancer cell lines. Cancer
Sci 96:170–175
O’Toole PW, Lane MC, Porwollik S (2000) Helicobacter pylori motility. Microbes Infect
2:1207–1214
(2000) Virulence factors of Helicobacter pylori responsible for gastric diseases in Mongolian
gerbil. J Exp Med 192:1601–1610
Osaki T, Matsuki T, Asahara T, Zaman C, Hanawa T, Yonezawa H, Kurata S, Woo TD,
Nomoto K, Kamiya S (2012) Comparative analysis of gastric bacterial microbiota in Mongo-
lian gerbils after long-term infection with Helicobacter pylori. Microb Pathog 53:12–18
Pappo J, Torrey D, Castriotta L, Savinainen A, Kabok Z, Ibraghimov A (1999) Helicobacter pylori
infection in immunized mice lacking major histocompatibility complex class I and class II
functions. Infect Immun 67:337–341
Parker GA, Gilmore CJ, Dubois A (1981) Spontaneous gastric ulcers in a rhesus monkey. Brain
Res Bull 6:445–447
Peek RM Jr, Wirth HP, Moss SF, Yang M, Abdalla AM, Tham KT, Zhang T, Tang LH, Modlin
IM, Blaser MJ (2000) Helicobacter pylori alters gastric epithelial cell cycle events and gastrin
secretion in Mongolian gerbils. Gastroenterology 118:48–59
Perry S, de Jong B, Solnick J, Sanchez M, Yang S, Lin P, Hansen L, Talat N, Hill P, Hussain R,
Adegbola R, Flynn J, Canfield D, Parsonnet J (2010) Infection with Helicobacter pylori is
associated with protection against tuberculosis. PLoS One 5:e8804
Philpott DJ, Belaid D, Troubadour P, Thiberge JM, Tankovic J, Labigne A, Ferrero RL (2002)
Reduced activation of inflammatory responses in host cells by mouse-adapted Helicobacter
pylori isolates. Cell Microbiol 4:285–296
Raghavan S, Fredriksson M, Svennerholm A-M, Holmgren J, Suri-Payer E (2003) Absence of
CD4+CD25+ regulatory T cells is associated with a loss of regulation leading to increased
pathology in Helicobacter pylori-infected mice. Clin Exp Immunol 132:393–400
Reindel JF, Fitzgerald AL, Breider MA, Gough AW, Yan C, Mysore JV, Dubois A (1999) An
epizootic of lymphoplasmacytic gastritis attributed to Helicobacter pylori infection in
cynomolgus monkeys (Macaca fascicularis). Vet Pathol 36:1–13
128:1229–1242
Rogers AB, Taylor NS, Whary MT, Stefanich ED, Wang TC, Fox JG (2005) Helicobacter pylori
but not high salt induces gastric intraepithelial neoplasia in B6129 mice. Cancer Res
65:10709–10715
Rossi G, Rossi M, Vitali CG, Fortuna D, Burroni D, Pancotto L, Capecchi S, Sozzi S, Renzoni G,
Braca G, Del Giudice G, Rappuoli R, Ghiara P, Taccini E (1999) A conventional beagle dog
model for acute and chronic infection with Helicobacter pylori. Infect Immun 67:3112–3120
Roth K, Kapadia S, Martin S, Lorenz R (1999) Cellular immune responses are essential for the
development of Helicobacter felis-associated gastric pathology. J Immunol 163:1490–1497
Saito H, Yamaoka Y, Ishizone S, Maruta F, Sugiyama A, Graham DY, Yamauchi K, Ota H,
Miyagawa S (2005) Roles of virD4 and cagG genes in the cag pathogenicity island of
Helicobacter pylori using a Mongolian gerbil model. Gut 54:584–590
Sato T, Takeuchi A (1982) Infection by spirilla in the stomach of the rhesus monkey. Vet Pathol 19
(Supp. 7):17–25
Sayi A, Kohler E, Hitzler I, Arnold I, Schwendener R, Rehrauer H, Muller A (2009) The CD4+ T
cell-mediated IFN-gamma response to Helicobacter infection is essential for clearance and
determines gastric cancer risk. J Immunol 182:7085–7101
Sayi A, Kohler E, Toller IM, Flavell RA, Müller W, Roers A, Müller A (2011) TLR-2-activated B
Schreiber S, Konradt M, Groll C, Scheid P, Hanauer G, Werling HO, Josenhans C, Suerbaum S
(2004) The spatial orientation of Helicobacter pylori in the gastric mucus. Proc Natl Acad Sci
U S A 101:5024–5029
Seok J, Warren HS, Cuenca AG, Mindrinos MN, Baker HV, Xu W, Richards DR, McDonald-
Smith GP, Gao H, Hennessy L, Finnerty CC, Lopez CM, Honari S, Moore EE, Minei JP,
Cuschieri J, Bankey PE, Johnson JL, Sperry J, Nathens AB, Billiar TR, West MA, Jeschke MG,
Klein MB, Gamelli RL, Gibran NS, Brownstein BH, Miller-Graziano C, Calvano SE, Mason
PH, Cobb JP, Rahme LG, Lowry SF, Maier RV, Moldawer LL, Herndon DN, Davis RW,
Xiao W, Tompkins RG, Inflammation, Host Response to Injury LSCRP (2013) Genomic
responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci
U S A 110:3507–3512
Shomer NH, Dangler CA, Whary MT, Fox JG (1998) Experimental Helicobacter pylori infection
induces antral gastritis and gastric mucosa-associated lymphoid tissue in guinea pigs. Infect
Immun 66:2614–2618
Solnick JV, O’Rourke J, Lee A, Tompkins LS (1994) Molecular analysis of urease genes from a
newly identified uncultured species of Helicobacter. Infect Immun 62:1631–1638
Solnick JV, Canfield DR, Yang S, Parsonnet J (1999) Rhesus monkey (Macaca mulatta) model of
Helicobacter pylori: noninvasive detection and derivation of specific pathogen free monkeys.
Lab Anim Sci 49:197–201
Solnick JV, Canfield DR, Hansen LM, Torabian SZ (2000) Immunization with recombinant
Helicobacter pylori urease in specific-pathogen-free rhesus monkeys (Macaca mulatta). Infect
Immun 68:2560–2565
Solnick JV, Hansen LM, Canfield DR, Parsonnet J (2001) Determination of the infectious dose of
Helicobacter pylori during primary and secondary infection in rhesus monkeys (Macaca
mulatta). Infect Immun 69:6887–6892
Solnick JV, Hansen LM, Canfield DR (2002) [14C]Urea breath test is not sensitive for detection of
acute Helicobacter pylori infection in rhesus monkeys (Macaca mulatta). Dig Dis Sci
47:298–303
Solnick JV, Chang K, Canfield DR, Parsonnet J (2003) Natural acquisition of Helicobacter pylori
infection in newborn rhesus macaques. J Clin Microbiol 41:5511–5516
Solnick JV, Hansen LM, Salama N, Boonjakuakul JK, Syvanen M (2004) Modification of
rhesus macaques. Proc Natl Acad Sci U S A 101:2106–2111
Helicobacter pylori infection in rhesus macaques is most consistent with oral-oral transmis-
Peek RM Jr, Boren T, Solnick JV (2010) Expression of the BabA adhesin during experimental
infection with Helicobacter pylori. Infect Immun 78:1593–1600
Suerbaum S, Kraft C, Dewhirst FE, Fox JG (2001) Helicobacter nemestrinae ATCC49369T is a
strain of Helicobacter pylori and H. nemestrinae is therefore a junior heterotypic synonym of
H. pylori. Int J Med Microbiol 291(Suppl 31):145
Sun YQ, Monstein HJ, Nilsson LE, Petersson F, Borch K (2003) Profiling and identification of
eubacteria in the stomach of Mongolian gerbils with and without Helicobacter pylori infection.
Syu LJ, El-Zaatari M, Eaton KA, Liu Z, Tetarbe M, Keeley TM, Pero J, Ferris J, Wilbert D,
Kaatz A, Zheng X, Qiao X, Grachtchouk M, Gumucio DL, Merchant JL, Samuelson LC,
Dlugosz AA (2012) Transgenic expression of interferon-gamma in mouse stomach leads to
inflammation, metaplasia, and dysplasia. Am J Pathol 181:2114–2125
Takashima M, Furuta T, Hanai H, Sugimura H, Kaneko E (2001) Effects of Helicobacter pylori
infection on gastric acid secretion and serum gastrin levels in Mongolian gerbils. Gut
48:765–773
Tan S, Noto JM, Romero-Gallo J, Peek RM Jr, Amieva MR (2011) Helicobacter pylori perturbs
iron trafficking in the epithelium to grow on the cell surface. PLoS Pathog 7:e1002050
Thalmaier U, Lehn N, Pfeffer K, Stolte M, Vieth M, Schneider-Brachert W (2002) Role of tumor
necrosis factor alpha in Helicobacter pylori gastritis in tumor necrosis factor receptor
1-deficient mice. Infect Immun 70:3149–3155
Tokieda M, Honda S, Fujioka T, Nasu M (1999) Effect of Helicobacter pylori infection on the
N-methyl-N0 -nitro-N-nitrosoguanidine-induced gastric carcinogenesis in Mongolian gerbils.
Carcinogenesis 20:1261–1266
Tsugane S, Sasazuki S (2007) Diet and the risk of gastric cancer: review of epidemiological
evidence. Gastric Cancer Off J Int Gastric Cancer Assoc Japan Gastric Cancer Assoc 10:75–83
Wandler AM, Guillemin K (2012) Transgenic expression of the Helicobacter pylori virulence
factor CagA promotes apoptosis or tumorigenesis through JNK activation in Drosophila. PLoS
Pathog 8:e1002939
Wang TC, Dangler CA, Chen D, Goldenring JR, Koh T, Raychowdhury R, Coffey RJ, Ito S,
Varro A, Dockray GJ, Fox JG (2000) Synergistic interaction between hypergastrinemia and
Helicobacter infection in a mouse model of gastric cancer. Gastroenterology 118:36–47
Watanabe T, Tada M, Nagai H, Sasaki S, Nakao M (1998) Helicobacter pylori infection induces
gastric cancer in Mongolian gerbils. Gastroenterology 115:642–648
Weis VG, Goldenring JR (2009) Current understanding of SPEM and its standing in the
preneoplastic process. Gastric Cancer Off J Int Gastric Cancer Assoc Japan Gastric Cancer
Assoc 12:189–197
Wirth HP, Yang M, Sanabria-Valentin E, Berg DE, Dubois A, Blaser MJ (2006) Host Lewis
phenotype-dependent Helicobacter pylori Lewis antigen expression in rhesus monkeys.
FASEB J 20:1534–1536
Yang I, Nell S, Suerbaum S (2013) Survival in hostile territory: the microbiota of the stomach.
FEMS Microbiol Rev 37:736–761
Yokota K, Kurebayashi Y, Takayama Y, Hayashi S, Isogai H, Isogai E, Imai K, Yabana T,
Yachi A, Oguma K (1991) Colonization of Helicobacter pylori in the gastric mucosa of
Mongolian gerbils. Microbiol Immunol 35:475–480
Zavros Y, Eaton KA, Kang W, Rathinavelu S, Katukuri V, Kao JY, Samuelson LC, Merchant JL
(2005) Chronic gastritis in the hypochlorhydric gastrin-deficient mouse progresses to adeno-
carcinoma. Oncogene 24:2354–2366
Chapter 12
Helicobacter pylori and the Host Immune
Response
Anne Müller and Mara L. Hartung
Abstract Helicobacter pylori is an ancient companion of humans and has

coevolved with the human race at least since its migration out of Africa. Conse-
quently, it is well adapted to its exclusive niche, the mucosal surface of the human
stomach, and has developed elaborate strategies to evade or suppress immunity and
establish persistent infection. This chapter will discuss the most recent findings
regarding innate immune recognition of H. pylori and the mechanisms that allow
the bacteria to avoid detection and subsequent killing by antimicrobial peptides and
other first-line defense mechanisms. Additional topics focus on the differences in
adaptive immune responses that may explain the broad spectrum of disease out-
comes in H. pylori-infected individuals, which range from a completely asymp-
tomatic carrier state to often fatal gastric cancer development. The immune cell
compartments driving H. pylori infection-associated immunopathology are
discussed along with their main effector mechanisms. Additionally, several strate-
gies employed by H. pylori to block the clonal expansion of specific effector T cells,
and to preferentially induce the differentiation of regulatory T cells, are introduced
in light of their putative role in establishing and maintaining persistent infection. A
final topic deals with the consequences of H. pylori-specific immunomodulation for
the risk of the carrier to develop immune-related disorders such as chronic inflam-
matory diseases of the lower bowel and certain allergic disease manifestations. A
potential protective effect of H. pylori on such immune disorders is discussed with
regard to the latest epidemiological findings in humans as well as experimental
studies in animal models.
Keywords Helicobacter pylori • Type IV secretion • Immunomodulation •

Adaptive immunity • Innate immune recognition • Virulence determinants •
Persistence
A. Müller (*) • M.L. Hartung

Institute of Molecular Cancer Research, University of Zürich, Winterthurerstr. 190, 8057
Zürich, Switzerland

DOI 10.1007/978-4-431-55936-8_12
300 A. Müller and M.L. Hartung
12.1 Introduction
Helicobacter pylori exclusively infects the human gastric mucosa, where it colo-
nizes the mucus and binds to gastric epithelial pit cells (Salama et al. 2013). The
mucus layer and gastric epithelial cell monolayer of the stomach mucosa thus form
the first host defense barrier against H. pylori. The host/H. pylori interaction at the
mucosal surface of the stomach is characterized by a fine balance of both pro- and
anti-inflammatory responses, which, in ~80 % of carriers, permits persistent infec-
tion while at the same time preventing clinically overt disease. H. pylori expresses
pathogen-associated molecular patterns (PAMPs) that evade detection by the host
innate immune system yet retain their biological function as structural components
of the bacterial cell wall and motility apparatus. Other evolutionary adaptations
allow the bacteria to suppress anti-Helicobacter immunity by directly interfering
with effector T cell activation, proliferation, and function and by indirectly
blocking T cells via the induction of highly suppressive regulatory T cells. The
development of peptic ulcer disease and gastric premalignant and malignant lesions
is now widely viewed to be the consequence of a misbalance in effector and
regulatory T cell responses to the infection that arises due to a specific genetic or
lifestyle predisposition of the host or a mismatch between the genetic makeup of
host and pathogen. The preferential induction of regulatory over effector T cells,
which is a hallmark of persistent (asymptomatic) H. pylori infection, exerts not only
local but also systemic effects that remain poorly understood. One of the conse-
quences of the systemic immunomodulation by H. pylori is the relative protection
of the infected fraction of the population from allergic and chronic inflammatory
diseases such as allergen-induced asthma, hay fever, ectopic dermatitis, inflamma-
tory bowel diseases, and celiac disease, all of which are immune-related disorders.
The purpose of the following chapters is the detailed discussion of the elaborate
molecular adaptations of H. pylori that allow it to evade and manipulate host
immune responses and to thereby establish persistent infection. The local and
systemic consequences of this host/pathogen interaction are also discussed.
12.2 Innate Immune Responses to H. pylori
As mentioned above, the gastric epithelial cell monolayer of the stomach mucosa
forms the first innate immune defense barrier against H. pylori and therefore has
been studied in great detail. The direct interaction of H. pylori with epithelial cells
results in the assembly of the type IV secretion system (T4SS) pilus (for more
details, see Chap. 4) and the subsequent exposure to virulence factors encoded on
the Cag pathogenicity island (cagPAI); among these, the CagL/integrin α5β1
interaction has proven to be directly responsible for the activation of NF-κB and
production of the neutrophil chemoattractant interleukin-8 (IL-8) (Gorrell
et al. 2012; Jimenez-Soto et al. 2009; Kwok et al. 2007; Shaffer et al. 2011). In
12 Helicobacter pylori and the Host Immune Response 301
line with the well-known pro-inflammatory/immunostimulatory activity of the

H. pylori T4SS, mutants lacking this activity colonize mice and gerbils at higher
levels than the corresponding wild-type strains because they are controlled less
efficiently; cagPAI mutants further cause significantly less preneoplastic immuno-
pathology in rodent models (Arnold et al. 2011b; Rieder et al. 2005). In human
carriers, cagPAI-positive strains (often identified serologically by their expression
of the cagPAI-encoded CagA protein) are clearly associated with more severe
disease and higher gastric cancer risk than cagPAI/CagA-negative strains (Blaser
et al. 1995; Huang et al. 2003). Several studies suggest that in humans, the
expression of cagPAI proteins appears to confer a colonization advantage through
the downregulation of antimicrobial peptides: for example, it has been demon-
strated that H. pylori reduces the expression of human β-defensin 1 in a T4SS- and
NF-κB-dependent manner, with colonization and gastric β-defensin 1 levels being
inversely correlated in H. pylori carriers (Patel et al. 2013). Another human
antimicrobial peptide, β-defensin 3, which is known to be highly active against
H. pylori, is initially induced by the infection in vitro in an epidermal growth factor
receptor (EGFR)- and mitogen-activated protein (MAP) kinase-dependent manner
but is then stably shut down through CagA-mediated activation of the Src homol-
ogy domain containing protein tyrosine phosphatase 2 (SHP2) and down-
modulation of the EGFR signaling pathway (Bauer et al. 2012a). The
downregulation of β-defensin 3 protein could indeed be confirmed in the gastric
mucosa of H. pylori-infected subjects (Bauer et al. 2012b). In line with the critical
role of the cagPAI-encoded T4SS in pro-inflammatory and immune escape mech-
anisms, its expression and function have been shown to be fine-tuned by DNA
rearrangements at direct repeats in the coding sequence of the CagY protein, an
essential component of the T4SS, which is subject to immune-driven selective
pressure in both mice and nonhuman primates (Barrozo et al. 2013).
Whereas cagPAI-induced responses are best characterized and understood in
gastric epithelial cells, most non-PAI-mediated interactions of H. pylori with the
host have been studied in cells of the innate immune system. Innate immune
recognition of H. pylori is unique in that it predominantly results in anti- rather
than pro-inflammatory responses; several pathogen-associated molecular patterns
(PAMPs) of H. pylori have further evolved to specifically avoid excessive inflam-
mation (Fig. 12.1). The lipopolysaccharide (LPS) constituent of the bacterial outer
membrane, which in enteropathogenic bacteria has strong immunostimulatory
properties and readily activates inflammatory signaling via toll-like receptor
4 (TLR4), has evolved in H. pylori to avoid TLR4 recognition (Cullen
et al. 2012; Moran et al. 1997). The relative bio-inactivity of H. pylori LPS has
been attributed to its tetra-acylation (whereas E. coli LPS is hexa-acylated) (Moran
et al. 1997) and to the removal of phosphate groups from the 10 and 40 positions of
the lipid A backbone, which generates LPS that has less negative charge, escapes
detection by TLR4, and resists binding by antimicrobial peptides (Cullen
et al. 2012). Mutants lacking the phosphatases required for lipid A modification
consequently fail to colonize mice (Cullen et al. 2012). A similarly elaborate
mechanism of immune evasion has been reported for H. pylori flagellin, which is
Fig. 12.1 H. pylori subverts and evades innate immune recognition. H. pylori produces several
PAMPs that have evolved to evade detection by pro-inflammatory TLRs. Examples include
H. pylori’s tetra-acylated LPS, which is bioinactive due to specific lipid A modifications that
prevent detection by TLR4. H. pylori flagella cannot be detected by TLR5 due to mutations in the
TLR5 binding site of flagellin. H. pylori DNA, enriched for the immunoregulatory sequence
TTTAGGG, as well as an as yet uncharacterized PAMP (and possibly H. pylori LPS) are detected
by TLRs 9 and 2, respectively; these TLRs preferentially activate anti-inflammatory signaling
pathways and IL-10 expression. 50 triphosphorylated RNA is detected by the cytosolic receptor
RIG-I, which activates the transcription factors IRF3 and IRF7 to induce type I IFN expression; H.
pylori RNA is likely also detected by TLR8 in endosomes. H. pylori’s fucosylated DC-SIGN
ligands suppress activation of the signaling pathways downstream of DC-SIGN and activate anti-
inflammatory genes. Note that not all depicted pattern recognition receptors are necessarily
expressed by the same cell type; a generic cell is shown here for simplicity. DD, death domain;
TIR, toll/interleukin-1 receptor domain; CARD, caspase activation and recruitment domain;
MyD88, myeloid differentiation primary response gene 88; DC-SIGN, DC-specific intercellular
adhesion molecule-3-grabbing non-integrin; SRC, steroid receptor coactivator
mutated in exactly those N-terminal positions that mediate binding to TLR5 in

Salmonella enterica flagellin (Andersen-Nissen et al. 2005; Gewirtz et al. 2004).
Additional interactions of H. pylori PAMPs with host innate immune receptors
include the detection of H. pylori DNA by the endosomally localized TLR9 (Otani
et al. 2012) and the binding of an as yet unidentified ligand to TLR2 (Fig. 12.1). The
interactions via TLR9 and TLR2 result in predominantly anti-inflammatory
responses. Lipofection of dendritic cells (DCs) with H. pylori DNA activates
TLR9 (Rad et al. 2009), with documented anti-inflammatory consequences in the
first months of infection in a mouse model (Otani et al. 2012). The putative
immunoregulatory properties of H. pylori DNA have in fact even been exploited
successfully for therapeutic purposes: oral administration of purified DNA
alleviates experimentally induced inflammatory bowel disease in mice (Luther

et al. 2011), a finding that has been attributed to the unique immunoregulatory
sequence 50 TTTAGGG that is overrepresented in the H. pylori genome (Owyang
et al. 2012). The predominant TLR activated by H. pylori is TLR2, which drives an
anti-inflammatory signature characterized by high expression levels of the regula-
tory cytokine interleukin-10 (IL-10) in DCs (Rad et al. 2009) (Fig. 12.1). Whereas
the H. pylori ligand for TLR2 remains unknown, it is clear from various studies
conducted in vitro and in vivo that TLR2 signaling counteracts H. pylori clearance
and promotes immune tolerance (Sayi et al. 2011; Sun et al. 2013). TLR2-deficient
mice control H. pylori and related Helicobacter species efficiently develop more
severe and accelerated immunopathology (Sayi et al. 2011; Sun et al. 2013). TLR2
proficiency of B cells and DCs is required for the differentiation of regulatory T
cells, the H. pylori-induced production of IL-10 and other tolerogenic responses
in vitro and in vivo, and likely accounts for the phenotype of TLR2/ mice (Rad
et al. 2009; Sayi et al. 2011; Sun et al. 2013). In addition to the mentioned PAMPs
and their receptors, H. pylori RNA has also been shown to mediate innate immune
recognition. 50 -triphosphorylated RNA of H. pylori is sensed by the endosomal
TLR8, as well as the cytoplasmic retinoic acid-inducible gene (RIG)-like helicase
receptor RIG-I, the latter leading to the production of type I interferons (IFNs) (Rad
et al. 2009). Whether this pathway of innate immune detection contributes to
H. pylori control or pathogenesis is currently not known.
In addition to the described immune escape and immunomodulatory mecha-
nisms involving TLRs, H. pylori has further evolved to bind the C-type lectin
receptor DC-specific intercellular adhesion molecule-3-grabbing non-integrin
(DC-SIGN); however, in contrast to other pathogens expressing (mannosylated)
DC-SIGN ligands, the fucosylated DC-SIGN ligands of H. pylori fail to activate the
signaling cascade downstream of this receptor and instead dissociate the respective
signaling complexes to suppress pro-inflammatory signaling (Gringhuis
et al. 2009). Anti-inflammatory consequences have also been reported of the
H. pylori-induced activation of the inflammasome. It is now clear that H. pylori
activates caspase-1 and induces the proteolytic processing of caspase-1-dependent
cytokines in an NLRP3- and ASC-dependent manner (Hitzler et al. 2012b; Kim
et al. 2013). Mature IL-1β and IL-18 are produced and secreted by DCs that have
been exposed to H. pylori (Hitzler et al. 2012b; Kim et al. 2013); transcriptional
activation of pro-IL-1β appears to depend on the cagPAI (Kim et al. 2013), whereas
pro-IL-18 is known to be constitutively expressed. Interestingly, the two caspase-1-
dependent cytokines fulfill opposing roles in the context of H. pylori control and
pathogenesis. Whereas IL-1β is absolutely required for the differentiation and
function of Th1 and Th17 cells and thus for the control of experimental infection
as well as for the development of gastritis and preneoplastic immunopathology
(Hitzler et al. 2012b; Kim et al. 2013), IL-18 is dispensible for immunity to
H. pylori (Hitzler et al. 2012b). The phenotypes of IL-1R and IL-1β-deficient
mouse strains are nicely in line with observations in humans, where promoter
polymorphisms in the IL-1β gene leading to increased steady-state production of
the cytokine predispose to an increased gastric cancer risk (El-Omar et al. 2000).
Quite to the contrary, mice lacking IL-18 or its receptor control Helicobacter
infections more efficiently because they fail to generate FoxP3-positive regulatory
T cells (Tregs) and to develop immune tolerance to the infection and, as a conse-
quence, exhibit overly active Th17 cells (Hitzler et al. 2012b; Oertli et al. 2012).
The defect in Treg differentiation of IL-18/IL-18R-deficient mice has been attrib-
uted to the failure of IL-18/ DCs to induce Treg differentiation (Oertli
et al. 2012). The differential properties of caspase-1-dependent cytokines in
H. pylori control and immunopathology are reminiscent of the functions of both
cytokines in the lower GI tract, especially in models of experimentally induced
colitis, which are driven in large part by IL-1β (Coccia et al. 2012) and restricted by
IL-18 (Zaki et al. 2010).
Collectively, the data generated in recent years on the various innate immune
responses to (and their manipulation by) H. pylori suggest that the bacterium
effectively prevents its clearance and promotes its persistence by both evading
innate immune detection and subsequent killing and by skewing innate immune
responses toward anti-inflammatory and regulatory signals. The postulated
>60,000 years of coexistence of H. pylori with its human host have provided the
selective pressure necessary to drive these evolutionary processes; the remarkable
genetic variability, high mutation rate, and natural competence of H. pylori provide
the genetic setting facilitating its host adaptation.
12.3 Anti-Helicobacter Adaptive Immune Responses Differ

in Symptomatic and Asymptomatic Carriers
Whereas approximately one-half of humanity is infected with H. pylori, only a

fraction of human carriers will develop H. pylori-related gastric or duodenal
disease. The remaining ~80 % of the infected population remain asymptomatic
for life and in fact may never know that they are colonized. Host genetic traits (see
Chap. 14 on host gene polymorphisms and their effects on disease outcome) and the
specific virulence factors expressed by the infecting H. pylori strain (see Chaps. 3,
4, 5, 6, and 7) have been discussed as critical modulators influencing disease
outcome. One important predictor and driver of disease that has emerged in recent
years from studies conducted in human carriers as well as in mouse models is the
severity and polarization of gastric H. pylori-specific T helper cell responses
(Arnold et al. 2011b; Harris et al. 2008; Robinson et al. 2008). Individuals with
peptic ulcer disease (PUD) have a threefold higher anti-H. pylori Th1 response and
sixfold higher Th2 response than asymptomatic carriers (Robinson et al. 2008). The
PUD patients at the same time exhibited a twofold lower Treg response than
asymptomatic carriers and significantly reduced IL-10 and transforming growth
factor (TGF)-β levels in the gastric mucosa; the authors concluded from this
imbalance that an inadequate Treg response was associated with and possibly
responsible for the development of H. pylori-associated disease (Robinson
et al. 2008). Another study with a similar objective and approach compared the anti-
H. pylori responses of children and adults and also found an inverse correlation
between the degree of gastritis and the numbers of gastric Tregs and production of
Treg-derived cytokines (Harris et al. 2008). These two seminal studies have con-
firmed and extended previous work showing that Tregs home to and accumulate in
the gastric mucosa of infected but not uninfected individuals, where they suppress
H. pylori-specific memory T cell responses (Lundgren et al. 2003, 2005a, b). Taken
together, the observational studies in humans imply that asymptomatic (healthy)
carriers predominantly launch Treg responses to H. pylori infection, which effec-
tively control immunopathology and promote persistent infection, whereas symp-
tomatic carriers presenting with disease exhibit T-effector-dominated responses
(Fig. 12.2). The studies in humans are corroborated by experimental studies in
mice, where Treg depletion improves infection control by deregulating Th1 and
Th17 responses and at the same time promotes the development of chronic
infection-associated gastritis, atrophy, and intestinal metaplasia (Arnold
et al. 2011b; Hitzler et al. 2011). Treg-derived IL-10 and TGF-β are each critical
for preventing T-effector cell-driven immunopathology (Arnold et al. 2011b),
suggesting that the expression levels of these cytokines in the gastric mucosa are
excellent indicators of disease outcome. Interestingly, the lack or neutralization of
IL-10 signaling is sufficient to clear H. pylori and to induce strong T cell-dependent
immunopathology (Ismail et al. 2003; Sayi et al. 2011).
Fig. 12.2 Symptomatic and asymptomatic carriers differ in terms of their anti-H. pylori T cell
responses. Observational studies comparing the T cell responses of peptic ulcer disease patients
and asymptomatic carriers (Robinson et al. 2008) and children vs. adults with relatively mild and
severe gastritis, respectively (Harris et al. 2008), have revealed that Treg/T-effector cell ratios
correlate with disease outcome. Asymptomatic carriers predominantly generate Treg responses to
the infection, which suppress Th1 and Th17 cells through the production of soluble cytokines IL-
10 and TGF-β and other immunosuppressive mechanisms. This scenario is modeled in C57BL/6
mice neonatally infected with H. pylori (Arnold et al. 2011b) In contrast, the gastric mucosa of
symptomatic carriers is infiltrated by Th17 and Th1 cells and exposed to high levels of the
signature cytokines IFN-γ, TNF-α, and IL-17, which drive chronic gastritis and promote disease
progression toward chronic atrophic gastritis and hyperplasia, intestinal metaplasia, ulcers, and
gastric cancer. Infection of adult C57BL/6 mice mirrors the scenario found in symptomatic
carriers. H. pylori colonization levels are generally higher in asymptomatic vs. symptomatic
carriers and directly proportional to gastric mucosal Treg numbers
H. pylori is typically acquired during early childhood, with the mother serving as
the main source of infection in populations with low prevalence (Weyermann
et al. 2009). The infection is generally contracted within the first 2 years of life
(Rothenbacher et al. 2000), i.e., at a time when the immune system is immature and
predisposed to develop immune tolerance rather than immunity to foreign dietary
and environmental antigens and the newly acquired microbiota (Arnold et al. 2005).
Animal models that take into account and aim to reflect the age at the time of
H. pylori acquisition have revealed that exposure to H. pylori during the neonatal
period leads to the development of Treg-mediated immune tolerance to H. pylori
(Arnold et al. 2011b). Although anti-H. pylori effector T cell responses are gener-
ated normally by the neonatally infected murine host, these are under the tight
control of Tregs (Fig. 12.2). Neonatally infected animals are protected against
infection-induced gastric immunopathology, not just in the weeks and months
following experimental infection but apparently for life (Arnold et al. 2011b).
The hallmarks of the neonatal infection model are reminiscent of the Treg-
predominant responses of infected children (Harris et al. 2008); it is interesting to
note in this context that children not only suffer less frequently from the conse-
quences of gastric colonization with H. pylori but also appear to benefit more in
terms of their reduced allergy risk (see below). Understanding the relative role of
T-effector vs. Treg responses in the balance of H. pylori clearance and immunopa-
thology is of immediate practical relevance in H. pylori vaccinology, as protective
immunity can only be achieved by vaccination strategies aimed at overcoming
immune counter-regulation (Becher et al. 2010; Hitzler et al. 2011) and should
ideally be sterilizing (see Chap. 24 on vaccine development against H. pylori).
12.4 Gastric Preneoplastic Immunopathology Is Driven by

T Helper Cell Responses and T Cell-Derived
Cytokines
It is now well accepted due to work conducted in experimental infection and

vaccine-induced protection models that the host control of H. pylori colonization
depends on CD4+ effector T cells, but not on B cells or antibodies (Akhiani
et al. 2002, 2004a, b; Ermak et al. 1998; Hitzler et al. 2011; Sayi et al. 2009;
Velin et al. 2005, 2009; Velin and Michetti 2010). Human volunteer infections
confirm that strong T cell responses and H. pylori clearance are tightly correlated
and possibly causally associated (Aebischer et al. 2008). Out of a total of 58 vol-
unteers who were challenged with live H. pylori in a seminal study published in
2008, 13 managed to clear the infection as judged by urea breath test; eight of these
had been vaccinated against H. pylori using the Salmonella typhimurium vaccine
strain Ty21a expressing H. pylori urease and five had cleared the challenge infec-
tion spontaneously (Aebischer et al. 2008). H. pylori-specific T helper cells were
detected in 9 of the 13 volunteers who cleared the infection successfully, but only in
6 of 45 who did not. While the study disappointingly provided little evidence for
vaccination-induced protective immunity, it did show convincingly that anti-
H. pylori T cell responses correlate well with clearance (Aebischer et al. 2008).
It is evident from studies using T cell-deficient mouse strains that CD4+ TCRα/β+
T cells are required for the control of H. pylori as well as for the induction of
immunopathology: mice specifically lacking CD4+ TCRα/β+ T cells either due to a
deletion of the major histocompatibility complex (MHC) II or to lack of the TCR
βchain fail to control experimental infections (in vaccinated as well as naive
mice) and are at the same time protected against infection-associated immunopa-
thology (Hitzler et al. 2011, 2012a; Akhiani et al. 2002; Ermak et al. 1998). It has
been postulated that the CD4+ T helper cell subsets and signature cytokines that
contribute to infection control in experimental models at the same time promote the
immunopathology that is a hallmark of immunocompetent hosts and that manifests
histologically as chronic (atrophic) gastritis (Hitzler et al. 2012a; Horvath
et al. 2012; Sayi et al. 2009; Shi et al. 2010; Stoicov et al. 2009). Considerable
early evidence suggests that Th1-polarized T cells are critical mediators of H. pylori
control and immunopathology. Th1 cells and their signature cytokine IFN-γ have
been implicated in vaccine-induced protective immunity and in infection-
associated gastritis: mice lacking the p40 subunit of the Th1-polarizing cytokine
IL-12 or the receptor for the Th1 signature cytokine IFN-γ fail to control H. pylori
upon challenge infection and IFN-γR/ mice further fail to develop gastritis
(Akhiani et al. 2002). Similarly, mice lacking Th1 cells due to the genetic ablation
of the lineage-defining transcription factor T-bet are protected from gastric atrophy
and the development of gastric cancer later in life (Stoicov et al. 2009). The
adoptive transfer of wild-type but not IFN-γ-deficient CD4+ T cells controls the
infection and induces preneoplastic pathology in H. pylori-infected T cell-deficient
recipients (Sayi et al. 2009). In hindsight, some of the early work must be
interpreted with caution: for example, it is now clear that the p40 subunit of
IL-12 is shared by the related cytokine IL-23, which is generally accepted to
drive Th17 but not Th1 responses (Cua et al. 2003). Consequently, p40/ mice
lack Th17 as well as Th1 cells (Cua et al. 2003). More recent studies have addressed
the relative contribution of Th1 and Th17 cells to infection control; neither p19/
nor p35/ mice – lacking the specific subunits of IL-23 and IL-12, i.e., deficient
for either Th17 or Th1 cells – were protected upon vaccination with the gold
standard cholera toxin adjuvant whole cell extract vaccine (Hitzler et al. 2011);
the contribution of Th17 cells to H. pylori control was also confirmed by other
investigators (Horvath et al. 2012). In addition to their role in H. pylori control,
Th17 cells also contribute to infection-induced immunopathology, as assessed in
mice lacking the IL-23-specific p19 subunit (Hitzler et al. 2012a; Horvath
et al. 2012) Whether the best-studied Th17 signature cytokine IL-17 mediates the
effects of Th17 cells remains controversial; neutralizing antibodies to IL-17 pre-
vents H. pylori control and immunopathology in vaccination models (Velin
et al. 2009) but have been reported to promote colonization in non-vaccinated,
experimentally infected mice (Shi et al. 2010). In line with the known reciprocal
negative regulation of Th1 and Th17 subsets, this effect has been attributed by some
investigators to higher Th1 cytokine expression in the absence of IL-17 signaling

(Otani et al. 2009). In summary, the work of many groups has documented beyond
doubt that the control of H. pylori and the resulting infection-induced gastric
immunopathology preceding the development of gastric cancer are inseparably
linked, at least in mouse models, and mediated by both Th1 and Th17 subsets of
T cells. Public health strategies aimed at reducing gastric cancer risk by eradicating
H. pylori in high-risk individuals or populations thus might benefit from combining
antibiotic treatment with T cell-targeting immunomodulatory therapies to acceler-
ate mucosal healing and improve treatment outcome also in individuals with
preexisting atrophy and metaplasia that do not currently benefit sufficiently from
eradication therapy alone (Rokkas et al. 2007; Wong et al. 2004).
12.5 H. pylori Suppresses Effector T Cell Responses

to Achieve Persistent Infection
Given the critical role of effector T cells (Th1 and Th17 subsets) in controlling
H. pylori, it is not surprising that the bacteria have evolved elaborate mechanisms of
suppressing human T cell activity, proliferation, and clonal expansion. One key
virulence factor/persistence determinant with T cell inhibitory properties is the
vacuolating cytotoxin VacA. VacA was initially identified due to its ability to
induce massive vacuolation in primary gastric epithelial cells and certain gastric
epithelial cell lines (Smoot et al. 1996; Harris et al. 1996) and to its association with
peptic ulcer disease (Atherton et al. 1995) (see Chap. 5 on VacA). It is also known
to induce apoptosis in gastric epithelial cells (Cover et al. 2003), presumably via
insertion into mitochondrial membranes followed by cytochrome C release
(Domanska et al. 2010). Its vacuolating and pro-apoptotic activity requires a stretch
of N-terminally encoded hydrophobic amino acids, which allow VacA to form
hexameric pores in artificial lipid bilayers as well as in endosomal, lysosomal, and
mitochondrial membranes of epithelial cells and phagocytes (McClain et al. 2001;
Czajkowsky et al. 1999). VacA is expressed by all H. pylori isolates in the form of
either the m1 or m2 allele, which differ in expression levels, vacuolating activity,
and association with disease (Atherton et al. 1999). Mouse studies have demon-
strated that VacA is not only a virulence but also a colonization factor, as mutants
lacking VacA are rapidly outcompeted by the wild type in mixed infections
(Salama et al. 2001) and colonized at significantly lower levels in single infections
(Oertli et al. 2013). In addition to its vacuolating and pro-apoptotic effects on
epithelial cells and in line with its critical role in colonization, VacA has been
shown to inhibit the activation and proliferation of T and of B cells (Fig. 12.3)
(Torres et al. 2007; Boncristiano et al. 2003; Gebert et al. 2003; Sundrud
et al. 2004). In human primary T cells, activation upon TCR engagement is blocked
at the level of the Ca2+/calmodulin-dependent phosphatase calcineurin and the
nuclear translocation of the transcription factor nuclear factor of activated T cells
Fig. 12.3 H. pylori impairs T cell-mediated immunity through the production and secretion of
VacA and GGT. All strains of H. pylori express the secreted virulence factors VacA and GGT to
directly inhibit T cell activation, proliferation, and effector functions. Hexameric VacA binds to
the β2 integrin subunit of the heterodimeric transmembrane receptor LFA-1; the receptor complex
is internalized upon protein kinase C-mediated serine/threonine phosphorylation of the β2 integrin
cytoplasmic tail. Cytoplasmic VacA prevents nuclear translocation of NFAT by inhibiting its
dephosphorylation by the Ca2+/calmodulin-dependent phosphatase calcineurin and thereby blocks
IL-2 production and subsequent T cell activation and proliferation. GGT arrests T cells in the G1
phase of the cell cycle and thus prevents their proliferation. Note that the direct effects of VacA on
T cells appear to be human specific. LFA-1, lymphocyte function-associated antigen-1; NFAT,
nuclear factor of activated T cells; GGT, γ-glutamyl transpeptidase; CnA, B, calcineurin A and B
subunits; CaM, calmodulin
(NFAT) (Boncristiano et al. 2003; Gebert et al. 2003). VacA activity on T cells
requires the same N-terminal hydrophobic region that also mediates vacuolization
(Sundrud et al. 2004) and binding to a surface receptor, the β2 integrin (CD18)
(Sewald et al. 2008), which associates with CD11a/αL to form the heterodimeric
lymphocyte function-associated antigen-1 (LFA-1) receptor (Fig. 12.3). VacA is
taken up as LFA1 is recycled in a PKC-mediated, phosphorylation-dependent
manner (Sewald et al. 2010). It seems that CD18 must be directly phosphorylated
by either PKCη or PKCζ in its cytoplasmic tail to initiate VacA endocytosis and
inhibition of NFAT target gene transactivation (Fig. 12.3) (Sewald et al. 2010).
Murine T cells are resistant to VacA because they do not express the receptor
(Sewald et al. 2008; Algood et al. 2007); rather, it appears that VacA promotes
persistence in mice through a mechanism involving its interaction with DCs (see
below) (Oertli et al. 2013). In humans, VacA’s inhibitory activity on T cells likely
prevents the clonal expansion of H. pylori-specific, antigen-activated T cells,
thereby interfering effectively with a critical branch of adaptive immune defense
against this infection.
In addition to VacA, all strains of H. pylori produce and secrete a second

immunomodulatory molecule known to interfere with T cell proliferation, the
gamma-glutamyl transpeptidase (GGT). GGT has enzymatic activity and catalyzes
the transfer of the γ-glutamyl moiety of glutamine or glutathione to amino acids,
allowing H. pylori to convert glutamine and glutathione into glutamate, which can
be taken up and incorporated into the TCA cycle. H. pylori mutants lacking GGT
fail to colonize mice (Chevalier et al. 1999; Oertli et al. 2013), and this phenotype
has been linked to the ability of GGT to prevent T cell proliferation (Gerhard
et al. 2005; Schmees et al. 2007) (Fig. 12.3). Other parameters of T cell activation
(NFAT translocation, cytokine production) are not affected by GGT; its inhibitory
effect on proliferation could be linked to cell cycle arrest in the G1 phase and to the
enzymatic activity of GGT (Gerhard et al. 2005; Schmees et al. 2007). Similar to
VacA, GGT also exerts strong immunomodulatory effects on DCs, which acquire
tolerogenic activity upon exposure to GGT-proficient H. pylori or the recombinant
protein (discussed in more detail below) (Oertli et al. 2013). The inclusion of both
VacA and GGT in experimental H. pylori vaccines (Malfertheiner et al. 2008)
indicates that the neutralization of H. pylori’s immunomodulatory properties is
widely seen as a promising intervention strategy. Further details on GGT function
can be obtained in Chaps. 6 and 7.
12.6 H. pylori Promotes Tolerogenic DC Functions

and Induces Regulatory T Cells
DCs were long known mostly for their essential role in immunity to intracellular
and extracellular pathogens, to which they contribute through their unique ability to
prime naive T cells to differentiate, to proliferate, and to acquire effector functions
such as cytotoxicity and cytokine production. It has become clear in recent years,
however, that DCs are also critically involved in the development of immune
tolerance to autoantigens, allergens, and harmless antigens of the commensal
human microflora (Maldonado and von Andrian 2010; Yogev et al. 2012). A
major pathway driving DC-mediated immune tolerance to self-, dietary, or envi-
ronmental antigens involves the thymus-independent, “peripheral” induction of
highly suppressive, “inducible” Tregs, which, like their thymus-derived “natural”
counterparts, typically express the lineage-defining transcription factor FoxP3, the
surface marker CD25, and an array of secreted and surface-exposed regulatory
molecules that may include IL-10, TGF-β, CTLA-4, PD1, GITR, and others. Tregs
efficiently block effector T cell responses by direct and indirect mechanisms, and
their prolonged dysregulation results in severe and often fatal autoimmunity
(Bollrath and Powrie 2013; Harrison and Powrie 2013). Persistent pathogens,
such as Mycobacterium tuberculosis and certain helminths, have evolved to selec-
tively recruit or activate Tregs to or in their preferred niche to promote chronicity
(McBride et al. 2013; Ottenhoff 2012; Maizels and Smith 2011). The same is true
for H. pylori, which preferentially induces Tregs (Robinson et al. 2008) and relies
on Treg-mediated immunosuppression to promote persistent infection (Arnold
et al. 2011b) (also see above). Although difficult to study meaningfully in humans,
the process of Treg induction and DC/Treg-mediated immune tolerance and sup-
pression has received substantial attention lately and several key mechanisms have
been elucidated. Multiple studies have shown in vitro and in vivo that H. pylori
specifically targets DCs to promote their tolerogenic (i.e., Treg-inducing) properties
and to at the same time subvert their immunogenic functions. Cultured murine and
human DCs that have been exposed to and have phagocytosed live H. pylori fail to
prime Th17 and Th1 responses and instead preferentially induce FoxP3 expression
and suppressive activity in cocultured naive T cells (Kao et al. 2010; Kim
et al. 2011; Oertli et al. 2012) (Fig. 12.4). This tolerogenic activity has been
attributed to the failure of H. pylori-exposed DCs to mature, i.e., to express high
levels of MHCII and co-stimulatory markers such as CD80, CD86, and CD40, as
well as T helper cell-differentiating and T helper cell-activating cytokines such as
IL-12 or IL-23 (Kaebisch et al. 2013; Oertli et al. 2012) (Fig. 12.4). This is true even
in the presence of strong maturation signals delivered via TLR-4 or TLR-9 engage-
ment, e.g., by E. coli LPS or CpG oligonucleotides (Oertli et al. 2012). The semi-
mature DCs that result from H. pylori exposure are characterized by high expres-
sion of MHCII yet virtually no or low expression of co-stimulatory markers;
similarly, these DCs fail to produce IL-12, IL-6, and TNF-α and instead secrete
large amounts of the anti-inflammatory cytokine IL-10 (Oertli et al. 2012)
(Fig. 12.4). Semi-mature and immature DCs have been implicated in Treg induction
and tolerogenic immune responses (Maldonado and von Andrian 2010); indeed,
H. pylori-exposed, semi-mature DCs are excellent inducers of Treg differentiation
in vitro and in vivo (Kao et al. 2010; Oertli et al. 2012), and their forced maturation
by LPS treatment is sufficient to break tolerance in vivo (Oertli et al. 2012).
Consistent with the experimental results, the gastric mucosa of H. pylori-infected
human carriers is populated by semi-mature DCs lacking co-stimulatory markers
(Oertli et al. 2012). The H. pylori virulence and persistence factors required for the
bacteria’s tolerogenic activity on DCs are under intense investigation, whereas two
secreted factors, the vacuolating cytotoxin VacA and the γ-glutamyl transpeptidase
GGT, have been implicated in the inhibition of murine DC maturation and
tolerogenic reprogramming (Kim et al. 2011; Oertli et al. 2012); the T4SS of
H. pylori seems to be equally or more important in human DCs (Kaebisch
et al. 2013).
Several studies have attempted to functionally address the contribution of
(tolerogenic) DCs to immune tolerance and to H. pylori-specific immunity in
experimental models (Kao et al. 2010). Interestingly, the depletion of CD11c+
DCs in a genetic model taking advantage of the CD11c-driven expression of the
diphtheria toxin receptor improves clearance of H. pylori upon challenge infection
of vaccinated mice (Hitzler et al. 2011). Whereas prior vaccination with H. pylori
extract in conjunction with either the gold standard cholera toxin adjuvant or a
novel mycobacteria-derived adjuvant (CAF01) slashed H. pylori burdens by two
orders of magnitude, this reduction could be further improved by another one to two
Fig. 12.4 The maturation status of dendritic cells (DCs) directs T helper cell differentiation.
Immature DCs with high phagocytic activity follow chemokine gradients to sites of microbial
colonization, where they actively sample antigen, which is then processed and presented in the
context of MHC class II molecules on the cell surface. (a) Simultaneous stimulation of phagocytic
DCs by recognition of PAMPs via cytosolic or membrane-bound pattern recognition receptors
promotes DC maturation, which results in upregulation of maturation markers and co-stimulatory
molecules (CD40, CD80, CD86) on the cell surface, as well as the production of T cell-
differentiating and T cell-activating, as well as other pro-inflammatory cytokines (IL-12, IL-23,
TNF-α, IL-6). Three signals (antigen recognition via the T cell receptor, co-stimulatory signals,
and soluble cytokine signals) are required for the differentiation of naive T cells into Th1 or Th17
cells, with high levels of IL-12 driving Th1 and high levels of IL-23 driving Th17 differentiation.
Both subsets are required for H. pylori control. (b) H. pylori exposure generates semi-mature DCs
with high MHC class II expression but no or little co-stimulation and low levels of Th1/Th17 cell-
differentiating cytokines, resulting in Treg differentiation and immunosuppression. An additional
marker of semi-mature, tolerogenic DCs is their expression and secretion of IL-10
orders of magnitude by the depletion of DCs (Hitzler et al. 2011). Similar effects
were obtained in experimental H. pylori infection models not involving prior
immunization (Hitzler et al. 2011; Oertli et al. 2012). In both settings, the depletion
of DCs not only reduced bacterial burdens but also boosted all relevant correlates of
(vaccine-induced) protective immunity, such as the recruitment of memory T cells,
mast cells, and neutrophils to the gastric mucosa, the priming of H. pylori-specific
Th1 and Th17 cells in the draining mesenteric lymph nodes, and the local produc-
tion of protective cytokines including IFN-γ and IL-17 (Hitzler et al. 2011; Oertli
et al. 2012). The results suggest that DCs are dispensible for immunity to H. pylori
and instead are an essential element of immunoregulation, preventing control of the
infection. Similar observations have been made in models of autoimmunity, in
which the depletion of DCs invariably aggravates rather than improves disease
severity (Yogev et al. 2012). A model of adoptive bone marrow-derived DC
transfer into H. pylori-infected mice also showed that the transfer of DCs that had
been exposed to H. pylori ex vivo, but not of naive DCs, efficiently induced Treg
differentiation in vivo (Kao et al. 2010). Tregs induced in vivo upon Hp-DC transfer
in turn suppressed anti-H. pylori-specific Th17 responses and their depletion or the
depletion of IL-10 and/or TGF-β, promoted H. pylori clearance (Kao et al. 2010).
All available in vivo data thus suggest that H. pylori effectively directs DCs to
acquire tolerogenic properties, which drive Treg differentiation and anti-
inflammatory cytokine production, suppress T-effector cell functions, and promote
persistent infection (Fig. 12.4).
12.7 H. pylori Protects Against Allergic and Chronic

Inflammatory Diseases Through the Induction
of Treg-Mediated Immune Tolerance
Public health in developed countries has been dominated by two major trends since
the second half of the twentieth century. The incidence of infectious diseases has
declined sharply in that time frame, whereas immunological disorders such as
multiple sclerosis (MS), inflammatory bowel disease (IBD), allergic asthma and
other allergic diseases, and type I diabetes have dramatically increased in incidence
over the same time period (Bach 2002). The incidence of infections with H. pylori
has paralleled those of other infectious agents, with childhood acquisition rates
dropping in the USA from >50 to 10 % between the beginning and the end of the
twentieth century (Blaser and Falkow 2009). In line with the carcinogenic proper-
ties of H. pylori infection (Parsonnet et al. 1991), a beneficial effect of this trend has
been the steady decline in gastric cancer rates and associated mortality in countries
from which H. pylori has disappeared (Forman 2005). The downsides (some
confirmed, some debated) of the loss of H. pylori in developed countries are
(a) the increase in esophageal diseases such as esophagitis, Barrett’s esophagus,
and esophageal cancer with the latter having increased in incidence by over sixfold
in the years from 1975 to 2000 alone (Pohl and Welch 2005) and (b) the increase in
asthma and allergies (Eder et al. 2006). Numerous epidemiological studies have
shown an inverse association of H. pylori infection with asthma and other allergies
with respiratory tract manifestations, which was particularly strong in children and
adolescents and in individuals with early onset allergies and asthma (Amberbir
et al. 2011; Blaser et al. 2008; Chen and Blaser 2007, 2008; Reibman et al. 2008).
Meta-analyses examining 14 and 19 published studies on the topic have confirmed
the general trend (Wang et al. 2013; Zhou et al. 2013). The chronic inflammatory
skin disease atopic dermatitis/eczema has also been inversely linked to H. pylori
infection in studies including over 3000 German schoolchildren and almost 2000
Japanese university students (Herbarth et al. 2007; Shiotani et al. 2008). This trend
was recently confirmed in a longitudinal study conducted on over 1000 Ethiopian
children (Amberbir et al. 2014). Similarly, H. pylori infection seems to confer
protection against IBD, as suggested by a recent meta-analysis of 23 articles
examining such a possible link (Luther et al. 2011). Only 27 % of IBD patients
had evidence of infection with H. pylori compared to 41 % of patients in the control
group (Luther et al. 2011). A large epidemiological survey conducted on 136,000
patients in the USA found a decreased risk of celiac disease in individuals infected
with H. pylori (Lebwohl et al. 2013).
Following up on the various observational studies in human populations, possi-
ble protective effects of experimental H. pylori infection have been examined in
animal models of allergic asthma and IBD (Fig. 12.5). In a murine model of allergic
asthma induced by ovalbumin or house dust mite antigen sensitization and chal-
lenge, H. pylori infection conferred protection against the airway hyperrespon-
siveness, bronchoalveolar eosinophilia, lung inflammation, and goblet cell
metaplasia that are hallmarks of asthma in humans and mice (Arnold
et al. 2011a). The protective effects were evident in animals that had been exper-
imentally infected during the neonatal period (Arnold et al. 2011a), i.e., at an age
when humans typically contract the infection from their mothers (Weyermann
et al. 2009). Asthma protection conferred by H. pylori was abolished by antibiotic
eradication therapy prior to allergen challenge and depended critically on Tregs
(Fig. 12.5); the systemic depletion of Tregs abrogated asthma protection, and pure
populations of Tregs were sufficient to transfer protection from neonatally infected
donors to naive recipients (Arnold et al. 2011a). These results are in line with the
finding that neonatal infection with H. pylori induces Treg-mediated immune
tolerance to the bacteria (Arnold et al. 2011b) and that children predominantly
launch Treg responses to H. pylori infection (Harris et al. 2008). Experimental
models of colitis in mice also confirm the trends seen in human IBD patients
(Fig. 12.5); for example, H. pylori infection effectively reduced the Th17-driven
colitis induced by Salmonella typhimurium infection (Higgins et al. 2010). The
protective agent of H. pylori in colitis appears to be its DNA, which exhibits a
strongly biased ratio of immunoregulatory-to-immunostimulatory sequences and –
when administered orally – protects against the development of acute or chronic
DSS-induced colitis (Luther et al. 2011). The protective effects of H. pylori DNA
were attributed to its activity on DCs, which prevents IL-12 and type I IFN
production and generally favors anti- over pro-inflammatory responses (Luther
et al. 2011; Owyang et al. 2012). Overall, the combined results from observational
Fig. 12.5 H. pylori exerts systemic immunomodulatory effects. H. pylori exclusively inhabits the
gastric mucosa, but has systemic immunomodulatory effects that manifest in the airways and
lower gastrointestinal tract. Tissue-resident DCs can sample H. pylori antigens in the gastric
mucosa and subsequently migrate to the stomach-draining and mesenteric lymph nodes (MLNs),
where they prime T cell (in particular Treg) responses. Alternatively, soluble antigens can be
transported via the lymph to the MLNs for presentation by resident DC populations. MLN-derived,
H. pylori-specific Tregs enter the circulation and accumulate not only in the gastric mucosa but
also at other mucosal surfaces of the body, such as those of the airways and lower bowel.
According to current models, pathogenic effector T cell populations (allergen-specific Th17 and
Th2 cells and colitogenic Th1 and Th17 cells) are suppressed by H. pylori-induced Tregs via
soluble mediators (such as IL-10) and contact-dependent mechanisms. Abbreviations used: Th1/2/
17, T helper cell subsets; Treg, regulatory T cell
studies in humans and interventional studies in mice suggest strongly that H. pylori
infection can protect against experimentally induced allergic asthma and IBD; the
protective mechanisms appear to involve Tregs and DCs, which are actively
induced and “tolerized” by H. pylori, respectively. It is likely that the protection
against allergic and chronic inflammatory diseases that is conferred by H. pylori is a
by-product of its immunomodulatory activity, which allows the bacteria to suppress
Th1 and Th17 responses and to establish and maintain persistent infection. Similar
effects have been reported for other persistent pathogens including helminths and
Mycobacterium tuberculosis, which have been proposed to suppress allergen-

specific immune responses in asthma through the induction of Tregs or through
immune deviation (Obihara et al. 2007; Barlan et al. 2006). The potential use of
microbial products and live attenuated bacteria or parasites in the treatment of
allergies and IBD has received increasing attention lately, and some (especially
helminth-derived) compounds are in clinical development (Torres et al. 2013).
Whether H. pylori may be exploited in a similar fashion remains to be seen in the
future.
Despite having received substantial attention for three decades, several aspects of
the H. pylori/host interaction remain poorly understood. Among them is the manip-
ulation of innate and adaptive immunity by H. pylori, a feature that is central to the
persistence of the bacteria and to the chronicity of H. pylori-associated diseases.
Much attention has focused on gaining a better understanding of the prerequisites of
successful vaccination aiming to prevent the primary infection (see Chap. 24),
whereas less is known about the steady-state interaction of an established
H. pylori infection with the host immune system. The asymptomatic carrier state
in particular is vastly understudied, and the genetic and lifestyle parameters affect-
ing the risk of developing H. pylori-associated gastric disease remain largely
enigmatic. Although it is now clear that gastric infection-induced lesions are
immunopathological in nature and at least in animal models can be attributed to
the detrimental effects of T helper cells and their signature cytokines on the gastric
mucosa, it appears likely that H. pylori toxins contribute to the mucosal damage.
Experimental research into the pathomechanisms active during H. pylori infections
is limited by the failure of most strains to colonize small rodents persistently and to
the differences in gastric physiology and immunology that exist between humans
and all currently used animal models (maybe with the exception of rhesus
macaques). Future avenues of research in the field of H. pylori immunobiology
will likely explore the relative contribution of direct (bacterially mediated) and
indirect (immunopathological) effects to gastric carcinogenesis and will shed more
light on the differential disease risk within and across human populations. Exciting
new insights are expected as more elaborate model systems (such as gastric
organoids, humanized mice, etc.) become available to the broader research com-
munity. Finally, future research directions will certainly take into account that
H. pylori – beside its role as a gastric pathogen – is also an ancient member of
our gastric microbiota that, together with other constituents of the microbiota of the
gastrointestinal tract, has shaped the evolution of the human mucosal immune
system.
References
Aebischer T, Bumann D, Epple HJ, Metzger W, Schneider T, Cherepnev G, Walduck AK,

Kunkel D, Moos V, Loddenkemper C, Jiadze I, Panasyuk M, Stolte M, Graham DY,
Zeitz M, Meyer TF (2008) Correlation of T cell response and bacterial clearance in human
volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination
with Ty21a-based Salmonella vaccines. Gut 57:1065–1072
Akhiani AA, Pappo J, Kabok Z, Schon K, Gao W, Franzen LE, Lycke N (2002) Protection against
Akhiani AA, Schon K, Franzen LE, Pappo J, Lycke N (2004a) Helicobacter pylori-specific
antibodies impair the development of gastritis, facilitate bacterial colonization, and counteract
resistance against infection. J Immunol 172:5024–5033
Akhiani AA, Schon K, Lycke N (2004b) Vaccine-induced immunity against Helicobacter pylori
infection is impaired in IL-18-deficient mice. J Immunol 173:3348–3356
Amberbir A, Medhin G, Erku W, Alem A, Simms R, Robinson K, Fogarty A, Britton J, Venn A,
Davey G (2011) Effects of Helicobacter pylori, geohelminth infection and selected commensal
bacteria on the risk of allergic disease and sensitization in 3-year-old Ethiopian children. Clin
Exp Allergy 41:1422–1430
Amberbir A, Medhin G, Abegaz WE, Hanlon C, Robinson K, Fogarty A, Britton J, Venn A, Davey
G (2014) Exposure to Helicobacter pylori infection in early childhood and the risk of allergic
disease and atopic sensitization: a longitudinal birth cohort study. Clin Exp Allergy
44:563–571
Andersen-Nissen E, Smith KD, Strobe KL, Barrett SL, Cookson BT, Logan SM, Aderem A (2005)
Evasion of toll-like receptor 5 by flagellated bacteria. Proc Natl Acad Sci U S A
102:9247–9252
Arnold B, Schuler T, Hammerling GJ (2005) Control of peripheral T-lymphocyte tolerance in
neonates and adults. Trends Immunol 26:406–411
Arnold IC, Dehzad N, Reuter S, Martin H, Becher B, Taube C, Müller A (2011a) Helicobacter
pylori infection prevents allergic asthma in mouse models through the induction of regulatory
T cells. J Clin Invest 121:3088–3093
Arnold IC, Lee JY, Amieva MR, Roers A, Flavell RA, Sparwasser T, Müller A (2011b) Tolerance
Atherton JC, Sharp PM, Cover TL, Gonzalez-Valencia G, Peek RM Jr, Thompson SA, Hawkey
CJ, Blaser MJ (1999) Vacuolating cytotoxin (vacA) alleles of Helicobacter pylori comprise
two geographically widespread types, m1 and m2, and have evolved through limited recom-
bination. Curr Microbiol 39:211–218
Bach JF (2002) The effect of infections on susceptibility to autoimmune and allergic diseases. N
Engl J Med 347:911–920
Barlan I, Bahceciler NN, Akdis M, Akdis CA (2006) Bacillus Calmette-Guerin, mycobacterium
bovis, as an immunomodulator in atopic diseases. Immunol Allergy Clin North Am
26:365–377
Barrozo RM, Cooke CL, Hansen LM, Lam AM, Gaddy JA, Johnson EM, Cariaga TA, Suarez G,
Peek RM Jr, Cover TL, Solnick JV (2013) Functional plasticity in the type IV secretion system
of Helicobacter pylori. PLoS Pathog 9:e1003189
Bauer B, Pang E, Holland C, Kessler M, Bartfeld S, Meyer TF (2012a) The Helicobacter pylori
virulence effector CagA abrogates human beta-defensin 3 expression via inactivation of EGFR
signaling. Cell Host Microbe 11:576–586
Bauer B, Wex T, Kuester D, Meyer T, Malfertheiner P (2012b) Differential expression of human
beta defensin 2 and 3 in gastric mucosa of Helicobacter pylori-infected individuals.
Becher D, Deutscher ME, Simpfendorfer KR, Wijburg OL, Pederson JS, Lew AM, Strugnell RA,
Walduck AK (2010) Local recall responses in the stomach involving reduced regulation and
expanded help mediate vaccine-induced protection against Helicobacter pylori in mice. Eur J
Immunol 40:2778–2790
Blaser MJ, Falkow S (2009) What are the consequences of the disappearing human microbiota?
Nat Rev Microbiol 7:887–894
Blaser MJ, Perez-Perez GI, Kleanthous H, Cover TL, Peek RM, Chyou PH, Stemmermann GN,
Nomura A (1995) Infection with Helicobacter pylori strains possessing cagA is associated with
an increased risk of developing adenocarcinoma of the stomach. Cancer Res 55:2111–2115
Blaser MJ, Chen Y, Reibman J (2008) Does Helicobacter pylori protect against asthma and
allergy? Gut 57:561–567
Bollrath J, Powrie FM (2013) Controlling the frontier: regulatory T-cells and intestinal homeo-
stasis. Semin Immunol 25:352–357
Boncristiano M, Paccani SR, Barone S, Ulivieri C, Patrussi L, Ilver D, Amedei A, D’Elios MM,
Telford JL, Baldari CT (2003) The Helicobacter pylori vacuolating toxin inhibits T cell
activation by two independent mechanisms. J Exp Med 198:1887–1897
Chen Y, Blaser MJ (2007) Inverse associations of Helicobacter pylori with asthma and allergy.
Arch Intern Med 167:821–827
Chen Y, Blaser MJ (2008) Helicobacter pylori colonization is inversely associated with childhood
asthma. J Infect Dis 198:553–560
gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice. Mol
Coccia M, Harrison OJ, Schiering C, Asquith MJ, Becher B, Powrie F, Maloy KJ (2012) IL-1beta
mediates chronic intestinal inflammation by promoting the accumulation of IL-17A secreting
innate lymphoid cells and CD4(+) Th17 cells. J Exp Med 209:1595–1609
Cua DJ, Sherlock J, Chen Y, Murphy CA, Joyce B, Seymour B, Lucian L, To W, Kwan S,
Churakova T, Zurawski S, Wiekowski M, Lira SA, Gorman D, Kastelein RA, Sedgwick JD
(2003) Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune
inflammation of the brain. Nature 421:744–748
Cullen TW, Giles DK, Wolf LN, Ecobichon C, Boneca IG, Trent MS (2012) Helicobacter pylori
versus the host: remodeling of the bacterial outer membrane is required for survival in the
gastric mucosa. PLoS Pathog 7:e1002454
Czajkowsky DM, Iwamoto H, Cover TL, Shao Z (1999) The vacuolating toxin from Helicobacter
pylori forms hexameric pores in lipid bilayers at low pH. Proc Natl Acad Sci U S A
96:2001–2006
Domanska G, Motz C, Meinecke M, Harsman A, Papatheodorou P, Reljic B, Dian-Lothrop EA,
Galmiche A, Kepp O, Becker L, Gunnewig K, Wagner R, Rassow J (2010) Helicobacter pylori
VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.
Eder W, Ege MJ, von Mutius E (2006) The asthma epidemic. N Engl J Med 355:2226–2235
El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, Herrera J,
Lissowska J, Yuan CC, Rothman N, Lanyon G, Martin M, Fraumeni JF Jr, Rabkin CS
(2000) Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature
404:398–402
Ermak TH, Giannasca PJ, Nichols R, Myers GA, Nedrud J, Weltzin R, Lee CK, Kleanthous H,
Monath TP (1998) Immunization of mice with urease vaccine affords protection against
Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class
II-restricted responses. J Exp Med 188:2277–2288
Forman D (2005) Re: the role of overdiagnosis and reclassification in the marked increase of
esophageal adenocarcinoma incidence. J Natl Cancer Inst 97:1013–1014
Gerhard M, Schmees C, Voland P, Endres N, Sander M, Reindl W, Rad R, Oelsner M, Decker T,
Mempel M, Hengst L, Prinz C (2005) A secreted low-molecular-weight protein from
Helicobacter pylori induces cell-cycle arrest of T cells. Gastroenterology 128:1327–1339
Gewirtz AT, Yu Y, Krishna US, Israel DA, Lyons SL, Peek RM Jr (2004) Helicobacter pylori
flagellin evades toll-like receptor 5-mediated innate immunity. J Infect Dis 189:1914–1920
Gorrell RJ, Guan J, Xin Y, Tafreshi MA, Hutton ML, McGuckin MA, Ferrero RL, Kwok T (2012)
A novel NOD1- and CagA-independent pathway of interleukin-8 induction mediated by the
Helicobacter pylori type IV secretion system. Cell Microbiol 15:554–570
Gringhuis SI, den Dunnen J, Litjens M, van der Vlist M, Geijtenbeek TB (2009) Carbohydrate-
specific signaling through the DC-SIGN signalosome tailors immunity to mycobacterium
tuberculosis, HIV-1 and Helicobacter pylori. Nat Immunol 10:1081–1088
Harris PR, Cover TL, Crowe DR, Orenstein JM, Graham MF, Blaser MJ, Smith PD (1996)
Helicobacter pylori cytotoxin induces vacuolation of primary human mucosal epithelial
cells. Infect Immun 64:4867–4871
Harris PR, Wright SW, Serrano C, Riera F, Duarte I, Torres J, Pena A, Rollan A, Viviani P,
Guiraldes E, Schmitz JM, Lorenz RG, Novak L, Smythies LE, Smith PD (2008) Helicobacter
pylori gastritis in children is associated with a regulatory T-cell response. Gastroenterology
134:491–499
Harrison OJ, Powrie FM (2013) Regulatory T cells and immune tolerance in the intestine. Cold
Spring Harb Perspect Biol 5:a018341
Herbarth O, Bauer M, Fritz GJ, Herbarth P, Rolle-Kampczyk U, Krumbiegel P, Richter M, Richter
T (2007) Helicobacter pylori colonisation and eczema. J Epidemiol Community Health
61:638–640
Higgins PD, Johnson LA, Luther J, Zhang M, Sauder KL, Blanco LP, Kao JY (2010) Prior
Helicobacter pylori infection ameliorates Salmonella typhimurium-induced colitis: mucosal
crosstalk between stomach and distal intestine. Inflamm Bowel Dis 17:1398–1408
Hitzler I, Oertli M, Becher B, Agger EM, Müller A (2011) Dendritic cells prevent rather than
promote immunity conferred by a helicobacter vaccine using a mycobacterial adjuvant.
Hitzler I, Kohler E, Engler DB, Yazgan AS, Muller A (2012a) The role of Th cell subsets in the
Immunol 3:142
Hitzler I, Sayi A, Kohler E, Engler DB, Koch KN, Hardt WD, Muller A (2012b) Caspase-1 has
both proinflammatory and regulatory properties in helicobacter infections, which are differen-
tially mediated by its substrates IL-1beta and IL-18. J Immunol 188:3594–3602
Horvath DJ Jr, Washington MK, Cope VA, Algood HMS (2012) IL-23 contributes to control of
chronic Helicobacter pylori infection and the development of T helper responses in a mouse
model. Front Immun 3:56
Huang JQ, Zheng GF, Sumanac K, Irvine EJ, Hunt RH (2003) Meta-analysis of the relationship
between cagA seropositivity and gastric cancer. Gastroenterology 125:1636–1644
Ismail HF, Fick P, Zhang J, Lynch RG, Berg DJ (2003) Depletion of neutrophils in IL-10(/)
mice delays clearance of gastric Helicobacter infection and decreases the Th1 immune
response to Helicobacter. J Immunol 170:3782–3789
Jimenez-Soto LF, Kutter S, Sewald X, Ertl C, Weiss E, Kapp U, Rohde M, Pirch T, Jung K, Retta
Kaebisch R, Mejias-Luque R, Prinz C, Gerhard M (2013) Helicobacter pylori cytotoxin-associated
gene A impairs human dendritic cell maturation and function through IL-10-mediated activa-
tion of STAT3. J Immunol 192:316–323
Kao JY, Zhang M, Miller MJ, Mills JC, Wang B, Liu M, Eaton KA, Zou W, Berndt BE, Cole TS,
Takeuchi T, Owyang SY, Luther J (2010) Helicobacter pylori immune escape is mediated by
dendritic cell-induced Treg skewing and Th17 suppression in mice. Gastroenterology
138:1046–1054
Kim JM, Kim JS, Yoo DY, Ko SH, Kim N, Kim H, Kim YJ (2011) Stimulation of dendritic cells
with Helicobacter pylori vacuolating cytotoxin negatively regulates their maturation via the
restoration of E2F1. Clin Exp Immunol 166:34–45
Kim DJ, Park JH, Franchi L, Backert S, Nunez G (2013) The Cag pathogenicity island and
interaction between TLR2/NOD2 and NLRP3 regulate IL-1beta production in Helicobacter
pylori-infected dendritic cells. Eur J Immunol 43:2650–2658
Lebwohl B, Blaser MJ, Ludvigsson JF, Green PH, Rundle A, Sonnenberg A, Genta RM (2013)
Decreased risk of celiac disease in patients with Helicobacter pylori colonization. Am J
Epidemiol 178:1721–1730
Lundgren A, Suri-Payer E, Enarsson K, Svennerholm AM, Lundin BS (2003) Helicobacter pylori-
specific CD4+ CD25 high regulatory T cells suppress memory T-cell responses to H. pylori in
infected individuals. Infect Immun 71:1755–1762
Lundgren A, Stromberg E, Sjoling A, Lindholm C, Enarsson K, Edebo A, Johnsson E, Suri-
Payer E, Larsson P, Rudin A, Svennerholm AM, Lundin BS (2005a) Mucosal FOXP3-
expressing CD4+ CD25 high regulatory T cells in Helicobacter pylori-infected patients. Infect
Immun 73:523–531
Lundgren A, Trollmo C, Edebo A, Svennerholm AM, Lundin BS (2005b) Helicobacter pylori-
specific CD4+ T cells home to and accumulate in the human Helicobacter pylori-infected
gastric mucosa. Infect Immun 73:5612–5619
Luther J, Owyang SY, Takeuchi T, Cole TS, Zhang M, Liu M, Erb-Downward J, Rubenstein JH,
Chen CC, Pierzchala AV, Paul JA, Kao JY (2011) Helicobacter pylori DNA decreases
pro-inflammatory cytokine production by dendritic cells and attenuates dextran sodium
sulphate-induced colitis. Gut 60:1479–1486
Maizels RM, Smith KA (2011) Regulatory T cells in infection. Adv Immunol 112:73–136
Maldonado RA, von Andrian UH (2010) How tolerogenic dendritic cells induce regulatory T cells.
Adv Immunol 108:111–165
Malfertheiner P, Schultze V, Rosenkranz B, Kaufmann SH, Ulrichs T, Novicki D, Norelli F,
Contorni M, Peppoloni S, Berti D, Tornese D, Ganju J, Palla E, Rappuoli R, Scharschmidt BF,
Del Giudice G (2008) Safety and immunogenicity of an intramuscular Helicobacter pylori
vaccine in noninfected volunteers: a phase I study. Gastroenterology 135:787–795
McBride A, Konowich J, Salgame P (2013) Host defense and recruitment of Foxp3(+) T regula-
tory cells to the lungs in chronic Mycobacterium tuberculosis infection requires toll-like
receptor 2. PLoS Pathog 9:e1003397
McClain MS, Cao P, Cover TL (2001) Amino-terminal hydrophobic region of Helicobacter pylori
vacuolating cytotoxin (VacA) mediates transmembrane protein dimerization. Infect Immun
69:1181–1184
Moran AP, Lindner B, Walsh EJ (1997) Structural characterization of the lipid A component of
Helicobacter pylori rough- and smooth-form lipopolysaccharides. J Bacteriol 179:6453–6463
Obihara CC, Kimpen JL, Beyers N (2007) The potential of Mycobacterium to protect against
allergy and asthma. Curr Allergy Asthma Rep 7:223–230
Oertli M, Sundquist M, Hitzler I, Engler DB, Arnold IC, Reuter S, Maxeiner J, Hansson M,
Taube C, Quiding-Jarbrink M, Muller A (2012) DC-derived IL-18 drives Treg differentiation,
murine Helicobacter pylori-specific immune tolerance, and asthma protection. J Clin Invest
122:1082–1096
mote gastric persistence and immune tolerance. Proc Natl Acad Sci U S A 110:3047–3052
Otani K, Watanabe T, Tanigawa T, Okazaki H, Yamagami H, Watanabe K, Tominaga K,
Fujiwara Y, Oshitani N, Arakawa T (2009) Anti-inflammatory effects of IL-17A on
Helicobacter pylori-induced gastritis. Biochem Biophys Res Commun 382:252–258
Otani K, Tanigawa T, Watanabe T, Nadatani Y, Sogawa M, Yamagami H, Shiba M, Watanabe K,
Tominaga K, Fujiwara Y, Arakawa T (2012) Toll-like receptor 9 signaling has anti-
inflammatory effects on the early phase of Helicobacter pylori-induced gastritis. Biochem
Biophys Res Commun 426:342–349
Ottenhoff TH (2012) New pathways of protective and pathological host defense to mycobacteria.
Trends Microbiol 20:419–428
Owyang SY, Luther J, Owyang CC, Zhang M, Kao JY (2012) Helicobacter pylori DNA’s anti-
inflammatory effect on experimental colitis. Gut Microbes 3:168–171
Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, Sibley RK
(1991) Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med
325:1127–1131
Patel SR, Smith K, Letley DP, Cook KW, Memon AA, Ingram RJ, Staples E, Backert S, Zaitoun
AM, Atherton JC, Robinson K (2013) Helicobacter pylori down regulates expression of human
beta-defensin 1 in the gastric mucosa in a type IV secretion-dependent fashion. Cell Microbiol
15:2080–2092
Pohl H, Welch HG (2005) The role of over diagnosis and reclassification in the marked increase of
esophageal adenocarcinoma incidence. J Natl Cancer Inst 97:142–146
Rad R, Ballhorn W, Voland P, Eisenacher K, Mages J, Rad L, Ferstl R, Lang R, Wagner H, Schmid
RM, Bauer S, Prinz C, Kirschning CJ, Krug A (2009) Extracellular and intracellular pattern
recognition receptors cooperate in the recognition of Helicobacter pylori. Gastroenterology
136:2247–2257
Reibman J, Marmor M, Filner J, Fernandez-Beros ME, Rogers L, Perez-Perez GI, Blaser MJ
(2008) Asthma is inversely associated with Helicobacter pylori status in an urban population.
PLoS One 3:e4060
128:1229–1242
Robinson K, Kenefeck R, Pidgeon EL, Shakib S, Patel S, Polson RJ, Zaitoun AM, Atherton JC
(2008) Helicobacter pylori-induced peptic ulcer disease is associated with inadequate regula-
tory T cell responses. Gut 57:1375–1385
Rokkas T, Pistiolas D, Sechopoulos P, Robotis I, Margantinis G (2007) The long-term impact of
Helicobacter pylori eradication on gastric histology: a systematic review and meta-analysis.
Helicobacter 12(Suppl 2):32–38
Rothenbacher D, Inceoglu J, Bode G, Brenner H (2000) Acquisition of Helicobacter pylori
infection in a high-risk population occurs within the first 2 years of life. J Pediatr 136:744–748
Salama NR, Otto G, Tompkins L, Falkow S (2001) Vacuolating cytotoxin of Helicobacter pylori
plays a role during colonization in a mouse model of infection. Infect Immun 69:730–736
Salama NR, Hartung ML, Muller A (2013) Life in the human stomach: persistence strategies of the
bacterial pathogen Helicobacter pylori. Nat Rev Microbiol 11:385–399
Sayi A, Kohler E, Toller IM, Flavell RA, Muller W, Roers A, Müller A (2011) TLR-2-activated B
Gerhard M (2007) Inhibition of T-cell proliferation by Helicobacter pylori gamma-glutamyl
transpeptidase. Gastroenterology 132:1820–1833
Sewald X, Gebert-Vogl B, Prassl S, Barwig I, Weiss E, Fabbri M, Osicka R, Schiemann M, Busch
DH, Semmrich M, Holzmann B, Sebo P, Haas R (2008) Integrin subunit CD18 Is the
T-lymphocyte receptor for the Helicobacter pylori vacuolating cytotoxin. Cell Host Microbe
3:20–29
Sewald X, Jimenez-Soto L, Haas R (2010) PKC-dependent endocytosis of the Helicobacter pylori
vacuolating cytotoxin in primary T lymphocytes. Cell Microbiol 13:482–496
components for pilus assembly at the bacteria-host cell interface. PLoS Pathog 7:e1002237
Shi Y, Liu XF, Zhuang Y, Zhang JY, Liu T, Yin Z, Wu C, Mao XH, Jia KR, Wang FJ, Guo H,
Flavell RA, Zhao Z, Liu KY, Xiao B, Guo Y, Zhang WJ, Zhou WY, Guo G, Zou QM (2010)
Helicobacter pylori-induced Th17 responses modulate Th1 cell responses, benefit bacterial
growth, and contribute to pathology in mice. J Immunol 184:5121–5129
Shiotani A, Miyanishi T, Kamada T, Haruma K (2008) Helicobacter pylori infection and allergic
diseases: epidemiological study in Japanese university students. J Gastroenterol Hepatol 23:
e29–e33
Smoot DT, Resau JH, Earlington MH, Simpson M, Cover TL (1996) Effects of Helicobacter
pylori vacuolating cytotoxin on primary cultures of human gastric epithelial cells. Gut
39:795–799
Sun X, Zhang M, El-Zataari M, Owyang SY, Eaton KA, Liu M, Chang YM, Zou W, Kao JY
(2013) TLR2 mediates Helicobacter pylori-induced tolerogenic immune response in mice.
PLoS One 8:e74595
Torres VJ, VanCompernolle SE, Sundrud MS, Unutmaz D, Cover TL (2007) Helicobacter pylori
Torres J, Danese S, Colombel JF (2013) New therapeutic avenues in ulcerative colitis: thinking out
of the box. Gut 62:1642–1652
Velin D, Michetti P (2010) Advances in vaccination against Helicobacter pylori. Expert Rev
Velin D, Bachmann D, Bouzourene H, Michetti P (2005) Mast cells are critical mediators of
vaccine-induced Helicobacter clearance in the mouse model. Gastroenterology 129:142–155
Velin D, Favre L, Bernasconi E, Bachmann D, Pythoud C, Saiji E, Bouzourene H, Michetti P
(2009) Interleukin-17 is a critical mediator of vaccine-induced reduction of Helicobacter
infection in the mouse model. Gastroenterology 136:2237–2246 e2231
Wang Q, Yu C, Sun Y (2013) The association between asthma and Helicobacter pylori: a meta-
analysis. Helicobacter 18:41–53
Wong BC, Lam SK, Wong WM, Chen JS, Zheng TT, Feng RE, Lai KC, Hu WH, Yuen ST, Leung
SY, Fong DY, Ho J, Ching CK (2004) Helicobacter pylori eradication to prevent gastric cancer
in a high-risk region of China: a randomized controlled trial. JAMA 291:187–194
Yogev N, Frommer F, Lukas D, Kautz-Neu K, Karram K, Ielo D, von Stebut E, Probst HC, van den
Broek M, Riethmacher D, Birnberg T, Blank T, Reizis B, Korn T, Wiendl H, Jung S, Prinz M,
Kurschus FC, Waisman A (2012) Dendritic cells ameliorate autoimmunity in the CNS by
controlling the homeostasis of PD-1 receptor(+) regulatory T cells. Immunity 37:264–275
Zaki MH, Boyd KL, Vogel P, Kastan MB, Lamkanfi M, Kanneganti TD (2010) The NLRP3
inflammasome protects against loss of epithelial integrity and mortality during experimental
colitis. Immunity 32:379–391
Zhou X, Wu J, Zhang G (2013) Association between Helicobacter pylori and asthma: a meta-
analysis. Eur J Gastroenterol Hepatol 25:460–468
Chapter 13
The Role of Inflammatory Responses
in Mouse Gastric Tumorigenesis
Hiroko Oshima, Mizuho Nakayama, and Masanobu Oshima
Abstract It has been established that chronic inflammation plays an important role
in cancer development. The expression of cyclooxygenase-2 (COX-2), a rate-
limiting enzyme for prostaglandin biosynthesis, is induced in most cancer tissues
and plays a key role in tumorigenesis. Helicobacter pylori infection causes atrophic
gastritis, which is associated with the induction of COX-2 expression and its
downstream product, prostaglandin E2 (PGE2), biosynthesis. Transgenic mice
expressing COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) in the
gastric mucosa show the generation of an inflammatory microenvironment via the
activation of the COX-2/PGE2 pathway. Notably, simultaneous activation of
canonical Wnt signaling and the COX-2/PGE2 pathway causes intestinal-type
gastric tumor development, although Wnt activation alone is not sufficient for
tumor formation. These results suggest that H. pylori infection-associated chronic
inflammation contributes to gastric tumorigenesis through activation of the COX-2/
PGE2 pathway. Using a gastric tumor mouse model (Gan mice), we found that the
inflammatory microenvironment induces the activation of epidermal growth factor
receptor (EGFR) signaling and promotes canonical Wnt signaling. Moreover,
infiltrated macrophages express tumor necrosis factor-α (TNF-α) in gastric tumors,
which plays an important role in tumor promotion through the induction of NADPH
oxidase organizer 1 (NOXO1) expression. NOXO1 contributes to the production of
reactive oxygen species (ROS) by the NOX1 complex, which is thought to be
important for the maintenance of stem cell properties. These studies indicate that
chronic inflammation promotes gastric tumorigenesis through a variety of mecha-
nisms. Accordingly, targeting an inflammatory microenvironment should be an
effective therapeutic or preventive strategy for gastric cancer.
Keywords Gastric cancer • Inflammation • COX-2 • PGE2 • EGFR • TNF-α • Wnt

signaling
H. Oshima (*) • M. Nakayama • M. Oshima

Division of Genetics, Cancer Research Institute, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan

DOI 10.1007/978-4-431-55936-8_13
326 H. Oshima et al.
13.1 Introduction
It has been reported that the regular use of nonsteroidal anti-inflammatory drugs
(NSAIDs) is associated with a reduced risk of developing gastrointestinal cancer
(Thun et al. 1991). The target molecules of NSAIDs are cyclooxygenase (COX)-1
and COX-2, the rate-limiting enzymes for prostaglandin biosynthesis. COX-2
expression is induced in inflammatory lesions and cancer tissues, whereas COX-1
is constitutively expressed in variety of organs. COX-2-derived PGE2 plays a key
role in both inflammation and cancer development, while COX-1-dependent PGE2
plays a housekeeping role such as protection of gastrointestinal mucosa. We
previously demonstrated that COX-2 expression is induced predominantly in stro-
mal cells including fibroblasts of human and mouse benign intestinal polyps
(Oshima et al. 1996; Sonoshita et al. 2002). Moreover, disruption of the genes
encoding COX-2 or the one of four PGE2 receptors (called EP2) in Adenomatous
polyposis coli (Apc) gene knockout mice (ApcΔ716 mice) resulted in significant
suppression of intestinal polyposis (Oshima et al. 1996; Sonoshita et al. 2001).
Number of intestinal polyps in the COX-2 gene-disrupted ApcΔ716 mice decreased
to about 20 % of the COX-2 wild-type ApcΔ716 mice. These results indicate that
COX-2/PGE2 signaling through the EP2 receptor plays a key role in intestinal
tumor development. In the intestinal tumors of ApcΔ716 mice, PGE2 signaling
contributes to angiogenesis by inducing the expression of angiogenic factors
including vascular endothelial growth factor (VEGF) and basic fibroblast growth
factor (bFGF) (Seno et al. 2002). It has also been reported that PGE2 signaling
activates peroxisome proliferator-activated receptor δ (PPARδ) in the intestinal
tumors of another Apc gene-mutant mice (ApcMin mice), resulting in suppression of
the apoptosis of tumor cells (Wang et al. 2004). They also demonstrated that
treatment of ApcMin mice with PGE2 caused significant promotion of intestinal
tumor development. Moreover, it has been shown that PGE2 signaling induces
DNA methylation in intestinal tumor epithelial cells, which can contribute to
tumorigenesis by silencing expression of tumor suppressor genes (Xia
et al. 2012). Recently, it has been demonstrated that PGE2 induces expression of
chemokine ligands with CXC motifs, CXCL1 and CXCL2, which further recruit
myeloid-derived suppressor cells (MDSCs) to intestinal tumors. MDSCs then
suppress antitumor immunity, resulting in the promotion of inflammation-
associated tumorigenesis in colon (Katoh et al. 2013). Taken together, these
findings indicate that the COX-2/PGE2 pathway promotes intestinal tumorigenesis
through a variety of mechanisms.
In contrast to colon cancer, the role of the COX-2/PGE2 pathway in gastric
tumorigenesis is still not fully understood, although COX-2 expression is widely
found in gastric cancer tissues (Saukkonen et al. 2001). Since Helicobacter pylori
infection induces COX-2 expression and PGE2 production, it is possible that the
COX-2/PGE2 pathway is one of the important mechanisms underlying H. pylori
infection-induced gastric tumorigenesis. To investigate the mechanism(s) by which
the COX-2/PGE2 pathway is involved in gastric tumorigenesis, we constructed a
13 The Role of Inflammatory Responses in Mouse Gastric Tumorigenesis 327
gastric tumor mouse model, Gan (Gastric neoplasia) mice. Gan mice develop
intestinal-type gastric tumors in the glandular stomach by the simultaneous activa-
tion of canonical Wnt signaling and the COX-2/PGE2 pathway in gastric mucosa
(Oshima et al. 2006, 2009; Oshima and Oshima 2010). Although genetic alterations
that activate Wnt signaling are not common in human gastric cancer cells (The
Cancer Genome Atlas Research Network 2014), activation of the Wnt signaling is
found in more than 50 % of cases suggesting a role of Wnt signaling in gastric
tumorigenesis (Oshima et al. 2006). Wnt signaling plays an important role in
maintenance of epithelial stem cells in undifferentiated status by increasing
“stemness.” In this chapter, we will review the role of COX-2/PGE2-associated
inflammation in gastric tumorigenesis, which was uncovered using the Gan mouse
model.
13.2 The Development of a Gastric Tumor Model:

Gan Mice
The eradication of H. pylori results in a significant reduction of COX-2 expression

and improvement of gastric atrophy, indicating that H. pylori infection induces the
COX-2/PGE2 pathway in the gastric mucosa (Wambura et al. 2004). On the other
hand, β-catenin accumulation was detected in about 50 % of gastric cancers
(Oshima et al. 2006). In the Wnt signaling-activated epithelial cells, β-catenin is
stabilized in cytoplasm and translocated to nuclei-forming complex with T-cell
factor (TCF), which leads to induction of Wnt-target gene expression. Based on this
information, we constructed compound transgenic mice, K19-Wnt1/C2mE mice
(Gan mice), that express Wnt1, COX-2, and mPGES-1 simultaneously in gastric
epithelial cells using the cytokeratin 19 gene (Krt19) promoter (Oshima et al. 2006,
2009; Oshima and Oshima 2010). K19 promoter is transcriptionally active in
gastric epithelial cells including undifferentiated epithelia (Brembeck et al. 2001;
Oshima et al. 2004). Wnt1 is a ligand for the Frizzled receptor that activates
canonical Wnt signaling, resulting in acquisition of stem cell property. An inducible
PGE-converting enzyme, mPGES-1, is expressed both in inflamed and cancer
tissues together with COX-2, resulting in increased levels of PGE2. Accordingly,
Wnt signaling and the COX-2/PGE2 pathways are activated simultaneously in the
Gan mouse gastric mucosa (Oshima et al. 2006, 2009; Oshima and Oshima 2010).
The activation of Wnt signaling alone in K19-Wnt1 mice that express only Wnt1 in
gastric epithelial cells causes the development of small preneoplastic lesions
consisting of dysplastic epithelial cells in the gastric mucosa, suggesting a role of
Wnt signaling activation in initiation of gastric tumorigenesis (Oshima et al. 2006)
(Fig. 13.1). On the other hand, the induction of the COX-2/PGE2 pathway in the
stomach in K19-C2mE mice results in the infiltration of inflammatory cells includ-
ing macrophages into the gastric mucosa and the development of mucous cell
metaplasia (Oshima et al. 2004). Consistently, induction of COX-2/PGE2 signaling
Fig. 13.1 Transgenic mouse models of gastric tumorigenesis. The transgenic vectors and repre-
sentative macroscopic and microscopic photographs of the stomach are shown for each transgenic
line. K19-Wnt1/C2mE (Gan) mice are compound transgenic mice bred from K19-Wnt1 to
K19-C2mE mice that express Wnt1 and COX-2/mPGES-1, respectively. The arrowhead and
inset in the K19-Wnt1 figure show a preneoplastic lesion initiated by Wnt signaling activation.
The arrowheads in the K19-C2mE figure show PGE2-induced inflammatory infiltration to submu-
cosal. The arrows in K19-Wnt1/C2mE indicate gastric tumors. The bars indicate 100 μm
(Reproduced from Oshima et al. Cancer Sci, 2009 with permission from Wiley-Blackwell)
in intestinal mucosa causes activation of infiltrated macrophages, which contributes

to generation of inflammatory microenvironment (Wang et al. 2014). Because
COX-2/PGE2 pathway is activated in most of gastrointestinal cancer tissues, it is
possible that COX-2/PGE2 signaling is responsible for elicitation of inflammatory
responses in these tumors. Importantly, simultaneous activation of Wnt signaling
and the COX-2/PGE2 pathway in the Gan mice causes intestinal-type gastric tumor
development with 100 % incidence (Oshima et al. 2006) (Fig. 13.1). These results
indicate that the cooperation of Wnt signaling activation and COX-2/PGE2
pathway-induced inflammatory responses causes gastric tumor development.
Microarray analyses indicated that the gene expression profiles of Gan mouse
tumors are similar to those of human intestinal-type gastric cancer (Itadani
et al. 2009). Accordingly, Gan mice recapitulate human intestinal-type gastric
cancer from the molecular mechanism to the characteristics of tumor histopathol-
ogy and gene expression profiles.
13.3 Tumor-Associated Macrophages in Gan Mouse

Gastric Tumors
Macrophages in tumor tissues are called as “tumor-associated macrophages” or

TAMs, and accumulating evidence has indicated that TAMs play an important role
in cancer development and malignant progression (Pollard 2009; Qian and Pollard
2010). In the Gan mouse gastric tumors, macrophage-trophic CC motif
chemokines, CCL2 and CCL8, are induced, and macrophages are accumulated in
gastric tumor stroma. These macrophages are activated and express inflammatory
cytokines including TNF-α and the interleukins IL-1β and IL-6 (Oshima
et al. 2011a). Expression of CCL2 and CCL8 and macrophage infiltration into the
gastric mucosa are also found in the gastritis tissues of COX-2/mPGES-1 transgenic
(K19-C2mE) mice (Oshima et al. 2004). Accordingly, it is possible that PGE2-
signaling plays a key role in TAM recruitment in gastric mucosa. Interestingly,
when Gan mice were maintained under germfree conditions, the inflammatory
responses and macrophage infiltration were significantly suppressed, although
COX-2/PGE2 pathway is continuously activated (Oshima et al. 2011a). Moreover,
gastric tumorigenesis was also suppressed in germfree Gan mice. These results
indicate that activation of COX-2/PGE2 pathway is not sufficient for generation of
inflammatory microenvironment, but both COX-2/PGE2 pathway and innate
immune response to bacterial infection are required. Moreover, such microenvi-
ronment is important for promotion of tumor development.
Bacterial infection is recognized by toll-like receptors (TLRs) that activate
innate immune responses. It has been shown that disruption of the Tlr2, Tlr4, or
Myd88 gene encoding the toll-like receptors TLR2 and TLR4 and the adapter
protein Myd88, respectively, results in significant suppression of the repair of
the injured intestinal mucosa, indicating that the innate immune response through
TLR signaling plays an important role in intestinal mucosal homeostasis

(Rakoff-Nahoum et al. 2004). Furthermore, disruption of Myd88 gene in ApcMin
mice as well as chemically induced hepatocellular carcinoma (HCC) mouse model
suppressed tumor development in these models, along with suppression of COX-2
and cytokine expression (Rakoff-Nahoum and Medzhitov 2007; Naugler
et al. 2007). Recently, several reports have shown a role for bacterial infection in
intestinal tumorigenesis. For example, Fusobacterium infection promotes ApcMin
mouse intestinal tumorigenesis by inducing inflammatory responses, and IL10/
mice showed more severe intestinal inflammation and a tumor phenotype when
treated with the chemical mutagen, azoxymethane (Allen-Veroce and Jobin 2014).
Accordingly, it is possible that the innate immune responses to bacterial infection
mediated by TLR/Myd88 signaling are also important for gastric mucosal homeo-
stasis and tumorigenesis. Epidemiological studies also showed that polymorphisms
in TLR4 gene were associated with increased risk of gastric cancer development,
suggesting a role of innate responses in gastric tumorigenesis (Hold et al. 2007;
El-Omar et al. 2008).
In the Gan mouse tumor tissues, macrophages are activated by innate immune
responses, and these express cytokines, chemokines, and growth factors, which
contribute to tumor cell proliferation. Macrophage depletion in Gan mouse tumors
by treatment with clodronate liposomes resulted in the induction of gastric tumor
cell apoptosis (Oshima et al. 2011a). The depletion of macrophages in ApcΔ716 mice
by crossing them with colony stimulating factor 1 gene (Csf1) mutant mice (op/op
mice: a mutant strain that shows decreased mature macrophages in peripheral
tissues because of Csf1 gene mutation) also resulted in significant suppression of
intestinal tumorigenesis (Oguma et al. 2008). Accordingly, it is possible that the
innate immune response triggers macrophage infiltration into the gastric mucosa,
which promotes tumor development.
Importantly, it has been established the role of innate immunity also in epithelial
cells in tumorigenesis. Expression of TLR2 in gastric tumors of Gan mice and
another gastric tumor model, gp130F/F mice, was increased significantly. Gp130 is a
co-receptor of IL-6 and IL-11, and gp130F/F mutation causes constitutive activation
of Stat3 signaling. Importantly, disruption of Tlr2 in gp130F/F mice resulted in
significant suppression of gastric tumorigenesis, indicating a role of TLR2 signaling
in tumor formation (Tye et al. 2012). More recently, it has been demonstrated that
TLR2-MyD88 signaling in the intestinal stem cells is required for maintenance of
stemness (Scheeren et al. 2014). Accordingly, it is possible that innate immune
responses in macrophages promote tumorigenesis through generation of inflamma-
tory microenvironment, whereas those in epithelial cells also accelerate tumor
development by acquirement of stemness.
13.4 The Mechanisms by Which Inflammation Promotes

Tumor Formation
13.4.1 EGFR Signaling Activation
cDNA microarray analyses of Gan mice and other transgenic mouse lines indicated
that the expression of ligands for the epidermal growth factor receptor (EGFR),
such as amphiregulin, epiregulin, and heparin-binding EGF-like growth factor
(HB-EGF), is significantly upregulated in both Gan mouse gastric tumors and in
K19-C2mE mouse gastritis tissues, indicating that these EGFR ligands are induced
via a COX-2/PGE2-associated inflammation-dependent mechanism (Oshima
et al. 2011b). Importantly, members of the ADAM (a disintegrin and
metalloproteinase) family proteases (ADAM8, ADAM9, and ADAM10) are also
upregulated in both Gan mouse tumors and K19-C2mE mouse gastritis. These
ADAM family members are important for tumorigenesis through the activation of
EGFR signaling by shedding from membrane-bound forms of precursor EGFR
ligands (Mochizuki and Okada 2007). Accordingly, one of the mechanisms by
which PGE2-associated inflammation promotes tumor formation is the activation of
EGFR signaling through the simultaneous induction of EGFR ligands and ADAM
proteases, which leads to acceleration of tumor cell proliferation (Fig. 13.2). Con-
sistently, treatment of Gan mice with an EGFR inhibitor significantly suppressed
gastric tumorigenesis, and combination treatment with an EGFR inhibitor and a
COX-2 inhibitor resulted in complete regression of gastric tumors (Oshima
et al. 2011b). These results suggest that such combination treatment may be an
effective strategy to prevent the development of inflammation-associated gastric
cancer or to treat existing gastric cancer.
13.4.2 Wnt Signaling Activation
Wnt signaling is important for the stem cell properties of epithelial cells, and thus,
constitutive activation of Wnt signaling causes gastrointestinal tumorigenesis. It
has been shown that β-catenin accumulation, a hallmark of Wnt signaling activa-
tion, is significantly increased in the invasion front and metastasized tumor cells of
human colon cancer, suggesting that increased Wnt signaling activation leads to the
malignant progression of colon cancer (Fodde and Brabletz 2007). In the gastric
preneoplastic lesions of the K19-Wnt1 mice that express Wnt signaling ligand Wnt1
in gastric epithelial cells, macrophages are infiltrated into the stroma surrounding
dysplastic epithelial cells, and β-catenin nuclear accumulation is enhanced in
dysplastic epithelial cells (Oguma et al. 2008). These results suggest that epithelial
Wnt signaling activity in preneoplastic lesions is enhanced by infiltrated macro-
phages. Activated macrophages express variety of inflammatory cytokines includ-
ing TNF-α, IL-1β, and IL-6. Importantly, stimulation of gastric cancer cells with the
Fig. 13.2 Possible mechanisms by which the inflammatory microenvironment promotes gastric
tumorigenesis. COX-2/PGE2 pathway and TLR/Myd88 signaling cooperatively generate inflam-
matory microenvironment by recruitment of macrophages and their activation. Such microenvi-
ronment promotes tumorigenesis through a variety of mechanisms, including EGFR activation
(left), Wnt signaling promotion (center left), ROS production (center right), and modulation of
oncomiR and tumor suppressor microRNA expression (right)
conditioned medium from lipopolysaccharide (LPS)-treated activated macrophages

caused increased Wnt signaling activity, which was mimicked by treating cells with
TNF-α (Oguma et al. 2008). Moreover, the infection of the stomachs of K19-Wnt1
mice with Helicobacter felis resulted in tumor development with increased Wnt
signaling activity in the tumor epithelial cells. These results indicate that activated
macrophages in tumor microenvironment promote the Wnt activity level of tumor
epithelial cells via TNF-α expression, which further contributes to tumor develop-
ment and progression (Fig. 13.2) (Oshima et al. 2004; Oguma et al. 2010).
In the inflammatory environment, macrophages express not only TNF-α but also
a variety of cytokines. Notably, other inflammatory cytokines have also been shown
to promote gastrointestinal tumorigenesis. For example, transgenic expression of
IL-1β in the mouse stomach leads to gastritis and gastric tumor development that
correlates with the recruitment of myeloid-derived suppressor cells (Tu et al. 2008).
Moreover, it has been reported that activation of Stat3, a transcription factor that
plays an important role in inflammatory responses, in intestinal epithelial cells

promotes colitis-associated tumorigenesis in mouse models (Bollrath et al. 2009;
Grivennikov et al. 2009). Stat3 is activated by IL-6 and IL-11 signaling through
their co-receptor gp130. Although IL-6 has shown to be responsible for Stat3-
dependent tumor promotion in these studies, it has been shown recently that IL-11
has a strong correlation with elevated Stat3 activation in gastrointestinal cancers,
and IL-11 has a prominent role during the progression of colon and gastric cancers
(Putoczki et al. 2013). Therefore, it is possible that the pro-inflammatory cytokine
network comprising TNF-α, IL-1β, IL-6, and IL-11 in the tumor microenvironment
promotes gastrointestinal tumorigenesis through a variety of mechanisms.
13.4.3 Noxo1 Expression and ROS Production
A polymorphism at the 308 position of the TNF-α gene promoter, the

TNF-α-308A allele, is associated with an increased risk of gastric cancer, with an
odds ratio of 1.9, suggesting a role for TNF-α in gastric tumorigenesis (El-Omar
et al. 2002; Machado et al. 2003). To further examine the role of TNF-α in gastric
cancer development other than via Wnt activation, we crossed Gan mice with the
TNF-α gene (Tnf) knockout mice and examined the gastric phenotype. Importantly,
gastric tumor development was significantly suppressed in Tnf / Gan mice, and
the mean gastric tumor volume in Tnf / Gan mice was decreased to 18 % of that
in Tnf +/+ Gan mice (Fig. 13.3) (Oshima et al. 2014). The major source of TNF-α in
the tumor tissue is bone marrow-derived cells (BMDCs), including macrophages.
Bone marrow transplantation from Tnf +/+ mice into Tnf / Gan mice restored the
gastric tumor phenotype, with significant infiltration of BMDCs in the tumor stroma
(Oshima et al. 2014). These results indicate that TNF-α signaling activation in
BMDCs, possibly macrophages, is important for gastric tumor promotion.
By cDNA microarray analyses using Tnf +/+ Gan and Tnf / Gan mouse tumors,
about 150 genes were selected as candidate TNF-α-dependent tumor-promoting
genes. Interestingly, characteristic genes for intestinal stem cells, including CD44 a
cell-surface glycoprotein and known as a receptor for hyaluronic acid, Prom1,
Sox9, and EphB3 (Itzkovitz et al. 2012), were included in the gene list. These
genes are expressed in intestinal stem cells, and some of their products have shown
to play a role in maintenance of stem cell property. Accordingly, it is possible that
TNF-α plays an important role in the maintenance tumor cells in undifferentiated
status. We thus compared TNF-α-dependent genes with the list of Lgr5-expressing
gastric stem cell-specific genes (Barker et al. 2012) and found Noxo1 that is
upregulated in both tumor cells and normal stem cells. Noxo1 is a NADPH oxidase
(NOX)-organizing protein 1, which is a component of the NOX1 complex, which
generates reactive oxygen species (ROS) (Adachi et al. 2008). It has been shown
that Nox1 expression is required for the transformation of Ras-activated cancer
cells, and Noxo1 is upregulated in colon cancers (Mitsushita et al. 2004; Juhasz
et al. 2009). Moreover, it has been reported that the small Rho GTPase Rac1,
Fig. 13.3 Suppression of gastric tumor development in Gan mice by TNF-α gene (Tnf) disruption.
a Representative macroscopic photographs of Tnf +/+, Tnf +/, and Tnf / Gan mouse gastric
tumors at 50 weeks of age. The bars indicate 5 mm. b Representative histological photographs of
the whole views of Tnf +/+ Gan (top) and Tnf / Gan mouse (bottom) gastric tumors. Note that
gastric tumor development was significantly suppressed by disruption of TNF-α gene. c The
gastric tumor size of the Tnf+/+ Gan, Tnf +/ Gan and Tnf / Gan mice relative to the mean
size of Tnf +/+ Gan mouse tumors (set at 100 %). Asterisks, P < 0.05 (Reproduced from Oshima
et al. Oncogene, 2014 with permission from Nature Publishing Group)
another component of the Nox1 complex, is activated by cooperation of Wnt

signaling and NF-κB activation in ApcMin mouse intestinal tumor cells, and acti-
vated Rac1 promotes ROS production of the NOX1 complex (Myant et al. 2013).
Increased ROS level by NOX1 complex has shown to be important for the stem cell
property of intestinal tumor cells. Taken together, these results suggest that TNF-α
signaling activates the NOX1 complex through induction of Noxo1 expression,
resulting in increased ROS production, which contributes to the maintenance of
tumor cells in undifferentiated status (Fig. 13.2).
On the other hand, it has been shown that a variant form of CD44 (CD44v) is
induced in Gan mouse gastric tumor cells by a PGE2-dependent mechanism
(Ishimoto et al. 2010), and CD44v is required for gastric tumorigenesis by the
suppression of ROS production by glutathione synthesis (Ishimoto et al. 2011).
Consistently, CD44 gene disruption in Gan mice resulted in significant suppression
of the gastric tumorigenesis. Accordingly, it is possible that strict regulation of the
ROS level is important for the stemness and survival of tumor cells, i.e., a basal
ROS level is required for stemness, but a high ROS level is toxic and decreases
tumor cell survival. However, the precise mechanisms underlying the involvement
of ROS in tumor development are still being investigated.
13.4.4 Other Mechanisms, MicroRNA
In Gan mouse gastric tumor tissues, expression of vascular endothelial growth

factor (VEGF) is increased significantly and angiogenesis is enhanced. We found
that VEGF expression is induced in stromal fibroblasts of Gan mouse tumor tissues
and that tumor cell-derived factors stimulate bone marrow-derived stromal cells to
express VEGF (Guo et al. 2008). It is possible that inflammatory signaling is
involved in angiogenesis in tumors.
It is known that expression of some microRNAs is affected by inflammatory
responses through activation of NF-κB or Stat3. MicroRNAs are single-stranded
small noncoding RNAs that regulate gene expression by posttranscriptional inter-
ference of specific mRNAs (Ambros 2004). Interestingly, several oncogenic
microRNAs, such as miR-21 and miR-155, are upregulated in both Gan mouse
gastric tumors and K19-C2mE gastritis tissues, suggesting that these microRNAs
are induced by inflammation-dependent manner (Kong et al. 2012). On the other
hand, tumor suppressor microRNAs, such as miR-7 and miR-145, are
downregulated in both Gan mouse tumors and K19-C2mE gastritis, suggesting
inflammation-dependent downregulation. Expression of miR-7 induces differenti-
ation of epithelial cells by inhibition of EGFR expression. Thus, downregulation of
miR-7 contributes to maintenance of epithelial cells in undifferentiated status.
Accordingly, it is possible that inflammatory responses promote gastric tumorigen-
esis by both inducing oncogene microRNAs (oncomiRs) and suppressing the
expression of tumor suppressor microRNAs (ts miRs) (Fig. 13.2).
H. pylori infection induces chronic gastritis, which further induces expression of

COX-2 and mPGES-1, resulting in enhanced PGE2 biosynthesis in the gastric
mucosa. Gan mice develop inflammation-associated gastric tumors by simulta-
neous activation of Wnt signaling and COX-2/PGE2 pathway, which recapitulates
molecular mechanisms of human gastric tumorigenesis. Wnt signaling activation
alone induces only limited preneoplastic lesions. Accordingly, H. pylori-induced
COX-2/PGE2 pathway plays an essential role in gastric tumorigenesis through
generation of inflammatory microenvironment. Using Gan mouse model, it has
been demonstrated that both COX-2/PGE2 pathway and innate immune responses
are cooperatively responsible for the generation of an inflammatory microenviron-
ment with macrophage infiltration and cytokine expression. Such a microenviron-
ment promotes gastric tumorigenesis through the activation of EGFR signaling that
causes increased proliferation, promotion of Wnt signaling activation, and enhance-
ment of NOX1 complex-dependent ROS production. Wnt activation and ROS
production are important for stemness and thus contribute to maintenance of
tumor cells in undifferentiated status. These results strongly suggest that targeting
the COX-2/PGE2 pathway-dependent inflammatory microenvironment will be an

effective therapeutic and preventive strategy for gastric cancer.
References
Adachi Y, Shibai Y, Mitsushita J, Shang WH, Hirose K, Kamata T (2008) Oncogenic Ras
upregulates NADPH oxidase 1 gene expression through MEK-ERK-dependent phosphoryla-
tion of GATA-6. Oncogene 27:4921–4932
Allen-Veroce E, Jobin C (2014) Fusobacterium and enterobacteriaceae: important players for
CRC? Immunol Lett 162:54–61
Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355
Barker N, van Oudenaarden A, Clevers H (2012) Identifying the stem cell of the intestinal crypt:
strategies and pitfalls. Cell Stem Cell 11:452–460
Bollrath J, Phesse TJ, von Burstin VA, Putoczki T, Bennecke M, Bateman T et al (2009) Gp130-
mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression
during colitis-associated tumorigenesis. Cancer Cell 15:91–102
Brembeck FH, Moffett J, Wang TC, Rustgi AK (2001) The keratin 19 promoter is potent for cell-
specific targeting of genes in transgenic mice. Gastroenterology 120:1720–1728
El-Omar EM, Rabkin CS, Gammon MD, Vaughan TL, Risch HA, Schoenberg JB et al (2002)
Increased risk of noncardia gastric cancer associated with proinflammatory cytokine gene
polymorphisms. Gastroenterology 123:1406–1407
El-Omar EM, Ng MT, Hold GL (2008) Polymorphisms in Toll-like receptor genes and risk of
cancer. Oncogene 27:244–252
Fodde R, Brabletz T (2007) Wnt/beta-catenin signaling in cancer stemness and malignant behav-
ior. Curr Opin Cell Biol 19:150–158
Grivennikov S, Karin E, Terzic J, Mucida D, Yu GY, Vallabhapurapu S et al (2009) IL-6 and Stat3
are required for survival of intestinal epithelial cells and development of colitis-associated
cancer. Cancer Cell 15:103–113
Guo X, Oshima H, Kitamura T, Taketo MM, Oshima M (2008) Stromal fibroblasts activated by
tumor cells promote angiogenesis in mouse gastric cancer. J Biol Chem 283:19864–19871
Hold GL, Rabkin CS, Chow WH, Smith MG, Gammon MD, Risch HA et al (2007) A functional
polymorphism of toll-like receptor 4 gene increases risk of gastric carcinoma and its pre-
cursors. Gastroenterology 132:905–912
Ishimoto T, Oshima H, Oshima M, Kai K, Torii R, Masuko T et al (2010) CD44+ slow-cycling
tumor cell expansion is triggered by cooperative actions of Wnt and prostaglandin E2 in gastric
tumorigenesis. Cancer Sci 101:673–678
Ishimoto T, Nagano O, Yae T, Tamada M, Motohara T, Oshima H et al (2011) CD44 variant
regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(-) and thereby
promotes tumor growth. Cancer Cell 19:387–400
Itadani H, Oshima H, Oshima M, Kotani H (2009) Mouse gastric tumor models with prostaglandin
E2 pathway activation show similar gene expression profiles to intestinal-type human gastric
cancer. BMC Genomics 10:615
Itzkovitz S, Lyubimova A, Blat IC, Maynard M, van Es J, Lees J et al (2012) Single-molecule
transcript counting of stem-cell markers in the mouse intestine. Nat Cell Biol 14:106–115
Juhasz A, Ge Y, Markel S, Chiu A, Matsumoto L, van Balgooy J et al (2009) Expression of
NADPH oxidase homologues and accessory genes in human cancer cell lines, tumours and
adjacent normal tissues. Free Radic Res 43:523–532
Katoh H, Wang D, Daikoku T, Sun H, Dey SK, DuBois RN (2013) CXCR2-expressing myeloid-
derived suppressor cells are essential to promote colitis-associated tumorigenesis. Cancer Cell
24:631–644
Kong D, Piao YS, Yamashita S, Oshima H, Oguma K, Fushida S et al (2012) Inflammation-

induced repression of tumor suppressor miR-7 in gastric tumor cells. Oncogene 31:3949–3960
Machado JC, Figueiredo C, Canedo P, Pharoah P, Carvalho R, Nabais S et al (2003) A
proinflammatory genetic profile increases the risk for chronic atrophic gastritis and gastric
carcinoma. Gastroenterology 125:364–371
Mitsushita J, Lambeth JD, Kamata T (2004) The superoxide-generating oxidase Nox1 is func-
tionally required for Ras oncogene transformation. Cancer Res 64:3580–3585
Mochizuki S, Okada Y (2007) ADAMs in cancer cell proliferation and progression. Cancer Sci
98:621–628
Myant KB, Cammareri P, McGhee EJ, Ridgway RA, Huels DJ, Cordero JB et al (2013) ROS
production and NF-κB activation triggered by RAC1 facilitate WNT-driven intestinal stem cell
proliferation and colorectal cancer initiation. Cell Stem Cells 12:761–773
Naugler WE, Sakurai T, Kim S, Maeda S, Kim K, Elsharkawy AM et al (2007) Gender disparity in
liver cancer due to sex differences in MyD88-dependent IL-6 production. Science
317:121–124
Oguma K, Oshima H, Aoki M, Uchio R, Naka K, Nakamura S et al (2008) Activated macrophages
promote Wnt signaling through tumor necrosis factor-alpha in gastric tumor cells. EMBO J
27:1671–1681
Oguma K, Oshima H, Oshima M (2010) Inflammation, tumor necrosis factor and Wnt promotion
in gastric cancer development. Future Oncol 6:515–526
Oshima H, Oshima M (2010) Mouse models of gastric tumors: Wnt activation and PGE2
induction. Pathol Int 60:599–607
Oshima M, Dinchuk JE, Kargman SL, Oshima H, Hancock B, Kwong E et al (1996) Suppression
of intestinal polyposis in ApcΔ716 knockout mice by inhibition of cyclooxygenase 2 (COX-2).
Cell 87:803–809
Oshima H, Oshima M, Inaba K, Taketo MM (2004) Hyperplastic gastric tumors induced by
activated macrophages in COX-2/mPGES-1 transgenic mice. EMBO J 23:1669–1678
Oshima H, Matsunaga A, Fujishita T, Ysukamoto T, Taketo MM, Oshima M (2006) Carcinogen-
esis in mouse stomach by simultaneous activation of the Wnt signaling and prostaglandin E2
pathway. Gastroenterology 131:1086–1095
Oshima H, Oguma K, Du YC, Oshima M (2009) Prostaglandin E2, Wnt, and BMP in gastric tumor
mouse models. Cancer Sci 100:1779–1785
Oshima H, Hioki K, Papivanova BK, Oguma K, Van Rooijen N, Ishikawa TO et al (2011a)
Prostaglandin E2 signaling and bacterial infection recruit tumor promoting macrophages to
mouse gastric tumors. Gastroenterology 140:596–607
Oshima H, Papivanova BK, Oguma K, Kong D, Ishikawa TO, Oshima M (2011b) Activation of
epithelial growth factor receptor signaling by the prostaglandin E2 receptor EP4 pathway
during gastric tumorigenesis. Cancer Sci 102:713–719
Oshima H, Ishikawa T, Yoshida GJ, Naoi K, Maeda Y, Naka K et al (2014) TNF-a/TNFR1
signaling promotes gastric tumorigenesis through induction of Noxo1 and Gna14 in tumor
cells. Oncogene 33:3820–3829
Pollard JW (2009) Trophic macrophages in development and disease. Nat Rev Immunol
9:259–270
Putoczki TL, Thiem S, Loving A, Busuttil RA, Wilson NJ, Ziegler PK et al (2013) Interleukin-11
is the dominant IL-6 family cytokine during gastrointestinal tumorigenesis and can be targeted
therapeutically. Cancer Cell 24:257–271
Qian BZ, Pollard JW (2010) Macrophage diversity enhances tumor progression and metastasis.
Cell 141:39–51
Rakoff-Nahoum S, Medzhitov R (2007) Regulation of spontaneous intestinal tumorigenesis
though the adaptor protein Myd88. Science 317:124–127
Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R (2004) Recognition of
commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell
118:229–241
Saukkonen K, Nieminen O, van Rees B, Vilkki S, Harkonen M, Juhola M et al (2001) Expression

of cyclooxygenase-2 in dysplasia of the stomach and in intestinal-type gastric adenocarcinoma.
Clin Cancer Res 7:1923–1931
Seno H, Oshima M, Ishikawa TO, Oshima H, Takaku K, Chiba T et al (2002) Cyclooxygenase 2-
and prostaglandin E2 receptor EP2-dependent angiogenesis in ApcΔ716 mouse intestinal
polyps. Cancer Res 62:506–511
Scheeren FA, Kuo AH, van Weele LJ, Cai S, Glykofridis I, Sikandar SS et al (2014) A cell-
intrinsic role for TLR2-MYD88 in intestinal and breast epithelia and oncogenesis. Nat Cell
Biol 16:1238–1248
Sonoshita M, Takaku K, Sasaki N, Sugimoto Y, Ushikubi F, Narumiya S et al (2001) Acceleration
of intestinal polyposis through prostaglandin receptor EP2 in ApcΔ716 knockout mice. Nat Med
7:1048–1051
Sonoshita M, Takaku K, Oshima M, Sugihara K, Taketo MM (2002) Cyclooxygenase-2 expres-
sion in fibroblasts and endothelial cells of intestinal polyps. Cancer Res 62:6846–6849
The Cancer Genome Atlas Network (2014) Comprehensive molecular characterization of gastric
adenocarcinoma. Nature 513:202–209
Thun MJ, Namboodiri MM, Heath CW Jr (1991) Aspirin use and reduced risk of fatal colon
cancer. NEJM 325:1593–1596
Tu S, Bhagat G, Cui G, Takaishi S, Kurt-Jones EA, Rickman B et al (2008) Overexpression of
interleukin-1beta induced gastric inflammation and cancer and mobilizes myeloid-derived
suppressor cells in mice. Cancer Cell 14:408–419
Tye H, Kennedy CL, Najdovska M, McLeod L, McCormack W, Hughes N et al (2012) STAT3-
driven upregulation of TlR2 promotes gastric tumorigenesis independent of tumor inflamma-
tion. Cancer Cell 22:466–478
Wambura C, Aoyama N, Shirasaka D, Kuroda K, Maekawa S, Ebara S et al (2004) Influence of
gastritis on cyclooxygenase-2 expression before and after eradication of Helicobactor pylori
infection. Eur J Gastroenterol Hepatol 16:969–979
Wang D, Wang H, Shi Q, Katkuri S, Walhi W, Desvergne B et al (2004) Prostaglandin E2
promotes colorectal adenoma growth via transactivation of the nuclear peroxisome prolifera-
tion-activated receptor δ. Cancer Cell 6:285–295
Wang D, Fu L, Ning W, Guo L, Sun X, Dey SK et al (2014) Peroxisome proliferator-activated
receptor δ promotes colonic inflammation and tumor growth. Proc Natl Acad Sci U S A
111:7084–7-89
Xia D, Wang D, Kim SH, Katoh H, DuBois RN (2012) Prostaglandin E2 promotes intestinal tumor
growth via DNA methylation. Nat Med 18:224–226
Chapter 14
Influence of Host Gene Polymorphisms
on Development of Gastroduodenal Diseases
Mairi H. McLean, Ruairidh Nicoll, Cheryl Saw, Georgina L. Hold,

and Emad M. El-Omar
Abstract Helicobacter pylori infection remains the commonest chronic bacterial

infection in the world and is associated with a variety of clinical outcomes that
range from simple asymptomatic gastritis to more serious conditions such as peptic
ulcer disease and gastric cancer. The key determinants of these outcomes are the
severity and distribution of H. pylori-induced gastritis. Host genetic factors play an
important role in influencing disease risk, but identifying candidate genes is a major
challenge that has to stem from a profound understanding of the pathophysiology of
the disease. In the case of H. pylori-associated disease, the initial search focused on
candidate genes that attenuate gastric physiology and lead to a destructive chronic
inflammatory response against the infection. In particular, certain cytokine and
innate immune response gene polymorphisms appear to influence risk of gastric
cancer and its precursor conditions. More recent genome-wide association studies
have identified novel genetic markers that show impressive associations with
gastric cancer risk but whose function remains unclear. Very recently, there has
been progress in identifying genetic risk markers for acquisition of H. pylori
infection, but there remains a lack of suitable markers for risk of peptic ulcer
disease. Future research agenda should focus on identifying the full genetic risk
profile for H. pylori-induced gastroduodenal disease. This will help target the
population most at risk by directing eradication therapy and closer follow-up to
the affected individuals.
Keywords Helicobacter pylori • Host genetics • Genetic polymorphisms • Gastric

cancer • MALT lymphoma • Peptic ulcer disease • Chronic inflammation
M.H. McLean • R. Nicoll • C. Saw • G.L. Hold • E.M. El-Omar (*)

Division of Applied Medicine, Institute of Medical Sciences, School of Medicine & Dentistry,
Aberdeen University, Foresterhill, Aberdeen AB25 2ZD, UK

DOI 10.1007/978-4-431-55936-8_14
340 M.H. McLean et al.
14.1 Introduction
Helicobacter pylori (H. pylori) is associated with a variety of clinical outcomes that
range from simple asymptomatic gastritis to serious diseases such as peptic ulcer
disease, gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma.
The basic pathogenesis of H. pylori-induced disease is the establishment of chronic
gastritis. The severity and distribution of this gastritis determines the clinical
outcome. There are three main gastric phenotypes that result from chronic
H. pylori infection: (1) the commonest by far is a mild pangastritis that does not
affect gastric physiology and is not associated with significant human disease; (2) a
corpus-predominant gastritis associated with gastric atrophy, hypochlorhydria and
increased risk of gastric cancer (the gastric cancer phenotype) (El-Omar
et al. 1997); and (3) an antral-predominant gastritis associated with high gastric
acid secretion and increased risk of duodenal ulcer disease (the DU phenotype)
(El-Omar et al. 1995). What is most curious is that the DU outcome is actually
protective against the gastric cancer outcome, and this conundrum needs to be
explained.
There is accumulating evidence that acid secretory capacity is crucial in deter-
mining the distribution and natural history of H. pylori infection (El-Omar
et al. 1995, 1997; Amieva 2008). In hosts with low secretory capacity (genetically
determined or secondary to pharmacologic inhibition), the organism is capable of
colonising a wider niche than would be possible in the presence of high volumes of
acid. Colonisation of a wider niche, including the corpus mucosa, leads to corpus
gastritis with resultant functional inhibition of acid secretion. This inhibition is
mediated by H. pylori-induced inflammatory cytokines such as the interleukin-1
beta (IL-1β) and tumour necrosis factor alpha (TNF-α), and the net effect is the
establishment of a more aggressive gastritis that accelerates the development of
gastric atrophy. Once atrophy develops, acid secretion is not only attenuated by the
functional inhibition caused by inflammatory mediators but by a more permanent
morphological change that is harder to reverse. This situation is very relevant to the
subgroup of humans who develop the gastric cancer phenotype in the presence of
chronic H. pylori infection.
The inflammatory response to H. pylori infection is modulated by cofactors,
including bacterial and host genes, and environmental factors. In this chapter, we
review the evidence for the role of host genetic factors in determining the risk of
gastroduodenal disease. We start by discussing the known host genetic factors
involved in acquiring the infection. We then discuss the published literature on
host genetic factors that predispose to specific disease outcomes such as peptic ulcer
disease, gastric cancer and MALT lymphoma.
14 Influence of Host Gene Polymorphisms on Development of Gastroduodenal Diseases 341
14.2 Host Genetics and Risk of Helicobacter pylori

Infection
Genetic association studies have been instrumental in aiding clinicians and

researchers in the study of infectious diseases, ranging from linkage mapping to
candidate gene studies and genome-wide association studies (GWAS). There are
numerous studies that have assessed genetic risk factors for HIV, malaria, leprosy
and tuberculosis. In this section, we summarise the most notable studies on the role
of host genetic factors relevant to H. pylori infection, an infection that is by far the
commonest globally. We start by discussing the candidate gene approach followed
by the more recent GWAS studies.
14.2.1 Candidate Gene Studies of Helicobacter pylori

Infection
At an epidemiologic level, there is little doubt that genetic factors influence

susceptibility to H. pylori infection. In a landmark study, Malaty and co-workers
assessed H. pylori status in 269 pairs of twins, including 36 monozygotic twin pairs
reared apart, 64 monozygotic twin pairs reared together, 88 dizygotic twin pairs
reared apart and 81 dizygotic twin pairs reared together (Malaty et al. 1994). The
probandwise concordance rate for H. pylori infection was higher in monozygotic
twin pairs (81 %) than in dizygotic twin pairs (63 %) (P ¼ 0.001). For twins reared
apart, the probandwise concordance rates for H. pylori infection were 82 % and
66 % for monozygotic and dizygotic twins, respectively (P ¼ 0.003). The correla-
tion coefficient was 0.66 for monozygotic twins reared apart, and it provides the
best single estimate of the relative importance of genetic effects (heritability) for
variation in the acquisition of H. pylori infection (Mandell et al. 2004).
Despite this genetic influence, the study of this heritability and its clinical
consequences is hampered by the fact that the H. pylori infection is acquired during
childhood, and it is almost impossible to prove exposure or acquisition with any
precision. This is largely due to the fact that the infection may be asymptomatic or
may be associated with a mild illness that could easily be confused with any
common childhood ailment. To complicate matters, it is never clear if absence of
the infection in an individual is due to lack of exposure or due to genetically
determined resistance to the infection. Unfortunately, these are difficult
confounding factors to dissect or control.
Tseng and colleagues attempted to study genetic susceptibility to H. pylori
infection using the Jamaica Mother Infant Cohort, a prospective follow-up of
children, designed to examine the transmission and natural history of certain
infections (Mandell et al. 2004; Tseng et al. 2006). Between January 1989 and
August 1990, 339 expectant mothers were enrolled from two prenatal clinics in
Kingston, Jamaica. Expectant mothers provided demographic, reproductive and
medical history data and blood samples at the time of delivery. For children,
physical examination and phlebotomy were performed at birth and repeated every
6 weeks for the first 6 months, then every 3 months until age 2 and every 6 months
thereafter. Single-nucleotide polymorphisms (SNPs) at 17 loci in 11 cytokine genes
(IL1A, IL1B, IL2, TNF, TLR4, IL4, IL6, IL10, IL10RA, IL12A and IL13) were
analysed. The only positive finding was that the IL1A-889 T allele, known to
express a higher level of cytokine IL-1α, was associated with a lower risk of
H. pylori infection among these children. The finding supports the hypothesis that
an upregulation of specific pro-inflammatory cytokines may protect against
H. pylori colonisation (Smith et al. 2003). However, a further study by Liou and
co-workers appears to contradict this hypothesis. They studied the role of promoter
polymorphisms in the interleukin-1β gene (IL1B) in healthy individuals with and
without H. pylori infection (Liou et al. 2007) and reported that a pro-inflammatory
genetic make-up increased the risk of having H. pylori infection. Overall, the
findings of these studies suggest that other host genetic factors, particularly in
genes related to the initial handling of H. pylori within the gastric mucosa, may
prove more relevant to the pathogenesis of this infection.
14.2.2 Tumour Necrosis Factor Alpha (TNF-α)
One of the most commonly studied genes regarding the acquisition of H. pylori
infection is the tumour necrosis factor-α (TNFA) gene. Hamajima and co-workers
studied 1374 Japanese subjects and reported a reduced odds ratio (OR) for H. pylori
seropositivity in individuals with TNFA-1031CC genotype as compared to those
with the TNFA-1031TT genotype (OR ¼ 0.43, 95 % CI ¼ 0.20–0.91), with the OR
adjusted for sex, age and recruitment source. In the same study, subjects with
TNFA-857CC and the aforementioned TNFA-1031CC genotypes showed the low-
est H. pylori seropositivity (38.2 % of 34 subjects), whereas those with the TNFA-
857TT and the TNFA-1031TT genotypes showed the highest seropositivity (66.7 %
of 42 subjects) (Hamajima et al. 2003).
However, in a similar study conducted in 2006 on a population of 963 Japanese
Brazilian individuals investigating the relationship between the TNFA-857TT and
TNFA-1031TT genotypes and H. pylori seropositivity, no significant association
was found between the two factors (Atsuta et al. 2006). Saijo and colleagues studied
410 Japanese transit company employees and showed that contrary to other studies,
the TNFA-857TT genotype could actually have a protective effect against chronic
H. pylori infection, with subjects with TNFA-857TT having a significantly lower
odds ratio for H. pylori seropositivity (OR ¼ 0.15, 95 % CI ¼ 0.03–0.59, P ¼ 0.007)
(Saijo et al. 2007). In yet another study by Abdiev and co-workers on an Uzbek
population of 167 participants, it was shown that subjects with the TNFA-1031TC
genotype had a significantly increased risk for anti-H. pylori IgG seropositivity
(OR ¼ 2.82, 95 % CI ¼ 1.05–7.57), as compared to TNFA-1031TT (Abdiev
et al. 2010), an interesting contradiction to the results from the Hamajima study.
The TNFA-308G/A promoter polymorphism has also been associated with

infection stratified by the cytotoxin-associated gene A (cagA) subtype of
H. pylori. A study conducted on a Korean population of 83 patients with known
gastric disease and 113 healthy controls showed that H. pylori infection was
strongly associated with the TNFA-308G/A polymorphism as compared to healthy
controls (OR ¼ 2.912, 95 % CI ¼ 1.08–7.84; p ¼ 0.034) (Yea et al. 2001). The same
polymorphism had an even stronger correlation with the H. pylori cagA-positive
infection as compared to the polymorphism in cagA-negative H. pylori infection
(OR ¼ 8.757, 95 % CI ¼ 1.413–54.262; p ¼ 0.019) and healthy controls
(OR ¼ 3.683; 95 % CI ¼ 1.343–10.101, p ¼ 0.011). However, these results were
not replicated in the Abdiev study, which showed no association between the TNFA
polymorphisms 857C/T and 1031C/T and the risk of anti-CagA IgG sero-
positivity or gastric atrophy (Abdiev et al. 2010).
From the data above, it can be concluded that the TNFA gene plays an important
role in deciding an individual’s susceptibility to H. pylori infection. However, the
conflicting results highlight the need for larger and adequately powered studies.
14.2.3 Toll-Like Receptors (TLRs)
The TLRs represent one of the major groups of pattern recognition receptors
(PRRs) within the host innate immune system, allowing cells of the innate immune
system to recognise pathogens by detecting molecules expressed across large
numbers of different pathogen species, the pathogen associated molecular patterns
(PAMPs).
14.2.3.1 TLR4
TLR4 is known to be involved in signal transduction pathways initiated by lipo-

polysaccharides (LPSs) mostly from Gram-negative bacteria. In a 2000 study
conducted by Arbour and co-workers, it was demonstrated that common missense
mutations (Asp299Gly and Thr399Ile) affecting the extracellular domain of the
TLR4 receptor can cause a reduced response to inhaled LPS in humans and that the
Asp299Gly mutation can also cause disruptions in LPS signalling as mediated by
TLR4 (Arbour et al. 2000). The above results clearly demonstrate that the varying
levels of LPS responsiveness between individuals can be attributed to TLR4 and
that genetic mutations can have an effect on an individual’s degree of response to
any form of environmental exposures. In 2001, a study by Maeda et al. showed that
macrophages from C3H/HeJ mice carrying point mutations in the Tlr4 gene showed
decreased activation of transcription factor nuclear factor kappa B (NF-κB) and
TNF-α secretion compared with C3H/HeN mouse macrophage when infected with
H. pylori (Maeda et al. 2001). This illustrates the importance of TLR4 mutations in
H. pylori-induced NF-κB activation in macrophages, which are crucial in mediating

the body’s inflammatory response to H. pylori infection. Additionally, Kawahara
and colleagues also showed that TLR4 is the primary receptor responsible for the
initiation of the gastric mucosal response to H. pylori infection because of the large
amount of TLR4 protein found on the plasma membrane of gastric pit cells in
guinea pigs as compared to the lack of TLR2 and TLR9 transcripts and insignificant
amounts of TLR2 protein present in the same region (Kawahara et al. 2001).
14.2.3.2 TLR2 and TLR5
In more recent years, various studies have identified other TLR proteins as the
primary factors for the initiation of the gastric mucosal response to H. pylori
infection. Bäckhed and co-workers showed that while all cell lines within the
gastric mucosa expressed TLR4, H. pylori itself could not be recognised by
TLR4 (Backhed et al. 2003). It has also been shown that macrophages from wild-
type and TLR4-deficient mice could produce a strong cytokine response involving
IL-6 and monocyte chemoattractant protein-1 (MCP-1) when stimulated by intact
H. pylori (Mandell et al. 2004), thus casting doubts on the popular hypothesis that
TLR4 is instrumental in the initiation of a host inflammatory response against
H. pylori infection.
Additionally, a study conducted by Smith and co-workers in 2003 on gastric
epithelial cells showed that H. pylori infection of the cultures caused NF-κB
activation in both HEK293 cells and MKN45 gastric epithelial cells transfected
with TLR2 and TLR5, but not TLR4 (Smith et al. 2003). These results were
replicated in a 2004 study by Mandell and colleagues on transfected HEK293
cells, where it was shown that human TLR2 expression was sufficient in inducing
a response to intact H. pylori bacteria, while TLR4 transfection was proven to be
insufficient (Mandell et al. 2004). It was also demonstrated in the same study that
macrophages from TLR2-deficient mice had a significant lack of response to
stimulation by intact H. pylori, as shown by the host’s failure to secrete cytokines
at 100:1 bacterium-to-macrophage ratios. Furthermore, HEK293 cells transfected
with TLR2 and TLR5 expression plasmids showed an induction of chemokine gene
expression by H. pylori infection. These results suggest that TLR2 and TLR5 have a
more significant role to play in influencing the ability of the host to recognise and
respond to H. pylori infection as compared to TLR4 as previously believed.
14.2.4 Susceptibility to H. pylori Infection and GWAS

Studies
The first GWAS study that explored the relationship between H. pylori sero-
positivity and host genetics was published by Mayerle and co-workers in 2013
(Mayerle et al. 2013). In this study, two independent GWAS studies were
conducted on two independent population-based cohorts from Northeastern Ger-
many (Study of Health in Pomerania, n ¼ 3830) and the Netherlands (Rotterdam
Study, n ¼ 7108). The main underlying objective of the study was to identify
genetic loci associated with H. pylori seroprevalence and as a result discover the
pathophysiology behind this association. H. pylori seroprevalence, used as an
indicator for previous or current infection, was defined as an anti-H. pylori IgG
equal to or greater than 20 U/mL. Individuals with the highest 25 % of the IgG titre
distribution were considered as case patients, and those in the lower 75 % of the IgG
titre distribution made up the control group in this study. In total, there were 2736
case patients and 8202 control patients, largely of European ancestry. Faecal
H. pylori antigen testing was used to determine the presence of infection in these
individuals. GWAS meta-analysis identified two genome-wide significant loci in
terms of their association to H. pylori seropositivity, namely, the TLR locus on 4p14
and the FCGR2A locus on 1q23.3. The lead SNP on the TLR locus with the lowest p
value was rs10004195 (OR ¼ 0.70, 95 % CI ¼ 0.65–0.76), closely followed by
rs4833095 (OR ¼ 0.70, 95 % CI ¼ 0.65–0.76). For the FCGR2A locus, the lead
SNP was rs368433 (OR ¼ 0.73, 95 % CI, 0.65–0.81).
Three different TLRs are located along the 4p14 region: TLR1, TLR6 and
TLR10. In an additional study conducted on 1763 participants from both cohorts,
analysis of whole-blood RNA gene expression profiling showed that among the
three TLR genes, only TLR1 was differentially expressed in relation to the
rs10004195 genotype (in the presence of the rs10004195-A allele [beta ¼ 0.23,
95 % CI ¼ 0.34 to 0.11]). Furthermore, analysis of TLR1, TLR6 and TLR10
mRNA amounts also showed that there was a specific and genotype-independent
transcriptional upregulation of TLR1 in the presence of H. pylori. These results
imply that the increase in TLR1 mRNA expression as a result of the rs10004195
SNP is strongly associated with an increased risk of H. pylori seropositivity.
14.2.4.1 Effects of TLR1 on H. pylori Infection
The mechanism for the relationship between increased TLR1 expression and a
higher H. pylori seroprevalence remains unexplained. However, TLR1 has been
shown to interact with TLR2 to form a heterodimer (Jin et al. 2007), which is
responsible for the initiation of cell signalling in response to the recognition of
tri-acylated lipopeptides from Gram-negative bacterial cell wall (Takeuchi
et al. 2002). This is particularly relevant to H. pylori infection as tri-acylated
lipopeptides can be found in the structure of H. pylori ligand A, allowing it to be

recognised by the TLR1-TLR2 complex. It has been suggested that the resulting
activation of the immune cascade could reduce the anti-inflammatory response of
the host against H. pylori and allow a persistent infection (El-Omar 2013). Another
explanation proposed recently is that the SNP at the TLR1 gene causes less
effective anti-inflammatory signalling initiated by the TLR1-TLR2 complex in
response to the presence of H. pylori, thus increasing the risk of persistent infection
(Guan et al. 2010).
The second genome-wide significant locus was identified as the FCGR2A locus
located on chromosome 1q23.3, the lead SNP being rs368433 A/G. This SNP is
located in an intron of FCGR2A encoding the Fcγ receptor 2a. Whole-blood RNA
gene expression profiling showed that this SNP was associated with a decrease in
FCGR2A expression in individuals carrying one or more minor alleles, thus
increasing the risk of H. pylori infection. However, further analysis of mRNA
amounts failed to show any significant statistical association between H. pylori
bacterial load and the expression of FCG2RA in whole blood.
14.2.4.2 Effect of FCGR2A on H. pylori Infection
As mentioned above, the rs368433 SNP at the 1q23 region has also been associated
with an increased risk of H. pylori seroprevalence. While the results were not as
significantly defined as that of the rs10004195 SNP and the mechanism of this
SNP’s effects is still unclear, there is evidence showing that polymorphisms in the
FCGR2A gene could possibly affect the individual’s ability to phagocytose bacte-
rial cells. Homozygotes for allelic polymorphisms in the FCGR2A gene were
shown to have polymorphonuclear leukocytes and monocytes which could less
effectively phagocytose and internalise erythrocytes coated with human IgG2
(Salmon et al. 1992). It is possible that the same effect can be applied to
H. pylori infection, where homozygotes for the rs368433 SNP could result in less
effective phagocytosis IgG2-opsonised bacteria by neutrophils, resulting in an
increased risk of infection.
While the study by Mayerle and co-workers is indeed ground-breaking, there
remain many unanswered questions in this field. Firstly, the study primarily focused
on H. pylori seropositivity, whereas H. pylori colonisation and symptomatic disease
were not assessed. As such, it is crucial that more studies are conducted to assess if
the same genetic polymorphisms are associated with symptomatic H. pylori infec-
tion, and these studies also have to fulfil the difficult task of measuring exposure
levels to H. pylori in the populations under study. Western populations have a low
H. pylori exposure pressure compared to South American and African populations
(Miwa et al. 2002; Prinz et al. 2006). Therefore, H. pylori seronegativity in the
reported results could have been due to the lack of exposure to H. pylori instead of a
protective genetic effect.
The translational benefits from the study of host genetics of H. pylori infection
are potentially impressive. For example, better understanding of the genetic basis of
risk might inform better development of antibiotics or indeed vaccines against the
infection.
14.3 Genetic Susceptibility to H. pylori-Induced Peptic

Ulcer Disease
The study of genetic susceptibility to peptic ulcers is restricted by their hetero-

geneity: (1) Gastric ulcers do not share the pathophysiology of duodenal ulcers. The
latter is associated with the classic antral-predominant pattern of gastritis and acid
hypersecretion, while the former is associated with a variety of phenotypes ranging
from DU-like (e.g. prepyloric ulcers) to gastric cancer-like (with gastric atrophy
and hypochlorhydria). Studies that have failed to take into account this rigorous
definition of phenotype may have been compromised. (2) Most studies published to
date have failed to exclude non-steroidal anti-inflammatory drug (NSAID)-induced
ulcers from both groups. The pathogenesis of NSAID ulcers is different from peptic
ulcers, and failing to exclude them may introduce significant bias. Finally, classic
H. pylori-induced peptic ulcers are now extremely rare, and very few research units
are able to muster adequately powered studies to address the issue. Perhaps a more
worthwhile research agenda would be to concentrate on understanding the genetic
determinants of NSAID-induced ulceration. These drugs are very commonly pre-
scribed, and their side effects continue to cause significant morbidity and mortality.
Very little is known about the host genetic factors that influence risk of side effects.
14.4 Role of Host Genetic Factors in Gastric Cancer
14.4.1 Single-Nucleotide Polymorphisms (SNPs)

in Candidate Genes
There are many examples in the literature of polymorphic genes increasing sus-
ceptibility to gastric cancer (El-Omar et al. 2008). This role was initially investi-
gated within chosen candidate genes based on knowledge of gastrointestinal disease
pathophysiology, with particular focus on gastric cancer as the most serious out-
come of H. pylori infection. Advances in technology for genetic sequencing now
allow simultaneous assessment of multiple previously unidentified SNPs within a
large number of genes, and this has revealed exciting novel insights into gastric
carcinogenesis. It is clear that genetic polymorphisms vary in accordance to eth-
nicity and, along with exposure to environmental risk factors, are a key player in
individual susceptibility to the development of gastric cancer.
SNPs within host inflammatory genes are a prime example of a candidate gene
approach, with emphasis on host response to H. pylori infection and pathological
consequences of this. IL-1β was the first pro-inflammatory cytokine gene to be

identified as an important candidate gene in gastric cancer susceptibility. It was
specifically chosen for its influence on gastric physiology, namely, upregulation in
response to H. pylori infection and potent inhibition of gastric acid secretion
(El-Omar 2001). Subsequent evidence of the pivotal role of this cytokine in gastric
tumourigenesis came from a transgenic mouse model displaying promoter-driven
targeted IL-1β overexpression in the stomach. This model provided evidence for a
crucial role for IL-1β in gastric premalignant pathology to overt gastric cancer in
the absence of H. pylori infection (Tu et al. 2008). When H. pylori colonisation was
introduced into this model, the pathological consequences were accelerated
reinforcing the importance of host-environment interaction.
Indeed, polymorphisms in the IL1 gene cluster (encoding IL-1α, IL-1β and
IL-1RN, the naturally occurring receptor antagonist) were shown to be associated
with increased risk of developing premalignant gastric atrophy and
hypochlorhydria in response to H. pylori infection within a Caucasian population
of gastric cancer relatives (El-Omar et al. 2000). These premalignant gastric
pathologies were associated with carriage of the IL1B-31*C or IL1B-511*T and
IL1RN*2/*2 genotype. This positive association remained true for the development
of non-cardia gastric cancer, with an estimated odds ratio of 1.6 (95 % CI, 1.2–2.2)
and 2.9 (95 % CI, 1.9–4.4) for carriage of IL1B-31*C/IL1B-511*T and IL1RN*2/
*2, respectively. This association has subsequently been validated in various
populations with different ethnicities (Kimang’a 2012; Zhao et al. 2012). However,
there are numerous accounts of interethnic and geographical variation with
conflicting reports of association in the literature. Overall, several large-scale
meta-analyses confirmed the positive association between IL1 markers and risk of
gastric cancer, especially in Caucasian populations (Camargo et al. 2006; Vincenzi
et al. 2008; Loh et al. 2009; He et al. 2011).
Following this finding, over the last decade, other potential candidate inflam-
matory cytokine genes, such as IL-8, IL-10 and TNFA, have been studied in the
context of gastric cancer risk. Most recently, this was examined in a Human
Genome Epidemiology (HuGE) systematic review and meta-analysis (Persson
et al. 2011). Persson and colleagues conducted a series of meta-analyses using a
predefined protocol and looked at the most studied polymorphisms in inflammatory
genes, including IL1B, IL1RN, IL8, IL10 and TNFA in the literature published over
a 16-year period (1990–2006). Gastric cancer was stratified on histological subtype
and anatomic subsite, H. pylori infection status and geographical location (Asian or
non-Asian study population) and by a quantitative index of study quality. There was
a consistent positive association between carriage of IL1RN*2 and increased risk of
gastric cancer, specific to non-Asian populations. This risk was seen for both
intestinal and diffuse cancers and, particularly, for distal cancers. In Asian
populations, reduced risk was observed in association with IL1B-31C carrier status.
When considering all the conflicting associations in the literature, it is essential to
recognise the importance of the tumour factors mentioned above in addition to
H. pylori infection status and ethnic origin of the population under study. Many of
these discrepant studies could be explained by variations in study design and

laboratory techniques.
Most recently, another cytokine gene under focus as a candidate gene was IL-17.
The IL-17 family of pro-inflammatory cytokines (IL-17A-F) is produced from
mainly Th17 cells but also other cell types in the inflammatory milieu, such as
NK T cells, neutrophils and CD8+ cytotoxic T (Tc17) cells. IL-17A has been
implicated in many inflammatory driven diseases, including autoimmunity and
cancer (Zou and Restifo 2010). Interest in the association between IL-17 gene
polymorphisms and gastric cancer arose in response to emerging evidence impli-
cating IL-17 in gastric cancer pathogenesis (Maruyama et al. 2010; Iida et al. 2011;
Meng et al. 2012; Zhuang et al. 2012). Several studies have now been published
assessing risk of gastric cancer in association with several polymorphic loci in the
IL17 genes in multiple ethnic populations, and overall, G to A transition at position-
187 in the IL17-A gene (rs22759133) has been associated with an increased risk
(Shibata et al. 2009; Qinghai et al. 2014; Rafiei et al. 2013; Zhang et al. 2014). It is
still unclear whether genetic variation in IL17A could impact host response to
H. pylori infection and be a driver for gastric cancer development. Overall, it has
been shown that IL-17-producing cells play a varied and important role in host
response to H. pylori infection (Kabir 2011). Do IL-17 polymorphisms impact
susceptibility to H. pylori infection? Current reports are conflicting, ranging from
no link between allelic carriage and H. pylori status (Rafiei et al. 2013) to a recent
report of a positive interaction with polymorphisms at multiple sites in the IL17A
gene and H. pylori infection in association with increased gastric cancer risk (p for
interaction 0.036).
14.4.2 Genome-Wide Association Studies
Novel susceptibility loci offering new molecular insights into gastric cancer devel-
opment have been identified using genome-wide association studies (GWAS). A
significant association between diffuse gastric cancer and polymorphic genetic
variation (rs2294008 and rs2976392) that fell within exon 1 of the PSCA (prostate
stem cell antigen) gene was identified using this technology, in a large Japanese
population (Sakamoto et al. 2008). Allelic differences at this site, G>A and C>T at
rs2976392 and rs2294008, respectively, interfered with transcription initiation and
conferred risk of diffuse gastric cancer with an adjusted odds ratio of 4.18 (95 % CI
of 2.88–6.21, p ¼ 1.5 1017), and this association was subsequently confirmed
within a Korean population of over 450 diffuse-type gastric cancer patients. The
authors went on to show that the PSCA protein is expressed in areas of the glandular
crypts. Crucially, this expression is in close proximity to stem cell progenitors, the
proposed initiator cell for this particular histological subtype of gastric cancer.
Furthermore, PSCA was downregulated at both the gene and protein level in gastric
cancer tissue specimens. The role for PSCA in the inhibition of epithelial cell
proliferation was highlighted by a series of in vitro experiments. This molecular
association was unexpected and highlights the power of newer genetic sequencing
technologies in contrast to the candidate gene approach. This association has now
been validated in several Asian population-based case-control studies (Matsuo
et al. 2009; Lu et al. 2010; Zeng et al. 2011; Wu et al. 2009). In a Caucasian
population, carriage of the rs2294008 T allele was associated with risk of chronic
atrophic gastritis (OR 1.5 (95 % CI 1.1–1.9)) in addition to overt non-cardia gastric
malignancy (OR 1.9 (95 % CI 1.3–2.8)) (Lochhead et al. 2011). Data from the EPIC
(European Prospective Investigation into Cancer and Nutrition) cohort confirmed
positive association of similar strength (OR 1.42 (95 % CI; 1.23–1.66) with carriage
of the T allele and both intestinal and diffuse histological subtypes of gastric cancer
(Sala et al. 2012). The relationship between gastric cancer risk and polymorphic
alleles in the PSCA gene remains true in several meta-analyses (Qiao and Feng
2012; Shi et al. 2012; Wang et al. 2012; Zhang et al. 2012; Gu et al. 2014). Does
carriage of the PSCA risk T allele impact on patient outcome? Two previous studies
assessed this and reported conflicting data. Wang and co-workers (Wang
et al. 2011) reported that carriage of the T allele conferred 25 % increased survival
rate (HR 0.75 (5 % CI 0.59–0.96)) for diffuse subtype gastric cancer, whereas the
other study reported a significantly worse survival outcome (OR 2.2 (95 % CI
1.22–3.69) (Lochhead et al. 2011). It is clear that further analysis is required in a
larger, more varied patient cohort to further explore this possibility.
The intestinal glycocalyx mucus layer is composed of tightly packed
glycosylated proteins from the mucin family that provides a physical barrier
protection between luminal contents and the host. The large group of mucin pro-
teins (MUC1-21) is either membrane bound or can be secreted. The physiological
role of MUC1 in particular has been of interest in the pathogenesis of gastrointes-
tinal disease as this protein is not simply an inert barrier. Instead MUC1 can
profoundly influence many important cell functions, such as cell growth, inhibition
of apoptosis via mitochondrial influences, influences on cell-cell adhesion, stimu-
lation of kinase-driven cell signalling pathways and interaction with several tran-
scription factors, such as the STATs and NF-κB, to impact on target downstream
expression. As such MUC1 has been termed an oncoprotein and has been impli-
cated in a number of cancers (Kufe 2009, 2012; Senapati et al. 2010; Boltin and Niv
2013). Interruption of the interaction between MUC1 and other proteins such as
β-catenin, ICAM and EGFR has been explored for therapeutic gain in the treatment
of cancer. As an alternative strategy, anti-MUC1 anticancer immunotherapy has
also been developed, reflecting its pivotal role in cancer pathogenesis (Kufe 2012;
Mukhopadhyay et al. 2011; Silva et al. 2001). Therefore, as one may expect, the
relevance of MUC1 in gastric cancer has been widely investigated, and it is
accepted that aberrant expression of this protein occurs in malignant gastric tissue.
Indeed, several studies have investigated MUC1 as an important gene associated
with gastric cancer risk in different geographical and ethnic populations, from both
a candidate gene approach and detection within susceptibility loci in GWAS studies
(Silva et al. 2001; Xu et al. 2009; Abnet et al. 2010; Jia et al. 2010; Saeki et al. 2011;
Zhang et al. 2011; Palmer et al. 2012; Song et al. 2014). Overall, the consensus from
these studies is that rs4072037 (G>A) polymorphism in exon 2 confers risk.
Specifically, carriage of the G allele at this site is protective. Presence of the G

allele was associated with a 28 % reduced risk of gastric cancer within a recent
meta-analysis that included over 6000 gastric cancer cases and 10,000 controls
across several geographical and ethnic populations (Zheng et al. 2013). From a
functional perspective, carriage of the A allele was associated with reduced protein
expression in gastric cancer tissue (Xu et al. 2009). Specifically, this polymorphism
affects gene promoter function and influences splice variants in gastric epithelium
(Saeki et al. 2011). With particular relevance to this review, it has been shown that
epithelial cell expression of the mucin proteins in the stomach inhibits the binding
of H. pylori to the host epithelial layer, therefore protecting the host from chronic
inflammatory activity in the underlying gastric mucosa (Guang et al. 2010; He
et al. 2014). MUC1 also negatively regulates signalling via TLRs in the respiratory
mucosa, and therefore this phenomenon is not confined to the gastrointestinal tract
(Ueno et al. 2008). In addition, it has been shown that muc1 regulates host
inflammatory response through inhibition of pro-inflammatory mediators in
response to H. pylori infection (Sheng et al. 2012). The emerging hypothesis is
that carriage of the A allele reduces protein expression of MUC1, allowing
H. pylori to directly interact with the gastric epithelium. This then creates a chronic
and persistent, unchecked inflammatory response in the host that could positively
influence cancer initiation and progression. However, this is not fully understood.
Indeed, Marin and colleagues showed that genetic variation in MUC family mem-
ber genes did not impact on clinical outcome and progression to overt gastric
malignancy in individuals with premalignant phenotype followed up over at least
a 12-year period (Marin et al. 2012). Interestingly, carriage of more than one of
these susceptibility-associated SNPs confers a cumulative risk effect, with greater
than eightfold increased risk of gastric cancer with carriage of both MUC1 and
PSCA risk alleles (Saeki et al. 2011). This may account for the high incidence of
gastric cancer in the Japanese population, despite relatively low H. pylori coloni-
sation, where more than 60 % of the population is believed to carry one or both
susceptibility SNPs (Saeki et al. 2013).
GWAS studies continue to reveal susceptibility loci associated with gastric
cancer risk (Abnet et al. 2010; Shi et al. 2011; Kang et al. 2014). Ongoing analysis
will no doubt uncover additional important polymorphic genes associated with
gastric cancer susceptibility. The crucial question is how these will fit functionally
into the pathogenesis of this disease and how this genetic susceptibility interacts
with environmental factors, such as H. pylori infection status to initiate and promote
this disease.
14.5 Genetic Associations with MALT Lymphoma
Marginal zone B-cell lymphomas of mucosa-associated lymphatic tissue (MALT),

also known as MALT lymphomas or MALTomas, are the predominant lymphoma
occurring within the stomach (Fischbach 2013). Infection with H. pylori is known
to be almost essential in the development of this rare malignancy, with 98.5 % of

cases being seropositive for H. pylori in one study (Eck et al. 1997). This relation-
ship is further strengthened by the fact that gastric MALT lymphoma can often be
successfully treated with H. pylori eradication therapy (Fischbach 2013). Inflam-
mation is thought to be the key mechanism by which H. pylori drives gastric MALT
lymphoma pathogenesis (Fischbach 2013). However, the majority of patients with
H. pylori infection do not develop gastric MALT lymphoma, and host genetic
factors are being recognised as increasingly important risk factors in determining
which patients with H. pylori infection go on to develop this rare malignancy. This
section summarises recent studies into host genetic factors associated with gastric
MALT lymphoma and the insights they offer into its pathogenesis.
14.5.1 Detoxification and Antioxidant Genes
The glutathione S-transferases (GST) are a major family of enzymes involved in the
conjugation of substrates to glutathione for the purpose of detoxification. They also
have an antioxidant function and neutralise ROS protecting against DNA damage
(Saeidnia and Abdollahi 2013). In a study published in 2003, Rollinson and
colleagues proposed that gene deletions at the GST T1 and GST M1 loci resulting
in a lack of the active protein would be associated with an increased risk of gastric
MALT lymphoma (Rollinson et al. 2003). They compared 66 cases of patients with
gastric MALT lymphoma from the North of England against 163 healthy controls
and found that the GST T1 null genotype was significantly associated with gastric
MALT lymphoma (57.6 % of cases versus 13.5 % of controls, OR 9.51, 95 % CI
4.57–19.81). This finding was replicated in a study published in 2004 by Wu and
co-workers, who compared 75 cases with MALT lymphoma against 321 healthy
controls of a Han Chinese background (Wu et al. 2004b). They also found a
significant association between the GST T1 null genotype and the risk of gastric
MALT lymphoma (57.3 % cases versus 43.0 % of controls, OR 1.8, 95 % CI
1.1–3.0), albeit with a reduced effect size. With regard to the GST M1 null
genotype, no significant associations were found in either Rollinson’s study
(62.1 % cases versus 54.5 % controls, OR 1.10, 95 % CI 0.59–2.07) or Wu’s
study (60 % cases versus 52 % controls, no statistics available).
The positive associations between the GST T1 null genotype show very different
effect sizes, with an odds ratio of 9.51 in the North of England cohort and an odds
ratio of 1.8 in the Han Chinese cohort. The likely explanation for this is the
difference in carrier rates for the GST T1 null genotype in the control populations.
The Han Chinese controls had a higher frequency of the GST T1 null genotype at
43 % compared to a carrier rate of 13.5 % in the North of England controls. This is
consistent with existing studies which show a higher carrier rate for this genotype in
Han Chinese populations (Wu et al. 2004b; Strange and Fryer 1999). These findings
offer interesting insights into gastric MALT lymphoma pathogenesis. As the
glutathione S-transferases play a major role in protecting DNA against damage, it
has been proposed that impaired antioxidant functioning in individuals carrying the
GST T1 null genotype would be subjected to higher rates of DNA damage enhanc-
ing the rate of MALT lymphoma pathogenesis. Supporting this theory are studies
showing an association between the GST T1 null genotype and other cancers,
including astrocytoma, meningioma, myelodysplasia (Rebbeck 1997) and other
lymphoproliferative disorders (Strange and Fryer 1999).
14.5.2 Cytokine and Cytokine Receptor Genes
14.5.2.1 Interleukin-1
Rollinson and co-workers first studied the association of IL1RN with gastric MALT
lymphoma. They compared 66 cases of gastric MALT lymphoma from the North
East of England against 163 healthy controls and found that the IL1RN 2/2 genotype
was significantly associated with gastric MALT lymphoma (33.9 % cases versus
8 % controls, OR 5.51, 95 % CI 2.16–14.07). A subsequent study by Wu et al. found
no associations between the IL1RN gene and gastric MALT lymphoma although
there were no cases positive for the 2/2 genotype and only three positive controls
(Rollinson et al. 2003). A further larger study by Hellmig and co-workers compar-
ing 153 MALT lymphoma patients with 344 controls all from the German-Austrian
Lymphoma Study group had more participants carrying the 2/2 genotype (9/153
cases versus 27/344 controls) but also failed to find any association between the
IL1RN 2/2 genotype and gastric MALT lymphoma (Hellmig et al. 2004). Hellmig
et al. suggested that the use of gastric biopsies as a source of DNA in Rollinson
et al.’s study could have resulted in contamination with tumour material leading to
an increased frequency of the 2/2 genotype in their case population (Hellmig
et al. 2004). Therefore, the evidence for the role of the IL1RN 2/2 genotype in
gastric MALT lymphoma is conflicted at present with the largest study available
showing no significant association.
With regard to the IL1B gene, two single-nucleotide polymorphisms of the C>T
type at positions 31 and 511 of the IL1B gene have been associated with
increased production of IL1B, and the IL1B 511 T allele has been associated
with an increased risk of gastric adenocarcinoma (El-Omar et al. 2000). These
polymorphisms have also been studied in gastric MALT lymphoma patients with
multiple case-control studies of the IL1B-31(Rollinson et al. 2003; Hellmig
et al. 2004; Wu et al. 2004a) and IL1B-511 (Wu et al. 2004a, b) gene poly-
morphisms failing to identify any significant associations.
14.5.2.2 Tumour Necrosis Factor (TNF)
The tumour necrosis factor (TNF) family of cytokines are another group of
pro-inflammatory cytokines, of which the best studied is TNF-α. TNF-α binds
two receptors: TNFR1 present on most tissues and TNFR2 present on cells of the
immune system. Polymorphisms in these genes have been associated with a range
of inflammatory conditions, and in a study published in 2004, Wu and co-workers
studied the associations of TNF-α, TNFR1 and TNFR2 gene polymorphisms with
gastric MALT lymphoma. In this case-control study, 70 patients of Han Chinese
background were compared with 210 healthy controls. The TNFA -857 T poly-
morphism was found to be significantly underrepresented in patients with gastric
MALT lymphoma (6.4 % cases versus 14.3 % controls, OR 0.33, 95 % CI
0.15–0.75) (Wu et al. 2004a). TNFA polymorphisms at positions -308, -863 and
-1031 were not found to be associated with gastric MALT lymphoma
(Wu et al. 2004a). Similarly, polymorphisms in position -383 of the TNFR1 gene
and codon 196 of the TNFR2 gene were not associated with gastric MALT
lymphoma (Wu et al. 2004a). These findings have not been replicated in other
studies. However, they are consistent with our existing understanding of gastric
MALT lymphoma pathogenesis. The TNFA-857 T gene polymorphism has previ-
ously been associated with reduced TNF-α expression in vitro as well as with a
reduced risk of Crohn’s disease (van Heel et al. 2002), one form of inflammatory
bowel disease, suggesting that it may be protective against gastric MALT lym-
phoma by reducing the inflammatory response to H. pylori.
14.5.3 Innate Immunity Genes
14.5.3.1 Toll-Like Receptor 4 and CD14
TLR4 is one of the best studied TLRs and binds lipopolysaccharide, an important
component of the bacterial cell wall. TLR4 has been recognised to play an impor-
tant role in H. pylori-induced gastritis. Gene polymorphisms have therefore been
proposed to play a role in the host genetics of gastric MALT lymphoma patho-
genesis. In a 2005 case-control study by Hellmig and co-workers, 87 patients with
gastric MALT lymphoma from the German-Austrian Lymphoma Study group were
compared against 344 controls positive for H. pylori (Hellmig et al. 2005). They
proposed that the TLR4 Asp299Gly allele, which leads to a diminished inflamma-
tory response to lipopolysaccharide, would be protective against gastric MALT
lymphoma and successfully demonstrated a protective association in this cohort
(4.6 % cases versus 11.6 % controls, p ¼ 0.019, OR 0.37, 95 % CI 0.13–1.03). A
follow-up study by Wu in 2006 attempted to replicate this finding but failed to
identify any Asp299Gly variants (Wu et al. 2006). A smaller study by Türe-
Ozdemir and colleagues published in 2008 studied 56 patients with gastric
MALT lymphoma and 51 H. pylori-infected controls and found no significant
association between carriers of the Asp299Gly allele and MALT lymphoma
(32.1 % cases versus 23.5 % controls, OR 1.54, 95 % CI 0.65–3.63) (Ture-Ozdemir
et al. 2008). Although the findings of Hellmig and co-workers have not been
replicated, they do have biological plausibility based on current understanding of

gastric MALT lymphoma pathogenesis.
Genetic polymorphisms of the gene coding for CD14, another pattern recogni-
tion receptor that acts as a co-receptor with TLR4 for lipopolysaccharide, have also
been investigated for associations with gastric MALT lymphoma. A case-control
study by Wu et al. investigated the -159C/T polymorphism of the CD14 gene. In
this study of 70 gastric MALT lymphoma patients and 210 healthy controls, no
significant association was found between the -159C/T polymorphism and gastric
MALT lymphoma (Wu et al. 2006). However, a smaller study by Türe-Ozdemir
later found a marginally significant association of the -159 T allele with gastric
MALT lymphoma (42.9 % cases versus 23.5 % controls, p 0.042, OR 2.44, 95 % CI
1.06–5.62) (Ture-Ozdemir et al. 2008). They proposed that the CD14 -159C/T
variant, which has been shown to lead to increased CD14 expression and TNF-α
production (Lin et al. 2005), leads to enhanced inflammatory reactions, which in the
case of H. pylori infection could lead to an increased risk of gastric MALT
lymphoma. This hypothesis has biological plausibility and remains consistent
with current understanding of gastric MALT lymphoma pathogenesis.
14.5.3.2 NOD1 and NOD2 Receptors
The NOD-like receptors are another important group of pattern recognition recep-
tor. NOD1 has been shown to be important in the immune response against
H. pylori infection (Viala et al. 2004). Subsequently Rosenstiel and colleagues
investigated the role of NOD1 and NOD2 gene polymorphisms in susceptibility to
gastric MALT lymphoma (Rosenstiel et al. 2006). In this study published in 2006,
83 patients with low-grade H. pylori-associated gastric lymphoma from the
German-Austrian Lymphoma Study group were compared with 428 H. pylori-
infected controls. They found that the rare R702W allele of the NOD2 gene was
associated with an increased risk of gastric MALT lymphomas (10.8 % cases versus
4.9 % controls, p 0.044, OR 2.4, 95 % CI 1.0–5.6) (Rosenstiel et al. 2006).
However, a smaller follow-up study by Türe-Ozdemir failed to replicate this finding
(7.1 % cases versus 11.8 % controls, OR 0.57, 95 % CI 0.15–2.18) (Ture-Ozdemir
et al. 2008). Rosenstiel and co-workers subsequently found, as part of the same
study, that this variant led to reduced NF-κB activation which would be consistent
with the proposal that this polymorphism is protective by limiting H. pylori-induced
gastritis (Rosenstiel et al. 2006).
14.5.3.3 Adaptive Immunity Genes
Studies have suggested that H. pylori-specific T cells may play an important role in
gastric MALT lymphoma pathogenesis. Genetic polymorphisms in genes coding
for important T-cell signalling proteins could therefore have a role to play in
determining which individuals with H. pylori infection go on to develop gastric
MALT lymphoma. Cytotoxic T lymphocyte antigen 4 (CTLA4), present on the

surface of T cells, negatively regulates T-cell activation by binding B7 molecules
on antigen-presenting cells. CD28, also present on the surface of T cells, is
structurally related to CTLA4 and binds B7 but on binding produces a strong
co-stimulatory signal leading to T-cell activation. Inducible co-stimulator (ICOS)
is another molecule expressed by T cells which is part of the CD28/CTLA4 family
and is also thought to play an important role in regulating T cells.
In a case-control study published by Cheng and co-workers, 62 patients with
gastric MALT lymphoma were compared against 250 healthy controls to investi-
gate the role of CTLA4, CD28 and ICOS gene polymorphisms in gastric MALT
lymphoma pathogenesis (Cheng et al. 2006). They found that the CTLA4 -318C/T
genotype was associated with a significantly lower risk of gastric MALT lymphoma
(4.8 % cases versus 16.0 % controls, OR 0.3, 95 % CI 0.1–0.9) and that the CTLA
49G/G genotype was associated with a significantly higher risk of gastric MALT
lymphoma (54.8 % cases versus 47.6 % controls, OR 4.1, 95 % CI 0.9–18.2)
(Cheng et al. 2006). However, when these polymorphisms were studied again
comparing against H. pylori-positive controls, only the CTLA 49G/G genotype
remained significantly associated with gastric MALT lymphoma (OR 6.4, 95 % CI
1.0–50.2) (Binion et al. 1997). No gene polymorphisms of the CD28 or ICOS genes
were found to be associated with gastric MALT lymphoma (Binion et al. 1997).
They propose that decreasing T-cell inhibition associated with the CTLA4 49G
allele leads to an enhanced inflammatory response to H. pylori resulting in an
increased susceptibility to gastric MALT lymphoma.
14.5.3.4 MALT1
Gastric MALT lymphomas are commonly found to have the t(11;18)(q21;q21)

translocation which involve genes MALT1 on chromosome 18 and API2 on
chromosome 11. This creates a fusion protein thought to be important in MALT
lymphoma pathogenesis, suggesting a possible role for germ line mutations of
MALT1 in gastric MALT lymphoma pathogenesis. A study by Hellmig and
co-workers published in 2009 compared 54 patients with high-grade lymphoma
against 344 healthy controls and found that the rare allele G of SNP 3 in the MALT1
gene was protective against high-grade gastric MALT lymphoma (OR 0.2, 95 % CI
0.1–0.6). However, as part of the same study, they failed to replicate the finding in
an independent cohort and concluded that there was no evidence that germ line
mutations in the MALT1 gene have a role to play in gastric lymphoma pathogenesis
(Hellmig et al. 2009).
The common thread in all these findings is that the main driving force in gastric
MALT lymphoma pathogenesis is inflammation, and that genetic polymorphisms
that reduce or enhance the inflammatory response to H. pylori will subsequently
reduce or enhance the risk of gastric MALT lymphoma.
The genetic revolution over the past decade has enabled scientists for the first time
to examine afresh a multitude of unanswered clinical problems. Defining host
genetic factors that control basic physiological processes will explain many of the
seemingly divergent phenotypic expressions of disease. Therefore, the most impor-
tant benefit to the study of host genetics is the better understanding of disease
pathogenesis. In the case of H. pylori infection, host genetics has helped in
confirming two very important facts: first is the essential role of the initial insult
in the form of the microbial challenge (in this case H. pylori), and second is the
important role of chronic inflammation with its long-term deleterious effects on
gastric physiology. As such, the most sensible and practical conclusions from this
knowledge are to either avoid getting the infection in the first place or to remove it
or ameliorate its effects once it is established.
The other benefit to studying host genetic factors is in being able to predict
clinical outcomes following certain exposures (microbial, chemical, dietary, phar-
macological, etc). For example, if we could define who might develop an atrophic,
hypochlorhydric response to H. pylori infection, this could form the basis for
genetic screening so that these individuals could be offered eradication therapy.
As things stand, this approach offers little benefit over simply checking for H. pylori
infection itself. The reason is that the currently identified genetic risk markers are
very common in the population and are not specific enough to act as predictors of
gastric cancer risk. It may be that future advances in affordable high-throughput
genotyping could uncover a much more extensive genetic profile that satisfies the
criteria for a screening test. If such a development were feasible, we must ensure
that our governments enact laws that protect individuals from being discriminated
against on the basis of their genetic heritage. These issues require a mature debate
that has to start now.
References
Abdiev S, Ahn KS, Khadjibaev A, Malikov Y, Bahramov S, Rakhimov B et al (2010) Helicobacter

pylori infection and cytokine gene polymorphisms in Uzbeks. Nagoya J Med Sci 72(3–4):
167–172
Abnet CC, Freedman ND, Hu N, Wang Z, Yu K, Shu X et al (2010) A shared susceptibility locus in
PLCE1 at 10q23 for gastric adenocarcinoma and esophageal squamous cell carcinoma.
Nat Genet 42(9):764–767
Amieva MR (2008) El–Omar EM. Host-Bacterial Interactions in Helicobacter pylori
Infection. Gastroenterology 134(1):306–323
Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN, Jones M et al (2000) TLR4 mutations are
associated with endotoxin hyporesponsiveness in humans. Nat Genet 25(2):187–191
Atsuta Y, Ito LS, Oba-Shinjo SM, Uno M, Shinjo SK, Marie SK et al (2006) Associations of TNF-A-
1031TT and -857TT genotypes with Helicobacter pylori seropositivity and gastric atrophy
among Japanese Brazilians. Int J Clin Oncol 11(2):140–145
Backhed F, Rokbi B, Torstensson E, Zhao Y, Nilsson C, Seguin D et al (2003) Gastric mucosal

recognition of Helicobacter pylori is independent of Toll-like receptor 4. J Infect Dis 187(5):
#2#829–836
Binion DG, West GA, Ina K, Ziats NP, Emancipator SN, Fiocchi C (1997) Enhanced leukocyte
binding by intestinal microvascular endothelial cells in inflammatory bowel disease. Gastro-
enterology 112(6):1895–1907
Boltin D, Niv Y (2013) Mucins in gastric cancer – an update. J Gastrointest Dig Syst 3(123):15519
Camargo MC, Mera R, Correa P, Peek RM Jr, Fontham ET, Goodman KJ et al (2006) Interleukin-
1beta and interleukin-1 receptor antagonist gene polymorphisms and gastric cancer: a meta-
analysis. Cancer Epidemiol Biomarkers Prev 15(9):1674–1687
Cheng TY, Lin JT, Chen LT, Shun CT, Wang HP, Lin MT et al (2006) Association of T-cell
regulatory gene polymorphisms with susceptibility to gastric mucosa-associated lymphoid tissue
lymphoma. J Clin Oncol 24(21):3483–3489
Eck M, Schmausser B, Haas R, Greiner A, Czub S, Muller-Hermelink H (1997) MALT-type
lymphoma of the stomach is associated with Helicobacter pylori strains expressing the
CagA protein. Gastroenterology 112(5):1482–1486
El-Omar EM (2001) The importance of interleukin 1beta in Helicobacter pylori associated disease.
Gut 48(6):743–747
El-Omar EM (2013) Helicobacter pylori susceptibility in the GWAS era. JAMA 309(18):
1939–1940
El-Omar EM, Penman ID, Ardill JE, Chittajallu RS, Howie C, McColl KE (1995) 
Helicobacter pylori infection and abnormalities of acid secretion in patients with duo-
denal ulcer disease. Gastroenterology 109(3):681–691
El-Omar EM, Oien K, El-Nujumi A, Gillen D, Wirz A, Dahill S et al (1997) Helicobacter pylori
infection and chronic gastric acid hyposecretion. Gastroenterology 113(1):15–24
El-Omar EM, Carrington M, Chow WH, McColl KEL, Bream JH, Young HA et al (2000)
Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 404:
398–402
El-Omar EM, Ng MT, Hold GL (2008) Polymorphisms in Toll-like receptor genes and risk of
cancer. Oncogene 27(2):244–252
Fischbach W (2013) Gastric mucosal-associated lymphoid tissue lymphoma. Gastroenterol Clin
North Am 42(2):371–380
Gu X, Zhang W, Xu L, Cai D (2014) Quantitative assessment of the influence of prostate stem cell
antigen polymorphisms on gastric cancer risk. Tumor Biol 35(3):2167–2174
Guan Y, Omueti-Ayoade K, Mutha SK, Hergenrother PJ, Tapping RI (2010) Identification of
novel synthetic toll-like receptor 2 agonists by high throughput screening. J Biol Chem
285(31):23755–23762
Guang W, Ding H, Czinn SJ, Kim KC, Blanchard TG, Lillehoj EP (2010) Muc1 cell surface mucin
attenuates epithelial inflammation in response to a common mucosal pathogen. J Biol Chem
285(27):20547–20557
Hamajima N, Shibata A, Katsuda N, Matsuo K, Ito H, Saito T et al (2003) Subjects with TNF-A-
857TT and-1031TT genotypes showed the highest Helicobacter pylori seropositive rate
compared with those with other genotypes. Gastric Cancer 6(4):230–236
He B, Zhang Y, Pan Y, Xu Y, Gu L, Chen L et al (2011) Interleukin 1 beta (IL1B) promoter
polymorphism and cancer risk: evidence from 47 published studies. Mutagenesis 26(5):
637–642
He C, Chen M, Liu J, Yuan Y (2014) Host genetic factors respond to pathogenic step-specific
virulence factors of Helicobacter pylori in gastric carcinogenesis. Mutat Res Rev
Mutat Res 759:14–26
Hellmig S, Vollenberg S, Goebeler-Kolve ME, Fischbach W, Hampe J, Folsch UR et al (2004)
IL-1 gene cluster polymorphisms and development of primary gastric B-cell lymphoma in
Helicobacter pylori infection. Blood 104(9):2994–2995
Hellmig S, Fischbach W, Goebeler-Kolve M, F€olsch UR, Hampe J, Schreiber S (2005) Associ-

ation study of a functional Toll-like receptor 4 polymorphism with susceptibility to
gastric mucosa-associated lymphoid tissue lymphoma. Leuk Lymphoma 46(6):869–872
Hellmig S, Bartscht T, Fischbach W, Ott SJ, Rosenstiel P, Klapper W et al (2009) Germline
variations of the MALT1 gene as risk factors in the development of primary gastric
B-cell lymphoma. Eur J Cancer 45(10):1865–1870
Iida T, Iwahashi M, Katsuda M, Ishida K, Nakamori M, Nakamura M et al (2011) Tumor-infiltrating
CD4 Th17 cells produce IL-17 in tumor microenvironment and promote tumor progression in
human gastric cancer. Oncol Rep 25(5):1271–1277
Jia Y, Persson C, Hou L, Zheng Z, Yeager M, Lissowska J et al (2010) A comprehensive analysis
of common genetic variation in MUC1, MUC5AC, MUC6 genes and risk of stomach cancer.
Cancer Causes Control 21(2):313–321
Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik S et al (2007) Crystal structure of the TLR1-
TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130(6):1071–1082
Kabir S (2011) The role of interleukin‐17 in the Helicobacter pylori induced infection and
immunity. Helicobacter 16(1):1–8
Kang M, Ding X, Xu M, Zhu H, Liu S, Wang M et al (2014) Genetic variation rs10484761 on 6p21.
1 derived from a genome-wide association study is associated with gastric cancer survival in a
Chinese population. Gene 536(1):59–64
Kawahara T, Teshima S, Oka A, Sugiyama T, Kishi K, Rokutan K (2001) Type I Helicobacter
pylori lipopolysaccharide stimulates toll-like receptor 4 and activates mitogen oxidase 1 in
gastric pit cells. Infect Immun 69(7):4382–4389
Kimang’a AN (2012) IL-1B-511 Allele T and IL-1RN-L/L play a pathological role in Helicobacter
pylori (H. pylori) disease outcome in the African population. Ethiop J Health Sci 22(3):163–169
Kufe DW (2009) Mucins in cancer: function, prognosis and therapy. Nat Rev Cancer 9(12):
874–885
Kufe DW (2012) MUC1-C oncoprotein as a target in breast cancer: activation of signaling pathways
and therapeutic approaches. Oncogene 32(9):1073–1081
Lin J, Yao YM, Huang ZH, Hou XX, Yu Y, Sheng ZY (2005) CD14 genomic polymorphism
influences CD14 expression in whole blood culture. Zhonghua Wai Ke Za Zhi 43(15):
1024–1027
Liou J, Lin J, Wang H, Huang S, Lee Y, Chiu H et al (2007) IL‐1B‐511 C!T polymorphism is
associated with increased host susceptibility to Helicobacter pylori infection in Chinese.
Helicobacter 12(2):142–149
Lochhead P, Frank B, Hold GL, Rabkin CS, Ng MT, Vaughan TL et al (2011) Genetic variation in the
 prostate stem cell antigen gene and upper gastrointestinal cancer in white individuals.
Gastroenterology 140(2):435–441
Loh M, Koh KX, Yeo BH, Song CM, Chia KS, Zhu F et al (2009) Meta-analysis of
genetic polymorphisms and gastric cancer risk: variability in associations according to race.
Eur J Cancer 45(14):2562–2568
Lu Y, Chen J, Ding Y, Jin G, Wu J, Huang H et al (2010) Genetic variation of PSCA gene is
associated with the risk of both diffuse‐and intestinal‐type gastric cancer in a Chinese popu-
lation. Int J Cancer 127(9):2183–2189
Maeda S, Akanuma M, Mitsuno Y, Hirata Y, Ogura K, Yoshida H et al (2001) Distinct mechanism
of Helicobacter pylori-mediated NF-kappa B activation between gastric cancer cells and
monocytic cells. J Biol Chem 276(48):44856–44864
Malaty HM, Engstrand L, Pedersen NL, Graham DY (1994) Helicobacter pylori infection: genetic
and environmental influences: a study of twins. Ann Intern Med 120(12):982–986
Mandell L, Moran AP, Cocchiarella A, Houghton JM, Taylor N, Fox JG et al (2004) Intact gram-
negative Helicobacter pylori, Helicobacter felis, and Helicobacter hepaticus bacteria activate
innate immunity via toll-like receptor 2 but not toll-like receptor 4 editor: FC Fang.
Infect Immun 72(11):6446–6454
Marin F, Bonet C, Munoz X, Garcia N, Pardo ML, Ruiz-Liso JM et al (2012) Genetic variation in
MUC1, MUC2 and MUC6 genes and evolution of gastric cancer precursor lesions in a long-
term follow-up in a high-risk area in Spain. Carcinogenesis 33(5):1072–1080
Maruyama T, Kono K, Mizukami Y, Kawaguchi Y, Mimura K, Watanabe M et al (2010) Distri-
bution of Th17 cells and FoxP3 (+) regulatory T cells in tumor‐infiltrating lymphocytes, tumor‐
draining lymph nodes and peripheral blood lymphocytes in patients with gastric cancer.
Cancer Sci 101(9):1947–1954
Matsuo K, Tajima K, Suzuki T, Kawase T, Watanabe M, Shitara K et al (2009) Association of
prostate stem cell antigen gene polymorphisms with the risk of stomach cancer in Japanese.
Int J Cancer 125(8):1961–1964
Mayerle J, den Hoed CM, Schurmann C, Stolk L, Homuth G, Peters MJ et al (2013) Identification
of genetic loci associated with Helicobacter pylori Serologic StatusGWAS of H. pylori
infection susceptibility. JAMA 309(18):1912–1920
Meng XY, Zhou CH, Ma J, Jiang C, Ji P (2012) Expression of interleukin-17 and its
clinical significance in gastric cancer patients. Med Oncol 29(5):3024–3028
Miwa H, Go MF, Sato N (2002) H. pylori and gastric cancer: the Asian enigma. Am J
Gastroenterol 97(5):1106–1112
Mukhopadhyay P, Chakraborty S, Ponnusamy MP, Lakshmanan I, Jain M, Batra SK (2011)
Mucins in the pathogenesis of breast cancer: implications in diagnosis, prognosis and therapy.
Biochim Biophys Acta (BBA)-Rev Cancer 1815(2):224–240
Palmer AJ, Lochhead P, Hold GL, Rabkin CS, Chow WH, Lissowska J et al (2012) Genetic
variation in C20orf54, PLCE1 and MUC1 and the risk of upper gastrointestinal cancers in
Caucasian populations. Eur J Cancer Prev 21(6):541–544
Persson C, Canedo P, Machado JC, El-Omar EM, Forman D (2011) Polymorphisms in inflamma-
tory response genes and their association with gastric cancer: a HuGE systematic review and
meta-analyses. Am J Epidemiol 173(3):259–270
Prinz C, Schwendy S, Voland P (2006) H. pylori and gastric cancer: shifting the global burden.
World J Gastroenterol 12(34):5458
Qiao L, Feng Y (2012) Genetic variations of prostate stem cell antigen (PSCA) contribute to the
risk of gastric cancer for Eastern Asians: a meta-analysis based on 16792 individuals.
Gene 493(1):83–91
Qinghai Z, Yanying W, Yunfang C, Xukui Z, Xiaoqiao Z (2014) Effect of interleukin-17A and
interleukin-17F gene polymorphisms on the risk of gastric cancer in a Chinese population.
Gene 537(2):328–332
Rafiei A, Hosseini V, Janbabai G, Ghorbani A, Ajami A, Farzmandfar T et al (2013) Poly-
morphism in the interleukin-17A promoter contributes to gastric cancer. World J Gastroenterol
WJG 19(34):5693
Rebbeck TR (1997) Molecular epidemiology of the human glutathione S-transferase genotypes
GSTM1 and GSTT1 in cancer susceptibility. Cancer Epidemiol Biomarkers Prev 6(9):733–743
Rollinson S, Levene AP, Mensah FK, Roddam PL, Allan JM, Diss TC et al (2003) Gastric
marginal zone lymphoma is associated with polymorphisms in genes involved in inflammatory
response and antioxidative capacity. Blood 102(3):1007–1011
Rosenstiel P, Hellmig S, Hampe J, Ott S, Till A, Fischbach W et al (2006) Influence of poly-
morphisms in the NOD1/CARD4 and NOD2/CARD15 genes on the clinical outcome of
Helicobacter pylori infection. Cell Microbiol 8(7):1188–1198
Saeidnia S, Abdollahi M (2013) Antioxidants: friends or foe in prevention or treatment of cancer:
the debate of the century. Toxicol Appl Pharmacol 271(1):49–63
Saeki N, Saito A, Choi IJ, Matsuo K, Ohnami S, Totsuka H et al (2011) A functional single
nucleotide polymorphism in Mucin 1 at chromosome 1q22, determines susceptibility
to diffuse-type gastric cancer. Gastroenterology 140(3):892–902
Saeki N, Ono H, Sakamoto H, Yoshida T (2013) Genetic factors related to gastric cancer
susceptibility identified using a genome‐wide association study. Cancer Sci 104(1):1–8
Saijo Y, Yoshioka E, Fukui T, Kawaharada M, Sata F, Sato H et al (2007) H. pylori seropositivity

and cytokine gene polymorphisms. World J Gastroenterol 13(33):4445
Sakamoto H, Yoshimura K, Saeki N, Katai H, Shimoda T, Matsuno Y et al (2008) Genetic
variation in PSCA is associated with susceptibility to diffuse-type gastric cancer. Nat Genet
40(6):730–740
Sala N, Mu~ noz X, Travier N, Agudo A, Duell EJ, Moreno V et al (2012) Prostate stem‐cell antigen
gene is associated with diffuse and intestinal gastric cancer in Caucasians: results from the
EPIC‐EURGAST study. Int J Cancer 130(10):2417–2427
Salmon JE, Edberg JC, Brogle NL, Kimberly RP (1992) Allelic polymorphisms of human Fc
gamma receptor IIA and Fc gamma receptor IIIB. Independent mechanisms for differences in
human phagocyte function. J Clin Invest 89(4):1274–1281
Senapati S, Das S, Batra SK (2010) Mucin-interacting proteins: from function to therapeutics.
Trends Biochem Sci 35(4):236–245
Sheng Y, Triyana S, Wang R, Das I, Gerloff K, Florin T et al (2012) MUC1 and MUC13
differentially regulate epithelial inflammation in response to inflammatory and infectious
stimuli. Mucosal Immunol 6(3):557–568
Shi Y, Hu Z, Wu C, Dai J, Li H, Dong J et al (2011) A genome-wide association study identifies
new susceptibility loci for non-cardia gastric cancer at 3q13. 31 and 5p13. 1. Nat Genet 43(12):
1215–1218
Shi D, Wang S, Gu D, Wu D, Wang M, Chu H et al (2012) The PSCA polymorphisms derived
from genome-wide association study are associated with risk of gastric cancer: a meta-analysis.
J Cancer Res Clin Oncol 138(8):1339–1345
Shibata T, Tahara T, Hirata I, Arisawa T (2009) Genetic polymorphism of interleukin-17A
 and -17F genes in gastric carcinogenesis. Hum Immunol 70(7):547–551
Silva F, Carvalho F, Peixoto A, Seixas M, Almeida R, Carneiro F et al (2001) MUC1 gene
polymorphism in the gastric carcinogenesis pathway. Eur J Hum Genet 9(7):548–552
Smith MF Jr, Mitchell A, Li G, Ding S, Fitzmaurice AM, Ryan K et al (2003) Toll-like receptor
(TLR) 2 and TLR5, but not TLR4, are required for Helicobacter pylori-induced NF-kappa B
activation and chemokine expression by epithelial cells. J Biol Chem 278(35):32552–32560
Song H, Kim HN, Kweon S, Choi J, Shim HJ, Cho SH et al (2014) Common genetic variants at
1q22 and 10q23 and gastric cancer susceptibility in a Korean population. Tumor Biol 35(4):
3133–3137
Strange RC, Fryer AA (1999) The glutathione S-transferases: influence of polymorphism on
cancer susceptibility. IARC Sci Publ 148:231–249
Takeuchi O, Sato S, Horiuchi T, Hoshino K, Takeda K, Dong Z et al (2002) Cutting edge: role of
Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol
169(1):10–14
Tseng F, Brown EE, Maiese EM, Yeager M, Welch R, Gold BD et al (2006) Polymorphisms in
cytokine genes and risk of Helicobacter pylori infection among Jamaican children.
Helicobacter 11(5):425–430
Tu S, Bhagat G, Cui G, Takaishi S, Kurt-Jones EA, Rickman B et al (2008) Overexpression of
interleukin-1β induces gastric inflammation and cancer and mobilizes myeloid-derived sup-
pressor cells in mice. Cancer Cell 14(5):408–419
Ture-Ozdemir F, Gazouli M, Tzivras M, Panagos C, Bovaretos N, Petraki K et al (2008) Asso-
ciation of polymorphisms of NOD2, TLR4 and CD14 genes with susceptibility to gastric
mucosa-associated lymphoid tissue lymphoma. Anticancer Res 28(6A):3697–3700
Ueno K, Koga T, Kato K, Golenbock DT, Gendler SJ, Kai H et al (2008) MUC1 mucin is a
negative regulator of toll-like receptor signaling. Am J Respir Cell Mol Biol 38(3):263–268
van Heel DA, Udalova IA, De Silva AP, McGovern DP, Kinouchi Y, Hull J et al (2002)
Inflammatory bowel disease is associated with a TNF polymorphism that affects an interaction
between the OCT1 and NF(-kappa)B transcription factors. Hum Mol Genet 11(11):1281–1289
Viala J, Chaput C, Boneca IG, Cardona A, Girardin SE, Moran AP et al (2004) Nod1 responds to
peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat Immunol
5(11):1166–1174
Vincenzi B, Patti G, Galluzzo S, Pantano F, Venditti O, Santini D et al (2008) Interleukin 1ß-511T
gene (IL1ß) polymorphism is correlated with gastric cancer in the Caucasian population:
results from a meta-analysis. Oncol Rep 20(5):1213–1220
Wang M, Bai J, Tan Y, Wang S, Tian Y, Gong W et al (2011) Genetic variant in PSCA predicts
survival of diffuse‐type gastric cancer in a Chinese population. Int J Cancer 129(5):1207–1213
Wang T, Zhang L, Li H, Wang B, Chen K (2012) Prostate stem cell antigen polymorphisms and
susceptibility to gastric cancer: a systematic review and meta-analysis. Cancer Epidemiol
Biomarkers Prev 21(5):843–850
Wu M, Chen L, Shun C, Huang S, Chiu H, Wang H et al (2004a) Promoter polymorphisms of
tumor necrosis factor‐α are associated with risk of gastric mucosa–associated lymphoid tissue
lymphoma. Int J Cancer 110(5):695–700
Wu MS, Shun CT, Huang SP, Cheng AL, Chen LT, Lin JT (2004b) Effect of interleukin-1beta and
glutathione S-transferase genotypes on the development of gastric mucosa-associated lym-
phoid tissue lymphoma. Haematologica 89(8):1015–1017
Wu MS, Cheng TY, Shun CT, Lin MT, Chen LC, Lin JT (2006) Functional polymorphisms of
CD14 and toll-like receptor 4 in Taiwanese Chinese with Helicobacter pylori-related
gastric malignancies. Hepatogastroenterology 53(71):807–810
Wu C, Wang G, Yang M, Huang L, Yu D, Tan W et al (2009) Two genetic variants in prostate stem
cell antigen and gastric cancer susceptibility in a Chinese population. Mol Carcinog 48(12):
1131–1138
Xu Q, Yuan Y, Sun L, Gong Y, Xu Y, Yu X et al (2009) Risk of gastric cancer is associated with
the MUC1 568 A/G polymorphism. Int J Oncol 35(6):1313
Yea SS, Yang YI, Jang WH, Lee YJ, Bae HS, Paik KH (2001) Association between TNF-alpha
promoter polymorphism and Helicobacter pylori cagA subtype infection. J Clin Pathol 54(9):
703–706
Zeng Z, Wu X, Chen F, Yu J, Xue L, Hao Y et al (2011) Polymorphisms in prostate stem cell
antigen gene rs2294008 increase gastric cancer risk in Chinese. Mol Carcinog 50(5):353–358
Zhang H, Jin G, Li H, Ren C, Ding Y, Zhang Q et al (2011) Genetic variants at 1q22 and 10q23
reproducibly associated with gastric cancer susceptibility in a Chinese population. Carcino-
genesis 32(6):848–852
Zhang T, Chen Y, Wang Z, Chen J, Huang S (2012) Effect of PSCA gene polymorphisms on
gastric cancer risk and survival prediction: a meta-analysis. Exp Ther Med 4(1):158–164
Zhang X, Zheng L, Sun Y, Zhang X (2014) Analysis of the association of interleukin-17 gene
polymorphisms with gastric cancer risk and interaction with Helicobacter pylori infection in a
Chinese population. Tumor Biol 35(2):1575–1580
Zhao J, Geng P, Li Z, Cui S, Zhao J, Wang L et al (2012) Associations between interleukin-1
polymorphisms and gastric cancers among three ethnicities. World J Gastroenterol WJG
18(47):7093
Zheng L, Zhu C, Gu J, Xi P, Du J, Jin G (2013) Functional polymorphism rs4072037 in MUC1
gene contributes to the susceptibility to gastric cancer: evidence from pooled 6,580 cases and
10,324 controls. Mol Biol Rep 40(10):5791–5796
Zhuang Y, Peng L, Zhao Y, Shi Y, Mao X, Chen W et al (2012) CD8 T cells that
produce interleukin-17 regulate myeloid-derived suppressor cells and are associated with
survival time of patients with gastric cancer. Gastroenterology 143(4):951–962.e8
Zou W, Restifo NP (2010) TH17 cells in tumour immunity and immunotherapy. Nat Rev Immunol
10(4):248–256
Part III
Helicobacter pylori-Associated Diseases
Chapter 15
Helicobacter pylori and Nonmalignant
Diseases
Doron Boltin and Yaron Niv
Abstract Infection with Helicobacter pylori initially leads to superficial gastritis

and usually progresses to chronic active gastritis. Although the vast majority of
chronically infected subjects remain asymptomatic, somewhere between 10 % and
15 % develop peptic ulcer disease. Most commonly, H. pylori infection is located in
the gastric antrum, where it acts to inhibit somatostatin release and thus stimulate
acid secretion, causing duodenal ulceration. Patients with pangastritis are
predisposed to developing gastric ulceration. Although population-based studies
show that patients with gastroesophageal reflux disease have a lower likelihood of
H. pylori infection, there is no robust evidence suggesting that eradication of
H. pylori leads to erosive esophagitis. Patients who lack endoscopic evidence of
disease such as peptic ulceration (i.e., functional dyspepsia) experience a greater
reduction in their dyspeptic symptoms following eradication of H. pylori as com-
pared to placebo. The pathophysiology of H. pylori-mediated dyspepsia in these
patients probably involves increased acid, decreased ghrelin, and altered gastric
emptying. Eradication of H. pylori in patients with functional dyspepsia has the
added benefit of preventing future peptic ulcer disease, especially in Asian
populations. H. pylori has been weakly linked to various nonmalignant conditions
ranging from halitosis to coronary heart disease. However, evidence in support of
actively seeking and treating H. pylori exists only for idiopathic thrombocytopenic
purpura and iron deficiency anemia. In epidemiological studies, H. pylori infection
has been shown to be inversely associated with Crohn’s disease and asthma.
Keywords Helicobacter pylori • Peptic ulcer • Gastroesophageal reflux •

Dyspepsia
D. Boltin (*) • Y. Niv

Department of Gastroenterology, Rabin Medical Center, Beilinson Campus, 39 Jabotinski
Street, Petah Tikva 49100, Israel

DOI 10.1007/978-4-431-55936-8_15
366 D. Boltin and Y. Niv
15.1 Introduction
Helicobacter pylori has been implicated as a precipitating factor, a perpetuating

factor, or a protective factor, in various nonmalignant diseases. H. pylori may exert
its effect via direct toxicity or via indirect paracrine, endocrine, neurocrine, or
immune pathways. Foremost, H. pylori is associated with the formation, compli-
cation, and recurrence of peptic ulcer disease, both as a solitary factor and in
conjunction with nonsteroidal anti-inflammatory drugs (NSAIDs). Testing for
H. pylori and subsequent eradication is therefore essential in patients with a history
of ulcer bleeding or prior to commencing long-term NSAID therapy. On the other
hand, evidence of a role for H. pylori in causing functional dyspepsia is less robust,
and the likelihood of symptomatic improvement following eradication varies
greatly across geographic region. H. pylori infection offers theoretical protection
against gastroesophageal reflux disease (GERD), owing to the hypochlorhydria
typically manifest in the setting of pangastritis. Indeed, the prevalence of infection
observed in subjects with both erosive and nonerosive GERD is reduced. Never-
theless, there is no evidence that GERD may develop following H. pylori eradica-
tion nor is there evidence that preexisting GERD or Barrett’s esophagus may
worsen. Epidemiological studies have found an inverse relationship between
H. pylori infection and atopic disease and Crohn’s disease, and various candidate
immune mechanisms have been explored. As yet, no causal relationship has been
established.
15.2 Peptic Ulcer Disease
15.2.1 Pathophysiology
Peptic ulcer disease (PUD) occurs when mucosal defense mechanisms are
overwhelmed by the destructive effects of acid and pepsin and most commonly
result from H. pylori infection or NSAIDs (Table 15.1). Although acute infection
with H. pylori causes hypochlorhydria, chronic H. pylori infection may cause either
hypo- or hyperchlorhydria. Whether H. pylori ultimately increases or decreases
gastric acid secretion depends upon the severity and distribution of gastritis (Schu-
bert and Peura 2008). Pangastritis usually results in decreased acid secretion. This is
probably mediated by inflammatory cytokines and bacterial toxins including the
vacuolating cytotoxin (VacA) gene product and cytotoxin-associated gene A
(CagA) product (Fig. 15.1). This pattern of gastritis is seen in approximately
85 % of subjects with chronic H. pylori infection and predisposes to gastric
ulceration. Conversely, 15 % of subjects with chronic H. pylori infection may
develop antral predominant gastritis, which is characterized by reduced antral
secretion of somatostatin. This in turn results in elevated basal and stimulated
15 Helicobacter pylori and Nonmalignant Diseases 367
Table 15.1 Etiology of Common

peptic ulcer disease
H. pylori infection
NSAIDs/aspirin
Uncommon
Hypersecretion
Zollinger–Ellison syndrome
Systemic mastocytosis
Medications (usually in combination with NSAIDs)
Bisphosphonates
Corticosteroids
Potassium chloride
5-Fluorouracil
Sirolimus
Mycophenolate mofetil
Infiltrative disease
Carcinoma/lymphoma
Sarcoidosis
Crohn’s disease
Infections
Helicobacter heilmannii
Herpes simplex virus
Cytomegalovirus
Tuberculosis
Syphilis
Ischemia
Atherosclerosis/embolic disease
Cameron ulcer
Cocaine
Severe systemic disease
gastrin secretion and increased corporal acid production. This pattern of gastritis
predisposes to duodenal ulceration.
15.2.2 Epidemiology
The population-based prevalence of peptic ulcer disease depends greatly on ethnic,

geographic, and socioeconomic factors, as well as the method of data collection. A
recent meta-analysis with pooled data from the USA, Europe, and Israel found an
annual PUD incidence of 0.03–0.19 % and a 1-year prevalence of 0.1–1.5 % (Sung
et al. 2009). The occurrence of PUD has been decreasing over the past few decades
in most Western populations. The prevalence of H. pylori infection in duodenal
ulcers was reported to be 84 % in studies published from 1999 to 2003 (Fig. 15.2),
Fig. 15.1 Regulation of gastric acid secretion. Acute infection with H. pylori activates CGRP
neurons to stimulate SST and thus inhibit gastrin secretion. In duodenal ulcer patients with chronic
antral H. pylori infection, the organism or cytokines released from the inflammatory infiltrate
inhibit SST and thus stimulate gastrin and hence acid secretion. When H. pylori colonizes the
gastric body, bacterial toxins such as VacA and CagA act to decrease acid secretion; however, the
precise mechanism is unknown
Fig. 15.2 Pie charts depicting the etiology of peptic ulcer disease. The percentages shown are
based on studies conducted in Western populations. Such a representation is clearly simplistic, as
H. pylori and NSAID use often coexist. Similarly, the relative proportion of each factor varies
according to age, ethnicity, and socioeconomic status
whereas in studies published from 2004 to 2008, the prevalence of H. pylori was
77 % (Gisbert and Calvet 2009). However, this trend might not be apparent in some
Asian populations, where the incidence of H. pylori gastric ulcer could be increas-
ing (Jang et al. 2008).
The declining incidence of PUD has occurred in parallel to a decline in H. pylori
infection rates. In areas of high H. pylori prevalence (e.g., in Asia), H. pylori causes
almost all uncomplicated duodenal ulcers and more than 80 % of gastric ulcers,
especially if the previous use of NSAIDs has been excluded (Gisbert et al. 1999). In
low-prevalence areas such as the USA, H. pylori accounts for a smaller proportion
of PUD (Table 15.2). However, evidence supporting this is derived from retrospec-
tive cohorts, which do not fully consider NSAID and proton pump inhibitor (PPI)
use (Jyotheeswaran et al. 1998; Ciociola et al. 1999).
15.2.3 Treatment of PUD
Eradication of H. pylori infection undoubtedly alters the natural history of PUD.

Several studies have shown that ulcer recurrence is very low or nonexistent 1 year
following eradication. This is in stark comparison with the natural recurrence rate
of over 70 % (Marshall et al. 1988). Following failure of H. pylori eradication, ulcer
recurrence may exceed 50 % (Fiocca et al. 1991). The details of medical treatment
for H. pylori infection are covered in Chap. 20.
15.2.4 Complications of PUD
15.2.4.1 Bleeding
Complications of PUD develop in 20–25 % of patients and include hemorrhage,

perforation, penetration, and obstruction. Bleeding is the most frequent complica-
tion and accounts for approximately 70 % of complicated PUD. Bleeding is the
major cause of PUD-associated morbidity and mortality. H. pylori-associated ulcers
are thought to confer a lower risk of bleeding compared to NSAID or idiopathic
ulcers (Vaira et al. 1997; Boltin et al. 2012). Gisbert and coworkers reviewed
32 studies and found H. pylori in 80 % of bleeding peptic ulcers which was lower
than the positivity rate in non-bleeding ulcers (Gisbert and Abraira 2006). However,
when delayed detection techniques are employed to determine H. pylori infection,
sensitivity is greatly improved, and the prevalence of H. pylori in bleeding ulcers
approaches that of non-bleeding ulcers. This was shown in an analysis of 71 studies
including 8496 subjects, where H. pylori was found to increase the risk of ulcer
bleeding by a factor of 1.79 (Sánchez-Delgado et al. 2011). CagA-positive strains of
H. pylori confer an even higher risk of ulcer bleeding (Stack et al. 2002).
Prospective long-term data indicate that the risk of rebleeding is virtually
eliminated following H. pylori eradication (Gisbert et al. 2012). Persistence of
H. pylori appears to be one of the most important factors causing rebleeding in
patients with ulcer bleeding. For this reason, PPI treatment is recommended until
the documented healing of a gastric ulcer, or until H. pylori eradication for
duodenal ulcers, but not beyond (Malfertheiner et al. 2012).
Table 15.2 Large studies (N 100) performed in Western countries evaluating Helicobacter
pylori prevalence in patients with duodenal ulcer (1999–2013)
Author Year Country Number of patients Prevalence of H. pylori (%)
Ciociola et al. 1999 The USA 2394 73
Gisbert et al. 1999 Spain 774 95
Higuchi et al. 1999 Japan 330 96
Tsuji et al. 1999 Japan 120 96
Aoyama et al. 2000 Japan 111 98
Arakawa et al. 2000 Japan 368 96
Meucci et al. 2000 Italy 317 92
Nishikawa et al. 2000 Japan 152 99
Bytzer et al. 2001 Denmark 276 88
Lutgen et al. 2001 France 152 79
Spaziani et al. 2001 Italy 240 75
Sugiyama et al. 2001 Japan 151 99
Xia et al. 2001 Hong Kong 599 79
Palli et al. 2002 The UK 275 90
Kamada et al. 2003 Japan 464 97
Arents et al. 2004 The Netherlands 254 89
Arroyo et al. 2004 Spain 472 96
Kato et al. 2004 Japan 100 83
Chu et al. 2005 Hong Kong 1343 70
Xia et al. 2005 Hong Kong 271 90
Ong et al. 2006 The UK 288 69
Pietroiusti et al. 2008 Italy 608 93
Chen et al. 2010 Taiwan 626 88.7
Ortega et al. 2010 Chile 5664 86.6
Li et al. 2010 China 1030 92.6
Musumba et al. 2012 The UK 386 66
Cekin et al. 2012 Turkey 222 84.9
Buzás et al. 2013 Hungary 4647 59.1
15.2.4.2 Perforation
H. pylori eradication is similarly beneficial following perforation of a peptic ulcer.

A large meta-analysis found that H. pylori eradication combined with operative
management resulted in a lower rate of ulcer recurrence at 1 year, compared to
surgery followed by PPI therapy alone (5.2 % vs. 35.2 %) (Tomtitchong
et al. 2012). Therefore, after surgical management of perforated peptic ulcer,
H. pylori eradication is recommended in all infected patients in order to prevent
ulcer relapse.
15.2.4.3 Gastric Outlet Obstruction
In a shift from the traditional approach of managing obstructing duodenal ulcers

surgically, most current guidelines recommended conservative treatment with
H. pylori eradication. A nonsurgical approach may also combine enteral nutritional
support and endoscopic dilatation. Surgery should be reserved for salvage treatment
in patients unresponsive to medical therapy (Gisbert and Pajares 2002).
15.2.5 H. pylori, Aspirin, and NSAIDs
Both H. pylori and NSAIDs are established risk factors for PUD and ulcer-related
bleeding. When these two factors are simultaneously present, the potential for
complications is compounded, even though it is impossible to know precisely the
individual contribution of each factor (Huang et al. 2002). H. pylori increases the
risk of NSAID-related mucosal injury (Lanza et al. 2009). So too, patients with
complicated PUD who continue to receive NSAIDs/aspirin following H. pylori
eradication have an increased incidence of rebleeding.
Naı̈ve patients, without prior PUD, who are set to commence long-term therapy
with NSAIDs/aspirin clearly benefit from H. pylori eradication. This is based on
large, well-designed randomized controlled trials (Chan et al. 1997). In subjects
already receiving long-term NSAIDs/aspirin, there is no obvious benefit in treating
H. pylori. A meta-analysis concluded that long-term PPI is more effective than
H. pylori eradication for the prevention of ulcer bleeding in patients already
receiving NSAIDS (Vergara et al. 2005). In this study, 2.6 % of subjects developed
PUD following eradication, whereas none of the patients receiving PPI developed
an ulcer, despite persistent H. pylori infection.
Patients receiving low-dose aspirin are at minimal risk of PUD and ulcer-related
bleeding. A prospective 10-year cohort study including 904 subjects receiving
low-dose aspirin compared three patient groups: H. pylori-positive patients with
bleeding ulcers who resumed aspirin following eradication, H. pylori-negative
patients with bleeding ulcers who resumed aspirin following ulcer healing, and
new users of aspirin without a history of ulcers (average risk) (Chan et al. 2013).
None of the patients received PPI treatment. The rate of ulcer bleeding in patients
with previous ulcer bleeding following eradication was similar to the average-risk
group (0.97 % and 0.66 %, respectively). Recurrent ulcer bleeding was highest in
aspirin users without prior H. pylori infection (5.22 %). In keeping with these
findings, the Maastricht IV/Florence Consensus Report recommends that patients
with a history of PUD should be tested for H. pylori and following eradication these
patients do not require long-term PPI (Malfertheiner et al. 2012). H. pylori-negative
patients should receive adequate antisecretory therapy if they have a history of ulcer
bleeding, since they are prone to ulcer bleeding with aspirin use.
15.3 Gastroesophageal Reflux Disease
The most commonly cited cause for GERD is transient relaxation of the lower
esophageal sphincter. Additional causes include a hypotensive sphincter, hiatal
hernia, and acid hypersecretion. Chronic H. pylori infection is commonly associ-
ated with hypochlorhydria, and therefore patients harboring H. pylori are theoret-
ically protected from GERD and may even exhibit an augmented response to proton
pump inhibitors. Following eradication of H. pylori, acid hypersecretion may occur
for up to 8 weeks due to increased parietal and ECL cell masses; however, these
changes are short-lived and GERD does not generally ensue (Gillen et al. 1999).
15.3.2 Epidemiology
GERD affects 25–40 % of the population. Population-based studies have consis-

tently found a lower prevalence of H. pylori infection among patients with GERD,
especially CagA-positive strains. A systematic review including 20 studies and
4134 patients found a lower prevalence of H. pylori in GERD subjects compared to
controls (OR 0.60, 95 % confidence interval, 0.47–0.78) (Raghunath 2003). This
disparity was most profound in Asian populations where subjects with GERD had
an even lower prevalence of H. pylori compared to their Western counterparts,
despite their higher background prevalence. Data from 1611 African American
patients with endoscopic evidence of esophagitis found that the prevalence of
H. pylori was 4 %. After adjusting for age and gender, the odds ratio of H. pylori
infection in erosive esophagitis was 0.06 (95 % confidence interval, 0.01–0.59;
p ¼ 0.01) (Ashktorab et al. 2012).
An association between H. pylori eradication and the subsequent development
GERD remains unsubstantiated (Table 15.3). A meta-analysis of ten randomized
controlled trials, comparing patients who received either H. pylori eradication or
placebo, found no significant difference in the incidence of reflux symptoms (17 %
vs. 23 %) or erosive esophagitis (5 % vs. 5.1 %) following treatment. In fact, a
sub-analysis found a significantly lower incidence of reflux symptoms in the
eradicated versus the placebo group (14 % vs. 25 %) (Saad et al. 2012). These
findings are consistent with current recommendation not to refrain from treating
helicobacter in patients with GERD (Malfertheiner et al. 2012).
Table 15.3 Randomized controlled trials comparing Helicobacter pylori treatment with no
treatment in symptomatic adults with gastroesophageal reflux disease (GERD)
GERD symptoms following
H. pylori eradication, n (%)
Number of Treatment
Author Year Country patients group Placebo
Befrits et al. 2000 Sweden 145 22/79 (27.8) 29/66 (43.9)
Bytzer et al. 2000 Denmark 276 7/83 (8.4) 5/85 (5.9)
Hamada et al. 2000 Japan 572 36/286 (12.6) 1/286 (0.3)
Vakil et al. 2000 The USA 242 41/178 (23.0) 12/64 (18.8)
Kim et al. 2001 Korea 452 26/233 (11.2) 8/144 (5.6)
Moayyedi et al. 2001 The UK 178 15/85 (17.6) 15/93 (16.1)
Schwizer et al. 2001 Switzerland 29 7/13 (53.8) 15/16 (93.8)
Laine et al. 2002 The USA 1165 33/361 (9.1) 27/172 (15.7)
Malfertheiner 2002 Germany 1362 121/993 (12.2) 88/369 (23.8)
et al.
Harvey et al. 2004 The UK 1558 169/787 (21.5) 170/771 (22.0)
Kuipers et al. 2004 The Netherlands 231 30/111 (27.0) 27/120 (22.5)
Wu et al. 2004 Hong Kong 104 15/53 (28.3) 8/51 (15.7)
Ott et al. 2005 Brazil 157 8/73 (11.0) 7/60 (11.7)
Pilotto et al. 2006 Italy 61 6/31 (19.4) 6/30 (20.0)
Jonaitis et al. 2010 Lithuania 181 17/119 (14.3) 2/31 (6.4)
Nam et al. 2010 Korea 10,102 25/548 (4.6) 22/1635 (1.3)
Rodrigues et al. 2012 Brazil 32 7/9 (77.8) 12/13 (92.3)
15.3.3 Proton Pump Inhibitors
Patients with chronic H. pylori infection typically exhibit low basal and stimulated
acid output compared to noninfected subjects (Fig. 15.1). Holtmann and coworkers
demonstrated that patients with H. pylori infection have accelerated healing of
erosive esophagitis when treated with PPIs, compared to uninfected patients. After
4 weeks of treatment, complete healing of esophageal erosions was seen in 86.6 %
of H. pylori-positive patients and 76.3 % of H. pylori-negative patients, p < 0.01
(Holtmann et al. 1999). The mechanism, via which H. pylori augments healing of
esophagitis in the presence of PPI, has not been explored. There is currently no
evidence that lower PPI doses are required in H. pylori-infected GERD patients in
order to induce or maintain remission of erosive esophageal disease (Schenk
et al. 1999).
Long-term acid suppression with proton pump inhibition, commonly prescribed
for GERD, causes the progressive loss of parietal glands. Patients with H. pylori
infection who are treated with PPIs may develop a corpus predominant atrophic
gastritis. This pattern of inflammation is distinct from the pangastritis typically seen
in infected patients who are not receiving PPIs (Moayyedi et al. 2000b; Kuipers
et al. 1996). In H. pylori-infected animal models, PPIs have been shown to
accelerate the progression of gastric cancer, although data in humans are lacking.
The Maastricht IV/Florence Consensus Report recommends eradication of
H. pylori in patients receiving chronic PPIs in order to heal gastritis and prevent
atrophic changes (Malfertheiner et al. 2012).
15.3.4 Complications of GERD

15.3.4.1 Barrett’s Esophagus
Barrett’s esophagus is a premalignant lesion of the distal esophagus, related to

chronic acid exposure. Histologically, Barrett’s esophagus is characterized by
metaplastic columnar epithelium which replaces the stratified squamous epithelium
which normally lines the distal esophagus. Seven studies including 1621 patients
with Barrett’s esophagus found a significantly lower prevalence of H. pylori
(49.1 %) compared to matched controls (57.7 %). The pooled odds ratio for
H. pylori infection in Barrett’s esophagus was 0.64 (95 % CI, 0.43–0.94;
p ¼ 0.03) (Rokkas et al. 2007). A negative correlation between H. pylori infection
and Barrett’s esophagus is similarly observed for CagA-positive strains (35.6 %
vs. 51.5 %; OR, 0.39; 95 % CI, 0.21–0.76; p < 0.01). Nevertheless, there is no
evidence to support withholding eradication of H. pylori in patients with Barrett’s
esophagus.
15.3.4.2 Esophageal Adenocarcinoma
It is tempting to attribute the recent increase in esophageal adenocarcinoma (EAC)

in Western countries to the declining prevalence of H. pylori. A review of 10 stud-
ies, including 737 patients with EAC, found that the prevalence of H. pylori in these
patients was lower compared to controls (34.3 % vs. 50.1 %; OR, 0.52; 95 % CI,
0.37–0.73; p < 0.01) (Rokkas et al. 2007). A negative correlation between H. pylori
infection and EAC is similarly observed for CagA-positive strains (26 % vs. 40 %
CagA positivity in EAC and controls, respectively; OR, 0.51; 95 % CI, 0.31–0.82;
p < 0.01). There is no evidence to support withholding eradication of H. pylori in
patients following resection of EAC.
15.4 Functional Dyspepsia
15.4.1 Classification
The Rome III committee defines functional dyspepsia (FD) as “the presence of one
or more dyspepsia symptoms that are considered to originate from the
gastroduodenal region, in the absence of any organic, systemic or metabolic disease

that is likely to explain the symptoms” (Drossman 2006). Dyspeptic symptoms can
be broadly characterized into two subgroups: the postprandial distress syndrome or
the epigastric pain syndrome. According to the Rome III consensus, H. pylori
infection does not preclude a diagnosis of FD, despite the fact that H. pylori is an
undisputed cause of mucosal inflammation. This has prompted a debate whether it
is really appropriate that H. pylori-associated dyspepsia be considered a functional
disease (Sugano 2011). Adding fuel to this debate, emerging endoscopic technol-
ogies enable the reliable diagnosis of H. pylori-associated chronic gastritis at the
time of endoscopy.
The precise mechanism via which H. pylori may cause postprandial distress or
epigastric pain is unknown; however, several pathways have been suggested:
1. Increased acid secretion. Patients with isolated antral H. pylori gastritis manifest
increased gastric acid secretion. This is probably mediated by a reduction in
somatostatin, a negative regulator of gastrin release. H. pylori-infected subjects
with FD manifest greater acid secretion in response to gastrin-releasing peptide,
compared to those without FD (el-Omar et al. 1993).
2. Decreased ghrelin secretion. Ghrelin is a peptide hormone produced by the P/D1
cells of the gastric fundus and is involved in hunger sensation, acid secretion,
and motility. Both gastric expression and serum levels of ghrelin are signifi-
cantly lower in H. pylori-positive subjects (Boltin and Niv 2012). Activation of
ghrelin receptors leads to increased levels of neuropeptide Y (NPY) and agouti-
related peptide which promote appetite. Low levels of ghrelin, as seen in
H. pylori infection, possibly mediate symptoms of early satiety and postprandial
fullness.
3. Altered gastric emptying. Chronic H. pylori infection leads to downregulation of
muscle-specific miRNA expression. In murine models this causes hyperplasia of
the muscularis mucosa leading to reduced gastric accommodation and acceler-
ated gastric emptying (Saito et al. 2011).
4. Mast cells. Increased numbers of antral mast cells have been noted in H. pylori-
infected subjects with FD. Although this may be involved in the pathogenesis of
FD, mast cell proliferation is unlikely to be mediated by H. pylori, as an
increased number of mast cells is also observed in FD patients without
H. pylori (Hall et al. 2003).
15.4.3 Epidemiology
About 20–30 % of the population reports persistent dyspepsia each year. Only a
minority of these patients is fully investigated; however, an organic cause is not
usually identified. Therefore, the remainder can be considered to have FD. The
incidence of FD is estimated at 1/100 person-years (Agréus et al. 1995).
15.4.4 Treatment
A recent systematic review identified 21 randomized controlled trials which have

examined the efficacy of H. pylori eradication for the treatment of FD. Overall,
H. pylori eradication is associated with a relative risk reduction of 10 %, for
dyspeptic symptoms, compared to placebo. The number needed to treat to cure
one case of dyspepsia is 14 (Moayyedi et al. 2011). Nevertheless, studies performed
in Western populations have inconsistent results, with eradication having a variable
impact on dyspeptic symptoms (Table 15.4) (Mazzoleni et al. 2011; Talley
et al. 1999; Hsu et al. 2001). Eradication may, however, be beneficial in preventing
the subsequent development of PUD in patients with epigastric pain. Studies in
Asian populations are more likely to show a benefit for H. pylori treatment in FD
(Gwee et al. 2009; Des Bruley Varannes et al. 2001; Lan et al. 2011). The cost-
effectiveness of eradication in FD depends upon the background prevalence of
H. pylori, the cost of treatment, as well as other factors. Therefore, although
eradication may be cost-effective in Asia and Europe, this is not necessarily true
in the USA (Moayyedi et al. 2000a).
15.5 Other Gastrointestinal Disease
15.5.1 Inflammatory Bowel Disease
Patients with Crohn’s disease (CD) have a disproportionately low prevalence of

H. pylori (Luther et al. 2010). The estimated relative risk of H. pylori infection in
CD is 0.64 (95 % CI, 0.54–0.75). Over the past few decades, the declining rates of
H. pylori carriage have mirrored the increasing prevalence of inflammatory bowel
disease. This cannot be fully explained by socioeconomic or other environmental
factors, since a similar relationship is not observed with ulcerative colitis or other
chronic diseases which feature immune dysfunction. Subjects with H. pylori infec-
tion exhibit a blunted Th1/Th17 immune response and have low tissue and serum
levels of proinflammatory cytokines such as interferon-γ (IFN-γ) (Fig. 15.3)
(Luther et al. 2011). On the other hand, patients with CD have an exaggerated
Th1/Th17 immune response. The factors involved in defining the nature of an
15
Table 15.4 Randomized controlled trials comparing Helicobacter pylori treatment with no treatment in adults with functional dyspepsia
Results
Number of Duration of Treatment
Author Year Country patients follow-up Outcome measure group n (%) Control n (%) p
Talley et al. 1999 Australasia 278 12 months Near-total relief of epigastric pain 32/133 (24) 31/142 (22) ns
and Europe
Hsu et al. 2001 Taiwan 161 12 months Symptom resolution 47/81 (58.0) 44/80 (55.0) ns
Des Bruley 2001 France 253 12 months Symptom resolution 55/129 (43) 38/124 (31) 0.048
Helicobacter pylori and Nonmalignant Diseases
Varannes et al.
Gwee et al. 2009 Singapore 82 12 months Symptom resolution 10/41 (24.4) 3/41 (7.3) 0.02
Lan et al. 2011 China 195 3 months Reduction rate (pretreatment–posttreatment 36/98 (36.7) 19/97 (19.6) <0.05
scores)/pretreatment score 100
Mazzoleni et al. 2011 Brazil 404 12 months 50 % symptom reduction 94/192 (49.0) 72/197 (36.5) 0.01
This table includes principal studies performed and is not intended to be exhaustive
377
Fig. 15.3 A proposed model of H. pylori’s effect on host immune regulation. Dendritic cells
(DCs) sample H. pylori antigens directly. DCs, in turn, secrete cytokines such as IL-10 to
upregulate Foxp3-positive regulatory T cells (Tregs). This upregulation skews the host immuno-
logic tone away from the IL-12 and IL-23-mediated inflammatory Th1/Th17 responses and leads
to decreased production of proinflammatory cytokines. This may theoretically confer protection
from the development of CD
individual’s Th1/Th17 immune response remain elusive. An individual’s particular

immune response might be modified by H. pylori at the time of infection, or it might
be an inherited trait which predisposes to either to chronic gastritis (following
exposure to H. pylori) or to future CD, at the two ends of the spectrum. Animal
models suggest the former – mice colonized with H. pylori are protected from
developing dextran sodium sulfate (DSS) – colitis, indicating that H. pylori mod-
ulates immune function. As more data becomes available on the immunoregulatory
function of H. pylori, we may find additional evidence to advocate postponing
eradication of H. pylori in children, with the added benefit of decreasing the
incidence of CD.
15.5.2 Halitosis
In the vast majority of cases (>90 %), halitosis is attributable to oral or nasopha-
ryngeal pathology. A connection between halitosis and either upper gastrointestinal
symptoms or endoscopic findings has not been conclusively proven (Tas

et al. 2011). Nevertheless, a theoretical basis exists, as well as abundant empirical
data, linking H. pylori to halitosis. Following Marshall’s historic ingestion of
H. pylori in his quest to prove Koch’s postulate, oral malodor was noted by his
colleagues (Marshall et al. 1985). Volatile sulfur compounds including hydrogen
sulfide (H2S) and methyl mercaptan (CH3SH), which are produced by certain
strains of H. pylori, are also the major components of oral malodor (Lee
et al. 2006). Katsinelos et al. reported that H. pylori eradication in FD led to a
sustained resolution of halitosis during long-term follow-up (Katsinelos
et al. 2007). Nevertheless, in the absence of robust data, H. pylori cannot be
considered a treatable cause of halitosis.
15.6 Non-gastrointestinal Disease
15.6.1 Asthma and Allergy
As described previously with respect to CD, H. pylori has the ability to influence the
maturation and direction of host immune pathways. H. pylori infection can induce
dendritic cells to generate regulatory T cells (Tregs) which subsequently protect
against asthma (Fig. 15.3) (Oertli and Müller 2012). The H. pylori virulence factor,
neutrophil-activating protein A (NapA), might protect against asthma by inhibiting
polarization of T helper (Th)-1 and inhibiting the allergic Th2 response. NapA and
Tregs are under investigation as novel asthma treatments. A meta-analysis of case–
control and cross-sectional studies by Zhou and coworkers, including over 28,000
subjects across three continents, found a slightly lower rate of H. pylori infection in
subjects with asthma (OR, 0.84; 95 % CI, 0.73–0.96; p ¼ 0.01) (Zhou et al. 2013).
Subgroup analysis, however, found that a significant difference in H. pylori prev-
alence between asthmatic and non-asthmatic subjects exists only in the USA and
not in Europe or Asia. Wang and coworkers published a similar meta-analysis but
also included cohort studies (Wang et al. 2013). Pooled data from all studies
revealed a significant inverse association between H. pylori infection and asthma
for both adults (OR, 0.88; 95 % CI, 0.71–1.08; p < 0.05) and children (OR, 0.81;
95 % CI, 0.72–0.91; p < 0.05). In both meta-analyses, CagA-positive H. pylori
strains seem to be even more protective against asthma than CagA-negative strains.
For more details we refer to Chap. 12.
15.6.2 Idiopathic Thrombocytopenic Purpura
The mechanism through which H. pylori causes idiopathic thrombocytopenic

purpura (ITP) probably involves cross mimicry between H. pylori and platelet
antigens. This may be specifically related to CagA strains of H. Pylori. It has been
demonstrated that platelet-associated IgG in ITP patients recognizes the CagA
antigen (Franchini and Veneri 2006). Another mechanism could involve the inter-
action between H. pylori and platelets through von Willebrand factor and IgG anti-
H. pylori antibody, leading to chronic platelet consumption. In a review of 16 stud-
ies involving 1126 subjects with ITP, H. pylori prevalence was 64 %. Following
successful eradication, a platelet response was seen in 53 %. However, the studies
included were heterogeneous and included mainly uncontrolled and anecdotal data
(Franchini and Veneri 2006). The Maastricht IV/Florence Consensus Report advo-
cates seeking and treating H. pylori in the setting of ITP (Malfertheiner et al. 2012).
15.6.3 Iron Deficiency Anemia
Possible pathogenic mechanisms for H. pylori causing iron deficiency anemia

include: occult blood loss secondary to erosive gastritis, decreased iron absorption
secondary to H. pylori-induced hypochlorhydria, and increased iron uptake and
utilization by H. pylori (Dubois and Kearney 2005). Although epidemiologic
studies support an association between H. pylori infection and low ferritin, only a
few small, uncontrolled case series and one small, randomized trial have shown
improvement in anemia following H. pylori treatment (Choe et al. 1999). The
available evidence suggests that H. pylori is most likely to be associated with
anemia in patients who are anyway predisposed to iron deficiency, such as
premenopausal women and children.
15.6.4 Others
H. pylori has been epidemiologically linked to a wide range of diseases including

Alzheimer’s disease, Parkinson’s disease, Raynaud’s phenomenon, scleroderma,
idiopathic urticaria, acne rosacea, migraines, thyroiditis, Guillain–Barré syndrome,
and coronary artery disease. The proposed mechanisms leading to these various
conditions range from systemic immune reactions, cross-reactivity of bacterial and
host proteins, and events secondary to gastric mucosal injury (Goodman
et al. 2006). Of these associations, the strongest link is for ischemic heart disease.
Specifically, CagA seropositivity has been significantly associated with acute
coronary events (OR, 1.34; 95 % CI, 1.15–1.58; p < 0.01) (Franceschi
et al. 2009). Nevertheless, the available evidence is still insufficient to make a
clear causal or therapeutic link.
There is a wealth of information regarding the epidemiology and pathophysiology

of H. pylori in the setting of nonmalignant diseases. The future direction of study in
the area of peptic ulcer disease is likely to involve emerging molecular technologies
such as micro-RNAs. Such research may focus on RNA silencing and posttransla-
tional regulation of the genes for proteins which mediate H. pylori virulence,
evasion of gastric mucosal defense apparatus, and subsequent ulcer formation.
Further study will also investigate the complex interaction between H. pylori and
the host immune system. This may lead to the recognition of a host
“immunophenotype” which predisposes to the development of peptic ulcer disease
in the presence of H. pylori or asthma or Crohn’s disease in its absence. Study of
H. pylori virulence factors will continue, including their role in peptic ulcer
formation. For example, the structure of the H. pylori proton-gated urea channel
was recently described. Future research into this channel might investigate gene
polymorphisms which affect function, interaction with host immunity, and even
therapeutic targeting of the channel. Another rapidly developing field of research
relates to the effect of H. pylori infection on the composition of the gastric
microbiome and intestinal microbiome as a whole. The functional role of the gastric
microbiome is entirely unclear, as is the effect of H. pylori-associated gastritis on
the composition of the intestinal microbiome. A better understanding of the inter-
action between H. pylori and the intestinal microbiome may be particularly relevant
in the setting of functional dyspepsia. In the area of GERD, future study may be
directed at identifying patients with a high risk of esophageal adenocarcinoma, in
whom H. pylori eradication might be withheld. Undeniably, H. pylori is relevant to
all of the highly prevalent nonmalignant diseases discussed. For this reason,
H. pylori in nonmalignant disease will continue to be the subject of research for
many years to come.
References
Agréus L, Svärdsudd K, Nyrén O, Tibblin G (1995) Irritable bowel syndrome and dyspepsia in the
general population: overlap and lack of stability over time. Gastroenterology 109(3):671–680
Ashktorab H, Entezari O, Nouraie M, Dowlati E, Frederick W, Woods A, Lee E, Brim H, Smoot
DT, Ghadyari F, Kamangar F, Razjouyan H (2012) Helicobacter pylori protection against
reflux esophagitis. Dig Dis Sci 57(11):2924–2928. doi:10.1007/s10620-012-2349-3
Boltin D, Niv Y (2012) Ghrelin, Helicobacter pylori and body mass: is there an association? Israel
Med Assoc J 14(2):130–132
Boltin D, Halpern M, Levi Z, Vilkin A, Morgenstern S, Ho SB, Niv Y (2012) Gastric mucin
expression in Helicobacter pylori-related, nonsteroidal anti-inflammatory drug-related and
idiopathic ulcers. World J Gastroenterol 18(33):4597–4603. doi:10.3748/wjg.v18.i33.4597
Chan FK, Sung JJ, Chung SC, To KF, Yung MY, Leung VK, Lee YT, Chan CS, Li EK, Woo J
(1997) Randomised trial of eradication of Helicobacter pylori before non-steroidal anti-
inflammatory drug therapy to prevent peptic ulcers. Lancet 350(9083):975–979
Chan FKL, Ching JYL, Suen BY, Tse YK, Wu JCY, Sung JJY (2013) Effects of Helicobacter
pylori infection on long-term risk of peptic ulcer bleeding in low-dose aspirin users. Gastro-
enterology 144(3):528–535. doi:10.1053/j.gastro.2012.12.038
Choe YH, Kim SK, Son BK, Lee DH, Hong YC, Pai SH (1999) Randomized placebo-controlled
trial of Helicobacter pylori eradication for iron-deficiency anemia in preadolescent children
and adolescents. Helicobacter 4(2):135–139
Ciociola AA, McSorley DJ, Turner K, Sykes D, Palmer JB (1999) Helicobacter pylori infection
rates in duodenal ulcer patients in the United States may be lower than previously estimated.
Am J Gastroenterol 94(7):1834–1840. doi:10.1111/j.1572-0241.1999.01214.x
Des Bruley Varannes S, Fléjou JF, Colin R, Zaı̈m M, Meunier A, Bidaut-Mazel C (2001) There are
some benefits for eradicating Helicobacter pylori in patients with non-ulcer dyspepsia. Aliment
Pharmacol Ther 15(8):1177–1185
Drossman DA (ed) (2006) Rome III the functional gastrointestinal disorders, 3rd edn. Degnon
Associates, McLean
DuBois S, Kearney DJ (2005) Iron-deficiency anemia and Helicobacter pylori infection: a review
of the evidence. Am J Gastroenterol 100(2):453–459. doi:10.1111/j.1572-0241.2005.30252.x
el-Omar E, Penman I, Dorrian CA, Ardill JE, McColl KE (1993) Eradicating Helicobacter pylori
infection lowers gastrin mediated acid secretion by two thirds in patients with duodenal ulcer.
Gut 34(8):1060–1065
Fiocca R, Solcia E, Santoro B (1991) Duodenal ulcer relapse after eradication of Helicobacter
pylori. Lancet 337(8757):1614
Franceschi F, Niccoli G, Ferrante G, Gasbarrini A, Baldi A, Candelli M, Feroce F, Saulnier N,
Conte M, Roccarina D, Lanza GA, Gasbarrini G, Gentiloni SN, Crea F (2009) CagA antigen of
Helicobacter pylori and coronary instability: insight from a clinico-pathological study and a
meta-analysis of 4241 cases. Atherosclerosis 202(2):535–542. doi:10.1016/j.atherosclerosis.
2008.04.051
Franchini M, Veneri D (2006) Helicobacter pylori -associated immune thrombocytopenia. Plate-
lets 17(2):71–77. doi:10.1080/09537100500438057
Gillen D, Wirz AA, Ardill JE, McColl KE (1999) Rebound hypersecretion after omeprazole and its
relation to on-treatment acid suppression and Helicobacter pylori status. Gastroenterology 116
(2):239–247
Gisbert JP, Abraira V (2006) Accuracy of Helicobacter pylori diagnostic tests in patients with
bleeding peptic ulcer: a systematic review and meta-analysis. Am J Gastroenterol 101
(4):848–863. doi:10.1111/j.1572-0241.2006.00528.x
Gisbert JP, Calvet X (2009) Review article: Helicobacter pylori-negative duodenal ulcer disease.
Aliment Pharmacol Ther 30(8):791–815. doi:10.1111/j.1365-2036.2009.04105.x
Gisbert JP, Pajares JM (2002) Review article: Helicobacter pylori infection and gastric outlet
obstruction – prevalence of the infection and role of antimicrobial treatment. Aliment
Pharmacol Ther 16(7):1203–1208
Gisbert JP, Blanco M, Mateos JM, Fernández-Salazar L, Fernández-Bermejo M, Cantero J, Pajares
JM (1999) H. pylori-negative duodenal ulcer prevalence and causes in 774 patients. Dig Dis
Sci 44(11):2295–2302
Gisbert JP, Calvet X, Cosme A, Almela P, Feu F, Bory F, Santolaria S, Aznárez R, Castro M,
Fernández N, Garcı́a-Grávalos R, Benages A, Ca~nete N, Montoro M, Borda F, Pérez-Aisa A,
Piqué JM (2012) Long-term follow-up of 1,000 patients cured of Helicobacter pylori infection
following an episode of peptic ulcer bleeding. Am J Gastroenterol 107(8):1197–1204. doi:10.
1038/ajg.2012.132
Goodman KJ, Joyce SL, Ismond KP (2006) Extragastric diseases associated with Helicobacter
pylori infection. Curr Gastroenterol Rep 8(6):458–464
Gwee K, Teng L, Wong RM, Ho K, Sutedja D, Yeoh K (2009) The response of Asian patients with
functional dyspepsia to eradication of Helicobacter pylori infection. Eur J Gastroenterol
Hepatol 21(4):417–424. doi:10.1097/MEG.0b013e328317b89e
Hall W, Buckley M, Crotty P, O’Morain CA (2003) Gastric mucosal mast cells are increased in
Helicobacter pylori-negative functional dyspepsia. Clin Gastroenterol Hepatol 1(5):363–369
Holtmann G, Cain C, Malfertheiner P (1999) Gastric Helicobacter pylori infection accelerates
healing of reflux esophagitis during treatment with the proton pump inhibitor pantoprazole.
Hsu PI, Lai KH, Tseng HH, Lo GH, Lo CC, Lin CK, Cheng JS, Chan HH, Ku MK, Peng NJ, Chien
EJ, Chen W, Hsu PN (2001) Eradication of Helicobacter pylori prevents ulcer development in
patients with ulcer-like functional dyspepsia. Aliment Pharmacol Ther 15(2):195–201
Huang JQ, Sridhar S, Hunt RH (2002) Role of Helicobacter pylori infection and non-steroidal anti-
inflammatory drugs in peptic-ulcer disease: a meta-analysis. Lancet 359(9300):14–22. doi:10.
1016/S0140-6736(02)07273-2
Jang HJ, Choi MH, Shin WG, Kim KH, Chung YW, Kim KO, Park CH, Baek IH, Baik KH, Kae
SH, Kim HY (2008) Has peptic ulcer disease changed during the past ten years in Korea? A
prospective multi-center study. Dig Dis Sci 53(6):1527–1531. doi:10.1007/s10620-007-0028-6
Jyotheeswaran S, Shah AN, Jin HO, Potter GD, Ona FV, Chey WY (1998) Prevalence of
Helicobacter pylori in peptic ulcer patients in greater Rochester, NY: is empirical triple
therapy justified? Am J Gastroenterol 93(4):574–578. doi:10.1111/j.1572-0241.1998.167_b.x
Katsinelos P, Tziomalos K, Chatzimavroudis G, Vasiliadis T, Katsinelos T, Pilpilidis I,
Triantafillidis I, Paroutoglou G, Papaziogas B (2007) Eradication therapy in Helicobacter
pylori-positive patients with halitosis: long-term outcome. Med Princ Pract 16(2):119–123.
doi:10.1159/000098364
Kuipers EJ, Lundell L, Klinkenberg-Knol EC, Havu N, Festen HP, Liedman B, Lamers CB, Jansen
JB, Dalenback J, Snel P, Nelis GF, Meuwissen SG (1996) Atrophic gastritis and Helicobacter
pylori infection in patients with reflux esophagitis treated with omeprazole or fundoplication.
N Engl J Med 334(16):1018–1022. doi:10.1056/NEJM199604183341603
Lan L, Yu J, Chen Y, Zhong Y, Zhang H, Jia C, Yuan Y, Liu B (2011) Symptom-based tendencies
of Helicobacter pylori eradication in patients with functional dyspepsia. World J Gastroenterol
17(27):3242–3247. doi:10.3748/wjg.v17.i27.3242
Lanza FL, Chan FKL, Quigley EMM (2009) Guidelines for prevention of NSAID-related ulcer
complications. Am J Gastroenterol 104(3):728–738. doi:10.1038/ajg.2009.115
Lee H, Kho H, Chung J, Chung S, Kim Y (2006) Volatile sulfur compounds produced by
Helicobacter pylori. J Clin Gastroenterol 40(5):421–426
Luther J, Dave M, Higgins PDR, Kao JY (2010) Association between Helicobacter pylori
infection and inflammatory bowel disease: a meta-analysis and systematic review of the
literature. Inflamm Bowel Dis 16(6):1077–1084. doi:10.1002/ibd.21116
Luther J, Owyang SY, Takeuchi T, Cole TS, Zhang M, Liu M, Erb-Downward J, Rubenstein JH,
Chen C, Pierzchala AV, Paul JA, Kao JY (2011) Helicobacter pylori DNA decreases
pro-inflammatory cytokine production by dendritic cells and attenuates dextran sodium
sulphate-induced colitis. Gut 60(11):1479–1486. doi:10.1136/gut.2010.220087
Malfertheiner P, Megraud F, O’Morain CA, Atherton J, Axon ATR, Bazzoli F, Gensini GF,
Gisbert JP, Graham DY, Rokkas T, El-Omar EM, Kuipers EJ (2012) Management of
Helicobacter pylori infection – the Maastricht IV/Florence consensus report. Gut 61
(5):646–664. doi:10.1136/gutjnl-2012-302084
Marshall BJ, Armstrong JA, McGechie DB, Glancy RJ (1985) Attempt to fulfil Koch’s postulates
for pyloric Campylobacter. Med J Aust 142(8):436–439
Marshall BJ, Goodwin CS, Warren JR, Murray R, Blincow ED, Blackbourn SJ, Phillips M, Waters
TE, Sanderson CR (1988) Prospective double-blind trial of duodenal ulcer relapse after
eradication of Campylobacter pylori. Lancet 2(8626–8627):1437–1442
Mazzoleni LE, Sander GB, Francesconi CFM, Mazzoleni F, Uchoa DM, de Bona LR, Milbradt
TC, von Reisswitz PS, Berwanger O, Bressel M, Edelweiss MI, Marini SS, Molina CG,
Folador L, Lunkes RP, Heck R, Birkhan OA, Spindler BM, Katz N, Colombo BS, Guerrieri
PP, Renck LB, Grando E, Hocevar de Moura B, Dahmer FD, Rauber J, Prolla JC (2011)
Helicobacter pylori eradication in functional dyspepsia: HEROES trial. Arch Intern Med 171
(21):1929–1936. doi:10.1001/archinternmed.2011.533
Moayyedi P, Soo S, Deeks J, Forman D, Mason J, Innes M, Delaney B (2000a) Systematic review
and economic evaluation of Helicobacter pylori eradication treatment for non-ulcer dyspepsia.
Dyspepsia Review Group. BMJ 321(7262):659–664
Moayyedi P, Wason C, Peacock R, Walan A, Bardhan K, Axon AT, Dixon MF (2000b) Changing
patterns of Helicobacter pylori gastritis in long-standing acid suppression. Helicobacter 5
(4):206–214
Moayyedi P, Soo S, Deeks JJ, Delaney B, Harris A, Innes M, Oakes R, Wilson S, Roalfe A,
Bennett C, Forman D (2011) WITHDRAWN: eradication of Helicobacter pylori for non-ulcer
dyspepsia. Cochrane Database Syst Rev 2:CD002096. doi:10.1002/14651858.CD002096.pub5
Oertli M, Müller A (2012) Helicobacter pylori targets dendritic cells to induce immune tolerance,
promote persistence and confer protection against allergic asthma. Gut Microbes 3
(6):566–571. doi:10.4161/gmic.21750
Raghunath A (2003) Prevalence of Helicobacter pylori in patients with gastro-oesophageal reflux
disease: systematic review. BMJ 326(7392):737. doi:10.1136/bmj.326.7392.737
Rokkas T, Pistiolas D, Sechopoulos P, Robotis I, Margantinis G (2007) Relationship between
Helicobacter pylori infection and esophageal neoplasia: a meta-analysis. Clin Gastroenterol
Hepatol 5(12):1413–1417.e1-2. doi:10.1016/j.cgh.2007.08.010
Saad AM, Choudhary A, Bechtold ML (2012) Effect of Helicobacter pylori treatment on gastro-
esophageal reflux disease (GERD): meta-analysis of randomized controlled trials. Scand J
Gastroenterol 47(2):129–135. doi:10.3109/00365521.2011.648955
Saito Y, Suzuki H, Tsugawa H, Suzuki S, Matsuzaki J, Hirata K, Hibi T (2011) Dysfunctional
gastric emptying with down-regulation of muscle-specific microRNAs in Helicobacter pylori-
infected mice. Gastroenterology 140(1):189–198. doi:10.1053/j.gastro.2010.08.044
Sánchez-Delgado J, Gené E, Suárez D, Garcı́a-Iglesias P, Brullet E, Gallach M, Feu F, Gisbert JP,
Calvet X (2011) Has H. pylori prevalence in bleeding peptic ulcer been underestimated? A
meta-regression. Am J Gastroenterol 106(3):398–405. doi:10.1038/ajg.2011.2
Schenk BE, Kuipers EJ, Klinkenberg-Knol EC, Eskes SA, Meuwissen SG (1999) Helicobacter
pylori and the efficacy of omeprazole therapy for gastroesophageal reflux disease. Am J
Gastroenterol 94(4):884–887. doi:10.1111/j.1572-0241.1999.982_e.x
Schubert ML, Peura DA (2008) Control of gastric acid secretion in health and disease. Gastroen-
terology 134(7):1842–1860. doi:10.1053/j.gastro.2008.05.021
Stack WA, Atherton JC, Hawkey GM, Logan RFA, Hawkey CJ (2002) Interactions between
Helicobacter pylori and other risk factors for peptic ulcer bleeding. Aliment Pharmacol Ther
16(3):497–506
Sugano K (2011) Should we still subcategorize helicobacter pylori-associated dyspepsia as
functional disease? J Neurogastroenterol Motil 17(4):366–371. doi:10.5056/jnm.2011.17.4.
366
Sung JJY, Kuipers EJ, El-Serag HB (2009) Systematic review: the global incidence and preva-
lence of peptic ulcer disease. Aliment Pharmacol Ther 29(9):938–946. doi:10.1111/j.1365-
2036.2009.03960.x
Talley NJ, Janssens J, Lauritsen K, Rácz I, Bolling-Sternevald E (1999) Eradication of
Helicobacter pylori in functional dyspepsia: randomised double blind placebo controlled
trial with 12 months’ follow up. The Optimal Regimen Cures Helicobacter Induced Dyspepsia
(ORCHID) Study Group. BMJ 318(7187):833–837
Tas A, K€ oklü S, Yüksel I, Başar O, Akbal E, Cimbek A (2011) No significant association between
halitosis and upper gastrointestinal endoscopic findings: a prospective study. Chin Med J 124
(22):3707–3710
Tomtitchong P, Siribumrungwong B, Vilaichone R, Kasetsuwan P, Matsukura N, Chaiyakunapruk
N (2012) Systematic review and meta-analysis: Helicobacter pylori eradication therapy after
simple closure of perforated duodenal ulcer. Helicobacter 17(2):148–152. doi:10.1111/j.1523-
5378.2011.00928.x
Vaira D, Menegatti M, Miglioli M (1997) What is the role of Helicobacter pylori in complicated
ulcer disease? Gastroenterology 113(6 Suppl):S78–S84
Vergara M, Catalán M, Gisbert JP, Calvet X (2005) Meta-analysis: role of Helicobacter pylori
eradication in the prevention of peptic ulcer in NSAID users. Aliment Pharmacol Ther 21
(12):1411–1418. doi:10.1111/j.1365-2036.2005.02444.x
analysis. Helicobacter 18(1):41–53. doi:10.1111/hel.12012
Zhou X, Wu J, Zhang G (2013) Association between Helicobacter pylori and asthma: a meta-
analysis. Eur J Gastroenterol Hepatol 25(4):460–468. doi:10.1097/MEG.0b013e32835c280a
Chapter 16
Helicobacter pylori and Gastrointestinal
Polyps
Robert M. Genta and Richard H. Lash
Abstract Polyps of the gastrointestinal tract are mucosal elevations that may have
a mechanical, developmental, inflammatory, or neoplastic pathogenesis. Therefore,
it is not surprising that there is no specific relationship between gastrointestinal
polyps and H. pylori infection. In the stomach, inflammatory and proliferative
responses, irrespective of their etiology, seem to favor the development of inflam-
matory polyps. Intact oxyntic glands, unaffected by inflammatory changes, seem to
be more vulnerable to focal dilatations induced by proton pump inhibitors, hence
the strong negative association of these nonneoplastic, noninflammatory polyps
with H. pylori gastritis. Gastric adenomas, precursors of adenocarcinoma, are likely
related to H. pylori infection by the same mechanisms that link this infection to
gastric cancer. In the colon, the indisputable association of H. pylori gastritis with
both hyperplastic and neoplastic polyps remains largely unexplained.
Keywords Helicobacter pylori • Fundic gland polyps • Gastric hyperplastic

polyps • Gastric adenomas • Gastric inflammatory fibroid polyps • Gastric
neuroendocrine tumors • Carcinoids • Duodenal adenomas • Colon polyps
16.1 Introduction
The aphorism attributed to Charles Mayo in 1933 “gastric cancer does not arise in a
healthy stomach” (Bockus 1968) could be paraphrased to “gastric polyps do not
arise in a healthy stomach.” An inflammatory background has traditionally been
considered necessary to favor the development of what (until two or three decades
ago) represented the vast majority of gastric polyps: hyperplastic-inflammatory
polyps, adenomas, and neuroendocrine proliferations (then referred to as carci-
noids). These three types of polyps were particularly likely to be found in patients
with atrophic gastritis: hyperplastic polyps and adenomas were more common in
R.M. Genta, M.D. (*) • R.H. Lash, M.D.

Miraca Life Sciences, 6655 North MacArthur Blvd, Irving, TX 75039, USA
Pathology and Medicine (Gastroenterology), University of Texas Southwestern Medical
Center at Dallas, Dallas, TX, USA

DOI 10.1007/978-4-431-55936-8_16
388 R.M. Genta and R.H. Lash
multifocal atrophic gastritis and carcinoids in autoimmune atrophic gastritis. As

long-standing Helicobacter pylori infection emerged as the cause of multifocal
atrophic gastritis, the former two lesions – one inflammatory and one neoplastic –
became intimately tied to H. pylori gastritis. Approximately 40 years ago, the
German pathologist Karl Elster described cystic lesions resulting from the dilata-
tion of oxyntic glands and proposed the name “fundic gland polyps.” These
polypoid lesions were soon found to be highly prevalent in patients with familial
polyposis syndromes, while their sporadic occurrence in the absence of familial
polyposis was rare. In the last two decades, however, not only have sporadic fundic
gland polyps become the most common type of gastric polyp but they have also
been found to arise almost exclusively in healthy uninfected stomachs. As
Helicobacter was being alternatively related and unrelated to gastric polyps,
another more intriguing association has emerged: colonic polyps of all types appear
to be more prevalent in patients with Helicobacter gastritis than in uninfected
subjects.
This chapter will explore the associations between H. pylori infection and polyps
of the gastrointestinal tract. We will discuss both inflammatory and neoplastic
polyps; however, we will not address the malignant transformation of polyps, as
this subject is covered in other chapters of this book.
16.2 Gastric Hyperplastic Polyps
Hyperplastic polyps are proliferations of the gastric foveolar cells and associated
stroma (lamina propria) (Shaib et al. 2013). Because of their prominent inflamma-
tory stromal component and because they do not resemble the serrated, stroma-poor
hyperplastic polyps of the colon, they have also been referred to as inflammatory
polyps. However, the use of two different names for the same entity has created
confusion, especially with inflammatory fibroid polyps; therefore, here we shall
adhere to the currently standard terminology of hyperplastic polyps. The typical
features of hyperplastic polyps, depicted in Fig. 16.1, include elongated, grossly
distorted, branching, and dilated hyperplastic foveolae lying in an edematous
stroma rich in vasculature and inflammatory cells with small, haphazardly distrib-
uted smooth muscle bundles. Hyperplastic polyps are equally common in men and
women and typically occur in the sixth and seventh decades (median age 66 years).
They are most frequently found in the antrum; are often multiple, sessile, or
pedunculated; and often appear erythematous endoscopically, occasionally with
erosions or even ulceration. Mutations of the tumor suppressor p53 gene, chromo-
somal aberrations, and microsatellite instability have all been detected in these
polyps, and between 1 % and 20 % of hyperplastic polyps have been reported to
harbor foci of dysplasia (Lauwers et al. 1993; Nogueira et al. 1999). The overall
prevalence of carcinoma in hyperplastic polyps is <2 %, and it is more frequent in
polyps larger than 2 cm (Murakami et al. 2001; Yao et al. 2002).
16 Helicobacter pylori and Gastrointestinal Polyps 389
Fig. 16.1 Gastric hyperplastic polyp. These lesions are characterized by elongated, grossly
distorted, branching, and dilated hyperplastic foveolae lying in an edematous stroma rich in
vasculature and inflammatory cells with small, haphazardly distributed smooth muscle bundles.
As in the polyp depicted here, the surface is frequently eroded. Hematoxylin and eosin, original
magnification 10
16.3 Association with H. pylori Infection
The classic association of gastric hyperplastic polyps has been with mucosal
atrophy, and gastric carcinomas have been known to be more likely to develop in
a stomach containing hyperplastic polyps. In an attempt to elucidate the reasons for
this association, Dirschmid and coworkers used the updated Sydney System (Dixon
et al. 1996) to evaluate the gastritis phenotype in 244 patients with hyperplastic
polyps (Dirschmid et al. 2006). In none of the 244 patients was the gastric mucosa
found to be normal. The most common disorder was autoimmune atrophic gastritis
(56 %). H. pylori gastritis was seen in 37 % of the patients, 56 % of whom had
corpus-predominant gastritis. Other forms of gastritis were rarely reported. The
authors interpreted autoimmune gastritis and all forms of H. pylori gastritis (present
in 89 % of their patients) as precancerous conditions and concluded that hyperplas-
tic polyps represent a strong predictor of the synchronous presence of preneoplastic
lesions. In an earlier study of 160 patients with hyperplastic polyps, in which more
rigorous criteria for the diagnosis of autoimmune atrophic gastritis were applied,
Abraham and colleagues evaluated the histopathologic background of the gastric

mucosa surrounding the polyps: 37 % of the patients had at least focal intestinal
metaplasia, while adenoma or low-grade flat epithelial dysplasia was found in 2 %
and synchronous or metachronous adenocarcinoma in 6 %. H. pylori gastritis
accompanied hyperplastic polyps in 25 % of the patients, reactive or chemical
gastropathy in 21 %, autoimmune gastritis in 12 %, and H. pylori-associated
multifocal atrophic gastritis in 8 %. Thus, a direct association with H. pylori or
atrophy was detected in 33 % and 20 % of the patients, respectively (Abraham
et al. 2001). Although these two studies disagree on the relative percentages, both
conclude that an inflammatory or reactive background greatly increases the risk of
developing a hyperplastic polyp.
Foveolar hyperplasia, long recognized as a prominent feature of chemical
(or reactive) gastropathy and, to a lesser extent, of H. pylori gastritis, initiates as
a hyper-proliferative response to tissue injury (erosions, perhaps even micro-
erosions, or ulcers) accompanied by increased cellular exfoliation (Dixon
et al. 1986). The initial step in the formation of hyperplastic polyps is foveolar
hyperplasia. Intermediate polyp forms known as “polypoid foveolar hyperplasia”
(Fig. 16.2) consist of localized elongated and prominent foveolae that stand out as
mucosal nodules, but without the edematous stroma, dilated glands, erosions, and
mixed inflammatory infiltrates that characterize classic hyperplastic polyps
(Carmack et al. 2009a). In the past two decades, in part because of the decline of
H. pylori infection and in part because of the widespread use of acid-suppressing
Fig. 16.2 Polypoid foveolar hyperplasia. Polypoid foveolar hyperplasia is frequently the initial
step in the formation of hyperplastic polyps and consists of localized elongated and prominent
foveolae that stand out as mucosal nodules, but without the edematous stroma, dilated glands,
erosions, and mixed inflammatory infiltrates that characterize classic hyperplastic polyps. Hema-
toxylin and eosin, original magnification 10
medications (histamine-2 receptor antagonists and proton pump inhibitors), the

prevalence of chronic gastritis has been declining, particularly in the industrialized
countries of Europe and North America and in the emerging economies of East
Asia, while reactive gastropathy appears to be on the rise. In a 2011 survey of more
than 500,000 North American patients who had gastric biopsy specimens evaluated
at a single pathology laboratory, only 20.1 % of the patients had gastritis (with or
without H. pylori), whereas 15.6 % had reactive gastropathy. The prevalence of
reactive gastropathy increased steadily with age, from 2 % in children younger than
10 years to more than 20 % in patients older than 80 years (Maguilnik et al. 2012).
In parallel with these shifting gastric pathologies, hyperplastic polyps have slipped
from being the most common type of gastric polyp encountered at endoscopy to
comprising less than 20 %,(Carmack et al. 2009b), and an increasing proportion of
hyperplastic polyps is found in the background of a reactive gastric mucosa with no
evidence of current or prior H. pylori infection. Taken together, hyperplastic polyps
are related to reactive and hyper-proliferative states of the gastric mucosa, but are
not specifically or even preferentially induced by H. pylori infection. Their associ-
ation with malignancy is low.
16.4 Gastric Adenomas (Raised Intraepithelial Neoplasia)
The most common neoplastic polyp of the stomach is an epithelial dysplastic

growth still commonly referred to as adenoma, in spite of the new nomenclature
(raised intraepithelial neoplasia) suggested by the World Health Organization
(WHO) (Lauwers et al. 2010). In Western Europe and North America, sporadic
gastric adenomas have become rare, accounting for <1 % of all gastric polyps
(Carmack et al. 2009b). This contrasts markedly with some East Asian regions,
where the incidence of gastric cancer remains high and gastric adenomas still
constitute approximately a quarter of all gastric polyps (Lauwers and Srivastava
2007; Nakamura and Nakano 1985).
Gastric adenomas occur with similar frequency in men and women, most
commonly in the sixth and seventh decade. Although they can be found anywhere
in the stomach, they are more often located in the antrum. Endoscopically they have
a velvety lobulated appearance and are usually solitary. Histologically, gastric
adenomas consist of elevated aggregates of dysplastic epithelial cells (Fig. 16.3),
hence the WHO’s term “raised intraepithelial neoplasia.”
Intestinal-type gastric cancer is a consequence of chronic H. pylori infection, which

is believed by some researchers to be a necessary but insufficient causative factor in
gastric carcinogenesis (Graham 2014; Hanada and Graham 2014). While the
Fig. 16.3 Gastric adenoma. These tumors consist of elevated aggregates of dysplastic epithelial
cells, typically starting at the surface. The currently suggested term by the World Health Organi-
zation Classification of Tumors is “raised intraepithelial neoplasia.” Hematoxylin and eosin,
original magnification 20
connotation of “necessary” is arguable, it is incontrovertible that decades of

H. pylori chronic active inflammation, epigenetic changes resulting in genetic
instability, and mucosal atrophy prepare the terrain for carcinogens or spontaneous
mutation to induce neoplasia. Carcinogens may be environmental, but a major
source is provided by the biotransformation of ingested or secreted products by
the overgrowth of other bacteria that are made possible by hypochlorhydria and
achlorhydria (Correa et al. 1979).
Gastric adenomas tend to arise in a background of atrophy and intestinal
metaplasia, similar to that typically associated with either H. pylori infection or
autoimmune gastritis (Abraham et al. 2001; Laxen et al. 1982). As in the colon,
gastric adenomas can be viewed as part of a sequence leading from dysplasia to
carcinoma. The larger the adenomatous polyp, the greater the probability it contains
foci of adenocarcinoma. Synchronous gastric adenocarcinomas have been reported
in up to 30 % of patients with adenomas containing foci of adenocarcinoma
(Abraham et al. 2001; Laxen et al. 1982). Finally, and perhaps most importantly,
gastric dysplasia and adenomas parallel the epidemiologic patterns of gastric
adenocarcinoma. In summary, gastric adenomas are neoplastic proliferations with
a high predictive value for the development of gastric intestinal-type adenocarci-
noma. Although gastric adenomas may arise in the context of other conditions
(familial polyposis, autoimmune atrophic gastritis), there is sufficient evidence to
implicate H. pylori infection as the most important risk factor for the development
of these polyps (Carmack et al. 2009b; Gotoda et al. 1999; Han et al. 2011; Haziri
et al. 2010; Kim et al. 2013; Laxen 1981; Lee et al. 2012; Nakamura and Nakano
1985; Saito et al. 2000).
16.6 Gastric Neuroendocrine Tumors (Carcinoids)
Carcinoids are neuroendocrine tumors derived from enterochromaffin-like (ECL)

cells. The term “carcinoid” was discarded in the most recent (2010) WHO classi-
fication of tumors in favor of “neuroendocrine tumor” (Solcia et al. 2010). In two
large studies (Germany in 1994 and the USA in 2008), gastric neuroendocrine
tumors comprised <2 % of gastric polypoid lesions (Carmack et al. 2009b; Stolte
et al. 1994).
Gastric neuroendocrine tumors are classified in three distinct types (Cockburn
et al. 2013). Type I represents 70–80 % of all gastric endocrine tumors. They are
associated with hypergastrinemia resulting from atrophic gastritis. Therefore, they
are more commonly found in elderly patients, particularly women with autoim-
mune (corpus-restricted) pernicious anemia, and may also be found in patients with
long-standing severe H. pylori-related multifocal atrophic gastritis. The pathogen-
esis of Type I neuroendocrine tumors begins with the destruction (or significant
loss) of corpus/fundus acid-secreting parietal cells, causing reduced gastric acid
production and loss of feedback inhibition of gastrin secretion by antral G cells. The
resulting hypergastrinemia stimulates the proliferation of ECL cells, progressing
from microscopic hyperplasia to multiple endoscopically visible nodules. These
tumors (Fig. 16.4) are small (<1 cm), confined to the oxyntic (corpus and fundic)
mucosa, and tend to be multiple, usually coexisting with background
enterochromaffin-like cell hyperplasia. Type I gastric neuroendocrine tumors tend
to be found incidentally, often in patients undergoing esophagogastroduodenoscopy
(EGD) as part of an evaluation for anemia. Histologically, they consist of nests or
ribbons of endocrine cells (small polygonal cells with round central nuclei featuring
“salt-and-pepper” chromatin) with a very low proliferation index (Cockburn
et al. 2013; Solcia et al. 1998).
Type II gastric neuroendocrine tumors are associated with hypergastrinemia
resulting from a gastrin-secreting tumor. They are frequently detected as part of
the workup for MEN-1 syndrome or for Zollinger-Ellison syndrome, and they are
the least common type, representing only 5–8 % of gastric neuroendocrine tumors
(Cockburn et al. 2013; Solcia et al. 1998). The unregulated secretion of gastrin by
the primary tumors causes a secondary neuroendocrine tumor (or tumors). The
prognosis of Type II tumors is intermediate, with metastasis in about 30 %.
Type III (sporadic) neuroendocrine tumors are not associated with hypergas-
trinemia or any other known predisposing condition, are generally solitary, arise in
otherwise healthy gastric mucosa, and are not accompanied by ECL-cell hyperpla-
sia. These tumors, which represent approximately 20 % of all gastric neuroendo-
crine tumors, are usually detected when they become symptomatic, either
Fig. 16.4 Type I gastric neuroendocrine tumor. These small lesions consist of nests or ribbons of
endocrine cells localized at the interface between mucosa and submucosa. The high-magnification
insert in the upper right corner shows the details of the small polygonal cells with round central
nuclei featuring “salt-and-pepper” chromatin and a very low proliferation index that forms the
nests and ribbons. Hematoxylin and eosin, original magnification 20; insert 40
secondary to mucosal erosion and blood loss or metastasis. They can occur any-
where in the stomach, are usually solitary, and have a generally poor prognosis,
with metastases around 70 % when larger than 2 cm (Rindi et al. 1999).
16.7 Association with Gastritis and H. pylori Infection
Type II and III neuroendocrine tumors have no relationship with gastritis. Type I
represents the progression of atrophy-induced ECL-cell hyperplasia. Autoimmune
gastritis is the condition that most commonly results in a degree of oxyntic mucosal
atrophy sufficient to alter the acid-gastrin axis and induce ECL-cell hyperplasia and
neoplasia (Neumann et al. 2013). However, a downregulation of gastric secretion
involving alterations of somatostatin and gastrin production may occur in advanced
stages of multifocal atrophic gastritis, a consequence of long-standing H. pylori
infection (Chu and Schubert 2013). Taken together, the pathogenesis of Type I
neuroendocrine proliferations is directly related to atrophy of the oxyntic mucosa
with severe hypochlorhydria or achlorhydria. An indirect relationship with
H. pylori exists only inasmuch as long-standing infection may cause extensive
atrophy that involves the gastric corpus.
16.8 Fundic Gland Polyps
Fundic gland polyps (FGPs) are the most common type of polyps detected at EGD
in Western countries. In a large 2008 pathologic study, fundic gland polyps were
diagnosed in approximately 6 % of patients who had an EGD, representing 74 % of
all gastric polyps submitted for histopathologic evaluation (Carmack et al. 2009b).
More recent data from the same database, with more than one million patients who
underwent EGD, shows a prevalence close to 8 % (Sonnenberg and Genta 2014).
Fundic gland polyps are usually multiple and small (less than 1 cm) and have a
smooth, glassy, sessile appearance under endoscopy. Histologically, they consist of
aggregates of dilated oxyntic glands lined by gastric foveolar epithelium
(Fig. 16.5). When first discovered, fundic gland polyps were believed to be
hamartomatous (Elster 1976); however, their association with proton inhibitor
use, confirmed in a number of studies, suggests that mechanisms related to the
suppression of acid secretion by proton pump inhibition may be involved in their
pathogenesis (el-Zimaity et al. 1997; Graham and Genta 2008; Raghunath
et al. 2005).
Fig. 16.5 Fundic gland polyp. These velvety elevations occur in the oxyntic mucosa and consist
of aggregates of dilated oxyntic glands with flattened parietal cells. The overlying surface
comprises normal gastric foveolar epithelium (Fig. 16.5). When first discovered, fundic gland
polyps were believed to have a hamartomatous origin. Hematoxylin and eosin, original magnifi-
cation 20
The relative rarity of fundic gland polyps in patients with H. pylori infection has
been noted in numerous studies (Cao et al. 2014; Dickey et al. 1996; Genta
et al. 2009; Kishikawa et al. 2014; Sakai et al. 1998; Samarasam et al. 2009;
Shand et al. 2002; Tazaki et al. 2011). In our database, the prevalence of
H. pylori in 61,295 patients with fundic gland polyps was 0.3 %, or 36 times less
common than in patients without fundic gland polyps. A report from Japan goes
even further in support of this inverse relationship by reporting that two patients
with multiple FGPs in normal fundic mucosa without inflammatory changes or
atrophy experienced a complete disappearance of their polyps after the acquisition
of H. pylori infection (Watanabe et al. 2002). Another Japanese study examined the
clinical importance of sporadic fundic gland polyps in a new light. Whereas most
such studies had focused on the positive predictive value of fundic gland polyps
with respect to the development of some other condition (and have regularly failed
to detect any such relationship), Kishikawa and colleagues correlated the presence
of fundic gland polyps to the serum pepsinogen levels, H. pylori antibody concen-
tration, and gastric juice pH in 375 subjects. Low risk for gastric cancer was defined
as normal serum pepsinogen and negative H. pylori antibodies. Fundic gland polyps
were found in 44 patients. The prevalence of a “low-risk” stomach in subjects with
and without FGPs was 98 % and 48 %, respectively. Multivariable logistic regres-
sion analysis indicated three variables as independent factors positively associated
with “low-risk” stomachs: FGPs (OR ¼ 38.6), reflux esophagitis (OR ¼ 4.8), and
age <60 years (OR ¼ 1.89). Gastric juice pH, which is associated with mucosal
atrophy, was significantly lower in subjects with than without FGPs. Sporadic FGPs
tend to be related to the least atrophic mucosa among non-gastric atrophy subjects
without H. pylori infection and can be used as predictors of a low risk of gastric
carcinogenesis. In summary, sporadic fundic gland polyps occur in otherwise
healthy gastric mucosa and, irrespective of their possible relationship with the use
of proton pump inhibitors, have a strong inverse association with H. pylori and
cancer.
16.10 Inflammatory Fibroid Polyps
Inflammatory fibroid polyps (also known as Vanek tumors) are rare lesions that
represent <0.1 % of all gastric polyps (Carmack et al. 2009b). Endoscopically they
are usually firm; solitary, sessile, or pedunculated; and often ulcerated. The histo-
logic features of these polyps are distinctive: they consist of submucosal prolifer-
ations of spindle cells, small vessels, and a conspicuous inflammatory infiltrate with
a predominance of eosinophils. Hence, these polyps have been occasionally (and
inaccurately) referred to as “eosinophilic granulomas” (Shaib et al. 2013).
16.11 Relationship with H. pylori Infection
Since the gastric mucosa adjacent to these polyps has consistently been reported as
being normal, it would seem unlikely that there is a relationship with H. pylori
gastritis. There are, however, case reports from Japan suggesting that inflammatory
fibroid polyps either disappeared or decreased in size after the successful eradica-
tion of H. pylori infection (Hirasaki et al. 2007; Nishiyama et al. 2003). These
authors speculate on but clearly fail to prove a possible role for H. pylori in the
pathogenesis of these polyps. In summary, no convincing association between
H. pylori infection and gastric inflammatory fibroid polyps has been established.
16.12 Other Gastric Polyps
Gastrointestinal stromal tumors, leiomyomas, and submucosal lipomas are uncom-

mon polyps that may occur anywhere in the gastrointestinal tract, including the
stomach. To our knowledge, no study has investigated their possible association
with H. pylori infection. Since these lesions do not arise in the mucosa, a relation-
ship with H. pylori would have little biological plausibility.
16.13 Duodenal Polyps
Although gastric foveolar metaplasia in the duodenum may have the endoscopic
appearance of a polyp, this lesion, whose association with H. pylori gastritis has
been suggested and later disputed (Genta et al. 2010; Tovey et al. 2004; Voutilainen
et al. 2003), is not a true proliferation, does not meet the histopathologic criteria for
polyp, and will not be discussed here.
Duodenal adenomas occur in patients with familial polyposis syndromes and as
sporadic lesions, usually in elderly patients. Sporadic duodenal adenomas are
diagnosed in approximately 0.1 % of patients who have duodenal biopsies, and
approximately 1 % of these have high-grade dysplasia. A recent large study has
confirmed the association of duodenal adenomas with colon neoplasia (Genta
et al. 2014). In our database (Miraca Life Sciences database), there was a small
but statistically significant inverse association of sporadic duodenal adenomas with
concurrent H. pylori gastritis (Genta et al. 2014). However, we suspect that the
likelihood that this represents a truly biologically relevant phenomenon is minimal,
and we can assume that there is no association between H. pylori infection and
duodenal adenomas.
16.14 Colonic Neoplasms
The relationship between H. pylori and colonic neoplasms has been investigated in
several studies. Two meta-analyses of previous publications have suggested that
infection with H. pylori confers a 1.4- to 1.6-fold increased risk for colon adenoma
or colon cancer (Zhao et al. 2008; Zumkeller et al. 2006). The studies included in
these meta-analyses relied on different variables to assess risk factors and the
occurrence of colonic neoplasms. Although the majority of studies used positive
serology as a marker for H. pylori infection, some also used the presence of gastritis
or other abnormal gastric histopathologies (Bae et al. 2009; Inoue et al. 2010;
Machida-Montani et al. 2007). The occurrence of colonic neoplasms was assessed
by the endoscopic diagnosis of adenomatous polyps, villous adenoma, adenocarci-
noma, or recurrence of adenomatous polyps after previous polypectomy (Fujimori
et al. 2005; Mizuno et al. 2005; Siddheshwar et al. 2001). All studies included
relatively small case populations, with the largest ones having evaluated fewer than
200 patients with adenomatous polyps.
In an attempt to provide evidence based on the analysis of large numbers of
patients, we used a national database of 156,000 patients who had undergone both a
colonoscopy and an EGD (Sonnenberg and Genta 2013). The database contained
information about the histopathology of the colonoscopy, as well as the EGD, with
detailed information about polyp size, number, and location. Our hypothesis was
that H. pylori gastritis would be a risk factor for all types of colonic neoplasms, and
that such risk would also affect the characteristics of the neoplasms with respect to
histology, number, size, and location. Our results showed that H. pylori gastritis
confers a significantly increased risk for colonic neoplasms. The risk applied to all
types of colonic neoplasms: non-advanced adenomas (OR 1.80, 95 % CI
1.69–1.92), villous adenomas, adenomas with high-grade dysplasia or adenomas
>1 cm (OR 1.97, 95 % CI 1.82–2.14), and adenocarcinomas (OR 2.35, 95 % CI
1.98–2.80). The risk also increased with advancing stage of the neoplasm from
hyperplastic and adenomatous polyps to tubulovillous adenoma, adenoma with
high-grade dysplasia, and adenocarcinoma. Such risk was not limited to H. pylori
gastritis but was found similarly in all other types of gastric histopathology related
to H. pylori infection, such as intestinal metaplasia, gastric adenoma, gastric
lymphoma, and gastric cancer, even in the absence of active H. pylori infection at
the time of the evaluation. In summary, all types of epithelial colon polyps are
strongly associated with H. pylori gastritis. Although the mechanisms of this
association remain in the realm of speculation, elevated gastrin levels in patients
with H. pylori gastritis have been frequently invoked as the most likely cause for
this association, (Singh et al. 2012), but this view is not supported by recent
evidence (Lahner et al. 2012; Selgrad et al. 2014).
In the gastrointestinal tract, polyps are mucosal elevations that owe their name to
the imagination of ancient observers, who were reminded of the head of an octopus
(“polypus” in Latin) lying on a flat surface. This purely morphologic term has no
histopathologic inferences and encompasses lesions of mechanical, developmental,
inflammatory, and neoplastic origin. Therefore, it is not surprising that there is no
univocal relationship between gastrointestinal polyps and H. pylori infection. In the
stomach, inflammatory and proliferative responses, irrespective of their etiology,
seem to favor the development of inflammatory polyps. Intact oxyntic glands,
unaffected by inflammatory changes, seem to be more vulnerable to focal dilata-
tions induced by proton pump inhibitors, hence the strong negative association of
these nonneoplastic, noninflammatory polyps with H. pylori gastritis. Gastric
adenomas, precursors of adenocarcinoma, are likely related to H. pylori infection
by the same mechanisms that link this infection to gastric cancer. In the colon, the
indisputable association of H. pylori gastritis with both hyperplastic and neoplastic
polyps remains largely unexplained.
References
Abraham SC, Singh VK, Yardley JH, Wu TT (2001) Hyperplastic polyps of the stomach:
associations with histologic patterns of gastritis and gastric atrophy. Am J Surg Pathol
25:500–507
Bae RC, Jeon SW, Cho HJ, Jung MK, Kweon YO, Kim SK (2009) Gastric dysplasia may be an
independent risk factor of an advanced colorectal neoplasm. World J Gastroenterol
15:5722–5726
Bockus HL (1968) Gastroenterology. Saunders, Philadelphia
Cao H, Qu R, Zhang Z, Kong X, Wang S, Jiang K, Wang B (2014) Sporadic fundic gland polyps
are not associated with proton pump inhibitors therapy but negatively correlate with
Helicobacter pylori infection in China. Chin Med J (Engl) 127:1239–1243
Carmack SW, Genta RM, Graham DY, Lauwers GY (2009a) Management of gastric polyps: a
pathology-based guide for gastroenterologists. Nat Rev Gastroenterol Hepatol 6:331–341
Carmack SW, Genta RM, Schuler CM, Saboorian MH (2009b) The current spectrum of gastric
polyps: a 1-year national study of over 120,000 patients. Am J Gastroenterol 104:1524–1532
Chu S, Schubert ML (2013) Gastric secretion. Curr Opin Gastroenterol 29:636–641
Cockburn AN, Morgan CJ, Genta RM (2013) Neuroendocrine proliferations of the stomach: a
pragmatic approach for the perplexed pathologist. Adv Anat Pathol 20:148–157
Correa P, Cuello C, Gordillo G, Zarama G, Lopez J, Haenszel W, Tannenbaum S (1979). The
gastric microenvironment in populations at high risk to stomach cancer. Natl Cancer Inst
Monogr 53:167–170
Dickey W, Kenny BD, McConnell JB (1996) Prevalence of fundic gland polyps in a western
European population. J Clin Gastroenterol 23:73–75
Dirschmid K, Platz-Baudin C, Stolte M (2006) Why is the hyperplastic polyp a marker for the
precancerous condition of the gastric mucosa? Virchows Arch 448:80–84
Dixon MF, O’Connor HJ, Axon AT, King RF, Johnston D (1986) Reflux gastritis: distinct
histopathological entity? J Clin Pathol 39:524–530
Dixon MF, Genta RM, Yardley JH, Correa P (1996) Classification and grading of gastritis. The
updated Sydney system. International workshop on the histopathology of gastritis, Houston
1994. Am J Surg Pathol 20:1161–1181
el-Zimaity HM, Jackson FW, Graham DY (1997) Fundic gland polyps developing during omep-
razole therapy. Am J Gastroenterol 92:1858–1860
Elster K (1976) Histologic classification of gastric polyps. Curr Top Pathol 63:77–93
Fujimori S, Kishida T, Kobayashi T, Sekita Y, Seo T, Nagata K, Tatsuguchi A, Gudis K, Yokoi K,
Tanaka N, Yamashita K, Tajiri T, Ohaki Y, Sakamoto C (2005) Helicobacter pylori infection
increases the risk of colorectal adenoma and adenocarcinoma, especially in women. J
Genta RM, Schuler CM, Robiou CI, Lash RH (2009) No association between gastric fundic gland
polyps and gastrointestinal neoplasia in a study of over 100,000 patients. Clin Gastroenterol
Hepatol 7:849–854
Genta RM, Kinsey RS, Singhal A, Suterwala S (2010) Gastric foveolar metaplasia and gastric
heterotopia in the duodenum: no evidence of an etiologic role for Helicobacter pylori. Hum
Pathol 41:1593–1600
Genta RM, Hurrell JM, Sonnenberg A (2014) Duodenal adenomas coincide with colorectal
neoplasia. Dig Dis Sci 59:2249–2254
Gotoda T, Saito D, Kondo H, Ono H, Oda I, Fujishiro M, Yamaguchi H (1999) Endoscopic and
histological reversibility of gastric adenoma after eradication of Helicobacter pylori. J
Gastroenterol 34(Suppl 11):91–96
Graham DY (2014) Helicobacter pylori eradication and metachronous gastric cancer. Clin
Graham DY, Genta RM (2008) Long-term proton pump inhibitor use and gastrointestinal cancer.
Curr Gastroenterol Rep 10:543–547
Han JS, Jang JS, Choi SR, Kwon HC, Kim MC, Jeong JS, Kim SJ, Sohn YJ, Lee EJ (2011) A study
of metachronous cancer after endoscopic resection of early gastric cancer. Scand J
Hanada K, Graham DY (2014) Helicobacter pylori and the molecular pathogenesis of intestinal-
type gastric carcinoma. Expert Rev Anticancer Ther 14:947–954
Haziri A, Juniku-Shkololli A, Gashi Z, Berisha D, Haziri A (2010) Helicobacter pylori infection
and precancerous lesions of the stomach. Med Arh 64:248–249
Hirasaki S, Matsubara M, Ikeda F, Taniguchi H, Suzuki S (2007) Gastric inflammatory fibroid
polyp treated with Helicobacter pylori eradication therapy. Intern Med 46:855–858
Inoue K, Fujisawa T, Haruma K (2010) Assessment of degree of health of the stomach by
concomitant measurement of serum pepsinogen and serum Helicobacter pylori antibodies.
Int J Biol Markers 25:207–212
Kim HH, Cho EJ, Noh E, Choi SR, Park SJ, Park MI, Moon W (2013) Missed synchronous gastric
neoplasm with endoscopic submucosal dissection for gastric neoplasm: experience in our
hospital. Dig Endosc 25:32–38
Kishikawa H, Kaida S, Takarabe S, Miyoshi J, Matsukubo T, Miyauchi J, Tanaka Y, Miura S,
Nishida J (2014) Fundic gland polyps accurately predict a low risk of future gastric carcino-
genesis. Clin Res Hepatol Gastroenterol 38:505–512
Lahner E, Sbrozzi-Vanni A, Vannella L, Corleto VD, di Giulio E, Delle FG, Annibale B (2012) No
higher risk for colorectal cancer in atrophic gastritis-related hypergastrinemia. Dig Liver Dis
44:793–797
Lauwers GY, Srivastava A (2007) Gastric preneoplastic lesions and epithelial dysplasia.
Gastroenterol Clin N Am 36:813–829, vi
Lauwers GY, Wahl SJ, Melamed J, Rojas-Corona RR (1993) p53 expression in precancerous
gastric lesions: an immunohistochemical study of PAb 1801 monoclonal antibody on adeno-
matous and hyperplastic gastric polyps. Am J Gastroenterol 88:1916–1919
Lauwers GY, Carneiro F, Graham DY, Curado MP, Franceschi S, Montgomery EA, Tatematsu M,
Hattori T (2010) Tumors of the stomach: gastric carcinoma. In: Bosman FT, Carneiro F,
Hruban RH, Theise ND (eds) WHO classification: tumors of the digestive system, 4th edn.
IARC, Lyon, pp 48–58
Laxen F (1981) Gastric polyps and gastric carcinoma. Ann Clin Res 13:154–155
Laxen F, Sipponen P, Ihamaki T, Hakkiluoto A, Dortscheva Z (1982) Gastric polyps; their
morphological and endoscopical characteristics and relation to gastric carcinoma. Acta Pathol
Microbiol Immunol Scand A 90:221–228
Lee IS, Park YS, Kim KC, Kim TH, Kim HS, Choi KD, Lee GH, Yook JH, Oh ST, Kim BS (2012)
Multiple synchronous early gastric cancers: high-risk group and proper management. Surg
Oncol 21:269–273
Machida-Montani A, Sasazuki S, Inoue M, Natsukawa S, Shaura K, Koizumi Y, Kasuga Y,
Hanaoka T, Tsugane S (2007) Atrophic gastritis, Helicobacter pylori, and colorectal cancer
risk: a case-control study. Helicobacter 12:328–332
Maguilnik I, Neumann WL, Sonnenberg A, Genta RM (2012) Reactive gastropathy is associated
with inflammatory conditions throughout the gastrointestinal tract. Aliment Pharmacol Ther
36:736–743
Mizuno S, Morita Y, Inui T, Asakawa A, Ueno N, Ando T, Kato H, Uchida M, Yoshikawa T, Inui
A (2005) Helicobacter pylori infection is associated with colon adenomatous polyps detected
by high-resolution colonoscopy. Int J Cancer 117:1058–1059
Murakami K, Mitomi H, Yamashita K, Tanabe S, Saigenji K, Okayasu I (2001) p53, but not
c-Ki-ras, mutation and down-regulation of p21WAF1/CIP1 and cyclin D1 are associated with
malignant transformation in gastric hyperplastic polyps. Am J Clin Pathol 115:224–234
Nakamura T, Nakano G (1985) Histopathological classification and malignant change in gastric
polyps. J Clin Pathol 38:754–764
Neumann WL, Coss E, Rugge M, Genta RM (2013) Autoimmune atrophic gastritis – pathogen-
esis, pathology and management. Nat Rev Gastroenterol Hepatol 10:529–541
Nishiyama Y, Koyama S, Andoh A, Kishi Y, Yoshikawa K, Ishizuka I, Yokono T, Fujiyama Y
(2003) Gastric inflammatory fibroid polyp treated with Helicobacter pylori eradication therapy.
Intern Med 42:263–267
Nogueira AM, Carneiro F, Seruca R, Cirnes L, Veiga I, Machado JC, Sobrinho-Simoes M (1999)
Microsatellite instability in hyperplastic and adenomatous polyps of the stomach. Cancer
86:1649–1656
Raghunath AS, O’Morain C, McLoughlin RC (2005) Review article: the long-term use of proton-
pump inhibitors. Aliment Pharmacol Ther 22(Suppl 1):55–63, 55–63
Rindi G, Azzoni C, La RS, Klersy C, Paolotti D, Rappel S, Stolte M, Capella C, Bordi C, Solcia E
(1999) ECL cell tumor and poorly differentiated endocrine carcinoma of the stomach: prog-
nostic evaluation by pathological analysis. Gastroenterology 116:532–542
Saito K, Arai K, Mori M, Kobayashi R, Ohki I (2000) Effect of Helicobacter pylori eradication on
malignant transformation of gastric adenoma. Gastrointest Endosc 52:27–32
Sakai N, Tatsuta M, Hirasawa R, Iishi H, Baba M, Yokota Y, Ikeda F (1998) Low prevalence of
Helicobacter pylori infection in patients with hamartomatous fundic polyps. Dig Dis Sci
43:766–772
Samarasam I, Roberts-Thomson J, Brockwell D (2009) Gastric fundic gland polyps: a clinico-
pathological study from North West Tasmania. ANZ J Surg 79:467–470
Selgrad M, Bornschein J, Kandulski A, Hille C, Weigt J, Roessner A, Wex T, Malfertheiner P
(2014) Helicobacter pylori but not gastrin is associated with the development of colonic
neoplasms. Int J Cancer 135:1127–1131
Shaib YH, Rugge M, Graham DY, Genta RM (2013) Management of gastric polyps: an
endoscopy-based approach. Clin Gastroenterol Hepatol 11:1374–1384
Shand AG, Taylor AC, Banerjee M, Lessels A, Coia J, Clark C, Haites N, Ghosh S (2002) Gastric
fundic gland polyps in south-east Scotland: absence of adenomatous polyposis coli gene
mutations and a strikingly low prevalence of Helicobacter pylori infection. J Gastroenterol
Hepatol 17:1161–1164
Siddheshwar RK, Muhammad KB, Gray JC, Kelly SB (2001) Seroprevalence of Helicobacter
pylori in patients with colorectal polyps and colorectal carcinoma. Am J Gastroenterol
96:84–88
Singh P, Sarkar S, Kantara C, Maxwell C (2012) Progastrin peptides increase the risk of
developing colonic tumors: impact on colonic stem cells. Curr Colorectal Cancer Rep
8:277–289
Solcia E, Rindi G, Paolotti D, Luinetti O, Klersy C, Zangrandi A, La RS, Capella C (1998) Natural
history, clinicopathologic classification and prognosis of gastric ECL cell tumors. Yale J Biol
Med 71:285–290
Solcia E, Arnold R, Capella C, Klimstra DS, Kloppel G, Komminoth P, Rindi G (2010) Tumours
of the stomach: neuroendocrine neoplasms of the stomach. In: Bosman FT, Carneiro F, Hruban
RH, Theise ND (eds) WHO classification of tumours of the digestive system, 2nd edn. IARC,
Lyon, pp 64–68
Sonnenberg A, Genta RM (2013) Helicobacter pylori is a risk factor for colonic neoplasms. Am J
Sonnenberg A, Genta RM (2014) Prevalence of benign gastric polyps in a large pathology
database. Dig Liver Dis 47(2):164–169
Stolte M, Sticht T, Eidt S, Ebert D, Finkenzeller G (1994) Frequency, location, and age and sex
distribution of various types of gastric polyp. Endoscopy 26:659–665
Tazaki S, Nozu F, Yosikawa N, Imawari M, Suzuki N, Tominaga K, Hoshino M, Suzuki S,
Hayashi K (2011) Sporadic fundic gland polyp-related adenomas occurred in non-atrophic
gastric mucosa without Helicobacter pylori infection. Dig Endosc 23:182–186
Tovey FI, Hobsley M, Kaushik SP, Pandey R, Kurian G, Singh K, Sood A, Jehangir E (2004)
Duodenal gastric metaplasia and Helicobacter pylori infection in high and low duodenal ulcer-
prevalent areas in India. J Gastroenterol Hepatol 19:497–505
Voutilainen M, Juhola M, Farkkila M, Sipponen P (2003) Gastric metaplasia and chronic inflam-
mation at the duodenal bulb mucosa. Dig Liver Dis 35:94–98
Watanabe N, Seno H, Nakajima T, Yazumi S, Miyamoto S, Matsumoto S, Itoh T, Kawanami C,
Okazaki K, Chiba T (2002) Regression of fundic gland polyps following acquisition of
Helicobacter pylori. Gut 51:742–745
Yao T, Kajiwara M, Kuroiwa S, Iwashita A, Oya M, Kabashima A, Tsuneyoshi M (2002)
Malignant transformation of gastric hyperplastic polyps: alteration of phenotypes, proliferative
activity, and p53 expression. Hum Pathol 33:1016–1022
Zhao YS, Wang F, Chang D, Han B, You DY (2008) Meta-analysis of different test indicators:
Helicobacter pylori infection and the risk of colorectal cancer. Int J Color Dis 23:875–882
Zumkeller N, Brenner H, Zwahlen M, Rothenbacher D (2006) Helicobacter pylori infection and
colorectal cancer risk: a meta-analysis. Helicobacter 11:75–80
Chapter 17
Helicobacter pylori Infection and Gastric
Cancer
Richard M. Peek Jr. and Lydia E. Wroblewski
Abstract Gastric cancer is widespread and remains a leading cause of cancer-

related death worldwide. Infection with Helicobacter pylori is one of the strongest
known risk factors for this malignancy. There is a high level of genetic diversity
among H. pylori strains, and bacterial virulence factors play an important role in
determining the risk of developing gastric adenocarcinoma following colonization
with H. pylori. Infection with strains that contain a cag pathogenicity island or type
s1 vacA alleles confers increased risk compared to infection with other strain types.
Additional risk factors for gastric cancer include dietary factors, such as a high-salt
diet and iron deficiency. H. pylori can interact with stem cell populations and in this
way may promote gastric carcinogenesis. Recent studies suggest that components
of the microbiome may also influence H. pylori-induced carcinogenesis. In this
review article, we discuss mechanisms by which H. pylori infection can lead to
gastric cancer.
Keywords Helicobacter pylori • Gastric adenocarcinoma • Type IV bacterial

secretion system
17.1 Introduction
17.1.1 Gastric Cancer
Gastric adenocarcinoma is the fourth most common cancer and the second leading
cause of cancer-related death worldwide, resulting in approximately 738,000 deaths
in 2008 (de Martel et al. 2012; Fuchs and Mayer 1995; Parkin et al. 2005). The
incidence rates of gastric adenocarcinoma can vary substantially in different
regions of the world, with the highest rates found in East Asia, Central America,
parts of South America, and Eastern Europe (de Martel et al. 2012; Forman and
Pisani 2008; Fuchs and Mayer 1995; Soerjomataram et al. 2012).
R.M. Peek Jr. (*) • L.E. Wroblewski

Division of Gastroenterology, Department of Medicine, Vanderbilt University School of
Medicine, Nashville, TN, USA

DOI 10.1007/978-4-431-55936-8_17
404 R.M. Peek Jr. and L.E. Wroblewski
Adenocarcinoma is the most common type of cancer that affects the stomach,
but other types of cancer can also arise, including lymphoma and leiomyosarcoma.
Two distinct variants of gastric adenocarcinoma can be differentiated histologi-
cally: diffuse-type gastric cancer, which consists of individually infiltrating neo-
plastic cells that do not form glandular structures, and intestinal-type
adenocarcinoma, which progresses through a series of well-defined histological
steps, first described in 1975 (Correa 1992) (Fig. 17.1). Intestinal-type adenocarci-
noma is initiated by the transition from normal mucosa to chronic superficial
gastritis; this is followed by the development of atrophic gastritis and intestinal
metaplasia, finally leading to dysplasia and adenocarcinoma (Correa 1996;
Sipponen and Marshall 2000) (Fig. 17.1). Intestinal-type adenocarcinoma affects
men twice as frequently as women, and it is thought that estrogen may confer some
protection in women (Correa and Houghton 2007; Hatakeyama 2004). Typically,
the diagnosis of gastric cancer is not established until late in the course of the
disease and is often not detected until invasion of the muscularis propria has
occurred. This delay in diagnosis likely contributes to poor survival rates; indeed,
5-year survival rates for gastric cancer in the USA are less than 15 % (Correa 2004).
It is therefore of great importance to gain a comprehensive understanding of the
factors that contribute to this malignancy and identify persons who are at the
greatest risk of developing gastric cancer with a goal of developing strategies to
prevent this devastating disease.
Over the past century, the incidence rates of gastric adenocarcinoma in devel-
oped countries have significantly decreased. This decline can primarily be attrib-
uted to a decline in intestinal-type adenocarcinomas in the distal stomach (Fuchs
and Mayer 1995; Howson et al. 1986). Currently, in the USA, distal gastric
adenocarcinoma is diagnosed most commonly in elderly persons and occurs more
frequently in African-Americans, Hispanic-Americans, and Native Americans than
among other ethnicities (Haile et al. 2012; Wu et al. 2007). While the incidence of
gastric adenocarcinomas of the distal stomach has been declining, the incidence
rates of proximal gastric adenocarcinomas as well as those originating in the
gastroesophageal junction have been increasing in both the USA and Europe
(Blot et al. 1991; Pera et al. 1993).
17.1.2 Helicobacter pylori
Histology studies performed decades ago indicated that intestinal-type gastric

adenocarcinoma of the distal stomach was preceded by gastric inflammation
(termed superficial gastritis) as well as several other histologic alterations, includ-
ing intestinal metaplasia (presence of intestinal-type epithelium in the stomach),
gastric atrophy (loss of specialized cell types such as parietal cells and chief cells),
and dysplasia (Correa 1992; Correa et al. 1976) (Fig. 17.1). In the early 1980s,
Robin Warren and Barry Marshall definitively identified Helicobacter pylori as a
cause of gastritis by culturing an organism that had been visualized for almost a
17 Helicobacter pylori Infection and Gastric Cancer 405
Fig. 17.1 Schematic representation showing the sequential steps of the precancerous cascade
initiated by infection with H. pylori. The risk of developing gastric cancer within 5 years of
diagnosis is indicated at each stage (de Vries et al. 2008) (Images provided courtesy of M. Blanca
Piazuelo)
century in gastric biopsies (Marshall and Warren 1984). In 1994, H. pylori was
recognized as a type I carcinogen by the WHO and is now considered to be the
strongest known risk factor for distal gastric adenocarcinoma (Fox and Wang 2007;
Polk and Peek 2010) and the most common etiologic agent of infection-related
cancers, which represent 5.5 % of the global cancer burden (Parkin et al. 2005).
H. pylori infection is usually acquired in childhood, and without antimicrobial
treatment, persists for the lifetime of the host, despite the harsh gastric environment
that it colonizes. Genetic studies indicate that H. pylori has colonized humans for at
least 58,000 years (Linz et al. 2007), and currently, approximately half of the
world’s population is infected with H. pylori. Persons colonized with H. pylori
reside in all regions of the world; however, the highest rates of colonization are
found in developing countries with lower rates in developed nations (Everhart
2000).
Among H. pylori-infected individuals, only 1–3 % develop gastric adenocarci-
noma (Peek and Crabtree 2006). Factors that influence pathologic outcomes of
H. pylori infection include strain-specific bacterial constituents, host genetic fac-
tors, alterations of the stem niche and host microbiota, and environmental influ-
ences including diet (Blaser and Berg 2001). In this chapter, we consider the
microbial factors, microbiome and stem cell alterations, as well as the dietary risk
factors that specifically modify microbial factors that are known to influence the
risk of H. pylori-associated gastric cancer. Host effectors are covered by El-Omar
and coworkers in Chap. 14.
17.2 Epidemiology Linking H. pylori Infection to Gastric

Cancer
A possible link between H. pylori and gastric cancer was debated for a number of
years; however, numerous studies throughout the world have shown that coloniza-
tion with H. pylori is associated with a significantly increased risk for developing
gastric adenocarcinoma. A prospective study of 1526 Japanese patients reported
that gastric cancer (both intestinal and diffuse types) developed in approximately
3 % of H. pylori-colonized persons compared with 0 % in uninfected persons over
8 years (Uemura et al. 2001).
Studies using stored blood samples to detect H. pylori by serology have also
supported the link between H. pylori infection and development of gastric cancer
(Nomura et al. 1991; Parsonnet et al. 1991). Results pooled from 12 geographically
diverse studies comprised of 1228 gastric cancer cases and 3406 controls estimate
that infection with H. pylori increased the risk for developing adenocarcinoma of
the distal stomach almost sixfold over baseline (Helicobacter and Cancer Collab-
orative 2001).
Recent evidence indicates that Western blot analysis is a more sensitive method
for detecting anti-H. pylori antibodies than enzyme-linked immunosorbent assay
(ELISA) (Plummer et al. 2014). Analysis of studies that employed Western blot
data only indicates that the worldwide attributable risk for H. pylori in non-cardia
gastric cancer is actually 89 % versus 75 % when ELISA assays were used and that
H. pylori infection accounts for 6.2 % of all cancers (Plummer et al. 2014).
Depending on the study, the degree to which H. pylori increases the risk for gastric
adenocarcinoma can vary and likely depends on numerous factors including,
patient age, selection of controls, and the site and stage of gastric cancer.
17.3 H. pylori Virulence Factors That Influence Gastric

Cancer Risk
There is a high level of genetic diversity among isolates of H. pylori harvested from
unrelated persons (Blaser and Berg 2001). Bacterial virulence factors play an
important role in determining the risk of developing gastric adenocarcinoma fol-
lowing colonization with H. pylori and will be discussed in the following sections.
17.3.1 The H. pylori cag Pathogenicity Island
One of the H. pylori determinants that clearly influences the risk of gastric cancer is
the cag pathogenicity island (cagPAI), a 40-kB DNA insertion element which
contains 27–31 genes, including cagA and genes which encode proteins that form
a type IV bacterial secretion system (T4SS). The cag T4SS exports CagA from
adherent H. pylori across the bacterial and epithelial membrane and into host cells
(Fischer et al. 2001; Kwok et al. 2007; Odenbreit et al. 2000; Shaffer et al. 2011).
Persons that are seropositive for both H. pylori and CagA harbor a 5.8-fold
increased risk of developing intestinal and diffuse gastric adenocarcinoma com-
pared with uninfected persons, whereas persons infected with CagA-negative
H. pylori are only at a 2.2-fold higher risk of developing distal gastric adenocarci-
noma compared to uninfected persons (Parsonnet et al. 1997). A meta-analysis of
studies examining cancer risk suggests that H. pylori strains harboring CagA
increase the risk of developing distal gastric adenocarcinoma twofold over the
risk incurred by CagA-negative strains of H. pylori (Huang et al. 2003).
Within the host cell, CagA can be tyrosine phosphorylated at glutamate-proline-
isoleucine-tyrosine-alanine (EPIYA) motifs. Four different EPIYA motifs (EPIYA-
A, EPIYA-B, EPIYA-C, or EPIYA-D) have been identified within CagA and can be
used as indicators of disease severity (Hatakeyama 2004; Higashi et al. 2005; Naito
et al. 2006). An increased number of CagA EPIYA-C sites are linked to an
increased risk of developing gastric cancer (Basso et al. 2008), and strains
containing the EPIYA-D motif induce more intense cellular morphologic aberra-
tions and higher levels of IL-8, an indicator of increased pathogenesis, in cell
culture than strains harboring C-type CagA (Argent et al. 2008; Hatakeyama 2004).
In rodent models of gastric cancer, cagA-isogenic mutant strains of H. pylori, or
strains defective in cag T4SS function, fail to induce gastric cancer (Franco
et al. 2008; Gaddy et al. 2013). Furthermore, transgenic mice expressing CagA
demonstrate increased gastric epithelial cell proliferation rates and carcinoma in the
absence of inflammation (Ohnishi et al. 2008). Thus CagA appears to function as a
microbial oncoprotein. Further details on CagA function can be obtained in
Chap. 4.
17.3.2 VacA
VacA is a toxin secreted by H. pylori and is another microbial constituent linked to

the development of gastric cancer (Boquet and Ricci 2012; Cover and Blanke
2005). All H. pylori strains possess vacA, but there are considerable differences
in vacA sequences among strains. The regions of greatest diversity are localized to
the 50 region of the gene, which encodes the signal sequence and amino-terminus of
the secreted toxin (allele types s1a, s1b, s1c, or s2), an intermediate region (allele
types i1 or i2), and a mid-region (allele types m1 or m2) (Atherton et al. 1995;
Rhead et al. 2007). Strains containing type s1 and m1 alleles are strongly associated
with gastric cancer (Atherton et al. 1995, 1997; Miehlke 2000 #197). Likewise,
strains containing a vacA i1 versus an i2 allele are also strongly associated with
gastric cancer. This association between type i1 alleles and gastric cancer may even
be stronger than the risk incurred by vacA s or m types or even cag status (Rhead
et al. 2007). In a recent study of a population in Belgium, vacA s1 and i1 genotypes
were determined to be better markers of gastric cancer than cagA genotypes
(Memon et al. 2014). Similar findings were reported in a British population
where the presence of the vacA i1 genotype was strongly associated with intestinal
metaplasia (Winter et al. 2014). Regardless of cagA positivity or vacA allele type,
the presence of a vacA i2 genotype is almost exclusively linked to the absence of
intestinal metaplasia (Winter et al. 2014).
17.3.3 BabA
Blood group antigen-binding adhesin (BabA) is another H. pylori constituent that

has been linked to the development of gastric cancer. BabA is an outer membrane
protein and is encoded by the babA2 gene which binds to fucosylated Lewisb
antigen (Leb) on the surface of gastric epithelial cells (Gerhard et al. 1999; Ilver
et al. 1998; Oliveira et al. 2003; Yu et al. 2002). BabA is more frequently expressed
by cag PAI-positive strains compared to cag PAI-negative strains, and the presence
of babA2 is associated with gastric cancer (Gerhard et al. 1999). When BabA is
found in conjunction with cagA and vacA s1 alleles, it is associated with an even
greater risk of developing more severe disease (Gerhard et al. 1999; Hennig
et al. 2004). In a recent study of a Swedish population, BabA was found to be
associated with adenocarcinoma of the gastric cardia (Song et al. 2014). H. pylori
BabA expression can be lost in H. pylori isolates retrieved from human clinical
samples as well as in macaque, mice, and gerbil animal models that are infected
with H. pylori (Solnick et al. 2004; Styer et al. 2010). Loss of BabA prevents
binding to Lewis B and may provide a mechanism through which H. pylori can
modify its outer membrane in order to adapt to changing conditions that occur
within an inflamed milieu (Solnick et al. 2004; Styer et al. 2010). Further details on
VacA function can be obtained in Chap. 5.
17.3.4 SabA
Sialic acid-binding adhesin (SabA) is another H. pylori adhesin which binds to the
carbohydrate structure sialyl-Lewisx, antigen expressed on gastric epithelium, and is
associated with increased gastric cancer risk (Yamaoka et al. 2006). Sialyl-Lewisx
expression is induced during chronic gastric inflammation, suggesting that H. pylori
modulates host cell glycosylation patterns to enhance attachment and colonization
(Mahdavi et al. 2002). SabA expression can rapidly be switched “on” or “off”
through phase variation to adapt to changes exerted by inflammation in the gastric
niche (Yamaoka et al. 2006), or SabA promoter activity can be fine-tuned by
altering the length of the thymine nucleotide repeat tract (Aberg et al. 2014).
Further details on BabA and SabA functions can be obtained in Chap. 6.
17.4 Dietary Risk Factors
The risk of gastric carcinoma is not only influenced by H. pylori strain constituents
but also by environmental factors such as diet. Diets that are high in salted, pickled,
smoked, or poorly preserved foods, those with a high meat content, and those with
low fruit and vegetable content are most commonly associated with an increased
risk for developing gastric cancer (Epplein et al. 2008; Gonzalez et al. 2006, 2012;
Kim et al. 2004, 2010; Ren et al. 2012; Tsugane and Sasazuki 2007). Within the
context of H. pylori infection, high dietary salt intake and low iron levels are most
highly associated with increased gastric cancer risk and will be discussed in further
detail in Sects. 4.1 and 4.2, respectively.
17.4.1 Salt
High dietary salt intake is associated with increased gastric cancer risk and has been
reported in numerous studies (Lee et al. 2003; Tsugane 2005; Tsugane and Sasazuki
2007). H. pylori infection in combination with a high-salt diet further increases
gastric cancer risk. Two independent investigations reported that H. pylori-infected
subjects consuming a high-salt diet exhibited an increased risk of gastric cancer
when compared to H. pylori-infected subjects who consumed lower levels of salt
(Lee et al. 2003; Shikata et al. 2006). A third study also identified a positive
association between H. pylori infection and levels of dietary salt (Beevers
et al. 2004).
In Mongolian gerbils, the combination of H. pylori infection and a high-salt diet
has been reported to exert synergistic effects on the development of premalignant
lesions or gastric cancer (Gamboa-Dominguez et al. 2007; Kato et al. 2006; Sun
et al. 2006). Gerbils maintained on a high-salt diet that are also infected with
H. pylori developed more severe gastric inflammation and exhibited increased

gastric epithelial proliferation compared to H. pylori-infected gerbils consuming a
regular diet (Gamboa-Dominguez et al. 2007). A high-salt diet in combination with
H. pylori infection has also been found to induce gastric tumors in an IL-10-
deficient mouse model. Of interest, H. pylori-induced pathogenesis in the presence
of a high-salt diet was prevented by administration of licorice extracts, possibly due
to antioxidant, anti-inflammatory, and antimutagenic effects of the extract (Park
et al. 2014).
The mechanisms by which salt increases the risk for developing gastric cancer in
humans are incompletely understood; however, one possibility is that salt may
modulate virulence gene expression in H. pylori. Recent work has demonstrated
that expression of H. pylori CagA, VacA, and UreA is significantly upregulated
when certain strains of H. pylori are cultured in a medium containing high salt
concentrations (Gancz et al. 2008; Loh et al. 2007) It is not entirely understood why
some strains respond to high salt and others do not; however, salt-responsive strains
of H. pylori more frequently harbor two copies of a 50 -TAATGA motif located
within the cagA promoter, while strains containing only a single copy of this motif
are less likely to upregulate CagA in response to salt (Loh et al. 2012).
17.4.2 Iron
In addition to salt, host iron levels have also been found to modulate the virulence
potential of H. pylori, and iron deficiency is associated with an increased risk for
gastric cancer. Iron deficiency is the most common nutritional disorder in the world
and can be a result of a diet deficient in iron, blood loss, or alternatively coloniza-
tion by certain H. pylori strains which has been associated with hemorrhagic
gastritis (Yip et al. 1997). Long-term colonization with H. pylori can further
exacerbate iron deficiency through the development of gastric atrophy which
leads to decreased acid secretion, reduced ascorbic acid levels, and decreased
iron absorption (Yip et al. 1997). Low iron levels in persons with H. pylori infection
are not reversed by iron supplementation but can be normalized following eradi-
cation of H. pylori (DuBois and Kearney 2005). A recent study suggests that iron
deficiency in H. pylori-infected persons accelerates the development of carcino-
genesis by increasing the virulence potential of H. pylori (Noto et al. 2013a).
Rodent models of H. pylori-induced gastric cancer have been used to investigate
potential relationships between iron, H. pylori, and gastric cancer risk. A frequently
used rodent model of H. pylori-induced carcinogenesis is the transgenic INS-GAS
mouse model. INS-GAS mice overexpress human gastrin, which results in
hypergastrinemia and after 20 months of age develop gastric cancer (Thomson
et al. 2012; Wang et al. 2000a). Infection with H. felis or H. pylori accelerates this
process, suggesting that persistently elevated gastrin levels synergize with
Helicobacter to augment cancer progression (Wang et al. 2000b). Using this
model, it has recently been demonstrated that infection of INS-GAS mice with
H. felis results in decreased serum iron concentrations, decreased transferrin satu-

ration and hypoferritinemia, and increased total iron binding capacity. Chronic
infection with H. felis also altered expression of host genes important in iron
metabolism and absorption such as hepcidin and transferrin receptor 1 (Thomson
et al. 2012). One limitation of studies utilizing H. felis is that virulence factors
present in H. pylori per se that are important in pathogenesis, such as the cagPAI,
cannot be studied
A recent study determined the effects of dietary iron depletion on the develop-
ment of H. pylori-induced carcinogenesis in gerbils (Noto et al. 2013). Infection of
gerbils with CagA-positive H. pylori induced more severe gastritis in iron-depleted
gerbils than in iron-replete gerbils. Furthermore, gastritis developed earlier in
H. pylori-infected iron-depleted gerbils compared to infected iron-replete gerbils
(Noto et al. 2013). H. pylori infection significantly increased the occurrence of
dysplasia and gastric adenocarcinoma in iron-depleted gerbils compared to iron-
replete gerbils (Noto et al. 2013). These effects were only present following
infection with CagA-positive H. pylori and were not seen in gerbils on a low iron
diet infected with an isogenic cagA-mutant strain. To investigate the molecular
mechanisms underlying these changes, the proteomes of H. pylori strains cultured
from gerbils fed an iron-depleted diet or an iron-replete diet were compared using
two-dimensional (2D) DIGE/mass spectrometry. Proteins involved in survival,
microbial adherence, and function of the cag T4SS were differentially regulated
when comparing H. pylori strains isolated from iron-depleted versus iron-replete
gerbils (Noto et al. 2013). CagA expression was significantly higher in H. pylori
cultured from iron-depleted gerbils, and H. pylori FlaA and FlaB, the major
flagellin subunits, were significantly upregulated in H. pylori isolated from the
iron-depleted gerbils (Noto et al. 2013). To further investigate differences between
these two types of H. pylori strains, gastric epithelial cells were cocultured with
H. pylori strains isolated from iron-depleted gerbils or iron-replete gerbils. Levels
of phosphorylated CagA (reflecting translocated CagA), the number of cag T4SS
pili, and IL-8 induction were significantly higher following coculture with strains
isolated from iron-depleted gerbils compared with strains isolated from iron-replete
gerbils (Noto et al. 2013). Collectively these data demonstrate that dietary iron
depletion significantly increases the severity of gastric inflammation and acceler-
ates the development of H. pylori-induced disease via augmentation of the cag
secretion system.
17.5 Gastric Stem Cells and H. pylori-Induced Gastric

Cancer
Stem cells are critical for regulating the self-renewing gastric epithelium and
maintaining homeostasis, and evidence from mouse models has indicated that
H. pylori can interact with stem cell populations. H. pylori directly attach to gastric
epithelial cells in transgenic mice that overexpress Leb (Falk et al. 1995; Guruge
et al. 1998), and genetic ablation of parietal cells in Leb-expressing transgenic mice
permits stem cell populations to expand, which is accompanied by a corresponding
expansion of H. pylori colonization and increased inflammation (Syder et al. 1999,
2003).
In vivo lineage tracing of the gastric epithelium has demonstrated that Lgr5
(leucine-rich repeat-containing G protein-coupled receptor 5)-positive cells are
self-renewing, multipotent stem cells capable of generating an entire antral gastric
gland (Barker et al. 2010). In the human stomach, H. pylori infection in patients
with gastric cancer leads to an increased population of Lgr5-positive epithelial cells
in the antrum compared to uninfected persons with cancer (Uehara et al. 2013).
Furthermore, in H. pylori-infected persons with cancer, Lgr5-positive cells are
more susceptible to DNA damage than Lgr5-negative cells (Uehara et al. 2013).
Lrig1 (Leucine-rich repeats and immunoglobulin-like domains 1) is a transmem-
brane protein that marks a distinct population of quiescent stem cells and functions
as a tumor suppressor (Powell et al. 2012). Lrig1 is expressed in the gastric
epithelium, and expression of Lrig1 is increased in the gastric epithelium of
H. pylori-infected versus uninfected mice suggesting that infection with H. pylori
increases this stem cell population as well (Noto et al. 2013).
Bone marrow-derived cells (BMDCs) are a heterogeneous population of cells
with the ability to differentiate into cells of diverse lineages. Studies in mice
infected with Helicobacter have demonstrated that BMDCs home to and engraft
in sites of chronic gastric inflammation and repopulate the endogenous tissue stem
cells (Houghton et al. 2004; Varon et al. 2012). Within an inflamed stomach,
BMDCs degenerate into adenocarcinoma, suggesting that gastric epithelial carci-
nomas can originate from marrow-derived sources (Houghton et al. 2004; Varon
et al. 2012).
A lack of molecular markers has hindered research into the possible interaction
of H. pylori with corpus stem cells; however, many of the factors that H. pylori is
known to modulate, such as the transcription factor NF-κB and reactive oxygen
species, have been found to be involved in maintaining stem cells and cancer stem
cells. It would therefore appear likely that the interaction of H. pylori with gastric
stem cells may potentiate gastric carcinogenesis (Cabarcas et al. 2011).
17.6 Microbiome
The stomach harbors a bacterial community with colonization densities ranging

from 101 to 103 colony-forming units/g which influence gastric homeostasis (Sheh
and Fox 2013). Recent progress in molecular techniques has provided evidence that
bacteria colonizing the gastric epithelium may influence H. pylori-associated path-
ogenesis. In transgenic INS-GAS mice, those harboring a complex microbiota
spontaneously develop gastric cancer (Thomson et al. 2012; Wang et al. 2000a).
However, in germfree INS-GAS mice, it takes over a year longer for gastric cancer
to develop (Lofgren et al. 2011). H. pylori-infected germfree INS-GAS mice

develop less severe lesions and are slower to progress to gastrointestinal
intraepithelial neoplasia than H. pylori-infected INS-GAS mice with a complex
microbiota (Lofgren et al. 2011). Furthermore, antimicrobial therapies have been
found to delay the progression to gastric cancer in both uninfected and H. pylori-
infected INS-GAS mice (Lee et al. 2009). Taken together these findings suggest
that components of the microbiome may exacerbate H. pylori-induced carcinogen-
esis. In support of this, a restricted microbiota containing only three species
(ASF356 Clostridium species, ASF361 Lactobacillus murinus, and ASF519
Bacteroides species) of commensal bacteria is sufficient to promote gastric cancer
in H. pylori-infected INS-GAS mice to the same extent as seen in H. pylori-infected
INS-GAS mice with a complex microbiota (Lertpiriyapong et al. 2014).
Extragastric constituents of the microbiota may also influence outcomes of
H. pylori-induced gastric cancer. Coinfection of C57BL/6 mice with H. bilis or
H. muridarum attenuated H. pylori-induced gastric pathology (Ge et al. 2011;
Lemke et al. 2009). In contrast, preexisting infection with H. hepaticus increases
H. pylori-induced gastric injury (Ge et al. 2011). Interestingly, new evidence
suggests that helminth infections prevent H. pylori-induced changes in the
microbiota of INS-GAS mice and may decrease the severity of H. pylori-induced
disease (Whary et al. 2014). While progress is being made in understanding the
complex interplay between the microbiota and H. pylori in the development of
gastric cancer in animal models, detailed molecular studies are still needed in well-
defined human populations to examine differences in the microbiota of H. pylori-
infected persons with and without gastric cancer (Sheh and Fox 2013).
Globally, gastric cancer leads to a high number of cancer-related deaths and

understanding the risk factors for this disease is of utmost importance in identifying
individuals that are most at risk of developing gastric cancer. Infection with
H. pylori is extremely common, and in some areas of the world, infection preva-
lence rates approach 100 %; however, 97–99 % of colonized persons will never
develop gastric cancer. The risk of developing gastric cancer is dependent on
numerous factors including H. pylori strain-specific virulence factors, the host
genotype, environmental factors such as diet, as well as alternations in stem cell
populations and the microbiome. Interactions among these factors affect the out-
come of long-term colonization of H. pylori; therefore, it is important to be able to
utilize results from mechanistic studies to identify people who are most at risk of
developing gastric cancer.
References
Aberg A, Gideonsson P, Vallstrom A, Olofsson A, Ohman C, Rakhimova L, Boren T, Engstrand L,

Brannstrom K, Arnqvist A (2014) A repetitive DNA element regulates expression of the
Helicobacter pylori sialic acid binding adhesin by a rheostat-like mechanism. PLoS Pathog
10:e1004234, http://www.ncbi.nlm.nih.gov/pubmed/24991812
Argent RH, Hale JL, El-Omar EM, Atherton JC (2008) Differences in Helicobacter pylori CagA
tyrosine phosphorylation motif patterns between western and east Asian strains, and influences
on interleukin-8 secretion. J Med Microbiol 57:1062–1067, http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_uids¼18719174
Atherton JC, Cao P, Peek RM Jr, Tummuru MK, Blaser MJ, Cover TL (1995) Mosaicism
in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types
with cytotoxin production and peptic ulceration. J Biol Chem 270:17771–17777, http://www.
ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_uids¼
7629077
Gastroenterology 112:92–99, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&
db¼PubMed&dopt¼Citation&list_uids¼8978347
Barker N, Huch M, Kujala P, van de Wetering M, Snippert HJ, van Es JH, Sato T, Stange DE,
Begthel H, van den Born M et al (2010) Lgr5(+ve) stem cells drive self-renewal in the stomach
and build long-lived gastric units in vitro. Cell Stem Cell 6:25–36, http://www.ncbi.nlm.nih.
gov/pubmed/20085740
Basso D, Zambon CF, Letley DP, Stranges A, Marchet A, Rhead JL, Schiavon S, Guariso G,
Ceroti M, Nitti D et al (2008) Clinical relevance of Helicobacter pylori cagA and vacA gene
polymorphisms. Gastroenterology 135:91–99, http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_uids¼18474244
Beevers DG, Lip GY, Blann AD (2004) Salt intake and Helicobacter pylori infection. J Hypertens
22:1475–1477, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼PubMed&
dopt¼Citation&list_uids¼15257168
Blaser MJ, Berg DE (2001) Helicobacter pylori genetic diversity and risk of human disease. J Clin
Invest 107:767–773, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼
PubMed&dopt¼Citation&list_uids¼11285290
Blot WJ, Devesa SS, Kneller RW, Fraumeni JF Jr (1991) Rising incidence of adenocarcinoma of
the esophagus and gastric cardia. JAMA 265:1287–1289, http://www.ncbi.nlm.nih.gov/
pubmed/1995976
Boquet P, Ricci V (2012) Intoxication strategy of Helicobacter pylori VacA toxin. Trends
Microbiol 20:165–174, http://www.ncbi.nlm.nih.gov/pubmed/22364673
Cabarcas SM, Mathews LA, Farrar WL (2011) The cancer stem cell niche – there goes the
neighborhood? Int J Cancer J Int Cancer 129:2315–2327, http://www.ncbi.nlm.nih.gov/
pubmed/21792897
Correa P (1992) Human gastric carcinogenesis: a multistep and multifactorial process – first
American Cancer Society award lecture on cancer epidemiology and prevention. Cancer Res
Correa P (1996) Helicobacter pylori and gastric cancer: state of the art. Cancer epidemiology,
biomarkers & prevention: a publication of the American Association for Cancer Research,
cosponsored by the Am Soc Prev Oncol 5:477–481 http://www.ncbi.nlm.nih.gov/entrez/query.
Correa P (2004) Is gastric cancer preventable? Gut 53:1217–1219, http://www.ncbi.nlm.nih.gov/
Correa P, Houghton J (2007) Carcinogenesis of Helicobacter pylori. Gastroenterology

Correa P, Cuello C, Duque E, Burbano LC, Garcia FT, Bolanos O, Brown C, Haenszel W (1976)
Gastric cancer in Colombia. III. Natural history of precursor lesions. J Natl Cancer Inst
57:1027–1035, http://www.ncbi.nlm.nih.gov/pubmed/1003539
Cover TL, Blanke SR (2005) Helicobacter pylori VacA, a paradigm for toxin multifunctionality.
Nat Rev Microbiol 3:320–332, http://www.ncbi.nlm.nih.gov/pubmed/15759043
de Martel C, Ferlay J, Franceschi S, Vignat J, Bray F, Forman D, Plummer M (2012) Global
burden of cancers attributable to infections in 2008: a review and synthetic analysis. Lancet
Oncol 13:607–615, http://www.ncbi.nlm.nih.gov/pubmed/22575588
de Vries AC, van Grieken NC, Looman CW, Casparie MK, de Vries E, Meijer GA, Kuipers EJ
(2008) Gastric cancer risk in patients with premalignant gastric lesions: a nationwide cohort
study in the Netherlands. Gastroenterology 134:945–952, http://www.ncbi.nlm.nih.gov/
pubmed/18395075
DuBois S, Kearney DJ (2005) Iron-deficiency anemia and Helicobacter pylori infection: a review
of the evidence. Am J Gastroenterol 100:453–459, http://www.ncbi.nlm.nih.gov/pubmed/
15667507
Epplein M, Nomura AM, Hankin JH, Blaser MJ, Perez-Perez G, Stemmermann GN, Wilkens LR,
Kolonel LN (2008) Association of Helicobacter pylori infection and diet on the risk of gastric
cancer: a case-control study in Hawaii. Cancer Causes Control: CCC 19:869–877, http://www.
18369531
Everhart JE (2000) Recent developments in the epidemiology of Helicobacter pylori.
Gastroenterol Clin North Am 29:559–578, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?
cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_uids¼11030073
Falk PG, Bry L, Holgersson J, Gordon JI (1995) Expression of a human alpha-1,3/4-
fucosyltransferase in the pit cell lineage of FVB/N mouse stomach results in production of
Leb-containing glycoconjugates: a potential transgenic mouse model for studying
Helicobacter pylori infection. Proc Natl Acad Sci U S A 92:1515–1519, http://www.ncbi.
nlm.nih.gov/pubmed/7878011
cells and induction of interleukin-8. Mol Microbiol 42:1337–1348, http://www.ncbi.nlm.nih.
gov/pubmed/11886563
Forman D, Pisani P (2008) Gastric cancer in Japan – honing treatment, seeking causes. N Engl J
Med 359:448–451, http://www.ncbi.nlm.nih.gov/pubmed/18669423
Fox JG, Wang TC (2007) Inflammation, atrophy, and gastric cancer. J Clin Invest 117:60–69,
http://www.ncbi.nlm.nih.gov/pubmed/17200707
virulence factors. Cancer Res 68:379–387, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?
Fuchs CS, Mayer RJ (1995) Gastric carcinoma. N Engl J Med 333:32–41, http://www.ncbi.nlm.
nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_uids¼7776992
Gaddy JA, Radin JN, Loh JT, Zhang F, Washington MK, Peek RM Jr, Algood HM, Cover TL
(2013) High dietary salt intake exacerbates Helicobacter pylori-induced gastric carcinogene-
sis. Infect Immun 81:2258–2267, http://www.ncbi.nlm.nih.gov/pubmed/23569116
Gamboa-Dominguez A, Ubbelohde T, Saqui-Salces M, Romano-Mazzoti L, Cervantes M,
Dominguez-Fonseca C, de la Luz Estreber M, Ruiz-Palacios GM (2007) Salt and stress
synergize H. pylori-induced gastric lesions, cell proliferation, and p21 expression in Mongo-
lian gerbils. Dig Dis Sci 52:1517–1526, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?
Gancz H, Jones KR, Merrell DS (2008) Sodium chloride affects Helicobacter pylori growth and
gene expression. J Bacteriol 190:4100–4105, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?
Ge Z, Feng Y, Muthupalani S, Eurell LL, Taylor NS, Whary MT, Fox JG (2011) Coinfection with
Enterohepatic Helicobacter species can ameliorate or promote Helicobacter pylori-induced
gastric pathology in C57BL/6 mice. Infect Immun 79:3861–3871, http://www.ncbi.nlm.nih.
gov/pubmed/21788386
Gerhard M, Lehn N, Neumayer N, Boren T, Rad R, Schepp W, Miehlke S, Classen M, Prinz C
adhesin. Proc Natl Acad Sci U S A 96:12778–12783, http://www.ncbi.nlm.nih.gov/entrez/
query.fcgi?cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_uids¼10535999
Gonzalez CA, Jakszyn P, Pera G, Agudo A, Bingham S, Palli D, Ferrari P, Boeing H, del
Giudice G, Plebani M et al (2006) Meat intake and risk of stomach and esophageal adenocar-
cinoma within the European Prospective Investigation Into Cancer and Nutrition (EPIC). J Natl
Cancer Inst 98:345–354, http://www.ncbi.nlm.nih.gov/pubmed/16507831
Gonzalez CA, Lujan-Barroso L, Bueno-de-Mesquita HB, Jenab M, Duell EJ, Agudo A,
Tjonneland A, Boutron-Ruault MC, Clavel-Chapelon F, Touillaud M et al (2012) Fruit and
vegetable intake and the risk of gastric adenocarcinoma: a reanalysis of the European Pro-
spective Investigation into Cancer and Nutrition (EPIC-EURGAST) study after a longer
follow-up. Int J Cancer J Int Cancer 131:2910–2919, http://www.ncbi.nlm.nih.gov/pubmed/
22473701
Guruge JL, Falk PG, Lorenz RG, Dans M, Wirth HP, Blaser MJ, Berg DE, Gordon JI (1998)
Epithelial attachment alters the outcome of Helicobacter pylori infection. Proc Natl Acad Sci
U S A 95:3925–3930, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼
Haile RW, John EM, Levine AJ, Cortessis VK, Unger JB, Gonzales M, Ziv E, Thompson P,
Spruijt-Metz D, Tucker KL et al (2012) A review of cancer in U.S. Hispanic populations.
Cancer Prev Res 5:150–163, http://www.ncbi.nlm.nih.gov/pubmed/22307564
Hatakeyama M (2004) Oncogenic mechanisms of the Helicobacter pylori CagA protein. Nat Rev
Cancer 4:688–694, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&
Helicobacter, and Cancer Collaborative, G (2001) Gastric cancer and Helicobacter pylori: a
combined analysis of 12 case control studies nested within prospective cohorts. Gut
Hennig EE, Mernaugh R, Edl J, Cao P, Cover TL (2004) Heterogeneity among Helicobacter pylori
strains in expression of the outer membrane protein BabA. Infect Immun 72:3429–3435, http://
www.ncbi.nlm.nih.gov/pubmed/15155649
Higashi H, Yokoyama K, Fujii Y, Ren S, Yuasa H, Saadat I, Murata-Kamiya N, Azuma T,
Hatakeyama M (2005) EPIYA motif is a membrane-targeting signal of Helicobacter pylori
virulence factor CagA in mammalian cells. J Biol Chem 280:23130–23137, http://www.ncbi.
nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_
uids¼15831497
Houghton J, Stoicov C, Nomura S, Rogers AB, Carlson J, Li H, Cai X, Fox JG, Goldenring JR,
Wang TC (2004) Gastric cancer originating from bone marrow-derived cells. Science
Howson CP, Hiyama T, Wynder EL (1986) The decline in gastric cancer: epidemiology of an
unplanned triumph. Epidemiol Rev 8:1–27, http://www.ncbi.nlm.nih.gov/pubmed/3533579
Huang JQ, Zheng GF, Sumanac K, Irvine EJ, Hunt RH (2003) Meta-analysis of the relationship
between cagA seropositivity and gastric cancer. Gastroenterology 125:1636–1644, http://
Ilver D, Arnqvist A, Ogren J, Frick IM, Kersulyte D, Incecik ET, Berg DE, Covacci A,
Engstrand L, Boren T (1998) Helicobacter pylori adhesin binding fucosylated histo-blood
group antigens revealed by retagging. Science 279:373–377, http://www.ncbi.nlm.nih.gov/

Kato S, Tsukamoto T, Mizoshita T, Tanaka H, Kumagai T, Ota H, Katsuyama T, Asaka M,
Tatematsu M (2006) High salt diets dose-dependently promote gastric chemical carcinogenesis
in Helicobacter pylori-infected Mongolian gerbils associated with a shift in mucin production
from glandular to surface mucous cells. Int J Cancer J Int Cancer 119:1558–1566, http://www.
16646055
Kim MK, Sasaki S, Sasazuki S, Tsugane S, Japan Public Health Center-based Prospective Study,
G (2004) Prospective study of three major dietary patterns and risk of gastric cancer in Japan.
Int J Cancer J Int Cancer 110:435–442, http://www.ncbi.nlm.nih.gov/pubmed/15095311
Kim HJ, Lim SY, Lee JS, Park S, Shin A, Choi BY, Shimazu T, Inoue M, Tsugane S, Kim J (2010)
Fresh and pickled vegetable consumption and gastric cancer in Japanese and Korean
populations: a meta-analysis of observational studies. Cancer Sci 101:508–516, http://www.
ncbi.nlm.nih.gov/pubmed/19860848
Konig W et al (2007) Helicobacter exploits integrin for type IV secretion and kinase activation.
Nature 449:862–866, http://www.ncbi.nlm.nih.gov/pubmed/17943123
Lee SA, Kang D, Shim KN, Choe JW, Hong WS, Choi H (2003) Effect of diet and Helicobacter
pylori infection to the risk of early gastric cancer. J Epidemiol 13:162–168, http://www.
12749604
Lee CW, Rickman B, Rogers AB, Muthupalani S, Takaishi S, Yang P, Wang TC, Fox JG (2009)
Combination of sulindac and antimicrobial eradication of Helicobacter pylori prevents pro-
gression of gastric cancer in hypergastrinemic INS-GAS mice. Cancer Res 69:8166–8174,
Lemke LB, Ge Z, Whary MT, Feng Y, Rogers AB, Muthupalani S, Fox JG (2009) Concurrent
Helicobacter bilis infection in C57BL/6 mice attenuates proinflammatory H. pylori-induced
gastric pathology. Infect Immun 77:2147–2158, http://www.ncbi.nlm.nih.gov/pubmed/
19223483
Lertpiriyapong K, Whary MT, Muthupalani S, Lofgren JL, Gamazon ER, Feng Y, Ge Z, Wang
TC, Fox JG (2014) Gastric colonisation with a restricted commensal microbiota replicates the
promotion of neoplastic lesions by diverse intestinal microbiota in the Helicobacter pylori
INS-GAS mouse model of gastric carcinogenesis. Gut 63:54–63, http://www.ncbi.nlm.nih.
gov/pubmed/23812323
van der Merwe SW et al (2007) An African origin for the intimate association between humans
and Helicobacter pylori. Nature 445:915–918, http://www.ncbi.nlm.nih.gov/entrez/query.
Lofgren JL, Whary MT, Ge Z, Muthupalani S, Taylor NS, Mobley M, Potter A, Varro A,
Eibach D, Suerbaum S et al (2011) Lack of commensal flora in Helicobacter pylori-infected
INS-GAS mice reduces gastritis and delays intraepithelial neoplasia. Gastroenterology
Loh JT, Torres VJ, Cover TL (2007) Regulation of Helicobacter pylori cagA expression in
response to salt. Cancer Res 67:4709–4715, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?
Loh JT, Friedman DB, Piazuelo MB, Bravo LE, Wilson KT, Peek RM Jr, Correa P, Cover TL
(2012) Analysis of Helicobacter pylori cagA promoter elements required for salt-induced
upregulation of CagA expression. Infect Immun 80:3094–3106, http://www.ncbi.nlm.nih.
gov/pubmed/22710874
Teneberg S, Karlsson KA et al (2002) Helicobacter pylori SabA adhesin in persistent infection
and chronic inflammation. Science 297:573–578, http://www.ncbi.nlm.nih.gov/entrez/query.

and peptic ulceration. Lancet 1:1311–1315, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?
Memon AA, Hussein NR, Miendje Deyi VY, Burette A, Atherton JC (2014) Vacuolating cytotoxin
genotypes are strong markers of gastric cancer and duodenal ulcer-associated Helicobacter
pylori strains: a matched case/control study. J Clin Microbiol 52:2984–2989, http://www.ncbi.
Miehlke S, Kirsch C, Agha-Amiri K, Günther T, Lehn N, Malfertheiner P, Stolte M, Ehninger G,
Bayerdörffer E (2000) The Helicobacter pylori vacA s1, m1 genotype and cagA is associated
with gastric carcinoma in Germany. Int J Cancer 87(3):322–327. PMID: 10897035
Naito M, Yamazaki T, Tsutsumi R, Higashi H, Onoe K, Yamazaki S, Azuma T, Hatakeyama M
(2006) Influence of EPIYA-repeat polymorphism on the phosphorylation-dependent biological
activity of Helicobacter pylori CagA. Gastroenterology 130:1181–1190, http://www.ncbi.nlm.
nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_
uids¼16618412
Nomura A, Stemmermann GN, Chyou PH, Kato I, Perez-Perez GI, Blaser MJ (1991) Helicobacter
pylori infection and gastric carcinoma among Japanese Americans in Hawaii. N Engl J Med
Noto JM, Khizanishvili T, Chaturvedi R, Piazuelo MB, Romero-Gallo J, Delgado AG, Khurana
SS, Sierra JC, Krishna US, Suarez G et al (2013) Helicobacter pylori promotes the expression
of Kruppel-like factor 5, a mediator of carcinogenesis, in vitro and in vivo. PLoS One 8:
e54344, http://www.ncbi.nlm.nih.gov/pubmed/23372710
Odenbreit S, Puls J, Sedlmaier B, Gerland E, Fischer W, Haas R (2000) Translocation of
Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science
Ohnishi N, Yuasa H, Tanaka S, Sawa H, Miura M, Matsui A, Higashi H, Musashi M, Iwabuchi K,
Suzuki M et al (2008) Transgenic expression of Helicobacter pylori CagA induces gastroin-
testinal and hematopoietic neoplasms in mouse. Proc Natl Acad Sci U S A 105:1003–1008,
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼PubMed&
Oliveira AG, Santos A, Guerra JB, Rocha GA, Rocha AM, Oliveira CA, Cabral MM, Nogueira
AM, Queiroz DM (2003) babA2- and cagA-positive Helicobacter pylori strains are associated
with duodenal ulcer and gastric carcinoma in Brazil. J Clin Microbiol 41:3964–3966, http://
Park JM, Park SH, Hong KS, Han YM, Jang SH, Kim EH, Hahm KB (2014) Special licorice
extracts containing lowered glycyrrhizin and enhanced licochalcone a prevented Helicobacter
pylori-initiated, salt diet-promoted gastric tumorigenesis. Helicobacter 19:221–236, http://
Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J Clin
Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, Sibley RK
(1991) Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med
Parsonnet J, Friedman GD, Orentreich N, Vogelman H (1997) Risk for gastric cancer in people
with CagA positive or CagA negative Helicobacter pylori infection. Gut 40:297–301, http://
www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_
uids¼9135515
Peek RM Jr, Crabtree JE (2006) Helicobacter infection and gastric neoplasia. J Pathol
Pera M, Cameron AJ, Trastek VF, Carpenter HA, Zinsmeister AR (1993) Increasing incidence of
adenocarcinoma of the esophagus and esophagogastric junction. Gastroenterology
Plummer M, Franceschi S, Vignat J, Forman D, de Martel C (2014) Global burden of gastric
cancer attributable to pylori. Int J Cancer J Int Cancer 136:487–490, http://www.ncbi.nlm.nih.
gov/pubmed/24889903
Polk DB, Peek RM Jr (2010) Helicobacter pylori: gastric cancer and beyond. Nat Rev Cancer
Powell AE, Wang Y, Li Y, Poulin EJ, Means AL, Washington MK, Higginbotham JN,
Juchheim A, Prasad N, Levy SE et al (2012) The pan-ErbB negative regulator Lrig1 is an
intestinal stem cell marker that functions as a tumor suppressor. Cell 149:146–158, http://www.
Ren JS, Kamangar F, Forman D, Islami F (2012) Pickled food and risk of gastric cancer – a
systematic review and meta-analysis of English and Chinese literature. Cancer epidemiology,
biomarkers & prevention: a publication of the American Association for Cancer Research,
cosponsored by the Am Soc Prev Oncol 21:905–915 http://www.ncbi.nlm.nih.gov/pubmed/
22499775
region, is associated with gastric cancer. Gastroenterology 133:926–936, http://www.ncbi.nlm.
nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_uids¼17854597
components for pilus assembly at the bacteria-host cell interface. PLoS Pathog 7:e1002237,
Sheh A, Fox JG (2013) The role of the gastrointestinal microbiome in Helicobacter pylori
pathogenesis. Gut Microbes 4:505–531, http://www.ncbi.nlm.nih.gov/pubmed/23962822
Shikata K, Kiyohara Y, Kubo M, Yonemoto K, Ninomiya T, Shirota T, Tanizaki Y, Doi Y,
Tanaka K, Oishi Y et al (2006) A prospective study of dietary salt intake and gastric cancer
incidence in a defined Japanese population: the Hisayama study. Int J Cancer J Int Cancer
Sipponen P, Marshall BJ (2000) Gastritis and gastric cancer. Western countries. Gastroenterol Clin
North Am 29:579–592, v-vi http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&
Soerjomataram I, Lortet-Tieulent J, Parkin DM, Ferlay J, Mathers C, Forman D, Bray F (2012)
Global burden of cancer in 2008: a systematic analysis of disability-adjusted life-years in
12 world regions. Lancet 380:1840–1850, http://www.ncbi.nlm.nih.gov/pubmed/23079588
Solnick JV, Hansen LM, Salama NR, Boonjakuakul JK, Syvanen M (2004) Modification of
rhesus macaques. Proc Natl Acad Sci U S A 101:2106–2111, http://www.ncbi.nlm.nih.gov/
pubmed/14762173
Song H, Michel A, Nyren O, Ekstrom AM, Pawlita M, Ye W (2014) A CagA-independent cluster
of antigens related to the risk of noncardia gastric cancer: associations between Helicobacter
pylori antibodies and gastric adenocarcinoma explored by multiplex serology. Int J Cancer J Int
Cancer 134:2942–2950, http://www.ncbi.nlm.nih.gov/pubmed/24259284
Peek RM Jr, Boren T et al (2010) Expression of the BabA adhesin during experimental
infection with Helicobacter pylori. Infect Immun 78:1593–1600, http://www.ncbi.nlm.nih.
gov/pubmed/20123715
Sun J, Aoki K, Zheng JX, Su BZ, Ouyang XH, Misumi J (2006) Effect of NaCl and Helicobacter
pylori vacuolating cytotoxin on cytokine expression and viability. World J Gastroenterol
Syder AJ, Guruge JL, Li Q, Hu Y, Oleksiewicz CM, Lorenz RG, Karam SM, Falk PG, Gordon JI
(1999) Helicobacter pylori attaches to NeuAc alpha 2,3Gal beta 1,4 glycoconjugates produced
in the stomach of transgenic mice lacking parietal cells. Mol Cell 3:263–274, http://www.ncbi.
Syder AJ, Oh JD, Guruge JL, O’Donnell D, Karlsson M, Mills JC, Bjorkholm BM, Gordon JI
(2003) The impact of parietal cells on Helicobacter pylori tropism and host pathology: an
analysis using gnotobiotic normal and transgenic mice. Proc Natl Acad Sci U S A
Thomson MJ, Pritchard DM, Boxall SA, Abuderman AA, Williams JM, Varro A, Crabtree JE
(2012) Gastric Helicobacter infection induces iron deficiency in the INS-GAS mouse. PLoS
One 7:e50194, http://www.ncbi.nlm.nih.gov/pubmed/23185574
Tsugane S (2005) Salt, salted food intake, and risk of gastric cancer: epidemiologic evidence.
Cancer Sci 96:1–6, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼
Tsugane S, Sasazuki S (2007) Diet and the risk of gastric cancer: review of epidemiological
evidence. Gastric Cancer: Off J Int Gastric Cancer Assoc Jpn Gastric Cancer Assoc 10:75–83,
Uehara T, Ma D, Yao Y, Lynch JP, Morales K, Ziober A, Feldman M, Ota H, Sepulveda AR
(2013) H. pylori infection is associated with DNA damage of Lgr5-positive epithelial stem
cells in the stomach of patients with gastric cancer. Dig Dis Sci 58:140–149, http://www.ncbi.
Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S, Yamakido M, Taniyama K,
Sasaki N, Schlemper RJ (2001) Helicobacter pylori infection and the development of gastric
cancer. N Engl J Med 345:784–789, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?
Varon C, Dubus P, Mazurier F, Asencio C, Chambonnier L, Ferrand J, Giese A, Senant-Dugot N,
Carlotti M, Megraud F (2012) Helicobacter pylori infection recruits bone marrow-derived cells
that participate in gastric preneoplasia in mice. Gastroenterology 142:281–291, http://www.
Wang J, Fan X, Lindholm C, Bennett M, O’Connoll J, Shanahan F, Brooks EG, Reyes VE, Ernst
PB (2000a) Helicobacter pylori modulates lymphoepithelial cell interactions leading to epi-
thelial cell damage through Fas/Fas ligand interactions. Infect Immun 68:4303–4311, http://
www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼Retrieve&db¼PubMed&dopt¼Citation&list_
uids¼10858249
Wang TC, Dangler CA, Chen D, Goldenring JR, Koh T, Raychowdhury R, Coffey RJ, Ito S,
Varro A, Dockray GJ et al (2000b) Synergistic interaction between hypergastrinemia and
Helicobacter infection in a mouse model of gastric cancer. Gastroenterology 118:36–47, http://
Whary MT, Muthupalani S, Ge Z, Feng Y, Lofgren J, Shi HN, Taylor NS, Correa P, Versalovic J,
Wang TC et al (2014) Helminth co-infection in Helicobacter pylori infected INS-GAS mice
attenuates gastric premalignant lesions of epithelial dysplasia and glandular atrophy and pre-
serves colonization resistance of the stomach to lower bowel microbiota. Microbes Infect/
Institut Pasteur 16:345–355, http://www.ncbi.nlm.nih.gov/pubmed/24513446
Winter JA, Letley DP, Cook KW, Rhead JL, Zaitoun AA, Ingram RJ, Amilon KR, Croxall NJ,
Kaye PV, Robinson K et al (2014) A role for the vacuolating cytotoxin, VacA, in colonization
and Helicobacter pylori-induced metaplasia in the stomach. J Infect Dis 210:954–963, http://
Wu X, Chen VW, Andrews PA, Ruiz B, Correa P (2007) Incidence of esophageal and gastric
cancers among Hispanics, non-Hispanic whites and non-Hispanic blacks in the United States:
subsite and histology differences. Cancer Causes Control: CCC 18:585–593, http://www.ncbi.

odenal disease. Gut 55:775–781, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd¼
Retrieve&db¼PubMed&dopt¼Citation&list_uids¼16322107
Yip R, Limburg PJ, Ahlquist DA, Carpenter HA, O’Neill A, Kruse D, Stitham S, Gold BD, Gunter
EW, Looker AC et al (1997) Pervasive occult gastrointestinal bleeding in an Alaska native
population with prevalent iron deficiency. Role of Helicobacter pylori gastritis. JAMA
Yu J, Leung WK, Go MY, Chan MC, To KF, Ng EK, Chan FK, Ling TK, Chung SC, Sung JJ
(2002) Relationship between Helicobacter pylori babA2 status with gastric epithelial cell
turnover and premalignant gastric lesions. Gut 51:480–484, http://www.ncbi.nlm.nih.gov/
pubmed/12235067
Chapter 18
Helicobacter pylori Infection and MALT
Lymphoma
Xavier Sagaert
Abstract Extranodal marginal zone lymphoma of mucosa-associated lymphoid

tissue (MALT), also known as MALT lymphoma, is an indolent B-cell
non-Hodgkin lymphoma, arising in lymphoid infiltrates that are induced by chronic
inflammation in extranodal sites. The stomach is the most commonly affected
organ, where MALT lymphomagenesis is clearly associated with Helicobacter
pylori gastroduodenitis. Outside the stomach, the role of infectious agents is less
clearly defined. In recent years, gastric MALT lymphoma became the focus of
attention because of the involvement of its genetic aberrations in the nuclear factor
kappa B (NF-κB) pathway, currently one of the most investigated pathways in the
fields of immunology and oncology. This chapter presents gastric MALT lym-
phoma as an outstanding example of the close pathogenic link between
Helicobacter pylori-induced chronic inflammation and tumour development. It
also presents gastric MALT lymphoma as one of the best models of how genetic
events initiate oncogenesis, determine tumour biology, dictate clinical behaviour
and represent viable therapeutic targets. Moreover, in view of the association of
gastric MALT lymphoma with deregulation of the NF-κB pathway, the latter
signalling pathway is also discussed in depth in both physiological and pathological
conditions.
Keywords Helicobacter pylori • MALT lymphoma • NF-κB pathway •

Carcinogenesis
18.1 Introduction
The marginal zone (MZ) represents a microanatomic compartment of the B follicle

and is especially well developed in lymphoid organs that are continuously exposed
to antigenic stimulation (e.g. spleen, mesenteric lymph nodes and mucosa-
associated lymphoid tissue or MALT) (Martin and Kearney 2002). The role of
X. Sagaert (*)
Department of Pathology, University Hospital K.U.Leuven, Minderbroederstraat 12, 3000
Leuven, Belgium

DOI 10.1007/978-4-431-55936-8_18
424 X. Sagaert
the splenic MZ has been well investigated. Splenic MZ B cells play a crucial role in
the immune response to T-cell-independent antigens, like polysaccharides of
encapsulated bacteria (Haemophilus influenza, Neisseria meningitides and Strepto-
coccus pneumoniae) (Pillai et al. 2005). As such, a suboptimal function of the
marginal zone, as seen in children under the age of 2 years or in splenectomised
individuals, dramatically increases vulnerability to infections with encapsulated
bacteria, and therefore vaccination in these groups is advised (Brigden and Pattullo
1999; Kruschinski et al. 2004). Marginal zone lymphomas (MZL) are believed to be
the neoplastic counterpart of the MZ. They account for approximately 8 % of B-cell
non-Hodgkin lymphomas, making it the third most frequent subtype after diffuse
large B-cell lymphoma (DLBCL) and follicular lymphoma. Based on their ana-
tomic location, the World Health Organisation (WHO) makes a distinction between
MALT lymphoma, splenic MZL and nodal MZL subtypes (Jaffe et al. 2008).
MALT lymphoma differs from its splenic and nodal counterpart as it arises in
organs that normally lack lymphoid tissue (like stomach, lung, salivary and lach-
rymal glands) but that have accumulated B cells in response to either chronic
infections or autoimmune processes (De Re et al. 2000; Ferreri et al. 2004; Hyjek
and Isaacson 1988; Hyjek et al. 1988; Lecuit et al. 2004; Roggero et al. 2000;
Stefanovic and Lossos 2009; Wotherspoon et al. 1991; Zucca et al. 2000). Sustained
(auto) antigenic stimulation not only triggers a polyclonal B-cell proliferation but
also attracts neutrophils to the site of inflammation, with the subsequent release of
reactive oxygen species (ROS). The latter are genotoxic and cause a wide range of
genetic abnormalities (Coussens and Werb 2002). Moreover, prolonged prolifera-
tion of B cells induced by chronic inflammation may also increase the risk of DNA
damage, like double-strand DNA breaks, due to the intrinsic genetic instability of B
cells during somatic hypermutation and class switch recombination (Goossens
et al. 1998). Several of these genotoxic events in MALT lymphomas have been
identified as either aneuploidy (trisomy 3, 7, 12 and 18) or disease-specific chro-
mosomal translocations, being t(1;14)(p22;q32), t(14;18)(q32;q21), t(11;18)(q21;
q21) and t(3;14)(p13;q32). Remarkably, the genes targeted by at least two of these
aneuploidies and at least three of these translocations participate in one and the
same pathway resulting in the activation of nuclear factor kappa B (NF-κB), which
is a key transcription factor in the immune response and has been the focus of
intense investigation over the past two decades (Bonizzi and Karin 2004). NF-κB
regulates the expression of a number of survival- and proliferation-related genes in
B cells (Ruefli-Brasse et al. 2003; Siebenlist et al. 2005), and as such, its constitu-
tive activation may result in uncontrolled B-cell proliferation and thus
lymphomagenesis (Packham 2008).
Here, we concentrate on MALT lymphomas at the most common site, the
stomach. Gastric MALT lymphoma accounts for 5 % of all gastric cancers and at
least 50 % of all gastric lymphomas, making it the most frequent lymphoma of the
gastrointestinal tract. Because of its association with Helicobacter pylori, gastric
MALT lymphoma represents a fascinating model of the close pathogenic link
between chronic inflammation and lymphoma development. In this review, we
will discuss the current insights into the pathogenesis of gastric MALT lymphomas
18 Helicobacter pylori Infection and MALT Lymphoma 425
and integrate this information into daily clinical practice as well as provide strat-
egies for new therapeutic targets.
18.2 Clinicopathological Features
Endoscopic biopsies remain the gold standard for the diagnosis of gastric MALT
lymphomas. Histologically, the tumour appears as a diffuse spread of neoplastic
lymphoma cells, surrounding reactive B follicles and invading epithelial structures
resulting in so-called lymphoepithelial lesions (Fig. 18.1). Diagnosis can be diffi-
cult, especially in cases where lymphoepithelial lesions are not prominent and/or
reactive B follicles cannot be recognised because of neoplastic colonisation.
Molecular techniques such as polymerase chain reaction (PCR) can further support
the diagnosis of a MALT lymphoma by identifying clonality of B cells, based on
the fact that all lymphoma cells have the same immunoglobulin gene
rearrangement. Most MALT lymphomas present as Ann Arbor stage IE disease
(extranodal disease limited to the site of origin). In 10–20 % of cases, regional
lymph nodes are involved (stage IIE), while bone marrow involvement occurs in
5–10 % (Montalban et al. 1995; Thieblemont et al. 2000). Multiple extranodal sites
are involved in 10 % of cases at the time of presentation (e.g. small intestine, colon,
the salivary gland and the splenic MZ). Consequently, extensive tumour staging
should be performed at diagnosis, especially at the above mentioned sites. The
disease is remarkably indolent and tends to remain localised for a long period of
time. Transformation into an aggressive DLBCL may occur. The 10-year survival
rate for gastric MALT lymphomas is approximately 90 %, with a disease-free
survival of approximately 70 % (Cogliatti et al. 1991). However, once transforma-
tion to DLBCL occurs, the 10-year survival rate falls to approximately 45 %
(Cogliatti et al. 1991). It is nowadays generally accepted that H. pylori eradication
with antibiotics is the first choice of therapy for localised gastric MALT lymphoma
(Zullo et al. 2009). The use of anti-infectious treatment in non-gastric locations is
still under investigation, although two studies discourage the use of antibiotics in
non-gastric MALT lymphoma (Grunberger et al. 2006a, b). Lack of response to
H. pylori eradication, stable disease at 1 year or disseminated disease at diagnosis is
usually considered an indication for chemo- or radiotherapy (Aviles et al. 2005;
Martinelli et al. 2005; Schmelz et al. 2005; Tsang et al. 2003). Surgery only has a
role in the treatment of gastric MALT lymphomas if local complications
(e.g. perforation) occur.
18.3 Gastric MALT Lymphoma Pathogenesis
Gastric MALT lymphoma development is a multistep progression from a reactive,

polyclonal to a neoplastic, monoclonal lymphoproliferation (Fig. 18.2).
426 X. Sagaert
Fig. 18.1 Morphologic and immunohistochemical features of a MALT lymphoma. (a) Histology
of a gastric MALT lymphoma (magnification 50): the tumour cells surround reactive B follicles
and invade gastric glandular epithelium, resulting in so-called lymphoepithelial lesions. (b)
Nuclear BCL10 expression is a typical feature of t(11;18)(q21;q21)- and (1;14)(p22;q32)-positive
MALT lymphomas (magnification 200). (c) t(14;18)(q32;q21)-positive MALT lymphomas are
characterised by perinuclear BCL10 expression (magnification 200)
18.3.1 Step 1: Acquisition of MALT
MALT lymphomas define a distinct category of infection-related lymphomas, in

which the infectious agent induces neoplastic lymphoid transformation by chron-
ically triggering the immune system and, as such, maintaining a protracted prolif-
erative status of lymphocytes. This is in contrast to the lymphotropic oncogenic
release of
chemotaxis of reactive oxygen
neutrophils species
H. pylori continuous
ACQUISITION stimulation & proliferation
OF MALT of B-lymphocytes
B-cells ACQUISITION
OF GENETIC ANOMALIES
CD40
CD40L
Recruitment &
activation of
T-cells
H. pylori
eradication therapy
decreased success rate
unknown MALT LYMPHOMA t(11;18)

mechanisme
others :
* t(1;14)
unknown * t(3;14)
DIFFUSE LARGE mechanisme MALT LYMPHOMA * t(14;18)
B-CELL LYMPHOMA (FOXP1 ?) * aneuploidy
Fig. 18.2 Hypothetical model for the pathogenesis of gastric MALT lymphoma. H. pylori
infection attracts B lymphocytes, T lymphocytes and neutrophils to the gastric mucosa. B-cell
proliferation is driven by CD40-CD40L interaction with H. pylori activated, reactive T cells as
well as by cytokines. The prolonged proliferative state of B cells as well as the release of reactive
oxygen species by neutrophils present in the area of chronic inflammation, induce additional
oncogenetic events which make the lymphoproliferation independent of antigenic stimulation.
t(11;18)(q21;q21)-positive MALT lymphomas are resistant to H. pylori therapy and do not evolve
to DLBCLs. Additional genetic alterations in t(11;18)(q21;q21)-negative MALT lymphomas may
ultimately result in transformation to a clinically aggressive DLBCL
viruses human herpesvirus 8 (HHV-8), human T-lymphotropic virus 1 (HTLV-1),

and Epstein-Barr virus (EBV), which directly infect and transform lymphoid cells
into tumour cells. Increasing evidence suggests that MZ lymphomas, and MALT
lymphomas in particular, are indeed proceeded by chronic antigenic stimulation. So
far, five microbial species are associated with MZ lymphoproliferations: H. pylori,
Campylobacter jejuni (C. jejuni), Chlamydia psittaci (C. psittaci), Borrelia
burgdorferi (B. burgdorferi) and hepatitis C virus (HCV); they are linked to gastric
MALT lymphoma, immunoproliferative small intestinal disease (IPSID), orbital
MALT lymphoma, cutaneous MALT lymphoma and splenic MZ lymphoma,
respectively (De Re et al. 2000; Ferreri et al. 2004; Lecuit et al. 2004; Roggero
et al. 2000; Wotherspoon et al. 1991; Zucca et al. 2000).
Of all MZ lymphomas, the infectious aetiology of gastric MALT lymphoma has
been documented the best. In healthy individuals, a thick layer of viscous mucus as
well as gastric acid limit bacterial colonisation of the stomach. However, H. pylori,
a Gram-negative bacterium also associated with peptic ulceration and gastric
carcinoma (Parsonnet et al. 1994), survives in the acid environment by secreting
428 X. Sagaert
a pH buffering urease and triggers lymphoid infiltration. There is now compelling

evidence that gastric MALT lymphoma is caused by H. pylori infection. First, the
prevalence of H. pylori in both the gastric mucosa and serum of gastric MALT
lymphoma patients is well above the infection frequency in other populations
(Wotherspoon et al. 1991; Parsonnet et al. 1994). Second, gastric MALT lymphoma
has the highest incidence in regions with endemic H. pylori infection (Doglioni
et al. 1992). Third, H. pylori triggers T-cell-mediated B-cell growth in vitro by
activating the CD40 pathway (Hussell et al. 1993a). Also, H. pylori eradication
therapy leads to complete lymphoma regression in about 80 % of the cases with
early stage disease (Bayerdorffer et al. 1995; Wotherspoon et al. 1993, 1994).
Finally, gastric MALT lymphomas can be induced in vivo in mice models by
prolonged H. pylori infection (Enno et al. 1995).
Remarkably, despite proliferation of tumour cells after H. pylori stimulation, the
gastric MALT lymphoma-derived immunoglobulin recognises various
autoantigens instead of H. pylori (Hussell et al. 1993b). As such, it may be
hypothesised that gastric MALT lymphoma arises from H. pylori-stimulated,
autoreactive B cells. Evidence for the role of an (auto)antigen in MALT
lymphomagenesis is also supported by sequence analysis studies of the immuno-
globulin heavy chain (IGH) gene in MALT lymphomas, which revealed the occur-
rence of somatic hypermutation with a pattern indicative of antigen selection
(Bahler et al. 1997; Du et al. 1996a; Hara et al. 2001; Qin et al. 1995). Also,
MALT lymphoma IGH genes were frequently derived from germline IGH genes
that are commonly involved in autoantibody production (Bahler et al. 1997; Du
et al. 1996b; Hara et al. 2001; Qin et al. 1995). In addition, ongoing mutation
(i.e. intraclonal variation) of the IGH gene was observed in MALT lymphoma,
emphasising the importance of continuing antigenic stimulation in the clonal B-cell
expansion (Qin et al. 1995; Du et al. 1996a). However, the rate of ongoing mutation
gradually declines during tumour progression, indicating that the role of direct
antigenic stimulation in MALT lymphomagenesis decreases during tumour evolu-
tion. This is probably due to the occurrence of ROS-induced genetic anomalies,
which make the lymphoproliferation progressively independent of antigenic
stimulation.
Conversely, 5–10 % of gastric MALT lymphomas are H. pylori negative. In
some of these cases, H. pylori infection may be undiagnosed. This is particularly the
case if H. pylori testing was only performed on the biopsy, as lymphomas may arise
in atrophic mucosa where H. pylori bacterial load is low. Therefore, a negative
H. pylori test in gastric MALT lymphoma should prompt reinvestigation by means
of the urea breath test, serological antibody test, biopsy immunostaining and/or
stool culture. It has been suggested that some H. pylori-negative gastric MALT
lymphomas are associated with H. heilmannii, and, interestingly, these lymphomas
have been shown to respond to antibiotic therapy (Morgner et al. 2000).
Outside the stomach, the role of infectious agents is less clearly defined. Asso-
ciations of previously mentioned bacteria (C. jejuni, B. burgdorferi, C. psittaci)
with MALT lymphoma have been described, based on epidemiological studies and
the detection of these bacteria in the affected tissue (Ferreri et al. 2004; Lecuit
et al. 2004; Roggero et al. 2000). Contrary to H. pylori, none of these infections
fulfil the criteria postulated by Koch (i.e. is the bacteria detectable in the host’s
tissue in early disease stage? can the bacteria be cultivated from the affected tissue?
can the bacteria induce the disease in animal models? can the bacteria be isolated
from sick animals?). However, new criteria have been established by recent molec-
ular advances that take into account the host specificity as well as putative
uncultivability of certain microbial organisms (Frederickx and Relman 1996;
Franco et al. 2004). Nevertheless, whether the observed association between
C. jejuni, B. burgdorferi and C. psittaci and MALT lymphoma is a proof of
causation remains to be investigated. Furthermore, it is well established that
autoimmune diseases increase the risk of developing non-gastric MALT lympho-
mas. Autoreactive B cells infiltrate the thyroid gland in Hashimoto thyroiditis and
the salivary glands in Sj€ogren syndrome and progressively organise into a MALT-
mimicking lymphoproliferation. Patients with Sj€ogren syndrome have a 44-fold
increased risk of developing a lymphoma, and MALT lymphomas account for
approximately 85 % of lymphomas in patients with Sj€ogren syndrome (Royer
et al. 1997). Patients with Hashimoto thyroiditis have a 70-fold increased risk of
thyroid lymphoma, and thyroiditis in the adjacent gland is present in 94 % of
thyroid lymphomas (Derringer et al. 2000).
18.3.2 Step 2: Acquisition of Genetic Abnormalities
Besides aneuploidy (trisomy 3, 7, 12 and 18), the chromosomal translocations t

(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21) and t(3;14)(p13;q32) all
occur with variable frequencies in gastric MALT lymphomas, resulting in API2-
MALT1, IGH-BCL10, IGH-MALT1 and IGH-FOXP1 rearrangements, respectively
(Baens et al. 2000; Streubel et al. 2003; Willis et al. 1999; Wlodarska et al. 2005).
T(11;18)(q21;q21) is the most common structural chromosomal abnormality in
MALT lymphomas. Remarkably, t(11;18)(q21;q21) is not found in other lym-
phoma types and its presence in MALT lymphoma correlates with the absence of
any further genetic aberrations (Muller-Hermelink 2003; Starostik et al. 2002).
Depending upon the study performed, it is present in 10–50 % of gastric MALT
lymphomas, whereas this translocation rarely occurs in non-gastric MALT lym-
phomas, with the exception of pulmonary MALT lymphomas (Baens et al. 2000;
Ye et al. 2003; Streubel et al. 2004). T(11;18)(q21;q21) fuses the amino-terminus of
the API2 gene (located at 11q21) to the carboxyl-terminus of the MALT1 gene
(located at 18q21), hereby creating the fusion protein API2-MALT1 (Dierlamm
et al. 1999; Akagi et al. 1999) (Fig. 18.3). The apoptosis inhibitor protein API2 is a
member of the inhibitor of apoptosis protein (IAP) family and inhibits the biolog-
ical activity of certain caspases (Roy et al. 1997). It contains three BIR (baculovirus
IAP repeat) domains, a CARD (caspase recruitment domain) motif and one RING
(really interesting new gene) finger motif. MALT1, classified as a paracaspase, is an
essential protein in the antigen receptor-mediated pathway that leads to NF-κB
430 X. Sagaert
Fig. 18.3 Structure of the API2 and MALT1 gene structure. Arrowheads indicate known
breakpoints and their frequencies. The breakpoints within the API2 gene almost consistently
occur in intron 7 (annotated now as intron 6 according to Ensembl gene ENSG00000023445),
whereas the breakpoints within the MALT1 coding DNA are more variable and are located in
introns 2, 4, 7 and 8, resulting, respectively, in an API2/exon7-MALT1/exon3, API2/exon7-
MALT1/exon5, API2/exon7-MALT1/exon8 and API2/exon7-MALT1/exon9 fusion products.
Abbreviations: BIR baculovirus inhibitor of apoptosis repeat, CARD caspase recruitment domain,
RING really interesting new gene, DD death domain, Ig-l immunoglobulin-like, p20 caspase-like
p20 domain, T6 TRAF6 binding site
activation and comprises an N-terminal death domain (DD), two immunoglobulin-

like domains and a C-terminal caspase-like domain (Uren et al. 2000). The
breakpoints in both API2 and MALT1 are well characterised (Baens et al. 2000;
Ye et al. 2003; Dierlamm et al. 1999; Remstein et al. 2000; Motegi et al. 2000;
Kalla et al. 2000; Liu et al. 2001a). All breakpoints are fused in-frame and
constitute a total of eight variant forms of the fusion transcripts, all of which
comprise the three intact BIR domains of the N-terminal API2 portion and the
intact caspase-like domain of the C-terminal MALT1 portion (Morgan et al. 1999).
The selection of specific domains of API2 and MALT1 to form a functional fusion
protein emphasises their synergetic importance in lymphomagenesis.
Presence of t(11;18)(q21;q21) in tumour tissue, either fresh frozen or paraffin
embedded, can be detected by reverse transcription-PCR (RT-PCR) and fluores-
cence in situ hybridisation (FISH). One or both of these techniques are now
routinely performed in most labs as the presence of t(11;18)(q21;q21) has direct
clinical compact : not only does it confirm the diagnosis of a MALT lymphoma,
moreover, t(11;18)(q21;q21)-positive gastric MALT lymphomas are more often
resistant to H. pylori eradication treatment and only rarely if ever evolve to a
DLBCL (Ye et al. 2003; Alpen et al. 2000; Liu et al. 2001b; Sugiyama
et al. 2001). Also, the presence of t(11;18)(q21;q21) in gastric MALT lymphomas
is significantly associated with infection by the CagA-positive H. pylori strains. The
latter generates a strong inflammatory response with release of interleukin-8 (IL-8),
a powerful chemokine for neutrophil activation and subsequent ROS secretion
(Ye et al. 2003). Therefore, it is tempting to hypothesise that t(11;18)(q21;q21) is
related to genotoxic oxidative stress caused by inflammatory responses in prema-
lignant MALT-like lesions, specifically in the mucosa of organs where an abundant
influx of exogenous environmental antigens is known to occur (e.g. stomach
and lung).
T(1;14)(p22;q32) and its variant t(1;2)(p22;p12) are present in approximately
5 % of MALT lymphomas, which tend to present with advanced stage of disease
and typically reveal additional genetic aberrations (Willis et al. 1999; Achuthan
et al. 2000; Wotherspoon et al. 1992). As a consequence of this translocation, the
BCL10 gene (located at 1p22) is brought under the control of the IGH gene
enhancer located at 14q32 (or the immunoglobulin light chain κ gene enhancer at
2p12 in the case of the variant translocation), resulting in overexpression of the
BCL10 transcript (Ye et al. 2000). BCL10 encodes a CARD-containing protein that
plays a key role in the antigen receptor signalling to NF-κB (Thome 2004).
Depending upon the study performed, t(14;18)(q32;q21) is demonstrated in
2–20 % of MALT lymphomas (Sagaert et al. 2006a; Streubel et al. 2005). Similar
to t(1;14)(q21;q21), this translocation is mediated by IGH and leads to MALT1
overexpression. In contrast to t(11;18)(q21;q21)-positive cases, t(14;18)(q32;q21)-
positive MALT lymphomas mainly occur outside the gastrointestinal or pulmonary
tract, presenting as tumours of the skin, breast, lachrymal glands, liver or salivatory
glands (Streubel et al. 2005; Remstein et al. 2004). It is hypothesised that this
polarisation reflects a distinct pathogenesis; that is that MALT lymphomas of the
stomach and lung are associated with an (un)known infectious agent while lym-
phomas arising in the salivary glands and ocular adnexa are often linked to
autoimmune disease. As such, genetic differences might reflect different exposures
to various inflammatory agents associated with MALT lymphomas in different
locations.
More recently, FOXP1 (forkhead-box protein P1 gene ; located at 3p13) was
identified as a new translocation partner of IGH, not only in MALT lymphomas but
also in DLBCL with mainly extranodal location (Wlodarska et al. 2005; Streubel
et al. 2005). In both lymphoma subtypes, the overall frequency of t(3;14)(p13;q32)
is relatively low (Wlodarska et al. 2005; Streubel et al. 2005; Sagaert et al. 2006b;
Haralambieva et al. 2006). Remarkably, a significant number of t(3;14)(p13;q32)-
negative MALT lymphomas and DLBCLs harbour strong nuclear FOXP1 expres-
sion, suggesting that mechanisms other than underlying FOXP1 rearrangements
can upregulate FOXP1 expression (e.g. trisomy 3). The significance of this nuclear
FOXP1 overexpression is still debated: two studies found FOXP1 to be associated
with inferior survival in DLBCLs (Banham et al. 2005; Barrans et al. 2004),
whereas this could not be confirmed in a third study (Hans et al. 2004). Also, two
studies found strong nuclear FOXP1 expression to be confined to (gastric) MALT
lymphomas that are at risk of transforming into a DLBCL with poor clinical
outcome (Sagaert et al. 2006a; Han et al. 2009). The FOXP1 gene encodes a
member of the FOX family of transcription factors, which is characterised by a
common DNA-binding winged helix or forkhead domain (Banham et al. 2001). It
was shown that FOXP1 is essential for B-cell maturation in the bone marrow, since
FOXP1-deficient mice showed an arrest in the transition from pro-B cell to pre-B
cell, probably due to diminished expression of the recombination-activating genes
RAG1 and RAG2 (Hu et al. 2006). However, so far, it is not clear yet how FOXP1
mediates signalling in the mature, peripheral B-cell pool and how FOXP1 could
contribute to MALT lymphomagenesis.
432 X. Sagaert
18.3.3 Step 3: Deregulation of the NF-κB Pathway
Mounting evidence links the oncogenic activity of t(11;18)(q12;q21), t(1;14)(p22;

q32) and t(14;18)(q32;q21) to aberrant NF-κB activation by API2-MALT1, BCL10
and MALT1, respectively. NF-κB was first described in B lymphocytes as a
transcription factor binding to the κB site of the immunoglobulin κ-light chain
enhancer. Soon after, it was found that NF-κB activity could be induced in all cell
types. To date, five members of the NF-κB transcription factor family have been
identified: RelA (p65), RelB, c-REL, NF-κB1 and NF-κB (Siebenlist et al. 2005).
After post-translation modification, the NF-κB1 and NF-κB2 precursor molecules
p105 and p100 are processed to p50 and p52, respectively. All 5 NF-κB family
members share a conserved REL homology domain for DNA binding, and they are
all essential for lymphocyte survival and activation. Two distinct NF-κB signalling
pathways have been identified: the canonical and non-canonical pathway (Li and
Verma 2002; Pomerantz and Baltimore 2002). The canonical pathway engages
RelA/p50 dimers and is triggered by viruses, bacterial lipopolysaccharides and
pro-inflammatory cytokines such as TNF-α (tumour-necrosis factor-α) and IL-1
(interleukin-1). In unstimulated B cells, inhibitory κB (IκB) proteins bind to the
NF-κB molecules RelA and p50, forming latent complexes that are present in the
cytoplasm. Activation of the canonical pathway induces polyubiquitination and
activation of Iκ-B kinase-γ (IKK-γ), also known as NF-κB essential modulator
(NEMO). This event results in the phosphorylation and subsequent proteasomal
degradation of IκB by the IKK catalytic subunit IKK-α. This allows the RelA/p50
dimers to translocate to the nucleus and mediate transcription of several
antiapoptotic genes, including BCL-XL and BCL-2, as well as pro-proliferative
genes, such as cyclin D2. Conversely, the non-canonical pathway engages RelB/
p52 dimers and is induced by a limited number of stimuli including BAFF (B-cell
activating factor), lymphotoxin-β and CD40L (CD40 ligand). In a NEMO-
independent manner, it leads to the phosphorylation of p100 by IKK-α and the
subsequent degradation of its carboxyl-terminal half, resulting in the nuclear
translocation of the p52/RelB dimers. Both pathways switch on a different set of
genes and therefore mediate distinct regulatory functions, one that is mostly
involved in innate immunity (canonical pathway) and the other in adaptive immu-
nity (non-canonical pathway) (Bonizzi and Karin 2004).
Although both pathways modulate MALT lymphomagenesis, most of our cur-
rent knowledge relates to the canonical pathway. The last decade has witnessed a
significant advance in our understanding of this pathway through the identification
of two key molecules, BCL10 and MALT1, which function downstream of the
antigen receptor and upstream of the IKK complex. Therefore, their overexpression
induced by fusion with the IGH enhancer in t(1;14)(p22;q32) and t(14;18)(q32;
q32), respectively, hints at a role for NF-κB deregulation in MALT
lymphomagenesis. Studies of knockout mice established that both BCL10 and
MALT1 are essential in transducing antigen receptor signals to activate the
NF-κB pathway (Ruefli-Brasse et al. 2003; Ruland et al. 2001, 2003). In vitro
experiments showed that overexpressed BCL10 activates NF-κB, whereas

overexpressed MALT1 can only do so after BCL10-induced oligomerisation
(Lucas et al. 2001). Also, BCL10-induced NF-κB activation is reduced in
MALT1-deficient cells, an effect that can only be reversed after reintroduction of
MALT1 expression (Ruefli-Brasse et al. 2003). Taken together, these data suggest
that physical and functional BCL10-MALT1 interaction is essential for optimal
NF-κB activation. The physical association between BCL10 and MALT1 involves
a short region downstream of the CARD motif of BCL10 and the immunoglobulin-
like domain of MALT1. This interaction is probably constitutive as endogenous
BCL10 and MALT1 can be co-immunoprecipitated from lysates of non-stimulated
B and T cells (Uren et al. 2000). In 2001, the upstream activator of the BCL10/
MALT1 complex was identified as CARMA1 (CARD, membrane-associated
guanylate kinase [MAGUK], protein 1), a 130 kDa CARD-containing protein that
is constitutively associated with cholesterol- and glycosphingolipid-enriched mem-
brane microdomains, termed lipid rafts (Gaide et al. 2001; Bertin et al. 2001;
Allister-Lucas et al. 2001). The following model of BCR (B-cell antigen
receptor)-induced activation of the canonical pathway fits the best with all currently
available data (Fig. 18.4). BCR ligation by an antigen initiates a tyrosine phos-
phorylation signalling cascade, culminating in the generation of second messengers
that activate PKC (protein kinase C) isoforms (Rawlings et al. 2006). PKC-β then
phosphorylates and structurally reconfigurates CARMA1, hereby exposing the
CARD motifs of CARMA1 and allowing interactions with its downstream compo-
nents (Rawlings et al. 2006). As such, CARMA1 recruits BCL10 and MALT1 to the
lipid rafts and binds BCL10 through CARD-CARD interaction (Gaide et al. 2002;
Wang et al. 2004). Subsequently, the high-molecular-weight BCL10/MALT1 olig-
omers interact with and induce oligomerisation of the TNF receptor-associated
factor 6 (TRAF6) via the carboxyl-terminus of MALT1 (Sun et al. 2004). As a
result, TRAF6 elicits the E3 ubiquitin ligase activity of its RING-E3-domain to
synthesise a Lys63-linked polyubiquitin chain on its target proteins, including
IKK-γ (NEMO) and TRAF6 itself. In contrast to Ly48-linked polyubiquitin chains,
this type of polyubiquitination does not induce the proteasomal degradation of
IKK-γ and TRAF6 but facilitates their interaction with and the activation of
TAK1 (TGF-β activating kinase). Activated TAK1 subsequently fully activates
the IKK complex via phosphorylation of its β-subunit, which results in IkB phos-
phorylation/degradation and nuclear translocation of the p65/p50 dimers.
As NF-κB regulates the transcription of pro-proliferative and antiapoptotic
genes in B cells, it is evident that its constitutive activation may result in
lymphomagenesis. In t(1;14)(p22;q32)-positive MALT lymphomas, characterised
by BCL10 overexpression, BCL10 is thought to oligomerise through its CARD
motif without the need for upstream signalling, thus triggering MALT1
oligomerisation and aberrant NF-κB activation. In t(14;18)(q32;q21)-positive
MALT lymphomas, in which MALT1 is overexpressed, the oligomerisation of
MALT1 with subsequent NF-κB activation is believed to be dependent on
BCL10, as MALT1 does not have a structural domain to mediate self-
oligomerisation, nor does its overexpression alone activate NF-κB in vitro (Uren
434 X. Sagaert
Fig. 18.4 Canonical NF-κB pathway. CARMA1 interacts with the antigen-activated B-cell
receptor in the lipid rafts and induces the oligomerisation of its downstream components BCL10
and MALT1 with TRAF6. The latter elicits its ubiquitin ligase activity, resulting in polyubiqui-
tination of IKK-γ (NEMO), which in turn phosphorylates IκB, hereby targeting IκB for phosphor-
ylation and proteasomal degradation. This allows the REL-A/p50 dimers to enter the nucleus and
mediate transcription of NF-κB-responsive genes
et al. 2000; Lucas et al. 2001). In t(11;18)(q21;q21)-positive MALT lymphomas, it

is believed that the fusion protein API2-MALT1 activates NF-κB directly by
increased IKK-γ polyubiquitination as demonstrated in cell lines and API2-
MALT1 transgenic mice (Zhou et al. 2005; Baens et al. 2006). This process
depends on the first BIR domain of API2 (Zhou et al. 2005; Baens et al. 2006).
Also, the API2-MALT1 fusion protein was reported to reside in the lipid rafts of
human B-cell lymphoma BJAB cells, which was associated with increased consti-
tutive NF-κB activity and resistance to Fas-induced apoptosis (Ho et al. 2005). In
fact, association of the MALT carboxyl-terminus with these lipid rafts, which is
mediated by the API2 portion, is sufficient to trigger NF-κB activation via enhanced
IKK-γ polyubiquitination activate (Baens et al. 2006). The oligomerisation of the
API2-MALT1 fusion protein and/or the association of its MALT1 domains with
downstream signalling molecules (e.g. TRAF6) might be eased by raft association,
in this way bypassing the normal antigen-induced oligomerisation of MALT1-
TRAF6 and thus ensuing constitutive NF-κB activation. In addition, it was shown
that API2-MALT1 induces transactivation of the API2 gene through NF-κB acti-
vation, hereby creating a positive feedback loop mechanism of self-activation by
upregulating its own expression in t(11;18)(q21;q21)-positive MALT lymphomas
(Hosokawa et al. 2005).
Finally, a remarkable feature of MALT lymphomas is the aberrant subcellular
BCL10 location in the tumour cells (Fig. 18.1). In normal lymphoid tissue, BCL10
is weakly expressed in the cytoplasm of MZ B cells (Ye et al. 2000). Conversely, t
(11;18)(q21;q21)- and (1;14)(p22;q32)-positive MALT lymphomas are marked by
a, respectively, moderate to strong nuclear BCL10 expression, while t(14;18)(q32;
q21)-positive MALT lymphomas are characterised by a perinuclear BCL10 loca-
tion (Ye et al. 2000, 2005; Sagaert et al. 2006b). While the significance of this
change in subcellular location is not yet known, it might be caused by disturbed
nucleocytoplasmic shuttling of BCL10 which is regulated by MALT1 (Nakagawa
et al. 2005). In the presence of t(1;14)(p22;q32), the overexpression of BCL10
results in nuclear BCL10 retention because of a shortage of MALT1 relative to
BCL10. In t(14;18)(q32;q21)-positive MALT lymphomas, the location of BCL10
remains cytoplasmic because all nuclear BCL10 is exported by MALT1. The
relative shortage of MALT1 due to the loss of one allele in t(11;18)(q21;q21)-
positive MALT lymphomas might be responsible for the nuclear retention of
BCL10 as the associated API2-MALT1 fusion is unable to export BCL10.
Gastric MALT lymphomagenesis is a multistep process, provoked by H. pylori

infection which induces genetic abnormalities and subsequent malignant transfor-
mation. As evident from the above discussion, the diagnosis of a gastric MALT
lymphoma, made by endoscopic biopsy, should prompt investigation for the pres-
ence of H. pylori and t(11;18)(q21;q21) to clarify the therapeutic approach. An
436 X. Sagaert
important aim in the treatment of MALT lymphomas is to prevent transformation

into an aggressive DLBCL, although the underlying mechanisms of that transfor-
mation are not known yet. Gastric MALT lymphoma-associated gene alterations,
including API2-MALT, IGH-BCL10 and IGH-MALT, result in constitutive activa-
tion of NF-κB. As such, pharmaceutical interference with the NF-κB pathway may
represent an attractive treatment strategy in the future.
18.5 Key Points
• Diagnosis of a gastric MALT lymphoma is made by morphologic analysis of the

endoscopic biopsy and supported by molecular techniques (PCR, FISH).
• Gastric MALT lymphoma is caused by H. pylori infection, and, therefore, every
diagnosis of a gastric MALT lymphoma should prompt a thorough investigation
for the presence of H. pylori.
• To date, gastric MALT lymphoma is the only malignancy in which antibiotics
are the first choice of therapy.
• It is important to screen for the (gastric) MALT lymphoma-specific translocation
t(11;18)(q21;q21) as its presence not only confirms the diagnosis of a MALT
lymphoma but also predicts resistance to H. pylori eradication treatment and
almost rules out evolution to a DLBCL.
• Gastric MALT lymphoma is characterised by a series of translocations, which
affects molecules involved in one and the same pathway leading to the activation
of NF-κB. Therefore, these molecules may represent attractive therapeutic
targets.
References
Achuthan R et al (2000) Novel translocation of the BCL10 gene in a case of mucosa associated
lymphoid tissue lymphoma. Genes Chromosomes Cancer 29:347–349
Akagi T et al (1999) A novel gene, MALT1 at 18q21, is involved in t(11;18) (q21;q21) found in
low-grade B-cell lymphoma of mucosa-associated lymphoid tissue. Oncogene 18:5785–5794
Allister-Lucas LM et al (2001) Bimp1, a MAGUK family member linking protein kinase C
activation to Bcl10-mediated NF-kappa B induction. J Biol Chem 276:30589–30597
Alpen B et al (2000) Translocation t(11;18) absent in early gastric marginal zone B-cell lymphoma
of MALT type responding to eradication of Helicobacter pylori infection. Blood 95:4014–4015
Aviles A, Nambo MJ, Neri N, Talavera A, Cleto S (2005) Mucosa-associated lymphoid tissue
(MALT) lymphoma of the stomach – results of a controlled clinical trial. Med Oncol 22:57–62
Baens M, Steyls A, Geboes K, Marynen P, Wolf-Peeters C (2000) The product of the t(11;18), an
API2-MLT fusion, marks nearly half of gastric MALT type lymphomas without large cell
proliferation. Am J Pathol 156:1433–1439
Baens M et al (2006) Selective expansion of marginal zone B cells in E mu-API2-MALT1 mice is
linked to enhanced I kappa B kinase gamma polyubiquitination. Cancer Res 66:5270–5277
Bahler DW, Miklos JA, Swerdlow SH (1997) Ongoing Ig gene hypermutation in salivary gland
mucosa-associated lymphoid tissue-type lymphomas. Blood 89:3335–3344
Banham AH et al (2001) The FOXP1 winged helix transcription factor is a novel candidate tumor
suppressor gene on chromosome 3p. Cancer Res 61:8820–8829
Banham AH et al (2005) Expression of the FOXP1 transcription factor is strongly associated with
inferior survival in patients with diffuse large B-cell lymphoma. Clin Cancer Res
11:1065–1072
Barrans SL, Fenton JAL, Banham A, Owen RG, Jack AS (2004) Strong expression of FOXP1
identifies a distinct subset of diffuse large B-cell lymphoma (DLBCL) patients with poor
outcome. Blood 104:2933–2935
Bayerdorffer E et al (1995) Regression of primary gastric lymphoma of mucosa-associated
lymphoid-tissue type after cure of Helicobacter-pylori infection. Lancet 345:1591–1594
Bertin J et al (2001) CARD11 and CARD14 are novel caspase recruitment domain (CARD)/
membrane-associated guanylate kinase (MAGUK) family members that interact with BCL10
and activate NF-kappa B. J Biol Chem 276:11877–11882
Bonizzi G, Karin M (2004) The two NF-kappa B activation pathways and their role in innate and
adaptive immunity. Trends Immunol 25:280–288
Brigden ML, Pattullo AL (1999) Prevention and management of overwhelming postsplenectomy
infection – an update. Crit Care Med 27:836–842
Cogliatti SB et al (1991) Primary B-cell gastric lymphoma – a clinicopathological study of
145 patients. Gastroenterology 101:1159–1170
Coussens LM, Werb Z (2002) Inflammation and cancer. Nature 420:860–867
De Re V et al (2000) Sequence analysis of the immunoglobulin antigen receptor of hepatitis C
virus-associated non-Hodgkin lymphomas suggests that the malignant cells are derived from
the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia. Blood
96:3578–3584
Derringer GA et al (2000) Malignant lymphoma of the thyroid gland – a clinicopathologic study of
108 cases. Am J Surg Pathol 24:623–639
Dierlamm J et al (1999) The apoptosis inhibitor gene API2 and a novel 18q gene, MLT, are
recurrently rearranged in the t(11;18)(q21;q21) associated with mucosa-associated lymphoid
tissue lymphomas. Blood 93:3601–3609
Doglioni C, Wotherspoon AC, Moschini A, Deboni M, Isaacson PG (1992) High-incidence of
primary gastric lymphoma in northeastern Italy. Lancet 339:834–835
Du M et al (1996a) Ongoing mutation in MALT lymphoma immunoglobulin gene suggests that
antigen stimulation plays a role in the clonal expansion. Leukemia 10:1190–1197
Du MQ et al (1996b) Intestinal dissemination of gastric malt lymphoma occurs following antigen
mediated tumour clonal expansion. Blood 88:2673
Enno A et al (1995) Maltoma-like lesions in the murine gastric-mucosa after long-term infection
with Helicobacter-felis – a mouse model of Helicobacter-pylori-induced gastric lymphoma.
Am J Pathol 147:217–222
Ferreri AJM et al (2004) Evidence for an association between Chlamydia psittaci and ocular
adnexal lymphomas. J Natl Cancer Inst 96:586–594
Franco EL et al (2004) Role and limitations of epidemiology in establishing a causal association.
Semin Cancer Biol 14:413–426
Fredricks DN, Relman DA (1996) Sequence-based identification of microbial pathogens: a
reconsideration of Koch’s postulates. Clin Microbiol Rev 9:18
Gaide O et al (2001) Carma1, a CARD-containing binding partner of Bcl10, induces Bcl10
phosphorylation and NF-kappa B activation. FEBS Lett 496:121–127
Gaide O et al (2002) CARMA1 is a critical lipid raft-associated regulator of TCR-induced
NF-kappa B activation. Nat Immunol 3:836–843
Goossens T, Klein U, Kuppers R (1998) Frequent occurrence of deletions and duplications during
somatic hypermutation: implications for oncogene translocations and heavy chain disease.
Proc Natl Acad Sci U S A 95:2463–2468
438 X. Sagaert
Grunberger B et al (2006a) Antibiotic treatment is not effective in patients infected with

Helicobacter pylori suffering from extragastric MALT lymphoma. J Clin Oncol 24:1370–1375
Grunberger B et al (2006b) ‘Blind’ antibiotic treatment targeting Chlamydia is not effective in
patients with MALT lymphoma of the ocular adnexa. Ann Oncol 17:484–487
Han SL et al (2009) FOXP1 expression predicts polymorphic histology and poor prognosis in
gastric mucosa-associated lymphoid tissue lymphomas. Dig Surg 26:156–162
Hans CP et al (2004) Confirmation of the molecular classification of diffuse large B-cell lym-
phoma by immunohistochemistry using a tissue microarray. Blood 103:275–282
Hara Y et al (2001) Immunoglobulin heavy chain gene analysis of ocular adnexal extranodal
marginal zone B-cell lymphoma. Invest Ophthalmol Vis Sci 42:2450–2457
Haralambieva E et al (2006) Genetic rearrangement of FOXP1 is predominantly detected in a
subset of diffuse large B-cell lymphomas with extranodal presentation. Leukemia 7:1300–1303
Ho L et al (2005) MALT1 and the API2-MALT1 fusion act between CD40 and IKK and confer
NF-kappa B-dependent proliferative advantage and resistance against FAS-induced cell death
in B cells. Blood 105:2891–2899
Hosokawa Y, Suzuki H, Nakagawa M, Lee TH, Seto M (2005) AP12-MALT1 fusion protein
induces transcriptional activation of the API2 gene through NF-kappa B binding elements:
evidence for a positive feed-back loop pathway resulting in unremitting NF-kappa B activa-
tion. Biochem Biophys Res Commun 334:51–60
Hu H et al (2006) Foxp1 is an essential transcriptional regulator of B cell development. Nat
Immunol 7:819–882
Hussell T, Isaacson PG, Crabtree JE, Spencer J (1993a) The response of cells from low-grade
B-cell gastric lymphomas of mucosa-associated lymphoid-tissue to Helicobacter-pylori. Lan-
cet 342:571–574
Hussell T, Isaacson PG, Crabtree JE, Dogan A, Spencer J (1993b) Immunoglobulin specificity of
low-grade B-cell gastrointestinal lymphoma of mucosa-associated lymphoid-tissue (Malt)
type. Am J Pathol 142:285–292
Hyjek E, Isaacson PG (1988) Primary B-cell lymphoma of the thyroid and its relationship to
Hashimotos thyroiditis. Hum Pathol 19:1315–1326
Hyjek E, Smith WJ, Isaacson PG (1988) Primary B-cell lymphoma of salivary-glands and its
relationship to myoepithelial sialadenitis. Hum Pathol 19:766–776
Jaffe E, Harris N, Stein H, Vardiman J (2008) World Health Organisation classification of
tumours: pathology and genetics: tumours of haemopoietic and lymphoid tissues. IAR Press,
Lyon
Kalla J et al (2000) Heterogeneity of the AP12-MALT1 gene rearrangement in MALT-type
lymphoma. Leukemia 14:1967–1974
Kruschinski C, Zidan M, Debertin AS, Von Horsten S, Pabst R (2004) Age-dependent develop-
ment of the splenic marginal zone in human infants is associated with different causes of death.
Hum Pathol 35:113–121
Lecuit M et al (2004) Immunoproliferative small intestinal disease associated with Campylobacter
jejuni. N Engl J Med 350:239–248
Li QT, Verma IM (2002) NF-kappa B regulation in the immune system. Nat Rev Immunol
2:725–734
Liu HX et al (2001a) Resistance of t(11;18) positive gastric mucosa-associated lymphoid tissue
lymphoma to Helicobacter pylori eradication therapy. Lancet 357:39–40
Liu HX et al (2001b) T(11;18)(q21;q21) is associated with advanced mucosa-associated lymphoid
tissue lymphoma that expresses nuclear BCL10. Blood 98:1182–1187
Lucas P et al (2001) Bcl10 and MALT1, independent targets of chromosomal translocation in malt
lymphoma, cooperate in a novel NF-kappa B signaling pathway. J Biol Chem
276:19012–19019
Martin F, Kearney JF (2002) Marginal-zone B cells. Nat Rev Immunol 2:323–335
Martinelli G et al (2005) Clinical activity of rituximab in gastric marginal zone non-Hodgkin’s

lymphoma resistant to or not eligible for anti-helicobacter pylori therapy. J Clin Oncol
23:1979–1983
Montalban C et al (1995) Gastric B-cell mucosa-associated lymphoid-tissue (Malt) lymphoma –
clinicopathological study and evaluation of the prognostic factors in 143 patients. Ann Oncol
6:355–362
Morgan JA et al (1999) Breakpoints of the t(11;18)(q21;q21) in mucosa-associated lymphoid
tissue (MALT) lymphoma lie within or near the previously undescribed gene MALT1 in
chromosome 18. Cancer Res 59:6205–6213
Morgner A et al (2000) Helicobacter heilmannii-associated primary gastric low-grade MALT
lymphoma: complete remission after curing the infection. Gastroenterology 118:821–828
Motegi M et al (2000) API2-MALT1 chimeric transcripts involved in mucosa-associated lym-
phoid tissue type lymphoma predict heterogeneous products. Am J Pathol 156:807–812
Muller-Hermelink HK (2003) Genetic and molecular genetic studies in the diagnosis of B-cell
lymphomas: marginal zone lymphomas. Hum Pathol 34:336–340
Nakagawa M et al (2005) MALT1 contains nuclear export signals and regulates cytoplasmic
localization of BCL10. Blood 106:4210–4216
Packham G (2008) The role of NF-kappa B in lymphoid malignancies. Br J Haematol 143:3–15
Parsonnet J et al (1994) Helicobacter-pylori infection and gastric lymphoma. N Engl J Med
330:1267–1271
Pillai S, Cariappa A, Moran ST (2005) Marginal zone B cells. Ann Rev Immunol 23:161–196
Pomerantz JL, Baltimore D (2002) Two pathways to NF-kappa B. Mol Cell 10:693–695
Qin YF et al (1995) Somatic hypermutation in low-grade mucosa-associated lymphoid tissue-type
B-cell lymphoma. Blood 86:3528–3534
Rawlings DJ, Sommer K, Moreno-Garcia ME (2006) The CARMA1 signalosome links the
signalling machinery of adaptive and innate immunity in lymphocytes. Nat Rev Immunol
6:799–812
Remstein ED, James CD, Kurtin PJ (2000) Incidence and subtype specificity of AP12-MALT1
fusion translocations in extranodal, nodal, and splenic marginal zone lymphomas. Am J Pathol
156:1183–1188
Remstein ED, Kurtin PJ, Einerson RR, Paternoster SF, Dewald GW (2004) Primary pulmonary
MALT lymphomas show frequent and heterogeneous cytogenetic abnormalities, including
aneuploidy and translocations involving API2 and MALT1 and IGH and MALT1. Leukemia
18:156–160
Roggero E et al (2000) Eradication of Borrelia burgdorferi infection in primary marginal zone
B-cell lymphoma of the skin. Hum Pathol 31:263–268
Roy N et al (1997) The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases.
Blood 90:2645
Royer B et al (1997) Lymphomas in patients with Sjogren’s syndrome are marginal zone B-cell
neoplasms, arise in diverse extranodal and nodal sites, and are not associated with viruses.
Blood 90:766–775
Ruefli-Brasse AA, French DM, Dixit VM (2003) Regulation of NF-kappa B-dependent lympho-
cyte activation and development by paracaspase. Science 302:1581–1584
Ruland J et al (2001) Bcl10 is a positive regulator of antigen receptor-induced activation of
NF-kappa B and neural tube closure. Cell 104:33–42
Ruland J, Duncan GS, Wakeham A, Mak TW (2003) Differential requirement for Malt1 in T and B
cell antigen receptor signaling. Immunity 19:749–758
Sagaert X et al (2006a) Forkhead box protein P1 expression in mucosa-associated lymphoid tissue
lymphomas predicts poor prognosis and transformation to diffuse large B-cell lymphoma. J
Clin Oncol 24:2490–2497
Sagaert X, Laurent M, Baens M, Wlodarska I, De Wolf-Peeters C (2006b) MALT1 and BCL10
aberrations in MALT lymphomas and their effect on the expression of BCL10 in the tumour
cells. Mod Pathol 19:225–232
440 X. Sagaert
Schmelz R et al (2005) Helyx study part I & II: treatment of low-grade gastric non-Hodgkin’s
lymphoma of mucosa-associated lymphoid tissue (MALT) type stages I & III, an interim
analysis. Gastroenterology 128:A295
Siebenlist U, Brown K, Claudio E (2005) Control of lymphocyte development by nuclear factor-
kappa B. Nat Rev Immunol 5:435–445
Starostik P et al (2002) Gastric marginal zone B-cell lymphomas of MALT type develop along
2 distinct pathogenetic pathways. Blood 99:3–9
Stefanovic A, Lossos IS (2009) Extranodal marginal zone lymphoma of the ocular adnexa. Blood
114:501–510
Streubel B et al (2003) T(14;18)(q32;q21) involving IGH and MALT1 is a frequent chromosomal
aberration in MALT lymphoma. Blood 101:2335–2339
Streubel B et al (2004) Variable frequencies of MALT lymphoma-associated genetic aberrations
in MALT lymphomas of different sites. Leukemia 18:1722–1726
Streubel B, Vinatzer U, Lamprecht A, Raderer M, Chott A (2005) T(3;4)(p14.1;q32) involving
IGH and FOXP1 is a novel recurrent chromosomal aberration in MALT lymphoma. Leukemia
19:652–658
Sugiyama T et al (2001) API2-MALT1 chimeric transcript is a predictive marker for the respon-
siveness of H-pylori eradication treatment in low-grade gastric MALT lymphoma. Gastroen-
terology 120:1884–1885
Sun LJ, Deng L, Ea CK, Xia ZP, Chen ZJJ (2004) The TRAF6 ubiquitin ligase and TAK1 kinase
mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol Cell 14:289–301
Thieblemont C et al (2000) Mucosa-associated lymphoid tissue lymphoma is a disseminated
disease in one third of 158 patients analyzed. Blood 95:802–806
Thome M (2004) CARMA1, BCL-10 and MALT1 in lymphocyte development and activation. Nat
Rev Immunol 4:348–359
Tsang RW et al (2003) Localized mucosa-associated lymphoid tissue lymphoma treated with
radiation therapy has excellent clinical outcome. J Clin Oncol 21:4157–4164
Uren AG et al (2000) Identification of paracaspases and metacaspases: two ancient families of
caspase-like proteins, one of which plays a key role in MALT lymphoma. Mol Cell 6:961–967
Wang D et al (2004) CD3/CD28 costimulation-induced NF-{kappa}B activation is mediated by
recruitment of protein kinase C-{theta}, Bcl10, and I{kappa}B kinase {beta} to the immuno-
logical synapse through CARMA1. Mol Cell Biol 24:164–171
Willis TG et al (1999) Bcl10 is involved in t(1;14)(p22;q32) of MALT B cell lymphoma and
mutated in multiple tumor types. Cell 96:35–45
Wlodarska I et al (2005) FOXP1, a gene highly expressed in a subset of diffuse large B-cell
lymphoma, is recurrently targeted by genomic aberrations. Leukemia 19:1299–1305
Wotherspoon AC, Ortizhidalgo C, Falzon MR, Isaacson PG (1991) Helicobacter-pylori-associated
gastritis and primary B-cell gastric lymphoma. Lancet 338:1175–1176
Wotherspoon AC, Pan LX, Diss TC, Isaacson PG (1992) Cytogenetic study of B-cell lymphoma of
mucosa-associated lymphoid-tissue. Cancer Genet Cytogenet 58:35–38
Wotherspoon AC et al (1993) Regression of primary low-grade B-cell gastric lymphoma of
mucosa-associated lymphoid-tissue type after eradication of Helicobacter-pylori. Lancet
342:575–577
Wotherspoon AC, Doglioni C, Deboni M, Spencer J, Isaacson PG (1994) Antibiotic-treatment for
low-grade gastric malt lymphoma. Lancet 343:1503
Ye HT et al (2000) BCL10 expression in normal and neoplastic lymphoid tissue – nuclear
localization in MALT lymphoma. Am J Pathol 157:1147–1154
Ye HT et al (2003) Variable frequencies of t(11;18)(q21;q21) in MALT lymphomas of different
sites: significant association with CagA strains of H pylori in gastric MALT lymphoma. Blood
102:1012–1018
Ye HT et al (2005) MALT lymphoma with t(14;18)(q32;q21)/IGH-MALT1 is characterized by
strong cytoplasmic MALT1 and BCL10 expression. J Pathol 205:293–301
Zhou HL, Du MQ, Dixit VM (2005) Constitutive NF-kappa B activation by the t(11;18)(q21;q21)
product in MALT lymphoma is linked to deregulated ubiquitin ligase activity. Cancer Cell
7:425–431
Zucca E et al (2000) Prevalence of Helicobacter pylori and hepatitis C virus infections among non
Hodgkin’s lymphoma patients in Southern Switzerland. Haematologica 85:147–153
Zullo A et al (2009) Eradication therapy for Helicobacter pylori in patients with gastric MALT
lymphoma: a pooled data analysis. Am J Gastroenterol 104:1932–1937
Chapter 19
Helicobacter pylori Infection in Children
Sibylle Koletzko and Francis Mégraud
Abstract Helicobacter pylori infection is acquired essentially in childhood and

may persist lifelong. The prevalence varies widely in the world according to the
socioeconomic status of the populations. It is very low in developed countries,
while almost all children become infected in the developing world. While the exact
routes of transmission are not fully determined, it appears that a gastro-oral (via
vomitus) or oral-oral (via saliva after regurgitation) is the most important, and it
occurs essentially, but not exclusively, from person to person inside the families.
The pathology observed in children is usually milder compared to that in adults
because of downregulation of the host immune response in the youngsters. Among
clinical features, peptic ulcer disease may be found as a consequence of H. pylori
infection but mostly in adolescents. Recurrent abdominal pain has multiple causes
and is not an indication to test noninvasively and treat H. pylori. The other diseases
caused by H. pylori are sideropenic anemia, chronic idiopathic thrombocytopenic
purpura, and growth retardation. Some animal data also point the benefit of
H. pylori infection as protector against atopic diseases in the youngest, especially
asthma. Diagnostic tests in children are those used in adulthood. However, at age
below 6 years, the urea breath test can lead to nonspecific results, and serology may
not be sensitive enough. Preference must be given to invasive testing. Treatment
should be tailored according to susceptibility testing.
Keywords Epidemiology • Pathogenesis • Clinical features • Diagnosis •

Treatment
S. Koletzko
Division of Paediatric Gastroenterology and Hepatology, Dr. von Hauner Children’s Hospital,
Ludwig-Maximilians-University of Munich, Munich, Germany
F. Mégraud (*)
Bacteriology Laboratory, University of Bordeaux, INSERM U853, 33000 Bordeaux, France

DOI 10.1007/978-4-431-55936-8_19
444 S. Koletzko and F. Mégraud
19.1 Introduction
Childhood is an important period of life for Helicobacter pylori infection because it

is the period of contamination. However, the consequences of this infection are
limited in early life and occur essentially at an older age, so it can be said that it is “a
pediatric infection with geriatric consequences.” Epidemiological studies are
lacking because of the difficulty in accessing the site of the infection for ethical
reasons. More recently, the development of the stool antigen test brought us
interesting data. Clinical studies were also carried out less frequently in children
than in adults because of a limited prevalence of the infection in western countries,
where technology is optimal. For these reasons, multicenter studies on diagnosis,
treatment, and clinical outcome were implemented, especially in Europe, under the
leadership of an H. pylori Pediatric Task Force resulting from the joint efforts of the
European Society for Pediatric Gastroenterology Hepatology and Nutrition and the
European Helicobacter Study Group. In this chapter, the particularities of H. pylori
infection will be reviewed with regard to its epidemiology, pathogenesis, clinical
manifestations, diagnosis, and treatment.
19.2 Epidemiology
19.2.1 Prevalence and Incidence
The first studies on the prevalence of H. pylori infection indicated a low prevalence
in children which progressively increased during the life span (Megraud
et al. 1989). However, it was quickly shown that H. pylori infection was not
progressively acquired over the years but that this curve corresponded to a cohort
phenomenon (Banatvala et al. 1993). Each age group (cohort) carries its own risk
linked to the socioeconomic conditions existing at the time of their youth. However,
in some developed countries like the Netherlands, it appears that the prevalence in
children has stabilized given the similar prevalence in subsequent birth cohorts (den
Hoed et al. 2011). Studies performed on cohorts of children and adults have
confirmed that the incidence (new cases per year) is higher in children and that
young children are more at risk than older ones.
The age of acquisition of H. pylori is not easy to determine because the infection
is essentially asymptomatic or paucisymptomatic and can be confounded with a
gastroenteritis. Incidence was monitored over 20-year period from ages 1–3 years
to ages 22–24 years, in the Bogalusa cohort from Louisiana, USA, at the end of the
twentieth century. The crude incidence rate decreased from 2.1 % at ages 4–5 years
to 1.5 % at ages 7–9 years and 0.3 % at ages 21–23 years (Malaty et al. 2002). It was
also shown in the Okinawan cohort followed for 10 years that the incidence in
children (2.7 %/year) was higher than in adults (Banatvala et al. 1994). The same
was observed in China (Mitchell et al. 1992) and in Chile (Russell et al. 1993).
19 Helicobacter pylori Infection in Children 445
Rowland and coworkers investigated 327 healthy children applying the 13C urea
breath test (UBT) for 4 years. At baseline, 28 children were already positive and
20 converted during follow-up. The highest incidence of 5.1 per 100 person years
was observed between 2 and 3 years of age and continuously declined to 0.7 by ages
7–8 years (Rowland et al. 2006).
Although most of the infections remain for a long time, it also happens that the
host can resolve them especially in the younger population. In the Bogalusa cohort,
2.2 % cleared the infection at ages 4–5 years vs. 0.2 % at ages 18–19 years (Malaty
et al. 2002). Transient H. pylori infections were also documented in Japan by Okuda
and coworkers using stool antigen test. Out of 16 children who became stool
antigen test positive during a year, only four remained persistent positives
(Okuda et al. 2007). These data confirmed those obtained by using serology in a
cohort of Native American children (Perez-Perez et al. 2003).
The prevalence of the infection in children is extremely diverse throughout the
world. In developing countries, it turns out to be high, while in developed countries
it is rare. However, there are some exceptions and, for example, in Europe, H. pylori
prevalence is still high in Portugal and some Eastern European countries. In a
cohort of adolescents (age of 13 years) from schools in Porto, the prevalence was
66.2 % with an incidence of 1.1 per year (Bastos et al. 2013b).
The prevalence of H. pylori infection also remains high in children of immi-
grants to developed countries as was shown in Belgium for children 0–9 years:
>30 % H. pylori positive vs. <5 % for those of European origin (Miendje Deyi
et al. 2011). However, in some areas, a dramatic change in the prevalence of
H. pylori infection is observed during childhood. For example, in St. Petersburg
(Russia), the prevalence among children younger than 5 years decreased from 30 to
2 % between 1995 and 2005, linked to improvement in the standard of living and an
increased use of antibiotics (Tkachenko et al. 2007).
19.2.2 Risk Factors
Risk factors for the acquisition of H. pylori are linked to the socioeconomic status in
a given country. It is likely that almost all European children were indeed infected
at the turn of the twentieth century when European countries still had a very low
hygiene level. The risk of infection then progressively decreased, and it was
amplified by the use of antibiotics which began after the Second World War.
Low socioeconomic status in terms of risk factors for families means low
income, small homes, large households, low education, and lack of facilities for
hygiene, all of which favor transmission of the bacterium.
In contrast, breastfeeding appears to be protective. IgG anti-H. pylori are present
in breast milk when the mother is H. pylori positive. Human milk contains many
anti-infectious factors such as lactoferrin, lysozyme, secretory IgA, and mucins
which may act even in infants of noninfected mothers (Chak et al. 2009; Carreira
et al. 2015).
The possibility of ethnic differences in susceptibility to the infection has been

proposed since the incidence of the infection is very different, for instance, between
Caucasian and African-Americans living in the same area (Malaty et al. 2002) or in
young women in a multiethnic European city (den Hollander et al. 2013), but this
difference is most likely linked to environmental conditions, especially the socio-
economic status.
A study of monozygotic and dizygotic twins reared together or separately was
the occasion to assess the importance of genetic effects on the acquisition of
H. pylori infection. A correlation coefficient of 0.66 for monozygotic twins reared
apart points to a positive, while moderate, effect of genetics (Malaty et al. 1994).
However, when the environmental conditions of these twins were studied, it
appeared that infected twins were raised in homes under poorer socioeconomic
conditions than those of their noninfected co-twins.
Looking at genome wide associations, a Toll-like receptor 1 single nucleotide
polymorphism was associated with H. pylori-positive serologic status (Mayerle
et al. 2013); these data must be confirmed in ethnic groups other than Caucasians.
For more details on the role of gene polymorphisms, we refer to Chap. 14.
19.2.3 Transmission
Transmission of H. pylori is essentially from person to person and mostly

intrafamilial and vertical through transmission, particularly in low prevalence
countries, while in high-prevalence settings, a horizontal transmission from outside
the family with infections of multiple strains within one person is common
(Schwarz et al. 2008). We can anticipate that about two-thirds of the transmission
occurs between family members and one-third outside. Among the parents, the role
of the mother appeared to be more important (Dominici et al. 1999). It was
confirmed in a German study (Weyermann et al. 2009).
But according to others, the transmission may also involve older siblings. The
risk of infection increases according to birth order in large families and is higher
when the age interval with the older siblings is low (Goodman and Correa 2000). In
another study, the number of siblings in a family rather than the birth order was
associated with H. pylori infection (Ford et al. 2007).
There is also the possibility of transmission from grandmothers. In Japan, where
the family structure is still traditional, they carried the third highest risk after
siblings and mothers (Urita et al. 2013).
Considering acquisition from outside home, a systemic review and meta-
analysis was carried out to evaluate childcare attendance as a risk factor for
acquiring infections especially in settings with a high prevalence of the infection
(Bastos et al. 2013a). In Portugal, for instance, when prevalence of the infection at
ages 4–5 years was 30.6 %, it significantly increased with cumulative time of
attendance in daycare centers/homes, from 13.2 % among those never attendees
to 40.2 % among those attending for >36 months ( p < 0.001) (Lunet et al. 2014).
In addition to epidemiological studies, molecular fingerprinting has also been

performed to prove the transmission of the same strain in families. The first studies
using ribotyping showed that the same strain was present in at least two family
members in three out of four families (Bamford et al. 1993). In Japan, using random
amplified polymorphic DNA (RAPD) fingerprinting, 76 % of 42 children showed
DNA fingerprint patterns identical to those of at least one of the respective family
members, essentially to the mother (69 %) and much less to the father (16 %)
(Konno et al. 2008).
The schema of intrafamilial transmission may be challenged in developing
countries. In Peru, also using RAPD fingerprinting, 30 % of the child-mother strain
pairs did match, as well as 18 % child-father and 32 % from siblings, suggesting
also transmission from unrelated individuals or via environmental sources (Herrera
et al. 2008). The corresponding figures in Bangladesh were 46 % for mothers and
15 % for fathers (Nahar et al. 2009). Raymond and coworkers studied H. pylori in
two French families with DNA microarrays: different matches of strains were
found, while in a Moroccan family of seven, five different strains were identified
(Raymond et al. 2008).
Our knowledge is also limited concerning the vehicle of transmission given the
absence of detectable environmental reservoirs and outbreak scenarios. To go from
one stomach to another stomach, the most probable route taken is the gastro-oral
transmission. Indeed, all vomitus samples from infected subjects grow H. pylori,
often in high quantities and even air samples obtained during vomiting (Parsonnet
et al. 1999).
This vehicle may therefore be responsible for H. pylori transmission among
siblings and in daycare centers if proper disinfection is not carried out.
In the USA, among 1752 noninfected household members, 30 new infections
occurred over a 3-month period, and 75 % were attributable to exposure to an
infected person with gastroenteritis especially because of vomiting (OR, 6) (Perry
et al. 2006).
H. pylori may also be present in the oral cavity after emesis (Parsonnet
et al. 1999) and following regurgitation in patients with gastroesophageal reflux
disease (GERD) (Young et al. 2000).
Its presence is more likely transient than permanent but may allow transmission
via saliva. It was shown that pre-mastication of food by mothers or wetting the
mammilla before breastfeeding with saliva was a risk factor of transmission to their
children (Albenque et al. 1990).
Stools do not seem to play a major role in the transmission of H. pylori, at least in
developed countries. Indeed, all elements from the stomach will be eliminated in
stools, but isolation of live H. pylori from stool samples commonly failed because
the bacterium rarely survives the bowel passage. After induced diarrhea in
H. pylori-positive subjects, growth of live H. pylori was obtained in only 22 % of
the stools (Parsonnet et al. 1999).
This result indicates that H. pylori can only be transmitted by stools in the
context of diarrheal diseases. Furthermore, it implies that fecal hygiene must not
be adequate in order to have a direct contact or to contaminate water. A number of
studies claimed that water can be positive for H. pylori. However, these results are
doubtful because most often, only one polymerase chain reaction (PCR) was
performed detecting DNA and the lack of direct growth of live H. pylori. In
water, as in the oral cavity and all specimens except the stomach, it is necessary
to use several PCRs based on different targets and perform growth to get the same
positive answer before making a conclusion.
The hypothesis that access to treated water and a sanitary sewage system that
reduces the incidence of H. pylori infection was tested in a cohort of children at the
US-Mexican border but did not provide a firm support for potential waterborne
transmission of H. pylori (Travis et al. 2010). Acquisition of H. pylori infection in
rhesus macaques is also most consistent with oral-oral transmission (Solnick
et al. 2006). The seroepidemiology of H. pylori infection and of hepatitis A argues
also against a common mode of transmission (Luzza et al. 1997).
In conclusion, H. pylori infection occurs essentially in childhood, mostly in the
family, the mother being the main source. Once established, H. pylori infection may
remain for life. Poor socioeconomic conditions are the main risk factor, and their
improvement leads to a decrease in the prevalence of the infection in successive
cohorts. A gastro-oral transmission via vomiting is the most likely.
19.3 Pathogenesis
The characteristics of H. pylori infection are dependent on the infecting strain and
on the host response and can be influenced by other environmental factors.
There are very few differences between the strains infecting children or adults.
The main known colonizing and pathogenic factors are the same, i.e., BabA, SabA,
cagPAI, VacA, and others (for more details, we refer to Chaps. 3, 4, 5, 6, and 7).
However, conversely, the host response can be different in children and in adults as
they evolve over time. Furthermore, the changing gastric environment in response
to infection may lead to changes in the expression of H. pylori virulence factors.
Finally, most of the environmental conditions which can influence the evolution of
the infection such as smoking, alcohol consumption, and drug intake are mostly
absent in children, leading to more “pure” pathologies compared to adults, making
it easier to study the relationship between putative diseases and virulence factors.
Harris and coworkers compared the differences in histology and T cell response
between 36 children younger than 12 years (50 % infected) and 79 adults (65 %
infected) in Chile (Harris et al. 2008). They observed a limited number of poly-
morphs and mononuclear cell infiltration, as well as an intact epithelium, in
children vs. adults, independently of the type of the infecting strains and the
bacterial load. They were able to demonstrate that the level of T regulatory cells
(Treg) and Treg cytokines (the transforming growth factor beta (TGF-β) signaling
pathway and interleukin-10 (IL-10)) is markedly increased in both infected and
noninfected children compared to adults, while interferon alpha (IFN-α) expression
was increased in adults. Then in children, it appears that Treg activity
downregulates the inflammatory response to H. pylori resulting in lower gastritis

scores compared to adults (Harris et al. 2008).
Similar results were obtained by Freire de Melo and coworkers. Treg-associated
cytokines were more predominant in children than in adults where Th17 cytokines
are mainly present. They concluded that the lower inflammatory infiltrates of the
gastric mucosa in children could be responsible for the children’s increased sus-
ceptibility to infection (Freire de Melo et al. 2012) and persistence of the bacteria
(Gil et al. 2014).
Freire de Melo and colleagues also studied the gastric concentrations of cyto-
kines representative of the innate and Th1 response. IL-1α and TNF-α concentra-
tions were significantly higher, while those of IL-2, IL12-p70, and IFN-γ were
lower in the infected children than in the infected adults, but the gastric concentra-
tions of these three latter Th1-associated cytokines increased with age in children
and correlated with an increased degree of gastritis (Freire de Melo et al. 2014).
Such a result, regarding IFN-γ secretion in the stomach of infected children, had
already been highlighted in the past (Bontems et al. 2003). The same authors also
showed that nuclear factor kappa-B (NF-κB) activation occurred essentially in
adults, possibly a consequence of a lower CD3+ and CD8+ T cell recruitment in
children (Bontems et al. 2014).
In China, Li and coworkers found that peptic ulcer disease (PUD) in children
was essentially due to the most virulent cagA+ EPIYA-ABD, while in adults, four
types of EPIYA motifs were identified (Li et al. 2009). Detailed explanation of
EPIYA and ABD type in cagA are discussed in Chap. 4.
Oleastro and coworkers studying children’s strains could also identify two
markers for PUD, jhp0562 involved in lipopolysaccharide biosynthesis and
jhp0870 an outer membrane protein (Oleastro et al. 2006).
In conclusion, the differences in pathophysiology between children and adults
are essentially due to the host innate and adaptive immune response rather than the
H. pylori strains. The lack of other environmental conditions which may influence
the outcome allows the observation of “pure” pathologies.
19.4 Clinical Manifestations
19.4.1 Peptic Ulcer Disease
Drumm and coworkers described in the New England Journal of Medicine in 1987,
for the first time convincingly, the association of primary gastritis and peptic
ulceration and H. pylori (at that time still called Campylobacter pylori) in children
(Drumm et al. 1987). In children with a normal histology, the bacteria could not be
identified. Since then, many studies in children and adults have identified H. pylori
as an important human pathogen that is responsible for the majority of cases with
PUD. With the decreasing prevalence of infected children, other pathogenic factors
than H. pylori are becoming predominant as causes for gastric and duodenal
ulcerations. A prospective, European multicenter, case-control study in 244 pediat-
ric patients with gastric or duodenal erosions (n ¼ 153) or ulcers (n ¼ 91) and two
age-matched controls for each from the same center was performed recently
(Bontems et al. 2013). Children receiving antimicrobials or acid-suppressive
drugs before endoscopy were excluded. H. pylori infection was detected more
frequently in cases than in controls (32.0 % versus 20.1 %) ( p ¼ 0.001), but in
two-thirds of the patients, erosions and ulcerations were due to other causes than
H. pylori.
H. pylori-related ulcers are rare in symptomatic children undergoing endoscopy.
In a large European study including 1322 H. pylori-infected children, gastric or
duodenal ulcers were found in only 3.5 % of children younger than 6 years of age, in
4.6 % between the ages of 6 and 11 years, and in 10.4 % of those older than 11 years
(Koletzko et al. 2006). In another multicenter study, only 64/454 (12.3 %) infected
children presented with gastric or duodenal ulcer and/or erosions (Oderda
et al. 2007). The low rate of PUD, particularly in children below 12 years of age,
may be related to a shorter disease duration, but it is more likely due to the absence
of additional risk factors for ulcerations such as smoking, alcohol consumption, and
regular intake of ulcerogenic drugs (e.g., nonsteroidal anti-inflammatory drugs
(NSAID)).
Duodenal ulcer patients have antral predominant, body-sparing, non-atrophic
gastritis with a highly stimulated acid production. Without clearance of the infec-
tion, the relapse rate of the ulceration is high even after healing with acid-
suppressive drug treatment. Meta-analyses of studies on adults consistently dem-
onstrate a reduced risk for bleeding ulcers after eradication of the H. pylori infec-
tion (Leodolter et al. 2001). Although no such data are available for infected
children, there is a clear recommendation that, in the presence of gastric or
duodenal ulceration, the organism should be eradicated independently of age
(Koletzko et al. 2011; Malfertheiner et al. 2012).
19.4.2 Recurrent Abdominal Pain
Recurrent abdominal pain as defined by Apley and Naish (1958) or weekly pain
occurs in 10–17 % of school-aged children (Schwille et al. 2009). Abdominal pain
and other complaints such as dyspepsia or nausea are nonspecific symptoms and
can be caused by different organic diseases related and unrelated to the gastroin-
testinal tract. Most affected children suffer from functional pain. Even 30 years
after the first publication by Marshall and Warren in the Lancet (Marshall and
Warren 1984), there is still controversy regarding whether, in the absence of PUD,
the infection causes abdominal symptoms in children. A few controlled studies on a
limited number of children with different study designs showed no difference
regarding improvement of dyspeptic symptoms after either successful or failed
triple therapy (Wewer et al. 2001) or placebo treatment (Ashorn et al. 2004). A
causal relationship between recurrent abdominal pain and non-ulcer H. pylori

infection in children can only be proved or disproved by randomized placebo-
controlled intervention studies. So far, only one such study has been performed on
children (Ashorn et al. 2004). No difference was found regarding dyspeptic symp-
toms after either successful triple therapy or proton pump inhibitor (PPI) treatment
only, even 12 months after treatment. As functional pain is highly responsive to
reassuring but also to placebo effect, the common improvement of symptoms in
H. pylori-treated patients may be unrelated to bacterial clearance. A meta-analysis
on 38 studies (23 case controls, 14 cross-sectional, and 1 prospective cohort study)
did not find an association between recurrent abdominal pain and H. pylori infec-
tion: pooled OR 1.21 [95 % CI, 0.82–1.78] in the case-control studies and pooled
OR 1.0 [0.761.31] in the cross-sectional studies (Spee et al. 2010). The absence of a
significant relationship between abdominal pain and the infection is consistent with
a previous meta-analysis (Macarthur 1999). The only significant association was
found in patients referred to a pediatric gastroenterologist. However, the wide
availability of noninvasive tests makes a referral bias of those with a positive test
result very likely. Since the meta-analysis of Spee and coworkers, there have been
further reports on the lack of relationship between H. pylori infection and abdom-
inal pain from industrialized and developing countries (Guariso et al. 2010;
Senbanjo et al. 2010; Cherian et al. 2010; Buonavolonta et al. 2011; Dore
et al. 2012).
In conclusion, the reported absence of an association between abdominal pain
and H. pylori infection supports the recommendations given by the European and
North American Societies of Pediatric Gastroenterology, Hepatology, and Nutrition
(ESPGHAN and NASPGHAN) that abdominal pain in children is not an indication
to test for H. pylori infection with a noninvasive test (Koletzko et al. 2011).
19.4.3 Extragastric Diseases
Extragastroduodenal disorders as a manifestation of H. pylori infection have been

looked for in many epidemiological cross-sectional studies, some case-control
studies, and only very few prospective interventional studies (Pacifico et al.
2014). Since H. pylori infections are not evenly distributed within a population or
a birth cohort but are highly related to socioeconomic factors or emigrant status, it is
very difficult to interpret positive findings from association studies. Factors such as
poor growth or iron deficiency (ID) are themselves highly related to socioeconomic
conditions. Only randomized prospective intervention trials can prove a causal
relationship between H. pylori infection and these health outcomes. This also
applies to potential beneficial effects of an H. pylori infection which have an
inverse relationship to childhood asthma and allergies, obesity, and inflammatory
bowel disease (Blaser et al. 2008; Amberbir et al. 2014; Chen and Blaser 2008;
Sonnenberg and Genta 2012; Francois et al. 2011). Trials by randomized infection
of infants or young children to evaluate the beneficial effect are not possible for
ethical reasons since the bacterium has been declared as a carcinogen. However, the
underlying mechanisms can be studied in animal models to elucidate whether

factors or pathogenic strains can be identified and transferred to children without
producing a lifelong infection and an increased risk for PUD and gastric cancer.
19.4.3.1 Sideropenic Anemia
Of all the extragastroduodenal manifestations, ID and iron deficiency anemia (IDA)

are those with the most available evidence to support a causal relationship. Pacifico
and coworkers recently reviewed all published studies performed in children
regarding ID and IDA and the possible biological mechanism (Pacifico
et al. 2014). Several pathways seem to be involved alone or in combination. Most
children with H. pylori gastritis only, in the absence of erosions or ulcerations, have
no evidence of occult blood loss. Hypothesized mechanisms are related to a reduced
acid output with a change to a higher gastric pH. This affects the reduction of the
ferric form (Fe +++) of the nonheme iron in the diet to the ferrous form (Fe ++) which
is more easily absorbed. Ascorbic acid is an important promoter of the reduction
process. At a pH of >5, an insoluble molecular complex between iron and ascorbic
acid is formed, making the iron unavailable for absorption. In Chilean children
undergoing upper endoscopy, H. pylori infection was more commonly found in
those with hypochlorhydria (pH > 4) compared with those with a lower gastric pH
(Harris et al. 2013). The link may be a higher gastric concentration of IL-1β which
is related to a higher pH and a higher frequency of ID and IDA. Queiroz and
colleagues demonstrated in Brazilian H. pylori-infected children that those with a
certain polymorphism in the IL1β receptor have higher IL-1β concentrations lead-
ing to more severe hypochlorhydria in the acute phase of the infection (Queiroz
et al. 2013b). Another possible mechanism may the absorption of lactoferrin bound
iron in the stomach by the bacteria and therefore a competition with the availability
for the host.
Epidemiological observational trials suffer from the coexistence of several risk
factors for ID and IDA which are also closely related to H. pylori infection in
childhood such as low hygiene and low socioeconomic status, crowding, helminth
infections and poor nutrition, and particularly low intake of fruit (vitamin C), meat,
and fish. Puberty normally accompanied by a growth spurt is a particular period in
life with a high demand in iron, especially in menstruating girls. For this reason, age
is a strong confounding factor for iron status which explains that H. pylori infection
was found to be a risk factor only in teenagers and not in infants and young children
(Muhsen et al. 2010; Choi 2003; Baggett et al. 2006). A recent multicenter study
from Brazil, Chile, and the UK investigated risk factors including H. pylori infec-
tion for the iron status (hemoglobin, mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), hematocrit (Hct,) transferrin saturation, and fer-
ritin) in children undergoing upper endoscopy (Queiroz et al. 2013a). Children with
mucosal lesions or intake of iron or other potentially interfering drug intake were
excluded. Higher age, female gender, being born in Brazil, and H. pylori infection
were independent variables for low ferritin and hemoglobin in a multiple linear
regression model.
Several intervention trials have been performed in children with and without ID
or IDA. In the absence of a depleted iron status, iron supplementation results in a
larger increase in iron stores in children without the infection or who are treated
successfully with eradication of the bacteria (Cardenas et al. 2011; Mahalanabis
et al. 2005; Xia et al. 2012). In another randomized clinical trial in Bangladesh, iron
plus eradication therapy was not better to improve iron status compared to iron
supplementation and placebo. No effect was seen in children receiving anti-
H. pylori therapy regarding the success of bacterial clearance or not (Sarker
et al. 2008). Another large intervention trial in Western Alaska included
H. pylori-infected children between 7 and 11 years of age (n ¼ 219) with ID. They
received either a 2-week anti-H. pylori therapy with 6 weeks of iron sulfate or iron
supplementation only. No difference was observed regarding iron status up to
14 months after treatment. After 40 months, 176 children were available for
reevaluation. Reinfection was very common (52 %). ID and IDA had improved
only in those who stayed uninfected during the observation period.
In conclusion, H. pylori infection may be considered as a risk factor for IDA in a
subgroup of children, particularly those living in developing countries. More
studies are required from developed countries where other interfering factors are
not or less prevalent. Considering the importance of iron for immunological,
cognitive, and other important functions in the growing child, it is recommended
that children with refractory IDA in which other causes have been ruled out or
treated may be tested for H. pylori infection and treated in case of a positive result
(Koletzko et al. 2011).
19.4.3.2 Chronic Immune Thrombocytopenic Purpura
Chronic Immune Thrombocytopenic Purpura (cITP) is an autoimmune disease with

platelet autoantibody formation resulting in platelet destruction and thrombocyto-
penia for at least 6 months. As in many other autoimmune diseases, infections
including H. pylori have been considered to trigger the autoimmune process. These
triggering factors may differ in pediatric patients vs. adult patients, and spontaneous
recovery occurs more commonly in children (~ one-third) compared to only 5 % in
adults. In low prevalence countries for H. pylori infection, most children with cITP
are not infected. For example, only 3 of 33 children in the Netherlands tested
positive for H. pylori (Neefjes et al. 2007) and none of the children in a Finnish
study (Rajantie and Klemola 2003). Therefore, most intervention trials have been
performed in high-prevalence countries with conflicting results. A weakness of the
studies is the lack of treatment in the H. pylori-negative control group. It is possible
that a 2-week therapy with PPI and two or three different antibiotics may have an
effect even in H. pylori-uninfected children by changing the luminal milieu or the
gut microbiome, and the transient or persistent positive effects on the platelet
counts may be unrelated to the clearance of the H. pylori infection.
In conclusion, there is no evidence that H. pylori infection plays a role in cITP in

developed countries and very little evidence in high-prevalence countries. Further
larger-scale randomized clinical trials including children with cITP from different
countries are required before a general screening for H. pylori infection can be
recommended.
19.4.3.3 Growth Retardation
The effect of H. pylori infection on growth has been assessed in many studies.
While most previous studies were not or inadequately controlled for potential
confounders affecting growth such as parental height, socioeconomic status, dietary
factors, and others, more recent studies have tried to account for this. If H. pylori
has a negative effect on growth, this can most likely be seen more easily when the
infection takes place within the first 6–12 months of life when the growth velocity is
highest. However, the type of feeding, breastfeeding or formula feeding, has a
strong influence on the growth pattern in the first year of life, but it also differs
markedly depending on the socioeconomic status.
The biological plausibility of early H. pylori infection affecting growth may be
related to the described transient hypochlorhydria during the acute infection which
increases the risk for intestinal infection with vomiting and diarrhea. H. pylori
gastritis may also change the ghrelin secretion from the gastric mucosa, conse-
quently affecting appetite and satiety. Most studies on the effect of H. pylori
infection on ghrelin and changes after spontaneous or treatment-related clearance
of the infection have been performed in children (Plonka et al. 2006; Pacifico
et al. 2008; Yang et al. 2012; Deng et al. 2012) and none on infants and toddlers.
Most but not all studies showed an increase in plasma ghrelin levels after successful
treatment. Most studies on the effect of H. pylori infection on growth have been
performed in developing countries on populations under poor socioeconomic con-
ditions with a high prevalence of both H. pylori infection and malnutrition during
early childhood, such as the Gambia (Thomas et al. 2004), Colombia (Bravo
et al. 2003; Mera et al. 2012; Goodman et al. 2011), Ecuador (Egorov
et al. 2010), and Peru (Jaganath et al. 2014). Results are controversial with some
showing a transient decrease in growth velocity but catch up growth not affecting
the height at the last follow-up (Thomas et al. 2004; Mera et al. 2012), while other
reported a persistently lower height in H. pylori infected compared to noninfected
children (Goodman et al. 2011; Egorov et al. 2010). Jaganath and coworkers
followed 183 infants from a peri-urban shanty town outside Lima from birth to
24 months of age. UBT was performed from 6 months of age. Almost all (>97 %)
became infected with H. pylori within the first 2 years and 77 % within the first
12 months of life (Jaganath et al. 2014). A low socioeconomic status increased the
risk of early infection until 12 months (HR 1.59, 95 % CI 1.1.6–2.19), while
breastfeeding had a preventive effect (HR 0.63, 95 % CI 0.40–0.96). The infection
was not independently associated with decreased growth ( p ¼ 0.58). However,
early infection in conjunction with several diarrheal episodes negatively affected
height at 24 months. It remains to be determined whether H. pylori infection itself

has an effect on growth either directly or indirectly or whether early infection is
only a marker for poor socioeconomic circumstances with a negative impact on
health and nutritional status.
19.4.4 Possible Beneficial Effects of H. pylori Infection
Many studies focused on the adverse health effects such as peptic ulcer disease or
gastric cancer and also on the effect of having an H. pylori-positive gastritis on
unspecific pain, dyspepsia, or gastric emptying. During the first 20 years of H. pylori
enthusiasm, hardly anybody asked the question whether the infection may have any
benefits for the host. Research by Linz and coworkers disclosed that H. pylori
migrated with the first humans about 60,000 years ago from East Africa and spread
over the world (Linz et al. 2007). Thus, for mankind, chronic H. pylori infection
was the normal situation with adaptation for thousands of years while not being
infected was the exception. The disappearance of one species from the stomach
may change the environment and the conditions for the remaining bacteria. The
immunological response may differ in relation to the H. pylori infectious status. As
pointed out above, a Treg response with high IL-10 and low IL-17 expression
predominates in children compared to adults (Freire de Melo et al. 2012). In a
mouse model using sensitization with ovalbumin (OVA), a protective effect has
been shown against asthma airway hyperresponsiveness and tissue inflammation
with eosinophils, Th2 cells, and Th17 cells through the induction of regulatory T
cell during early H. pylori infection (Arnold et al. 2011). The protective effect was
most robust when mice were infected as neonates compared to later in life and was
abrogated by antibiotic eradication of H. pylori. Systemic Treg depletion abolished
asthma protection; conversely, the adoptive transfer of purified Treg populations
was sufficient to transfer protection from infected donor mice to uninfected recip-
ients. These results gave experimental evidence for a beneficial effect of H. pylori
infection on the development of allergen-induced asthma.
In humans, only epidemiological data are available. A meta-analysis including
nine cross-sectional, seven case-control, and three cohort studies showed a risk
reduction of the infection on children but not adult onset asthma (OR, 0.81 (95 % CI
0.72–0.91) versus 0.88 (0.71–1.08)) (Wang et al. 2013). A recent meta-analysis
including 16 studies confirmed the reduced odds of atopy by H. pylori infection,
particularly in those with raise allergen-specific IgE (OR ¼ 0.75; 95 % CI
0.62–0.92, p < 0.01, seven studies) (Taye et al. 2015). In a recent study from a
low-income birth cohort in Ethiopia, 863 children were followed for up to 5 years
for incidence and prevalence of allergic disease and sensitization (Amberbir
et al. 2014). H. pylori infection was assessed by the stool antigen test and was
found to be positive in 25 % at both time points, in 21 % at age 5 years only, and in
17 % at age 3, but not at age 5. After adjustment for possible confounders, H. pylori
infection at age 3 years reduced the risk of incident eczema between ages 3 and
5 years (adjusted OR, 0.25; 0.007–0.92). Sensitization was inversely related to

H. pylori infection. A similar negative association between H. pylori infection
could be shown with other so-called immune-mediated disorders such as inflam-
matory bowel disease (IBD) (Sonnenberg and Genta 2012; Roka et al. 2014).
Although these association studies are good for generating hypothesis and are
biologically plausible and/or supported by experiments in animals, they are not
sufficient to prove a causal relationship. However, as long as there is the plausibility
that an early infection may be beneficial for the long-term immune response, the
indication to treat a child for the indication should be based on solid grounds to
balance the risk-benefit ratio.
19.5 Diagnosis
The numerous tests used to diagnose H. pylori infection in adults may also be used
in children. However, some noninvasive tests like UBT and serology have
limitations.
19.5.1 Invasive Tests
The endoscopic aspect of gastric mucosa can orientate itself toward the presence of
H. pylori. Antral nodularity is a specific feature of the infection but with a poor
sensitivity and positive predictive value (42 %) (Prasad et al. 2008). Gastric
biopsies can be obtained for histology, culture, molecular tests, and rapid urease
test (RUT). According to the guidelines, two biopsies from the antrum and two from
the corpus are necessary for histology (for the Sydney system classification) and
one of each for culture. In a series of children in Brazil, histological features
indicate a moderate to severe chronic active gastritis, more frequent and of higher
grade in the antrum than in the corpus with a topographic distribution of mostly
pangastritis (62 %), followed by antral gastritis (32 %) and corpus only gastritis
(6 %). No significant atrophy or intestinal metaplasia was present (Carvalho
et al. 2012). In contrast to what occurs in adults, in children, H. pylori may be
present without inflammatory stigmata in the case of an early stage of infection.
Molecular tests correspond essentially to real-time PCR. This test allows detec-
tion of H. pylori with an excellent sensitivity and specificity and also detection of
the mutations associated with H. pylori resistance to macrolides if the target is the
23S rDNA.
To render the procedure less invasive, especially in asymptomatic children, an
alternative in getting biopsies is to use the string test to obtain gastric juice. The
material is suitable for culture and molecular tests (Goncalves et al. 2013).
19.5.2 Noninvasive Tests
19.5.2.1 Urea Breath Test (UBT)
The UBT has its limitations in young children. A special device is needed to obtain
breath air (mask with unidirectional valve with a breath bag), the 75 mg dose of 13C
urea in adults can be decreased to 50 mg (Bazzoli et al. 2000), and the test meal
consisting of citric acid can be replaced by orange juice to be acceptable to children.
More importantly, a problem of specificity has been highlighted in infants and
children under 6 years of age (Imrie et al. 2001; Leal et al. 2011b). Kindermann and
coworkers proposed to define a gray zone rather than a cutoff for children less than
6 years of age (Kindermann et al. 2000).
Some authors proposed to normalize the UBT results for CO2 production in
children by calculating the “urea hydrolysis rate” to improve the performances
(Elitsur et al. 2009). The accuracy was not improved in children under 6 years of
age suggesting that factors other than endogenous CO2 production could be respon-
sible (Yang et al. 2008).
The question of UBT specificity was addressed essentially in developed coun-
tries and on small numbers of cases. More recently, Queiroz and coworkers
performed a large study in South America (Brazil and Peru) where they evaluated
the agreement between stool antigen test and UBT and indeed found UBT reliable
in infants and toddlers. Discrepant results did occur in 5.1 % of the samples. They
also found that delta over baseline (DOB) values of UBT increased with age
between birth and 2 years (Queiroz et al. 2013c).
19.5.2.2 Antibody-Based Tests
Serology
Serology in adults using ELISA was considered as a diagnostic test with limited
specificity. The main reason was because all of the results were aggregated in the
systematic reviews, independently of the kit used. Indeed, there is a great hetero-
geneity between kits available as pointed out by Leal and coworkers (Leal
et al. 2008); some have poor performances, but a few have excellent sensitivity
and specificity (Burucoa et al. 2013).
When serology is used in young children, it is also supposed to lack sensitivity
(Leal et al. 2008). Indeed, the serological response is mounted progressively, and
the amount of specific IgG may be below the usual threshold of detection. It may be
worth having special cutoffs for young children. Furthermore, at this age given that
we may be faced with a primo-infection, it is important to look for specific IgM. In
contrast to ELISA, Western blots showed high overall performances and heteroge-
neity in a meta-analysis of ten pediatric studies (Leal et al. 2008).
The measurement of specific IgG in urine and saliva has been carried out.
Unfortunately the lack of sensitivity does not allow to recommend them (Leal
et al. 2008; Okuda et al. 2013).
19.5.2.3 Stool Antigen Test
The stool antigen test appears to be a test well suited to children, especially infants
and young children. Current kits using monoclonal antigens have shown good
sensitivity and specificity, while tests based on polyclonal antibodies were inferior
(Leal et al. 2011a; Zhou et al. 2014). Indeed, sensitivity could theoretically be
altered in the case of diarrhea, leading to lower concentration of antigens in stools
than usual and specificity from the eventual presence of helicobacters other than
H. pylori. However, this does not appear to be common.
In contrast, the rapid immunochromatographic tests currently on the market show
a lower accuracy, and there are problems with interpretation (Prell et al. 2009).
19.5.2.4 PCR from Stool Samples
The real-time PCR developed for detecting H. pylori in gastric biopsies and
clarithromycin resistance can also be applied to stool specimens. The limitation is
the difficulty in obtaining DNA without Taq polymerase inhibitors which corre-
spond to polysaccharides from vegetable origin and follow DNA during the extrac-
tion (Monteiro et al. 2001). So while this method has an excellent specificity, the
sensitivity is unsatisfactory (Lottspeich et al. 2007; Vecsei et al. 2010).
A multicenter study was carried out in Europe to compare the accuracy of
noninvasive tests using a combination of invasive tests as the reference. Concerning
the 316 children recruited (133 H. pylori positive including children below 6 years),
the best results were obtained for UBT (96.8 % accuracy) followed by serology
(91.5 %) and stool antigen test (87 %). The performance of antibody detection in
urine was not good (Megraud 2005). In contrast, a literature review concluded that
the best noninvasive methods in children were immunoblot and stool antigen test
(Guarner et al. 2010).
The current recommendation is not to test and treat using a noninvasive test for
the diagnosis, because in children, gut symptoms are very unspecific, and causes
other than H. pylori infection are more likely than H. pylori infection itself. In cases
with significant complaints and suggestion of organic disease such as GERD, it is
important to perform an upper digestive endoscopy to carry out a global exploration
(Koletzko et al. 2011). For H. pylori diagnosis, it is recommended to perform a
histological examination and another test. Because of the high antibiotic resistance
of strains infecting children and the lack of treatment options overcoming this
problem such as the bismuth-based regimen, it is also highly recommended to
perform a culture and susceptibility testing or at least a molecular-based test to
determine resistance to clarithromycin in order to tailor the therapy accordingly.
19.6 Treatment
To treat H. pylori infection, the regimens have followed those successively pro-
posed for adults with some particularities. The PPI-based triple therapies have been
applied, especially omeprazole-amoxicillin-nitroimidazole for a week.
With the PPI-clarithromycin-amoxicillin combination for a week, the added
value of PPI was essential. The results per protocol (PP) were 80 % with PPI but
only 10 % without PPI (Gottrand et al. 2001).
Bismuth-based triple therapies have also been used in children. They included
essentially amoxicillin-nitroimidazole as antibiotics. In a recent review, the PP
result was 86 % (Pacifico et al. 2012). The addition of a PPI did not improve the
results. The pediatric European registry for treatment of H. pylori (PERTH) showed
a better efficacy of bismuth-based triple therapies (77 % eradication) compared to
PPI-based triple therapies (64 % eradication) (Oderda et al. 2007).
These last years, 10-day sequential therapy emerged as an alternative to previous
regimens, also in children (Francavilla et al. 2005). In a meta-analysis, Horvath
et al. found a higher eradication rate when compared to standard clarithromycin-
based triple therapy (78 % vs. 71 %) (Horvath et al. 2012). All of these studies were
faced with the problems of diversity in the posology and length of treatment, a small
number of children in each arm, and no clear randomization procedure, and
therefore, they are subject to criticism. Furthermore, data on susceptibility testing
are missing in most studies, even though this is by far the main risk factor for
treatment failure. With the current knowledge, a 10-day sequential therapy cannot
be recommended as first-line treatment without prior antibiotic susceptibility
testing.
A large prospective multicenter study, carried out in Europe from 1999 to 2002,
pointed out an overall resistance rate to clarithromycin of 20 %, higher in boys, in
children less than 6 years old, and in patients from Southern Europe. The resistance
rate to metronidazole, which has less impact on the treatment outcome, was indeed
23 % (Koletzko et al. 2006). Ten years later, a survey was conducted on adults but
also included children. The clarithromycin resistance rate was 31.8 %, and for
metronidazole it was 25.7 % (Megraud et al. 2013). A similar trend was also found
in other countries like South Korea where over a 20-year period, clarithromycin
resistance increased from 6.9 to 18.2 % and metronidazole resistance decreased
from 32.8 to 27.3 % (Seo et al. 2013).
The other aspect to consider is safety, and all current regimens lead to adverse
events, e.g., diarrhea, bad taste, etc. Fortunately, it is seldom that the patients must
stop the treatment. However, these factors may contribute to poor compliance with
drug intake, which is after antibiotic resistance, the major risk factor for low
eradication rates of H. pylori.
Another potential negative effect not explored is the impact on the intestinal
microbiome. The intestinal microbiome is very important for various diseases, and
the resilience capacity most likely varies between individuals and according to the
different antibiotics used.
Current guidelines (Koletzko et al. 2011) recommend as first-line therapy either

(1) the standard triple therapy (PPI-amoxicillin-clarithromycin or nitroimidazole)
tailored to results from prior antibiotic susceptibility testing or (2) the bismuth-
based triple therapy with amoxicillin. Given the importance of antibiotic resistance
as a first factor for failure and the fact that bismuth-based drugs are not licensed for
use in pediatric patients in many countries, the logical consequence is to prescribe a
tailored treatment based on antimicrobial susceptibility testing. In children with a
fully susceptible strain or resistance to metronidazole, a 10- or 14-day triple therapy
using PPI, amoxicillin, and clarithromycin is recommended. In case of
clarithromycin resistance, metronidazole should be used instead. Dosage should
be calculated on body weight with PPI 1–1,5 mg/kg (max. 60 mg), amoxicillin
60–70 mg/kg (max. 3 g), clarithromycin 20–25 mg/kg (max. 1 g), and metronida-
zole 20–25 mg/kg (max 1 g) per day divided in two doses. In case of double
resistance against both antibiotics, a 14-day high-dose regimen with PPI, amoxi-
cillin, and metronidazole was successful in 73 % of those who complied to the
regimen (Schwarzer et al. 2011).
19.7 Conclusion
H. pylori infection in children remains a challenge in many aspects. The long-term

impact of early infection on the immune response with possible reduction of
immune-mediated disorders is still unclear. This needs to be balanced against an
increased risk for later development of PUD and gastric cancer. Animal experi-
ments indicate that not only the infection itself but also the time point of infection,
infancy versus childhood, may play an important role for long-term health. The
chronic infection itself is rarely cause of symptoms; the risk for PUD is low before
puberty and for H. pylori-related gastric malignancy nonexistent. In contrast,
treatment options are less compared to adults, and the same treatment regimens
seem less effective during childhood. For these reasons, testing for H. pylori in
children should be restricted to those who will benefit from eradication therapy and
should include biopsies for antibiotic susceptibility to guide treatment.
References
Albenque M, Tall F, Dabis F, Megraud F (1990) Epidemiological study of Helicobacter pylori

transmission from mother to child in Africa. Rev Esp Enferm Dig 78(Suppl 1):P-93
Amberbir A, Medhin G, Abegaz WE, Hanlon C, Robinson K, Fogarty A, Britton J, Venn A, Davey
G (2014) Exposure to Helicobacter pylori infection in early childhood and the risk of allergic
disease and atopic sensitization: a longitudinal birth cohort study. Clin Exp Allergy
44:563–571
Apley J, Naish N (1958) Recurrent abdominal pains: a field survey of 1,000 school children. Arch
Dis Child 133:165–170
Arnold IC, Dehzad N, Reuter S, Martin H, Becher B, Taube C, Muller A (2011) Helicobacter
pylori infection prevents allergic asthma in mouse models through the induction of regulatory
T cells. J Clin Invest 121:3088–3093
Ashorn M, Rago T, Kokkonen J, Ruuska T, Rautelin H, Karikoski R (2004) Symptomatic response
to Helicobacter pylori eradication in children with recurrent abdominal pain: double blind
randomized placebo-controlled trial. J Clin Gastroenterol 38:646–650
Baggett HC, Parkinson AJ, Muth PT, Gold BD, Gessner BD (2006) Endemic iron deficiency
associated with Helicobacter pylori infection among school-aged children in Alaska. Pediat-
rics 117:e396–e404
Bamford KB, Bickley J, Collins JS, Johnston BT, Potts S, Boston V, Owen RJ, Sloan JM (1993)
Helicobacter pylori: comparison of DNA fingerprints provides evidence for intrafamilial
infection. Gut 34:1348–1350
Banatvala N, Mayo K, Megraud F, Jennings R, Deeks JJ, Feldman RA (1993) The cohort effect
and Helicobacter pylori. J Infect Dis 168:219–221
Banatvala N, Kashiwagi S, Abdi Y, Hayashi J, Hardie JM, Feldman RA (1994) H. pylori
seroconversion and seroreversion in an Okinawan cohort followed for 10 years. Am J
Gastroenterol 89(8):1300 (Abst No. 1362)
Bastos J, Carreira H, La Vecchia C, Lunet N (2013a) Childcare attendance and Helicobacter pylori
infection: systematic review and meta-analysis. Eur J Cancer Prev 22:311–319
Bastos J, Peleteiro B, Pinto H, Marinho A, Guimaraes JT, Ramos E, La Vecchia C, Barros H,
Lunet N (2013b) Prevalence, incidence and risk factors for Helicobacter pylori infection in a
cohort of Portuguese adolescents (EpiTeen). Dig Liver Dis 45:290–295
Bazzoli F, Cecchini L, Corvaglia L, Dall’Antonia M, De Giacomo C, Fossi S, Casali LG, Gullini S,
Lazzari R, Leggeri G, Lerro P, Valdambrini V, Mandrioli G, Marani M, Martelli P, Miano A,
Nicolini G, Oderda G, Pazzi P, Pozzato P, Ricciardiello L, Roda E, Simoni P, Sottili S, Zagari
RM (2000) Validation of the 13C-urea breath test for the diagnosis of Helicobacter pylori
infection in children: a multicenter study. Am J Gastroenterol 95:646–650
Bontems P, Robert F, Van Gossum A, Cadranel S, Mascart F (2003) Helicobacter pylori modu-
lation of gastric and duodenal mucosal T cell cytokine secretions in children compared with
adults. Helicobacter 8:216–226
Bontems P, Kalach N, Vanderpas J, Iwanczak B, Casswall T, Koletzko S, Oderda G, Martinez-
Gomez MJ, Urruzuno P, Kindermann A, Sykora J, Veres G, Roma-Giannikou E,
Pehlivanoglu E, Megraud F, Cadranel S (2013) Helicobacter pylori infection in European
children with gastro-duodenal ulcers and erosions. Pediatr Infect Dis J 32:1324–1329
Bontems P, Aksoy E, Burette A, Segers V, Deprez C, Mascart F, Cadranel S (2014) NF-kappaB
activation and severity of gastritis in Helicobacter pylori-infected children and adults.
Bravo LE, Mera R, Reina JC, Pradilla A, Alzate A, Fontham E, Correa P (2003) Impact of
Helicobacter pylori infection on growth of children: a prospective cohort study. J Pediatr
Gastroenterol Nutr 37:614–619
Buonavolonta R, Miele E, Russo D, Vecchione R, Staiano A (2011) Helicobacter pylori chronic
gastritis in children: to eradicate or not to eradicate? J Pediatr 159:50–56
Burucoa C, Delchier JC, Courillon-Mallet A, de Korwin JD, Megraud F, Zerbib F, Raymond J,
Fauchere JL (2013) Comparative evaluation of 29 commercial Helicobacter pylori serological
kits. Helicobacter 18:169–179
Cardenas VM, Prieto-Jimenez CA, Mulla ZD, Rivera JO, Dominguez DC, Graham DY, Ortiz M
(2011) Helicobacter pylori eradication and change in markers of iron stores among non-iron-
deficient children in El Paso, Texas: an etiologic intervention study. J Pediatr Gastroenterol
Nutr 52:326–332
Carreira H, Bastos A, Peleteiro B, Lunet N (2015) Breast-feeding and Helicobacter pylori
infection: systematic review and meta-analysis. Public Health Nutr 18(3):500–520
Carvalho MA, Machado NC, Ortolan EV, Rodrigues MA (2012) Upper gastrointestinal histopath-
ological findings in children and adolescents with nonulcer dyspepsia with Helicobacter pylori
infection. J Pediatr Gastroenterol Nutr 55:523–529
Chak E, Rutherford GW, Steinmaus C (2009) The role of breast-feeding in the prevention of
Helicobacter pylori infection: a systematic review. Clin Infect Dis 48:430–437
Chen Y, Blaser MJ (2008) Helicobacter pylori colonization is inversely associated with childhood
asthma. J Infect Dis 198:553–560
Cherian S, Burgner DP, Cook AG, Sanfilippo FM, Forbes DA (2010) Associations between
Helicobacter pylori infection, co-morbid infections, gastrointestinal symptoms, and circulating
cytokines in African children. Helicobacter 15:88–97
Choi JW (2003) Does Helicobacter pylori infection relate to iron deficiency anaemia in prepu-
bescent children under 12 years of age? Acta Paediatr 92:970–972
den Hoed CM, Vila AJ, Holster IL, Perez-Perez GI, Blaser MJ, de Jongste JC, Kuipers EJ (2011)
Helicobacter pylori and the birth cohort effect: evidence for stabilized colonization rates in
childhood. Helicobacter 16:405–409
den Hollander WJ, Holster IL, den Hoed CM, van Deurzen F, van Vuuren AJ, Jaddoe VW,
Hofman A, Perez Perez GI, Blaser MJ, Moll HA, Kuipers EJ (2013) Ethnicity is a strong
predictor for Helicobacter pylori infection in young women in a multi-ethnic European city. J
Deng ZH, Chu B, Xu YZ, Zhang B, Jiang LR (2012) Influence of Helicobacter pylori infection on
ghrelin levels in children. World J Gastroenterol 18:5096–5100
Dominici P, Bellentani S, Di Biase AR, Saccoccio G, Le Rose A, Masutti F, Viola L, Balli F,
Tiribelli C, Grilli R, Fusillo M, Grossi E (1999) Familial clustering of Helicobacter pylori
infection: population based study. BMJ 319:537–540
Dore MP, Fanciulli G, Tomasi PA, Realdi G, Delitala G, Graham DY, Malaty HM (2012)
Gastrointestinal symptoms and Helicobacter pylori infection in school-age children residing
in Porto Torres, Sardinia, Italy. Helicobacter 17:369–373
Drumm B, Sherman P, Cutz E, Karmali M (1987) Association of Campylobacter pylori on the
gastric mucosa with antral gastritis in children. N Engl J Med 316:1557–1561
Egorov AI, Sempertegui F, Estrella B, Egas J, Naumova EN, Griffiths JK (2010) The effect of
Helicobacter pylori infection on growth velocity in young children from poor urban commu-
nities in Ecuador. Int J Infect Dis 14:e788–e791
Elitsur Y, Tolia V, Gilger MA, Reeves-Garcia J, Schmidt-Sommerfeld E, Opekun AR,
El-Zimaity H, Graham DY, Enmei K (2009) Urea breath test in children: the United States
prospective, multicenter study. Helicobacter 14:134–140
Ford AC, Forman D, Bailey AG, Goodman KJ, Axon AT, Moayyedi P (2007) Effect of sibling
number in the household and birth order on prevalence of Helicobacter pylori: a cross-
sectional study. Int J Epidemiol 36:1327–1333
Francavilla R, Lionetti E, Castellaneta SP, Magista AM, Boscarelli G, Piscitelli D, Amoruso A, Di
Leo A, Miniello VL, Francavilla A, Cavallo L, Ierardi E (2005) Improved efficacy of 10-Day
sequential treatment for Helicobacter pylori eradication in children: a randomized trial.
Francois F, Roper J, Joseph N, Pei Z, Chhada A, Shak JR et al (2011) The effect of H. pylori
eradication on meal-associated changes in plasma ghrelin and leptin. BMC Gastroenterol 11:37
Freire de Melo F, Rocha AM, Rocha GA, Pedroso SH, de Assis Batista S, Fonseca de Castro LP,
Carvalho SD, Bittencourt PF, de Oliveira CA, Correa-Oliveira R, Magalhaes Queiroz DM
(2012) A regulatory instead of an IL-17 T response predominates in Helicobacter pylori-
associated gastritis in children. Microbes Infect 14:341–347
Freire de Melo F, Rocha GA, Rocha AM, Teixeira KN, Pedroso SH, Pereira Junior JB, Fonseca de
Castro LP, Cabral MM, Carvalho SD, Bittencourt PF, de Oliveira CA, Queiroz DM (2014) Th1
immune response to H. pylori infection varies according to the age of the patients and
influences the gastric inflammatory patterns. Int J Med Microbiol 304:300–306
Gil JH, Seo JW, Cho MS, Ahn JH, Sung HY (2014) Role of Treg and TH17 cells of the gastric
mucosa in children with Helicobacter pylori gastritis. J Pediatr Gastroenterol Nutr 58:252–258
Goncalves MH, Silva CI, Braga-Neto MB, Fialho AB, Fialho AM, Queiroz DM, Braga LL (2013)
Helicobacter pylori virulence genes detected by string PCR in children from an urban
community in northeastern Brazil. J Clin Microbiol 51:988–989
Goodman KJ, Correa P (2000) Transmission of Helicobacter pylori among siblings. Lancet
355:358–362
Goodman KJ, Correa P, Mera R, Yepez MC, Ceron C, Campo C, Guerrero N, Sierra MS, Bravo LE
(2011) Effect of Helicobacter pylori infection on growth velocity of school-age Andean
children. Epidemiology 22:118–126
Gottrand F, Kalach N, Spyckerelle C, Guimber D, Mougenot JF, Tounian P, Lenaerts C,
Roquelaure B, Lachaux A, Morali A, Dupont C, Maurage C, Husson MO, Barthelemy P
(2001) Omeprazole combined with amoxicillin and clarithromycin in the eradication of
Helicobacter pylori in children with gastritis: a prospective randomized double-blind trial. J
Pediatr 139:664–668
Guariso G, Meneghel A, Dalla Pozza LV, Romano C, Dall’Oglio L, Lombardi G, Conte S,
Calacoci M, Campanozzi A, Nichetti C, Piovan S, Zancan L, Facchin P (2010) Indications
to upper gastrointestinal endoscopy in children with dyspepsia. J Pediatr Gastroenterol Nutr
50:493–499
Guarner J, Kalach N, Elitsur Y, Koletzko S (2010) Helicobacter pylori diagnostic tests in children:
review of the literature from 1999 to 2009. Eur J Pediatr 169:15–25
Harris PR, Wright SW, Serrano C, Riera F, Duarte I, Torres J, Pena A, Rollan A, Viviani P,
Guiraldes E, Schmitz JM, Lorenz RG, Novak L, Smythies LE, Smith PD (2008) Helicobacter
pylori gastritis in children is associated with a regulatory T-cell response. Gastroenterology
134:491–499
Harris PR, Serrano CA, Villagran A, Walker MM, Thomson M, Duarte I, Windle HJ, Crabtree JE
(2013) Helicobacter pylori-associated hypochlorhydria in children, and development of iron
deficiency. J Clin Pathol 66:343–347
Herrera PM, Mendez M, Velapatino B, Santivanez L, Balqui J, Finger SA, Sherman J, Zimic M,
Cabrera L, Watanabe J, Rodriguez C, Gilman RH, Berg DE (2008) DNA-level diversity and
relatedness of Helicobacter pylori strains in shantytown families in Peru and transmission in a
developing-country setting. J Clin Microbiol 46:3912–3918
Horvath A, Dziechciarz P, Szajewska H (2012) Meta-analysis: sequential therapy for Helicobacter
pylori eradication in children. Aliment Pharmacol Ther 36:534–541
Imrie C, Rowland M, Bourke B, Drumm B (2001) Limitations to carbon 13-labeled urea breath
testing for Helicobacter pylori in infants. J Pediatr 139:734–737
Jaganath D, Saito M, Gilman RH, Queiroz DM, Rocha GA, Cama V, Cabrera L, Kelleher D,
Windle HJ, Crabtree JE, Checkley W (2014) First detected Helicobacter pylori infection in
infancy modifies the association between diarrheal disease and childhood growth in Peru.
Kindermann A, Demmelmair H, Koletzko B, Krauss-Etschmann S, Wiebecke B, Koletzko S
(2000) Influence of age on 13C-urea breath test results in children. J Pediatr Gastroenterol
Nutr 30:85–91
Koletzko S, Richy F, Bontems P, Crone J, Kalach N, Monteiro ML, Gottrand F, Celinska-Cedro D,
Roma-Giannikou E, Orderda G, Kolacek S, Urruzuno P, Martinez-Gomez MJ, Casswall T,
Ashorn M, Bodanszky H, Megraud F (2006) Prospective multicentre study on antibiotic
resistance of Helicobacter pylori strains obtained from children living in Europe. Gut
55:1711–1716
Koletzko S, Jones NL, Goodman KJ, Gold B, Rowland M, Cadranel S, Chong S, Colletti RB,
Casswall T, Elitsur Y, Guarner J, Kalach N, Madrazo A, Megraud F, Oderda G (2011)
Evidence-based guidelines from ESPGHAN and NASPGHAN for Helicobacter pylori infec-
tion in children. J Pediatr Gastroenterol Nutr 53:230–243
Konno M, Yokota S, Suga T, Takahashi M, Sato K, Fujii N (2008) Predominance of mother-to-

child transmission of Helicobacter pylori infection detected by random amplified polymorphic
DNA fingerprinting analysis in Japanese families. Pediatr Infect Dis J 27:999–1003
Leal YA, Flores LL, Garcia-Cortes LB, Cedillo-Rivera R, Torres J (2008) Antibody-based
detection tests for the diagnosis of Helicobacter pylori infection in children: a meta-analysis.
PLoS One 3:e3751
Leal YA, Cedillo-Rivera R, Simon JA, Velazquez JR, Flores LL, Torres J (2011a) Utility of stool
sample-based tests for the diagnosis of Helicobacter pylori infection in children. J Pediatr
Leal YA, Flores LL, Fuentes-Panana EM, Cedillo-Rivera R, Torres J (2011b) 13C-urea breath test
for the diagnosis of Helicobacter pylori infection in children: a systematic review and meta-
Leodolter A, Kulig M, Brasch H, Meyer-Sabellek W, Willich SN, Malfertheiner P (2001) A meta-
analysis comparing eradication, healing and relapse rates in patients with Helicobacter pylori-
associated gastric or duodenal ulcer. Aliment Pharmacol Ther 15:1949–1958
Li J, Ou Z, Wang F, Guo Y, Zhang R, Zhang J, Li P, Xu W, He Y (2009) Distinctiveness of the
cagA genotype in children and adults with peptic symptoms in South China. Helicobacter
14:248–255
van der Merwe SW, Yamaoka Y, Graham DY, Perez-Trallero E, Wadstrom T, Suerbaum S,
Achtman M (2007) An African origin for the intimate association between humans and
Helicobacter pylori. Nature 445:915–918
Lottspeich C, Schwarzer A, Panthel K, Koletzko S, Russmann H (2007) Evaluation of the novel
Helicobacter pylori ClariRes real-time PCR assay for detection and clarithromycin suscepti-
bility testing of H. pylori in stool specimens from symptomatic children. J Clin Microbiol
45:1718–1722
Lunet N, Peleteiro B, Bastos J, Correia S, Marinho A, Guimaraes JT, La Vecchia C, Barros H
(2014) Child day-care attendance and Helicobacter pylori infection in the Portuguese birth
cohort Geracao XXI. Eur J Cancer Prev 23:193–198
Luzza F, Imeneo M, Maletta M, Paluccio G, Giancotti A, Perticone F, Focan A, Pallone F (1997)
Seroepidemiology of Helicobacter pylori infection and hepatitis A in a rural area: evidence
against a common mode of transmission. Gut 41:164–168
Macarthur C (1999) Helicobacter pylori infection and childhood recurrent abdominal pain: lack of
evidence for a cause and effect relationship. Can J Gastroenterol 13:607–610
Mahalanabis D, Islam MA, Shaikh S, Chakrabarty M, Kurpad AV, Mukherjee S, Sen B, Khaled
MA, Vermund SH (2005) Haematological response to iron supplementation is reduced in
children with asymptomatic Helicobacter pylori infection. Br J Nutr 94:969–975
Malaty HM, Engstrand L, Pedersen NL, Graham DY (1994) Helicobacter pylori infection: genetic
and environmental influences. A study of twins. Ann Intern Med 120:982–986
Malaty HM, El-Kasabany A, Graham DY, Miller CC, Reddy SG, Srinivasan SR, Yamaoka Y,
Berenson GS (2002) Age at acquisition of Helicobacter pylori infection: a follow-up study
from infancy to adulthood. Lancet 359:931–935
Malfertheiner P, Megraud F, O’Morain CA, Atherton J, Axon AT, Bazzoli F, Gensini GF, Gisbert
JP, Graham DY, Rokkas T, El-Omar EM, Kuipers EJ (2012) Management of Helicobacter
pylori infection – the Maastricht IV/Florence consensus report. Gut 61:646–664
and peptic ulceration. Lancet 1:1311–1315
Mayerle J, den Hoed CM, Schurmann C, Stolk L, Homuth G, Peters MJ, Capelle L,
Zimmermann K, Rivadeneira F, Gruska S, Volzke H, de Vries AC, Volker U, Teumer A,
van Meurs JB, Steinmetz I, Nauck M, Ernst F, Weiss FU, Hofman A, Zenker M, Kroemer HK,
Prokisch H, Uitterlinden AG, Lerch MM, Kuipers EJ (2013) Identification of genetic loci
associated with Helicobacter pylori serologic status. JAMA 309:1912–1920
Megraud F (2005) Comparison of non-invasive tests to detect Helicobacter pylori infection in

children and adolescents: results of a multicenter European study. J Pediatr 146:198–203
Megraud F, Brassens-Rabbe MP, Denis F, Belbouri A, Hoa DQ (1989) Seroepidemiology of
Campylobacter pylori infection in various populations. J Clin Microbiol 27:1870–1873
Megraud F, Coenen S, Versporten A, Kist M, Lopez-Brea M, Hirschl AM, Andersen LP,
Goossens H, Glupczynski Y (2013) Helicobacter pylori resistance to antibiotics in Europe
and its relationship to antibiotic consumption. Gut 62:34–42
Mera RM, Bravo LE, Goodman KJ, Yepez MC, Correa P (2012) Long-term effects of clearing
Helicobacter pylori on growth in school-age children. Pediatr Infect Dis J 31:263–266
Miendje Deyi VY, Vanderpas J, Bontems P, Van den Borre C, De Koster E, Cadranel S, Burette A
(2011) Marching cohort of Helicobacter pylori infection over two decades (1988–2007):
combined effects of secular trend and population migration. Epidemiol Infect 139:572–580
Mitchell HM, Li YY, Hu PJ, Liu Q, Chen M, Du GG, Wang ZJ, Lee A, Hazell SL (1992)
Epidemiology of Helicobacter pylori in southern China: identification of early childhood as
the critical period for acquisition. J Infect Dis 166:149–153
Monteiro L, Gras N, Vidal R, Cabrita J, Megraud F (2001) Detection of Helicobacter pylori DNA
in human feces by PCR: DNA stability and removal of inhibitors. J Microbiol Methods
45:89–94
Muhsen K, Barak M, Henig C, Alpert G, Ornoy A, Cohen D (2010) Is the association between
Helicobacter pylori infection and anemia age dependent? Helicobacter 15:467–472
Nahar S, Kibria KM, Hossain ME, Sultana J, Sarker SA, Engstrand L, Bardhan PK, Rahman M,
Endtz HP (2009) Evidence of intra-familial transmission of Helicobacter pylori by PCR-based
RAPD fingerprinting in Bangladesh. Eur J Clin Microbiol Infect Dis 28:767–773
Neefjes VM, Heijboer H, Tamminga RY (2007) H. pylori infection in childhood chronic immune
thrombocytopenic purpura. Haematologica 92:576
Oderda G, Shcherbakov P, Bontems P, Urruzuno P, Romano C, Gottrand F, Gomez MJ, Ravelli A,
Gandullia P, Roma E, Cadranel S, De Giacomo C, Canani RB, Rutigliano V, Pehlivanoglu E,
Kalach N, Roggero P, Celinska-Cedro D, Drumm B, Casswall T, Ashorn M, Arvanitakis SN
(2007) Results from the pediatric European register for treatment of Helicobacter pylori
(PERTH). Helicobacter 12:150–156
Okuda M, Miyashiro E, Booka M, Tsuji T, Nakazawa T (2007) Helicobacter pylori colonization in
the first 3 years of life in Japanese children. Helicobacter 12:324–327
Okuda M, Kamiya S, Booka M, Kikuchi S, Osaki T, Hiwatani T, Maekawa K, Fukuda Y (2013)
Diagnostic accuracy of urine-based kits for detection of Helicobacter pylori antibody in
children. Pediatr Int 55:337–341
Oleastro M, Monteiro L, Lehours P, Megraud F, Menard A (2006) Identification of markers for
Helicobacter pylori strains isolated from children with peptic ulcer disease by suppressive
subtractive hybridization. Infect Immun 74:4064–4074
Pacifico L, Anania C, Osborn JF, Ferrara E, Schiavo E, Bonamico M, Chiesa C (2008) Long-term
effects of Helicobacter pylori eradication on circulating ghrelin and leptin concentrations and
body composition in prepubertal children. Eur J Endocrinol 158:323–332
Pacifico L, Osborn JF, Anania C, Vaira D, Olivero E, Chiesa C (2012) Review article: bismuth-
based therapy for Helicobacter pylori eradication in children. Aliment Pharmacol Ther
35:1010–1026
Pacifico L, Osborn JF, Tromba V, Romaggioli S, Bascetta S, Chiesa C (2014) Helicobacter pylori
infection and extragastric disorders in children: a critical update. World J Gastroenterol
20:1379–1401
Parsonnet J, Shmuely H, Haggerty T (1999) Fecal and oral shedding of Helicobacter pylori from
healthy infected adults. JAMA 282:2240–2245
Perez-Perez GI, Sack RB, Reid R, Santosham M, Croll J, Blaser MJ (2003) Transient and
persistent Helicobacter pylori colonization in Native American children. J Clin Microbiol
41:2401–2407
Perry S, de la Luz Sanchez M, Yang S, Haggerty TD, Hurst P, Perez-Perez G, Parsonnet J (2006)
Gastroenteritis and transmission of Helicobacter pylori infection in households. Emerg Infect
Dis 12:1701–1708
Plonka M, Bielanski W, Konturek SJ, Targosz A, Sliwowski Z, Dobrzanska M, Kaminska A,
Sito E, Konturek PC, Brzozowski T (2006) Helicobacter pylori infection and serum gastrin,
ghrelin and leptin in children of Polish shepherds. Dig Liver Dis 38:91–97
Prasad KK, Thapa BR, Sharma AK, Nain CK, Singh K (2008) Reassessment of diagnostic value of
antral nodularity for Helicobacter pylori infection in children. Minerva Gastroenterol Dietol
54:1–6
Prell C, Osterrieder S, Lottspeich C, Schwarzer A, Russmann H, Ossiander G, Koletzko S (2009)
Improved performance of a rapid office-based stool test for detection of Helicobacter pylori in
children before and after therapy. J Clin Microbiol 47:3980–3984
Queiroz DM, Harris PR, Sanderson IR, Windle HJ, Walker MM, Rocha AM, Rocha GA, Carvalho
SD, Bittencourt PF, de Castro LP, Villagran A, Serrano C, Kelleher D, Crabtree JE (2013a)
Iron status and Helicobacter pylori infection in symptomatic children: an international multi-
centered study. PLoS One 8:e68833
Queiroz DM, Rocha AM, Melo FF, Rocha GA, Teixeira KN, Carvalho SD, Bittencourt PF, Castro
LP, Crabtree JE (2013b) Increased gastric IL-1beta concentration and iron deficiency param-
eters in H. pylori infected children. PLoS One 8:e57420
Queiroz DM, Saito M, Rocha GA, Rocha AM, Melo FF, Checkley W, Braga LL, Silva IS, Gilman
RH, Crabtree JE (2013c) Helicobacter pylori infection in infants and toddlers in South
America: concordance between [13C]urea breath test and monoclonal H. pylori stool antigen
test. J Clin Microbiol 51:3735–3740
Rajantie J, Klemola T (2003) Helicobacter pylori and idiopathic thrombocytopenic purpura in
children. Blood 101:1660
Raymond J, Thiberge JM, Kalach N, Bergeret M, Dupont C, Labigne A, Dauga C (2008) Using
macro-arrays to study routes of infection of Helicobacter pylori in three families. PLoS One 3:
e2259
Roka K, Roubani A, Stefanaki K, Panayotou I, Roma E, Chouliaras G (2014) The prevalence of
Helicobacter pylori gastritis in newly diagnosed children with inflammatory bowel disease.
Rowland M, Daly L, Vaughan M, Higgins A, Bourke B, Drumm B (2006) Age-specific incidence
of Helicobacter pylori. Gastroenterology 130:65–72; quiz 211
Russell RG, Wasserman SS, O’Donnoghue JM, Taylor DN, Boslego J, Moreno JG, Hopkins RJ,
DeTolla LJ, Morris JG Jr (1993) Serologic response to Helicobacter pylori among children and
teenagers in northern Chile. Am J Trop Med Hyg 49:189–191
Sarker SA, Mahmud H, Davidsson L, Alam NH, Ahmed T, Alam N, Salam MA, Beglinger C,
Gyr N, Fuchs GJ (2008) Causal relationship of Helicobacter pylori with iron-deficiency
anemia or failure of iron supplementation in children. Gastroenterology 135:1534–1542
Schwarz S, Morelli G, Kusecek B, Manica A, Balloux F, Owen RJ, Graham DY, van der Merwe S,
Achtman M, Suerbaum S (2008) Horizontal versus familial transmission of Helicobacter
pylori. PLoS Pathog 4:e1000180
Schwarzer A, Urruzuno P, Iwanczak B, Martinez-Gomez MZ, Kalach N, Roma-Giannikou E,
Liptay S, Bontem P, Buderus S, Wenzl TG, Koletzko S (2011) New effective treatment
regimen for children infected with a double-resistant Helicobacter pylori strain. J Pediatr
Schwille IJ, Giel KE, Ellert U, Zipfel S, Enck P (2009) A community-based survey of abdominal
pain prevalence, characteristics, and health care use among children. Clin Gastroenterol
Hepatol 7:1062–1068
Senbanjo I, Akinbami A, Diaku-Akinwumi I, Oshikoya K, Adeyemo T, Dada O, Dosunmu A,
Oshinaike O (2010) Helicobacter pylori infection among a pediatric population with sickle cell
disease. J Natl Med Assoc 102:1095–1099
Seo JH, Jun JS, Yeom JS, Park JS, Youn HS, Ko GH, Baik SC, Lee WK, Cho MJ, Rhee KH (2013)
Changing pattern of antibiotic resistance of Helicobacter pylori in children during 20 years in
Jinju, South Korea. Pediatr Int 55:332–336
Helicobacter pylori infection in rhesus macaques is most consistent with oral-oral transmis-
Sonnenberg A, Genta RM (2012) Low prevalence of Helicobacter pylori infection among patients
with inflammatory bowel disease. Aliment Pharmacol Ther 35:469–476
Spee LA, Madderom MB, Pijpers M, van Leeuwen Y, Berger MY (2010) Association between
Helicobacter pylori and gastrointestinal symptoms in children. Pediatrics 125:e651–e669
Taye B, Enquselassie F, Tsegaye A, Medhin G, Davey G, Venn A (2015 May) Is H. pylori
infection inversely associated with atopy? A systematic review and meta-analysis. Clin Exp
Allergy 45:882–890
Thomas JE, Dale A, Bunn JE, Harding M, Coward WA, Cole TJ, Weaver LT (2004) Early
Helicobacter pylori colonisation: the association with growth faltering in The Gambia. Arch
Dis Child 89:1149–1154
Tkachenko MA, Zhannat NZ, Erman LV, Blashenkova EL, Isachenko SV, Isachenko OB, Graham
DY, Malaty HM (2007) Dramatic changes in the prevalence of Helicobacter pylori infection
during childhood: a 10-year follow-up study in Russia. J Pediatr Gastroenterol Nutr
45:428–432
Travis PB, Goodman KJ, O’Rourke KM, Groves FD, Sinha D, Nicholas JS, VanDerslice J,
Lackland D, Mena KD (2010) The association of drinking water quality and sewage disposal
with Helicobacter pylori incidence in infants: the potential role of water-borne transmission. J
Water Health 8:192–203
Urita Y, Watanabe T, Kawagoe N, Takemoto I, Tanaka H, Kijima S, Kido H, Maeda T,
Sugasawa Y, Miyazaki T, Honda Y, Nakanishi K, Shimada N, Nakajima H, Sugimoto M,
Urita C (2013) Role of infected grandmothers in transmission of Helicobacter pylori to
children in a Japanese rural town. J Paediatr Child Health 49:394–398
Vecsei A, Innerhofer A, Binder C, Gizci H, Hammer K, Bruckdorfer A, Riedl S, Gadner H, Hirschl
AM, Makristathis A (2010) Stool polymerase chain reaction for Helicobacter pylori detection
and clarithromycin susceptibility testing in children. Clin Gastroenterol Hepatol 8:309–312
Wewer V, Andersen LP, Paerregaard A, Gernow A, Hansen JP, Matzen P, Krasilnikoff PA (2001)
Treatment of Helicobacter pylori in children with recurrent abdominal pain. Helicobacter
6:244–248
Xia W, Zhang X, Wang J, Sun C, Wu L (2012) Survey of anaemia and Helicobacter pylori
infection in adolescent girls in Suihua, China and enhancement of iron intervention effects by
H. pylori eradication. Br J Nutr 108:357–362
Yang HR, Ko JS, Seo JK (2008) Does the diagnostic accuracy of the 13C-urea breath test vary with
age even after the application of urea hydrolysis rate? Helicobacter 13:239–244
Yang YJ, Sheu BS, Yang HB, Lu CC, Chuang CC (2012) Eradication of Helicobacter pylori
increases childhood growth and serum acylated ghrelin levels. World J Gastroenterol
18:2674–2681
Young KA, Akyon Y, Rampton DS, Barton SG, Allaker RP, Hardie JM, Feldman RA (2000)
Quantitative culture of Helicobacter pylori from gastric juice: the potential for transmission. J
Zhou X, Su J, Xu G, Zhang G (2014) Accuracy of stool antigen test for the diagnosis of
Helicobacter pylori infection in children: a meta-analysis. Clin Res Hepatol Gastroenterol
38:629–638
Part IV
Treatment of Helicobacter pylori
Chapter 20
Helicobacter pylori Therapy
Javier Molina-Infante and David Y. Graham
Abstract Eradication of Helicobacter pylori infection provides symptomatic ben-

efit for many non-ulcer dyspepsia patients, heals and prevents recurrence of peptic
ulcer disease, reduces the risk of development of gastric cancer, and can be a
curative therapy for gastric mucosa-associated lymphoid tissue lymphoma. An
effective therapy is defined as one achieving at least a 90 % eradication rate with
the first attempt. Cure rates of H. pylori with triple therapy have declined to
unacceptable levels worldwide, mostly due to increasing clarithromycin resistance
rates. As such, first-line treatment should be chosen upon local prevalence of
H. pylori antimicrobial resistance or, if unavailable, empirically based on a com-
bination of using only regimens that have proven to be reliably excellent locally and
knowledge of prior use of antibiotics by the patient. The preferred empirical choices
are currently 14-day concomitant therapy and 14-day bismuth quadruple therapy,
using high-dose proton pump inhibitor therapy. Rescue therapy after failure erad-
ication regimens should be tailored based on antimicrobial susceptibility testing
and, if not available, chosen upon which treatments were used initially (e.g.,
bismuth or non-bismuth quadruple therapy) and the prevalence of fluoroquinolone
resistance locally. Where available, furazolidone- and rifabutin-containing thera-
pies are likely to be successful as rescue therapy.
Keywords Helicobacter pylori • Eradication • Drug resistance • Triple • Non-

bismuth quadruple • Concomitant • Sequential • Bismuth quadruple • Rescue
therapy
J. Molina-Infante
Department of Gastroenterology, Hospital San Pedro de Alcantara, C/Pablo Naranjo s/n, 10003
Cáceres, Spain
D.Y. Graham (*)
Department of Medicine, Michael E. DeBakey VA Medical Center, and Baylor College of
Medicine, 2002 Holcombe Blvd (111D), Houston, TX 77030, USA

DOI 10.1007/978-4-431-55936-8_20
472 J. Molina-Infante and D.Y. Graham
20.1 Introduction
Helicobacter pylori (H. pylori) is a worldwide infection that affects millions of

people. This microorganism, discovered ~30 years ago, is the main cause of
gastritis, gastroduodenal ulcer disease, and gastric cancer. Over the last 20 years,
the most recommended treatment for eradication of H. pylori by all international
guidelines has been the so-called standard triple therapy, consisting of a proton
pump inhibitor (PPI) plus the combination of two antibiotics (clarithromycin plus
amoxicillin or metronidazole) (Chey and Wong 2007; Fock et al. 2009;
Malfertheiner et al. 2012). However, the effectiveness of triple therapy dramatically
declined a decade ago to unacceptably low levels, largely related to the develop-
ment of resistance to clarithromycin (Graham and Fischbach 2010). The general
lack of antibiotic susceptibility testing has limited the adoption of susceptibility
test-guided therapy, and, therefore, the scientific community has just recently taken
on the task of exploring novel empiric therapeutic schemes to overcome antibiotic
resistance. However, an ideal universal regimen to treatment has not been identified
yet, largely because of large geographical variations in antibiotic resistance. Effec-
tiveness of treatment is determined by the details of the regimen (the drugs used,
their doses, formulations, the frequency of administration, the duration of therapy,
etc.), host-related parameters, such as compliance with therapy and genetic differ-
ences in metabolism of the drugs, especially the PPI used, and the local pattern of
antimicrobial resistance. Some general rules have been established, the most
important of which is that one can expect similar, if not identical, results of a
regimen anywhere if the pattern of resistance is the same (Graham et al. 2014).
20.2 Definition of a Successful Eradication Regimen
For an infectious disease where one can expect to cure essentially 100 % of cases
with susceptible organisms (such as H. pylori), an optimal regimen is defined as a
regimen that reliably cures at least 90–95 % of infections with susceptible strains
(Graham et al. 2014). As with most bacterial infectious diseases, an appropriate
therapy should be devised based on antimicrobial susceptibility testing. However,
susceptibility testing is seldom available for H. pylori therapy and therapies are
mostly prescribed empirically. Regardless of the lack of antimicrobial data, the
therapeutic goal should be the same, so eradication therapies can be currently
classified according to their cure rates on a per-protocol analysis: excellent
(>95 % success), good (>90 % success), borderline acceptable (85–89 % success),
or unacceptable (<85 % success) (Graham et al. 2014) Using less stringent thera-
peutic thresholds will only lead to implementation of suboptimal therapies in
clinical practice and subsequent selection of H. pylori resistant strains (Graham
2009, 2010). In addition, rescue drugs after H. pylori therapy failure might be
unavailable (bismuth, tetracycline, furazolidone) or may potentially lead to serious
20 Helicobacter pylori Therapy 473
side effects (rifabutin), so the clinician should be sure of using the most effective
first “hit” to eradicate the bacteria (Graham et al. 2014).
20.3 Difficulties to Eradicate H. pylori Infection
Unlike the bulk of bacterial infections, H. pylori resides in the stomach, which is an
acid antinatural environment for microorganisms. Acute H. pylori infection may
result in hypochlorhydria, which is thought to facilitate survival of the organism
and colonization of the stomach (Schubert and Peura 2008). Moreover, several
bacteria-, environmental-, and drug-related factors may also account for difficulties
associated with the cure of the infection. Upon this unique picture, H. pylori
infection should be distinctly treated by means of a combination of acid-
suppressive agents and several antibiotics. By far, the most important factor
impairing the efficacy of eradication regimens is the presence or development of
H. pylori genetic mutations conferring antimicrobial resistance (Graham
et al. 2014).
20.3.1 Bacteria-Related Factors

20.3.1.1 H. pylori Genotypical Resistance to Antimicrobial Drugs
Resistance to bismuth is not thought to occur in H. pylori, and acquired resistance to

either amoxicillin or tetracycline (after previous exposure to these antibiotics) is
rare in most regions. Accordingly, they can be reused despite failure of an eradi-
cation regimen where they were prescribed, as we do with amoxicillin and bismuth
in clinical practice (Graham and Fischbach 2010). Clarithromycin is a typical
macrolide, and one can expect cross resistance to occur such that prior or frequent
use of other macrolides, such as erythromycin or azithromycin, which results in a
high prevalence of clarithromycin resistance (Megraud et al. 2013). Resistance to
clarithromycin has increased in most regions over time and empiric triple therapy is
strongly discouraged when clarithromycin resistance rate is prevalent (Chey and
Wong 2007; Fock et al. 2009; Malfertheiner et al. 2012). Treatment success falls
below 90 % at 7 days with about 5 % clarithromycin resistance and with about 8 %
with 14-day therapy. At 15–20 % resistance population, results of 70–75 % are
expected. The highest frequency of clarithromycin resistance in adults (40 %) has
been reported in southern and central European countries (Megraud et al. 2013),
although it is also fairly common in other settings such as the USA, Mexico, Japan,
Russia, Turkey, and Iran, usually over 15 % (Graham et al. 2014). In general, one
should consider that a high rate of clarithromycin resistance is present, unless
proven otherwise. Metronidazole resistance remains around 20–40 % in Western
countries but is often as high as 60–80 % in countries such as China, India, Iran, and
Central and South America (Graham and Fischbach 2010). Resistance to

fluoroquinolones (e.g., levofloxacin, moxifloxacin, sitafloxacin) is rapidly increas-
ing worldwide (e.g., Europe 14 % in 2013) (Megraud et al. 2013). Of note,
resistance to clarithromycin, fluoroquinolones, or rifabutin cannot be overcome
by increasing the dose or duration (Graham et al. 2014). As such, previous exposure
to macrolides or fluoroquinolones (due to previous eradication therapies or
ear-nose-throat, respiratory, or urinary tract infections) in patients carrying
H. pylori infection increases the likelihood of harboring antibiotic-resistant strains,
and alternative antibiotic schemes should be sought. On the contrary, metronidazole
resistance can be at least partially overcome by means of higher doses and longer
duration of therapies, especially when used as a part of a bismuth-containing
quadruple therapy (Graham et al. 2014).
20.3.1.2 H. pylori Phenotypic Resistance to Antimicrobial Drugs
Eradication treatment may fail even when the organism genetically remains sus-
ceptible to the antibiotic (Graham and Fischbach 2010). This is most commonly
seen with amoxicillin. This form of reversible resistance is termed phenotypic
antibiotic resistance, and it is thought to be due to the presence of nonreplicating
population of organisms. Bacteria usually oscillate between a nonreplicating (phe-
notypically resistant) and replicating state (phenotypically susceptible), during
which they cannot and can be eradicated, respectively. (Graham and Fischbach
2010). As such, short doses, short duration, or extended dosing interval of antibiotic
drugs may limit the presence of antibiotics during these susceptibility periods.
20.3.1.3 High Bacterial Load (Inoculum Effect)
The total number of H. pylori in the stomach is very high, resulting in an inoculum
effect, which stands for the decreasing efficacy of an antibiotic with increasing
bacterial density (Graham and Fischbach 2010). For example, if the spontaneous
mutation rate for a particular resistance was 1 in 10 million and there were
50 million organisms present, it would be statistically likely that a small population
of resistant organisms would always be present. In fact, resistance usually develops
because of the outgrowth of a small existing population of resistant organisms
(Graham and Fischbach 2010). Strategies aiming to overcome H. pylori high
bacterial load include increasing the dose and duration of antibiotic therapy,
combining several antibiotics (one of which will probably kill the resistant organ-
isms) and pretreatment with PPI and bismuth, reducing the bacterial load (which
would make survival of the minor populations less likely).
20.3.2 Environmental-Related Factors
The natural pH in the acid gastric environment remains between 1 and 2, especially
during fasting and starting meal ingestion (Schubert and Peura 2008). H. pylori
becomes phenotypically resistant with a pH range between 3 and 6, and this is
thought to be the main reason why acid-suppressive agents, along with antibiotics,
are often indispensable in H. pylori therapy. Increasing gastric pH to 6 or 7, by
means of PPI therapy, allows the bacteria to enter the replicative state, where they
become susceptible to specific antibiotics such as amoxicillin and clarithromycin
(Graham and Fischbach 2010). Therefore, insufficient acid suppression, due to
either low doses or rapid/extensive PPI metabolism, may predispose microorgan-
isms to a phenotypically resistant state, and eradication regimens may fail in spite
of genetic susceptibility to prescribed antibiotics.
20.3.3 Drug-Related Factors
Amoxicillin and clarithromycin are antibiotics that require microbial replication to

kill the organisms. As such, powerful acid suppression (high-dose PPI) and ade-
quate antibiotic doses, dosing intervals, and durations are key to avoid H. pylori
entering in a non-replicative state, where antibiotics are less effective (Graham and
Fischbach 2010). Clarithromycin must bind to ribosomes in order to kill H. pylori.
Acquired resistance is associated with failure to bind to ribosomes, such that
resistance cannot be overcome by increasing the dose or duration. Likewise,
resistance to fluoroquinolones (i.e., levofloxacin, moxifloxacin) is not responsive
to changes in dose or duration. Metronidazole resistance can be partially overcome
by increasing the dose and duration, especially with bismuth-containing therapies
(Graham et al. 2014). In contrast, bismuth resistance does not occur, and resistance
to either amoxicillin or tetracycline is rare in most regions.
20.4 Pivotal Considerations for an Optimal Therapeutic

Decision-Making
20.4.1 Choice of Antibiotic Therapy
The strongest predictor of H. pylori treatment failure using a regimen proven to be

effective elsewhere is antimicrobial resistance. From a microbiological standpoint,
treatment results are best when regimens are used to treat patients with organisms
susceptible to the antimicrobials chosen, but this approach is currently limited by
the unavailability of H. pylori culture (invasive procedure) or of molecular testing
in stools or gastric biopsies (expensive, time consuming, and limited largely to

clarithromycin) in most of the cases (Graham et al. 2014; Molina-Infante and
Gisbert 2014). Another choice would be using bismuth quadruple therapy; owing
to tetracycline resistance is negligible and metronidazole resistance can be partially
overcome by increasing doses and duration. Nonetheless, this approach is limited to
regions where bismuth salts and tetracycline are both available (Megraud 2012;
Graham et al. 2014). The launch of Pylera®, the three-in-one capsule containing
bismuth, tetracycline, and metronidazole, which decreases the pill burden and
theoretically might improve compliance, is one option where it is available (see
below under specific therapies). Therefore, one often must choose antibiotic ther-
apy, empirically making the best approach being to use regimens that have proven
to be reliably excellent locally. That choice should take advantage of knowledge of
local resistance patterns, clinical experience, and especially patient history. The
history of the patient’s prior antibiotic use and any prior therapies will help identify
which antibiotics are likely to be successful and those where resistance is probable
(Graham et al. 2014). All of these crucial variables in the decision-making process
for an optimal first-line eradication therapy are summarized in Fig. 20.1.
20.4.2 Optimization of Therapies
In this era of antibiotic resistance, all therapies should be optimized (Graham

et al. 2014; Molina-Infante and Gisbert 2014). We recommend high-dose PPI
(i.e., 40 mg of omeprazole or equivalent b.i.d. (bis in die or twice a day)) and a
14-day duration in order to ensure the greatest effectiveness. A 14-day regimen has
proven to achieve higher eradication rates for triple, bismuth quadruple, and
non-bismuth quadruple sequential and concomitant therapy (Salazar et al. 2012;
Yuan et al. 2013; Liou et al. 2013; Graham et al. 2014; Molina-Infante and Gisbert
2014). PPI therapy should be given at doses guaranteeing effective and prolonged
gastric acid suppression, since H. pylori becomes phenotypically resistant and less
susceptible to amoxicillin and clarithromycin when the pH in their microenviron-
ment is lower than 6 and higher than 3 (Graham and Fischbach 2010). All PPIs are
metabolized by cytochrome P450 (CYP) 2C19. Four different genotypes have been
described: ultrarapid metabolizer, rapid metabolizer, intermediate metabolizer, and
poor metabolizer. Plasma PPI levels and intragastric pHs during PPI treatment are
inversely related to CYP2C19 genotype, so they are lowest in the more extensive or
rapid metabolizer group and highest among the poor metabolizers (Graham and
Fischbach 2010). Subsequently, several meta-analyses have shown eradication
rates that are inversely related to the ability to metabolize the PPIs (e.g., the
ultrarapid and rapid metabolizer groups have lower eradication rate compared to
other groups) (Zhao et al. 2008; Tang et al. 2013). The prevalence of CYP2C19
rapid metabolizers has been shown to be highest in Europe and North America
(56–81 %), while the proportion is lower (27–38 %) in the Asian population. As
Susceptibility testing available Susceptibility testing not available
Tailored therapy Consider for empirical therapy :
1) Local antimicrobial resistance patterns. If not

available, clinical experience
2) Prior use of macrolides or nitroimidazoles
Low to intermediate-risk patients High-risk patients
CLA-R >15-20% and/or MET-R < 40% CLA-R >15-20% and/or MET-R > 50%
(dual resistance likely < 15%) (dual resistance likely > 15%)
Not prior antibiotic use Prior antibiotic use
14-day non-bismuth quadruple 14-day bismuth quadruple

concomitant therapy therapy or
or best locally effective treatment
best locally effective treatment
FAILURE FAILURE
14-day bismuth
quadruple therapy Tailored therapy
If unavailability of bismuth salts/tetracycline or susceptibility

testing, consider:
- Flouroquinolone-containing regimens (if not used previously)

- Furazolidone-containing quadruple therapy
- Rifabutin-containing triple therapy
Fig. 20.1 Recommended approach for Helicobacter pylori therapy
such, it is conceivable that all patients, especially in Europe and North America,
should routinely receive higher-dose PPI twice a day (dosing interval) therapy
in order to achieve similar effects in either rapid and poor metabolizers (Graham
et al. 2014; Molina-Infante and Gisbert 2014). In this regard, two other recent
meta-analyses have demonstrated that, at standard doses, esomeprazole and
rabeprazole provide better overall H. pylori eradication rates, especially in
CYP2C19 rapid metabolizers (McNicholl et al. 2012; Tang et al. 2013). Available
first-line regimens with preferred drug doses and dosing intervals are summarized
in Table 20.1.
Table 20.1 Current first-line therapeutic recommendations in the era of increasing clarithromycin
and metronidazole resistance
Preferred doses and dosing
intervals Caveat
14-day bismuth-containing Bismuth salts q.i.d. Availability
classical quadruple therapy PPI (double doses) b.i.d. Complexity
Tetracycline 500 mg q.i.d. Side effects
Nitroimidazole 500 mg t.i.d. Compliance
14-day bismuth-containing qua- PPI (double doses) bid Availability
druple therapy using Pylera® Pylera 3 pills q.i.d. Cost
Relatively low tetracycline
doses
14-day non-bismuth quadruple PPI (double doses) b.i.d. Cure rates 90 %
concomitant therapy Amoxicillin 1 g b.i.d. if dual resistance rate
Clarithromycin 500 mg b.i.d. 15 %
Nitroimidazole 500 mg b.i.d.
14-day non-bismuth quadruple 7 days Cure rates <90 %
hybrid therapy PPI (double doses) b.i.d. if dual resistance rate >9 %
Amoxicillin 1 g b.i.d.
7 days
PPI (double doses) b.i.d.
Amoxicillin 1 g b.i.d.
Clarithromycin 500 mg b.i.d.
14-day non-bismuth quadruple 7 days Cure rates <90 %
sequential therapy PPI (double doses) b.i.d. if dual resistance rate >5 %
Amoxicillin 1 g b.i.d. Not recommended as an
7 days empirical therapy
PPI (double doses) b.i.d.
Clarithromycin 500 mg b.i.d.
14-day triple therapy PPI (double doses) b.i.d. Cure rates <90 % if
Amoxicillin 1 g b.i.d. clarithromycin resistance
>15 %
Clarithromycin 500 mg b.i.d. Not recommended as an
empirical therapy
Dual resistance rate: H. pylori microorganism resistant to both clarithromycin and metronidazole
20.5 First-Line Therapy
The preferred empirical choices should be currently 14-day bismuth quadruple

therapy or 14-day concomitant therapy, depending on local conditions and patient
history of antibiotic use and possible drug allergies (Fig. 20.1) (Graham et al. 2014).
Sequential therapy is no longer recommended as it contains the same drugs as
concomitant therapy and is more complicated, and concomitant therapy will always
be equal or superior to it. Of note, none of the non-bismuth quadruple regimens is

infallible, and sequential, hybrid, and concomitant therapy have all been shown to
fail (<90 % eradication) if the rate of dual clarithromycin- and metronidazole-
resistant strains is >5 %, >9 %, or >15 %, respectively (Graham et al. 2014). As
such, empirical non-bismuth quadruple therapies would likely be poor choices in
settings with documented high clarithromycin and metronidazole resistance or with
documented high resistance (e.g., <50 % to clarithromycin or metronidazole) in
some specific high-risk patients (e.g., women in whom metronidazole has been used
for Trichomonas infections, immigrants from developing countries, patients who
previously failed sequential or PPI-clarithromycin-metronidazole triple therapy,
and those in whom the risk of acquired metronidazole resistance is high) (Graham
et al. 2014).
20.5.1 Triple Therapy (Currently Considered as Obsolete

as an Empiric Therapy)
Despite many still current recommendations, triple therapy should be no longer

prescribed on an empirical basis. Its empiric use should be strictly restricted to a
minority of settings where clarithromycin resistance is established by culture
known to be low (e.g., Northern Europe or Thailand (Megraud et al. 2013;
Vilaichone et al. 2013) or where cure rates >90 % have been documented in
clinical practice (Prasertpetmanee et al. 2013)). When used, it should be given for
14 days with double-dose PPI twice a day, since this can increase eradication rates
by 10 % (Malfertheiner et al. 2012).
20.5.2 Non-bismuth Quadruple Therapies

20.5.2.1 Sequential Therapy (Currently Considered Obsolete as an
Empiric Therapy)
Sequential therapy was developed in Italy in 2000 as a replacement for triple

therapy. It initially consisted of 5 days of PPI therapy plus amoxicillin, followed
by a further 5 days of PPI with two other antibiotics, usually clarithromycin and a
nitroimidazole (Zullo et al. 2000; De Francesco et al. 2001). Meta-analyses
conducted between 2007 and 2009, pooling mostly Italian evidence, confirmed
the advantage of 10-day sequential (cure rates >90 %) over 7- or 10-day triple
therapy (Zullo et al. 2007; Jafri et al. 2008; Tong et al. 2009; Gatta et al. 2009). In
2012 and 2013, two updated meta-analyses (Horvath et al. 2012; Yoon et al. 2013)
and two systematic reviews (Zullo et al. 2013a; Kate et al. 2013), including studies
on sequential therapy from Asia, Europe, and Latin America, showed that the mean
eradication rates were dramatically lower (79–84 %) than those reported in early
Italian trials. These poor results were confirmed in a global meta-analyses in 2013
with overall cure rates of 84 % (95 % CI 82.1–86.4 %) (Gatta et al. 2013). Further
analysis showed that the Achilles heels for sequential therapy was metronidazole
resistance and dual clarithromycin resistance (Liou et al. 2013; Graham et al. 2014).
Because concomitant therapy is effective in isolated metronidazole or
clarithromycin resistance, it will thus always be equal or superior to sequential
therapy in regions where sequential therapy is effective. We conclude that sequen-
tial therapy is an obsolete regimen.
20.5.2.2 Concomitant Therapy
The concept of a “non-bismuth quadruple regimen” or “concomitant” regimen

consists of converting standard triple therapy to a quadruple therapy by the addition
of 500 mg of metronidazole or tinidazole twice daily. This therapeutic regimen
resurfaced in 2010 as an alternative therapy to triple and sequential therapy. Meta-
analyses have consistently shown its advantage over triple therapy (Essa et al. 2009;
Gisbert and Calvet 2012b). Indeed, several recent studies (2010–2013) have eval-
uated its effectiveness in Latin America, Asia (Thailand, Japan, Taiwan, China, and
Korea), and Europe (Spain, Greece, Italy, and Turkey). Regardless of the duration
of the therapy, all studies showed intention-to-treat cure rates from 85 to 94 %, with
the exceptions of four studies conducted in regions where dual resistance was high
(Latin America, Korea, and Turkey) or where the duration of therapy (Latin
America, Italy) tested was too short (Molina-Infante and Gisbert 2014).
The efficacy of 14-day concomitant therapy is not impaired by neither
clarithromycin- nor metronidazole-isolated resistance, but it is expected to fall
below 90 % in regions where the prevalence of dual clarithromycin and
metronidazole-resistant strains is >15 % (Graham et al. 2014). Currently, 14-day
concomitant therapy should be the preferred non-bismuth quadruple therapy as it
has shown to be the most effective to overcome antibiotic resistance. When dealing
with dual clarithromycin- and metronidazole-resistant strains, concomitant therapy
has shown a remarkable advantage over sequential therapy (Georgopoulos 2013b;
Molina-Infante and Gisbert 2014). However, it is important to stress that it should
not be recommended in settings with high rates of metronidazole resistance
(>50–60 %) along with high clarithromycin resistance (i.e., Latin America, Tur-
key, and Korea) or in populations at high risk of dual resistance (i.e., following
clarithromycin or metronidazole treatment failures) (Graham et al. 2014; Molina-
Infante and Gisbert 2014). These recommendations are in agreement with accept-
able to excellent results (86–95 % eradication rates) in Greece, Spain, and Italy in
Southern Europe (Georgopoulos et al. 2012, 2013a; Molina-Infante et al. 2012;
Zullo et al. 2013b; Molina-Infante et al. 2013; McNicholl et al. 2014; De Francesco
et al. 2014) and Thailand, Taiwan, China, and Japan in Asia (Wu et al. 2010;
Kongchayanun et al. 2012; Huang et al. 2012; Yanai et al. 2012; Hsu et al. 2014),
where clarithromycin ranges from low (9 %) to high (40 %), but metronidazole
resistance remains relatively low (<30–40 %). As a matter of fact, if metronidazole

resistance remains stable in Southern Europe, clarithromycin resistance would need
to exceed 50 % to undermine 14-day concomitant therapy. These successful
eradication results for concomitant therapy have not been replicated in settings
with high rates of dual clarithromycin- and metronidazole-resistant H. pylori
strains, such as Turkey (Toros et al. 2011; Sharara et al. 2014), Korea (Lim
et al. 2013; Heo et al. 2014) and possibly Latin America (Greenberg et al. 2011),
where the duration was also too short.
20.5.2.3 Hybrid (Sequential-Concomitant) Therapy
Hybrid sequential-concomitant regimen is a therapeutic innovation which includes

a PPI plus amoxicillin for 14 days, adding clarithromycin and a nitroimidazole
for the final 7 days (Hsu et al. 2011). In other words, it is a 7-day first dual phase
(PPI + amoxicillin), followed by a 7-day quadruple phase (PPI + amoxicillin +
clarithromycin + nitroimidazole). Several recent studies conducted between 2011
and 2014 have consistently shown cure rates 86 % in Taiwan (Hsu et al. 2011; Wu
et al. 2014), Iran (Sardarian et al. 2013), Spain, and Italy (Molina-Infante
et al. 2013; Zullo et al. 2013b), but less satisfactory results have been reported in
other studies conducted in Italy (De Francesco et al. 2014) and Korea
(Oh et al. 2014). Hybrid therapy has been shown to be non-inferior to concomitant
therapy, besides improved safety, convenience, or better compliance. It could be
considered in the same populations where concomitant therapy is recommended;
however, 14-day hybrid therapy is expected to fall below 90 % when
clarithromycin-metronidazole resistance exceeds 9 % (Graham et al. 2014). A
recent first meta-analysis on hybrid therapy has not shown relevant differences in
terms of efficacy with the sequential and concomitant therapy (Wang et al. 2014).
Therefore, further studies are required to validate this therapy in settings with
different patterns of resistance.
20.5.3 Bismuth-Quadruple Therapy
This is the oldest effective therapy and a resurfacing one on account of increasing
failure of clarithromycin-containing therapies. Using this regimen at full doses and
for 14 days, one can expect 95 % or greater treatment success irrespective the level
of metronidazole resistance (Graham et al. 2014; Salazar et al. 2012). The main
disadvantages of this therapy are its complexity and frequent side effects, both of
which may hamper compliance with therapy. In addition, there are often issues with
the availability of bismuth salts or tetracycline. Generally, doxycycline has proven
not to be an adequate substitute for tetracycline (Graham et al. 2014). This is likely
the main reason why we lack more evidence on this therapy over the last decade.
Because of the relative high rate of side effects, optimization is needed in terms of
formulations, forms of bismuth, doses, and dosing intervals. Recent studies

conducted in Italy (Dore et al. 2011) and China (Liang et al. 2013) have shown
similar success rates using twice a day bismuth and full q.i.d. (quarter in die or four
times a day) antibiotic doses. In this context, bismuth-based quadruple therapy in its
most recent galenic formulation, bismuth subcitrate potassium, metronidazole, and
tetracycline (BMT, sold under license as Pylera®) has been suggested as a possible
first-line therapeutic option (Malfertheiner et al. 2011). However, its use is cur-
rently limited by high cost and the fact that only a 10-day regimen is available in a
prepackaged form. The dose of tetracycline is 1500 mg, which is lower than usually
recommended. Head to head comparisons with standard bismuth therapy and with
b.i.d. dosing are needed, especially in population with high rates of metronidazole
resistance.
20.6 Rescue Therapy
Rescue therapy is defined as therapy after two treatment failures with two different
regimens. Even with the current most effective treatment regimens, a variable
proportion of patients will fail to eradicate H. pylori infection at the first attempt
(Marin et al. 2013; Graham et al. 2014). Most will be cured by using the alternate
regimen among the two different preferred quadruple regimens (Marin et al. 2013;
Graham et al. 2014). Despite the number of studies, the optimal retreatment
regimen has not yet been defined. If susceptibility testing can be obtained, one
can almost always identify a regimen that will prove effective. Our therapeutic
target, similarly to first-line regimens, should be at least 90 % cure rates. The
empiric choice of a rescue treatment primarily depends on which treatment was
used initially (e.g., bismuth or non-bismuth quadruple therapy) and the local rate of
fluoroquinolone resistance. The available regimens, with preferred doses and inter-
val dosing, are summarized in Table 20.2.
After failure of a clarithromycin- or metronidazole-containing treatment, clini-
cians should assume that H. pylori is likely resistant to the antibiotic previously
used, so it is not appropriate to repeat the same antibiotics. An exception to this rule
is amoxicillin, which rarely induces acquired resistance. In the absence of
pretreatment antibiotic resistance testing, the most commonly used empirical strat-
egies for a second-line therapy are bismuth quadruple therapy and, where not
available, a fluoroquinolone-containing therapy. This latter should only be consid-
ered if no fluoroquinolone, including ciprofloxacin, was used before (Gisbert and
Morena 2006; Marin et al. 2013; Graham et al. 2014). Furazolidone quadruple
therapy (where available) and rifabutin triple therapy are typically reserved as
salvage therapies of last resort. A recommended therapeutic algorithm for
H. pylori rescue therapy, after failure, is displayed in Fig. 20.1.
Table 20.2 Current rescue therapeutic recommendations, in the era of increasing fluoroquinolone
resistance, in patients with high risk of acquired clarithromycin and/or metronidazole resistance
after failure of first eradication regimen
Preferred doses and dosing
intervals Caveat
14-day fluoroquinolone triple PPI (double doses) b.i.d. Cure rates 90 %
therapy Amoxicillin 1000 mg b.i.d. if levofloxacin resistance
Levofloxacin 500 mg 12 %
14-day bismuth-containing flu- Bismuth salts 240 mg b.i.d. Cure rates 90 %
oroquinolone quadruple PPI (double doses) b.i.d. if levofloxacin resistance
therapy Amoxicillin 1000 mg b.i.d. 25 %
Levofloxacin 250 mg b.i.d.
14-day bismuth quadruple PPI (double doses) b.i.d. Availability
concomitant therapy Bismuth salts 240 mg b.i.d. Side effects
Plus a combination of 2 Complexity
antibiotics among
Amoxicillin 1 g t.i.d./b.i.d. Compliance
Nitroimidazole 500 mg t.i.d. Potential genotoxic and
Tetracycline 500 mg q.i.d. carcinogenetic effects of
Furazolidone 100 mg t.i.d. furazolidone
14-day rifabutin-containing PPI (double doses) b.i.d. Cost
therapy Amoxicillin 1000 mg t.i.d. Potential severe side effects
(myelotoxicity and
hepatotoxicity)
Rifabutin 300 mg Risk of development of resis-
tance in M. Tuberculosis
20.6.1 Fluoroquinolone-Containing Therapies
Levofloxacin is a fluoroquinolone with a broad spectrum of activity against

H. pylori. Fluoroquinolone-based therapy has been proposed in several interna-
tional guidelines as a rescue therapy (Chey and Wong 2007; Fock et al. 2009;
Malfertheiner et al. 2012). However, prevalence of fluoroquinolone resistance has
increased rapidly in recent years, mostly due to the widespread use of levofloxacin
for ear, nose and throat, respiratory tract, and urinary infections. Recent studies
have reported high levofloxacin resistance rates, ranging from 63 % in China to
14 % in Europe (Graham et al. 2014). In line with these data, a recent review
revealed a weighted efficacy of 76 % (Marin et al. 2013), whereas several meta-
analyses have shown that 7-day fluoroquinolone triple therapy (including PPI,
amoxicillin, and levofloxacin) provides cure rates of typically <80 % and extending
the duration to 10 days for improved outcome, but the treatment success typically
remained typically below 90 % (Gisbert and Morena 2006; Marin et al. 2013;
Gisbert et al. 2013). Treatment success >90 % requires 14-day fluoroquinolone
triple therapy. However, treatment success will fall below 90 % with 14-day
fluoroquinolone triple therapy when fluoroquinolone resistance rates exceed

approximately 12 % (Chuah et al. 2012) and with 14-day bismuth-containing
fluoroquinolone quadruple therapy in areas where fluoroquinolone resistance
exceeds 25 % (Liao et al. 2013). As such, awareness of local resistance rates or
close monitoring of cure rates are mandatory in order to promptly detect inefficacy
of these therapies. The role of newer fluoroquinolones, such as sitafloxacin and
gemifloxacin, to overcome fluoroquinolone resistance, needs to be validated in
further studies.
20.6.2 Bismuth-Quadruple Therapy, Including

Furazolidone-Containing Regimens
Classical bismuth quadruple regimen has been also proposed in several interna-
tional guidelines as a rescue therapy (Chey and Wong 2007; Fock et al. 2009;
Malfertheiner et al. 2012). In a recent review, its weighted efficacy was 77 %
(including different durations, drug doses, and interval dosing) (Marin et al. 2013).
Concerns with this therapy are complexity, patient adherence, and availability.
Meta-analyses comparing fluoroquinolone-based and bismuth quadruple therapies
for rescue therapy did not find significant differences between both therapies
regarding efficacy, albeit fluoroquinolone therapy was significantly better tolerated
and induced significantly fewer side effects. Importantly, the results with both
regimens were unacceptably low due to poor choices of doses, duration, and the
presence of resistance (Saad et al. 2006; Marin et al. 2013).
Bismuth quadruple therapy, however, is the regimen with the most unanswered
questions regarding what are the optimal doses and frequencies of drug adminis-
tration. A recent study from China has evaluated the efficacy of bismuth quadruple
therapies (PPI, bismuth salts, and two antibiotics) with different antibiotic combi-
nations in patients with previous eradication treatment failure (Liang et al. 2013).
They used for all four evaluated regimens twice a day bismuth (220 mg b.i.d.) and
full doses and adequate dosing intervals for the antibiotics (amoxicillin 1000 t.i.d.
(tres in die or three times a day), tetracycline 500 mg q.i.d., metronidazole 400 mg
q.i.d., and furazolidone 100 mg t.i.d.). Cure rates were excellent (>90 %), regard-
less of the presence of clarithromycin, levofloxacin, or metronidazole resistance,
with the best results for the furazolidone-containing regimens. Of note, these
bismuth quadruple regimens were equally effective for patients allergic to penicil-
lin, combining tetracycline with either metronidazole or furazolidone. However,
furazolidone is seldom available in developed countries, and although it has been
declared a class C carcinogen (meaning there is no evidence that it is a carcinogen),
concern about possible genotoxic and carcinogenetic effects is often voiced. This
study, however, importantly highlights that bismuth quadruple therapy compliance
and efficacy can be improved through optimization of therapy.
20.6.3 Rifabutin-Containing Therapy
Rifabutin is a rifamycin-S derivative, which shares many of the properties of

rifampin (rifampicin). Rifabutin is commonly used to treat Mycobacterium avium
and Mycobacterium intracellulare, and it has shown utility against H. pylori, seeing
as the in vitro sensitivity is high and prevalence rate of rifabutin resistance is very
low, only about 1 %. A recent systematic review disclosed that mean H. pylori
eradication rate with rifabutin-containing rescue regimens was 73 % (Gisbert and
Calvet 2012a). Respective cure rates for second-line, third-line, and fourth/fifth-line
rifabutin therapies were 79, 66, and 70 %. The most prescribed regimen has been a
triple therapy combining PPI, amoxicillin, and rifabutin. The most effective dose is
300 mg/day and the ideal length remains unclear, although 10–14-day regimens are
generally recommended. The most successful cure rates (90 %) have been
reported using high-dose PPI (pantoprazole 80 mg t.i.d.), high-dose amoxicillin
(1 or 1.5 g t.i.d.), and rifabutin (150 mg b.i.d.) (Borody et al. 2006). The main
disadvantages of this drug are its high cost, uncommon but relevant adverse effects
(mainly myelotoxicity and hepatotoxicity), and the potential development of resis-
tance to M. tuberculosis in populations with a high prevalence of tuberculosis.
Owing to all these reasons, most consider it as one of the therapeutic options of last
resort.
H. pylori infection has proven challenging to eradicate due to bacteria-, environ-

mental-, and drug-associated factors. Antibiotic resistance and patient adherence
are the critical factors responsible for eradication treatment failure. Due to lack of
updated reliable data regarding antimicrobial susceptibility, most eradication ther-
apies are empirically prescribed. An optimal H. pylori regimen is defined as one
that reliably achieves at least 90–95 % of infections at first attempt. First-line
empiric triple therapy for H. pylori infection has become ineffective in most
settings worldwide because of clarithromycin resistance rates. Therefore, the
choice of therapy may depend on the patient’s previous antibiotic treatment, local
patterns of antibiotic resistance, and drug availability. Optimization of all eradica-
tion regimens (including duration, PPI/antibiotic doses, and dosing intervals) is key
to maximize efficacy. Currently, the most effective first-line eradication regimens
are 14-day bismuth and non-bismuth concomitant quadruple therapies. No trial has
compared the efficacy of both regimens yet. Pylera®, the three-in-one capsule
containing bismuth, tetracycline, and metronidazole, requires taking 14 capsules
daily, whereas the Chinese quadruple therapy with b.i.d. bismuth/PPI and 500 mg
capsules of tetracycline and metronidazole requires only 10 daily. Research to
improve tolerability and adherence with bismuth quadruple therapy is warranted.
The golden rule for choice of treatment is only to use what works locally
(>90–95 % success) and to closely monitor its effectiveness over time. Rescue
regimens should also target at least 90 % cure rates. Whenever possible, rescue
therapy should be based on antimicrobial susceptibility test data. The empiric
choice of rescue therapy primarily depends on which treatment was used initially
(e.g., bismuth or non-bismuth quadruple therapy) and the local rate of fluoroquin-
olone resistance. Furazolidone- and rifabutin-containing regimens might be also
effective as rescue treatments.
References
Borody TJ, Pang G, Wettstein AR, Clancy R, Herdman K, Surace R, Llorente R, Ng C (2006)
Efficacy and safety of rifabutin-containing ‘rescue therapy’ for resistant Helicobacter pylori
infection. Aliment Pharmacol Ther 23:481–488
Chey WD, Wong BC (2007) American College of Gastroenterology guideline on the management
of Helicobacter Pylori infection. Am J Gastroenterol 102:1808
Chuah SK, Tai WC, Hsu PI, Wu DC, Wu KL, Kuo CM, Chiu YC, Hu ML, Chou YP, Kuo YH,
Liang CM, Chiu KW, Hu TH (2012) The efficacy of second-line anti Helicobacter pylori
therapy using an extended 14-day levofloxacin/amoxicillin/proton-pump inhibitor treatment –
a pilot study. Helicobacter 17:374–381
De Francesco V, Zullo A, Hassan C, Faleo D, Ierardi E, Panella C, Morini S (2001) Two new
treatment regimens for Helicobacter pylori eradication: a randomised study. Dig Liver Dis
33:676–679
De Francesco V, Hassan C, Ridola L, Giorgio F, Ierardi E, Zullo A (2014) Sequential, concomitant
and hybrid first-line therapies for H. pylori eradication: a prospective, randomized study. J Med
Dore MP, Farina V, Cuccu M, Mameli L, Massarelli G, Graham DY (2011) Twice-a-day bismuth-
containing quadruple therapy for Helicobacter pylori eradication: a randomized trial of 10 and
14 days. Helicobacter 16:295–300
Essa AS, Kramer JR, Graham DY, Treiber G (2009) Meta-analysis: four-drug, three antibiotic,
non-.bismuth containing “concomitant therapy” versus triple therapy for Helicobacter pylori
eradication. Helicobacter 14:109–118
Fock KM, Katelaris P, Sugano K, Leong Ang T, Hunt R, Talley NJ, Lam SK, Xiao SD, Tan HJ,
Wu CY, Jung HC, Hoang BH, Kachintorn U, Goh KL, Chiba T, Rani AA, Second Asia-Pacific
Conference (2009) Second Asia-pacific consensus guidelines for Helicobacter pylori infection.
J Gastroenterol Hepatol 24:1587–1600
Gatta L, Vakil N, Leandro G, Di Mario F, Vaira D (2009) Sequential therapy or triple therapy for
Helicobacter pylori infection: systematic review and meta analysis of randomized controlled
trials in adults and children. Am J Gastroenterol 104:3069–3079
Gatta L, Vakil N, Vaira D, Scarpignato C (2013) Global eradication rates for Helicobacter pylori
infection: systematic review and meta-analysis of sequential therapy. BMJ 28:1801–1809
Georgopoulos S, Papastergiou V, Xirouchakis E, Laudi F, Papantoniou N, Lisgos P, Spiliadi C,
Fragou P, Skorda L, Karatapanis S (2012) Evaluation of a four drug, three antibiotic,
nonbismuth-containing “concomitant” therapy as first-line Helicobacter pylori eradication
regimen in Greece. Helicobacter 17:49–53
Georgopoulos SD, Xirouchakis E, Martinez-Gonzalez B, Sgouras DN, Spiliadi C, Mentis AF,
Laoudi F (2013a) Clinical evaluation of a ten-day regimen with esomeprazole, metronidazole,
amoxicillin, and clarithromycin for the eradication of Helicobacter pylori in a high
clarithromycin resistance area. Helicobacter 18:459–467
Georgopoulos SD, Xirouchakis E, Mentris A (2013b) Is there a nonbismuth quadruple therapy that
can reliably overcome bacterial resistance? Gastroenterology 145:1496–1497
Gisbert JP, Calvet X (2012a) Review article: rifabutin in the treatment of refractory Helicobacter
pylori infection. Aliment Pharmacol Ther 35:209–221
Gisbert JP, Calvet X (2012b) Update on non-bismuth quadruple (concomitant) therapy for
eradication of Helicobacter pylori infection. Clin Exp Gastroenterol 5:23–34
Gisbert JP, Morena F (2006) Systematic review and meta-analysis: levofloxacin based rescue
regimens after Helicobacter pylori treatment failure. Aliment Pharmacol Ther 23:35–44
Gisbert JP, Pérez-Aisa A, Bermejo F, Castro-Fernández M, Almela P, Barrio J, Cosme A,
Modolell I, Bory F, Fernández-Bermejo M, Rodrigo L, Ortu~ no J, Sánchez Pobre P,
Khorrami S, Franco A, Tomas A, Guerra I, Lamas E, Ponce J, Calvet X, H. pylori Study
Group of the Asociacion Espa~nola de Gastroenterologı́a (Spanish Gastroenterology Associa-
tion) (2013) Second-line therapy with levofloxacin after failure of treatment to eradicate
Helicobacter pylori infection: time trends in a Spanish Multicenter Study of 1000 patients. J
Clin Gastroenterol 47:130–135
Graham DY (2009) Efficient identification and evaluation of effective Helicobacter pylori ther-
apies. Clin Gastroenterol Hepatol 7:145–148
Graham DY (2010) Helicobacter pylori eradication therapy research: ethical issues and descrip-
tion of results. Clin Gastroenterol Hepatol 8:1032–1036
Graham DY, Fischbach L (2010) Helicobacter pylori treatment in the era of increasing antibiotic
resistance. Gut 59:1143–1153
Graham DY, Lee YC, Wu MS (2014) Rational Helicobacter pylori therapy: evidence based
medicine rather than medicine based evidence. Clin Gastroenterol Hepatol 12:177–186
Greenberg ER, Anderson GL, Morgan DR, Morgan DR, Torres J, Chey WD, Bravo LE,
Dominguez RL, Ferreccio C, Herrero R, Lazcano-Ponce EC, Meza-Montenegro MM,
Pe~na R, Pe~na EM, Salazar-Martinez E, Correa P, Martinez ME, Valdivieso M, Goodman
GE, Crowley JJ, Baker LH (2011) 14-day triple, 5 day concomitant, and 10-day sequential
therapies for Helicobacter pylori infection in seven Latin American sites: a randomised trial.
Lancet 378:507–514
Heo J, Jeon SW, Jung JT, Kwon JG, Kim EY, Lee DW, Seo HE, HA CY, Kim HJ, Kim ES, Park
KS, Cho KB, Lee SH, Jang BI, Daegu-Gyeongbuk Gastrointestinal Study Group (2014) A
randomised clinical trial of 10 day concomitant therapy and standard triple therapy for
Helicobacter pylori eradication. Dig Liver Dis 46:1980–1984
Horvath A, Dziechciarz P, Szajewska H (2012) Meta-analysis: sequential therapy for Helicobacter
pylori eradication in children. Aliment Pharmacol Ther 36:534–541
Hsu PI, Wu DC, Wu YC, Graham DY (2011) Modified sequential Helicobacter pylori therapy:
proton pump inhibitor and amoxicillin for 14 days with clarithromycin and metronidazole
added as a quadruple (hybrid) therapy for the final 7 days. Helicobacter 16:139–145
Hsu PI, Wu DC, Chen WC, Tseng HH, Yu HC, Wang HM, Kao SS, Lai KH, Chen A, Tsay FW
(2014) Randomized controlled trial comparing 7-day triple, 10 day sequential, and 7-day
concomitant therapies for Helicobacter pylori infection. Antimicrob Agents Chemother
58:5936–5942
Huang YK, Wu MC, Wang SS, Kuo CH, Lee YC, Chang LL, Wang TH, Chen YH, Wang WM,
Wu DC, Kuo FC (2012) Lansoprazole based sequential and concomitant therapy for the first-
line Helicobacter pylori eradication. J Dig Dis 13:232–238
Jafri NS, Hornung CA, Howden CW (2008) Meta-analysis: sequential therapy appears superior to
standard therapy for Helicobacter pylori infection in patients naive to treatment. Ann Intern
Med 148:923–931
Kate V, Kalayarasan R, Ananthakrishnan N (2013) Sequential therapy versus standard triple-drug
therapy for Helicobacter pylori eradication: a systematic review of recent evidence. Drugs
73:815–824
Kongchayanun C, Vilaichone RK, Pornthisarn B, Amornsawadwattana S, Mahachai V (2012)

Pilot studies to identify the optimum duration of concomitant Helicobacter pylori eradication
therapy in Thailand. Helicobacter 17:282–285
Liang X, Xu X, Zheng Q, Zhang W, Sun Q, Liu W, Xiao S, Lu H (2013) Efficacy of bismuth
containing quadruple therapies for clarithromycin-, metronidazole-and fluoroquinolone resis-
tant Helicobacter pylori infections in a prospective study. Clin Gastroenterol Hepatol
11:802–807
Liao J, Zheng Q, Liang X, Zhang W, Sun Q, Liu W, Xiao S, Graham DY, Lu H (2013) Effect of
fluoroquinolone resistance on 14-day levofloxacin triple and triple plus bismuth quadruple
therapy. Helicobacter 18:373–377
Lim JH, Lee DH, Choi C, Lee ST, Kim N, Jeong SH, Kim JW, Hwang JH, Park YS, Lee SH, Shin
CM, Jo HJ, Jang ES, Song I, Jung HC (2013) Clinical outcomes of two-week sequential and
concomitant therapies for Helicobacter pylori eradication: a randomized pilot study.
Liou JM, Chen CC, Chen MJ, Chen CC, Chang CY, Fang YJ, Lee JY, Hsu SJ, Luo JC, Chang WH,
Hsu YC, Tseng CH, Tseng PH, Wang HP, Yang UC, Shun CT, Lin JT, Lee YC, Wu MS,
Taiwan Helicobacter Consortium (2013) Sequential versus triple therapy for the first-line
treatment of Helicobacter pylori: a multicentre, open-label, randomised trial. Lancet
381:205–213
Malfertheiner P, Bazzoli F, Delchier JC, Celinski K, Giguere M, Riviere M, Megraud F, Pylera
Study Group (2011) Helicobacter pylori eradication with a capsule containing bismuth
subcitrate potassium, metronidazole, and tetracycline given with omeprazole versus
clarithromycin-based triple therapy: a randomised, open label, non inferiority, phase 3 trial.
Lancet 377:905–913
Malfertheiner P, Megraud F, O’Morain CA, Atherton J, Axon AT, Bazzoli F, Gensini GF, Gisbert
JP, Graham DY, Rokkas T, El-Omar EM, Kuipers EJ, European Helicobacter Study Group
(2012) Management of Helicobacter pylori infection – the Maastricht IV/Florence Consensus
Report. Gut 61:646–664
Marin AC, McNicholl AG, Gisbert JP (2013) A review of rescue regimens after clarithromycin-
containing triple therapy failure (for Helicobacter pylori eradication). Expert Opin
Pharmacother 14:843–861
McNicholl AG, Linares PM, Nyssen OP, Calvet X, Gisbert JP (2012) Meta analysis: esomeprazole
or rabeprazole vs. first-generation pump inhibitors in the treatment of Helicobacter pylori
McNicholl AG, Marin AC, Molina-Infante J, Castro M, Barrio J, Ducons J, Calvet X, de la
Coba C, Montoro M, Bory F, Perez-Aisa A, Forne M, Gisbert JP; on behalf of the participant
centers (2014) Randomised clinical trial comparing sequential and concomitant therapies for
Helicobacter pylori eradication in routine clinical practice. Gut 63:244–249
Megraud F (2012) The challenge of Helicobacter pylori resistance to antibiotics: the comeback of
bismuth-based quadruple therapy. Ther Adv Gastroenterol 5:103–109
Megraud F, Coenen S, Versporten A, Kist M, Lopez-Brea M, Hirschl AM, Andersen LP,
Goossens H, Glupczynski Y, Study Group Participants (2013) Helicobacter pylori resistance
to antibiotics in Europe and its relationship to antibiotic consumption. Gut 62:34–42
Molina-Infante J, Gisbert JP (2014) Optimizing clarithromycin-containing therapy for
Helicobacter pylori in the era of antibiotic resistance. World J Gastroenterol 10:10338–10347
Molina-Infante J, Pazos-Pacheco C, Vinagre-Rodriguez G, Perez-Gallardo B, Due~ nas-Sadornil C,
Hernandez-Alonso M, Gonzalez-Garcia G, Mateos Rodriguez JM, Fernandez-Bermejo M,
Gisbert JP (2012) Nonbismuth quadruple (concomitant) therapy: empirical and tailored effi-
cacy versus standard triple therapy for clarithromycin susceptible Helicobacter pylori and
versus sequential therapy for clarithromycin-resistant strains. Helicobacter 17:269–276
Molina-Infante J, Romano M, Fernandez-Bermejo M, Federico A, Gravina AG, Pozzati L, Garcia-
Abadia E, Vinagre-Rodriguez G, Martinez-Alcala C, Hernandez-Alonso M, Miranda A,
Iovene MR, Pazos-Pacheco C, Gisbert JP (2013) Optimized nonbismuth quadruple therapies
cure most patients with Helicobacter pylori infection in populations with high rates of
antibiotic resistance. Gastroenterology 145:121–128
Oh DH, Lee DH, Kang KK, Park YS, Shin CM, Kim N, Yoon H, Hwang JH, Jeoung SH, Kim JW,
Jang ES, Jung HC (2014) The efficacy of hybrid therapy as first line regimen for Helicobacter
pylori infection compared with sequential therapy. J Gastroenterol Hepatol 29:1171–1176
Prasertpetmanee S, Mahachai V, Vilaichone RK (2013) Improved efficacy of proton pump
inhibitor – amoxicillin – clarithromycin triple therapy for Helicobacter pylori eradication in
low clarithromycin resistance areas or for tailored therapy. Helicobacter 18:270–273
Saad RJ, Schoenfeld P, Kim HM, Chey WD (2006) Levofloxacin-based triple therapy versus
bismuth-based quadruple therapy for persistent Helicobacter pylori infection: a meta-analysis.
Am J Gastroenterol 101:488–496
Salazar CO, Cardenas VM, Reddy RK, Dominguez DC, Snyder LK, Graham DY (2012) Greater
than 95% success with 14-day bismuth quadruple anti-Helicobacter pylori therapy: a pilot
study in US Hispanics. Helicobacter 17:382–390
Sardarian H, Fakheri H, Hosseini V, Taqhvaei T, Maleki I, Mokhtare M (2013) Comparison of
hybrid and sequential therapies for Helicobacter pylori eradication in Iran: a prospective
randomized trial. Helicobacter 18:129–134
Schubert ML, Peura DA (2008) Control of gastric acid secretion in health and disease. Gastroen-
terology 134:1842–1860
Sharara AI, Sarkis FS, El-Halabi MM, Malli A, Mansour NM, Azar C, Eloubeidi MA, Mourad FH,
Barada K, Sukkarieh I (2014) Challenging the dogma: a randomized trial of standard vs. half-
dose concomitant nonbismuth quadruple therapy for Helicobacter pylori infection. U Eur
Gastroenterol J 2:179–188
Tang HL, Li Y, Hu YF, Xie HG, Zhai SD (2013) Effects of CYP2C19 loss-of function variants on
the eradication of H. pylori infection in patients treated with proton pump inhibitor-based triple
therapy regimens: a meta analysis of randomized clinical trials. PLoS One 8:e62162
Tong JL, Ran ZH, Shen J, Xiao SD (2009) Sequential therapy vs. standard triple therapies for
Helicobacter pylori infection: a meta-analysis. J Clin Pharm Ther 34:41–53
Toros AB, Ince AT, Kesici B, Saglam M, Polat Z, Uygun A (2011) A new modified concomitant
therapy for Helicobacter pylori eradication in Turkey. Helicobacter 16:225–228
Vilaichone RK, Gumnarai P, Ratanachu-Ek T, Mahachai V (2013) Nationwide survey of
Helicobacter pylori antibiotic resistance in Thailand. Diagn Microbiol Infect Dis 77:346–349
Wang B, Wang YH, Lv ZF, Xiong HF, Wang H, Yang Y, Xie Y (2014) Efficacy and safety of
hybrid therapy for Helicobacter pylori infection: a systematic review and meta-analysis.
Helicobacter. doi:10.1111/hel.12180. [Epub ahead of print]
Wu DC, Hsu PI, Wu JY, Opekun AR, Kuo CH, Wu IC, Wang SS, Chen A, Hung WC, Graham DY
(2010) Sequential and concomitant therapy with 4 drugs are equally effective for eradication of
H. pylori infection. Clin Gastroenterol Hepatol 8:36–41
Wu JY, Hsu PI, Wu DC, Graham DY, Wang WM (2014) Feasibility of shortening 14-day hybrid
therapy while maintaining an excellent Helicobacter pylori eradication rate. Helicobacter
19:207–213
Yanai A, Sakamoto K, Akanuma M, Ogura K, Maeda S (2012) Non bismuth quadruple therapy for
first-line Helicobacter pylori eradication: a randomized study in Japan. World J Gastrointest
Pharmacol Ther 3:1–6
Yoon H, Lee DH, Kim N, Park YS, Shin CM, Kang KK, Oh DH, Jang DK, Chung JW (2013)
Meta-analysis: is sequential therapy superior to standard triple therapy for Helicobacter pylori
infection in Asian adults? J Gastroenterol Hepatol 28:1801–1809
Yuan Y, Ford AC, Khan KJ, Gisbert JP, Forman D, Leontiadis GI, Tse F, Calvet X, Fallone C,
Fischbach L, Oderda G, Bazzoli F, Moayyedi P (2013) Optimum duration of regimens for
Helicobacter pylori eradication. Cochrane Database Syst Rev 12:CD008337
Zhao F, Wang J, Yang Y, Wang X, Shi R, Xu Z, Huang Z, Zhang G (2008) Effect of CYP2C19
genetic polymorphisms on the efficacy of proton pump inhibitor based triple therapy for
Helicobacter pylori eradication: a meta-analysis. Helicobacter 13:532–541
Zullo A, Rinaldi V, Winn S, Meddi P, Lionetti R, Hassan C, Ripani C, Tomaselli G, Attili AF

(2000) A new highly effective short-term therapy schedule for Helicobacter pylori eradication.
Aliment Pharmacol Ther 14:715–718
Zullo A, De Francesco V, Hassan C, Morini S, Vaira D (2007) The sequential therapy regimen for
Helicobacter pylori eradication: a pooled-data analysis. Gut 56:1353–1357
Zullo A, Hassan C, Ridola L, De Francesco V, Vaira D (2013a) Standard triple and sequential
therapies for Helicobacter pylori eradication: an update. Eur J Intern Med 24:16–19
Zullo A, Scaccianoce G, De Francesco V, Ruggiero V, D’Ambrosio P, Castorani L, Bonfrate L,
Vannella L, Hassan C, Portincasa P (2013b) Concomitant, sequential, and hybrid therapy for
H. pylori eradication: a pilot study. Clin Res Hepatol Gastroenterol 37:647–650
Chapter 21
Management of H. pylori Infection in Europe
Peter Malfertheiner and Michael Selgrad
Abstract Over the last decades, Helicobacter pylori (H. pylori) has been recog-
nized as the main risk factor for various gastroduodenal diseases. This knowledge
has dramatically changed the clinical management of H. pylori infection and related
gastroduodenal diseases. Nowadays, H. pylori-related diseases, such as peptic ulcer
disease and MALT lymphoma, can be cured by H. pylori eradication therapy.
Furthermore, H. pylori eradication has the potential to prevent gastric cancer. In
this chapter, we have selected the most significant aspects of H. pylori infection. We
reviewed indications for H. pylori eradication therapy and data about diagnosis and
therapy of H. pylori infection. New treatment regimens or modifications of
established therapies have been developed with the goal to overcome increasing
antibiotic resistance rates that can be regarded as the main reason for treatment
failure. Finally, more and more evidence has been gathered that prevention of
gastric cancer is feasible by eradication of H. pylori. This approach is likely to be
highly effective in reducing the incidence of gastric cancer.
Keywords H. pylori • Diagnostic strategies • Treatment options
21.1 Introduction
Management of H. pylori in Europe needs to consider specific regional/national

differences concerning prevalence of the infection and related diseases, environ-
mental, lifestyle, and health-care systems factors. The prevalence of H. pylori
infection varies among European countries and even among different regions in
the same country (Bastos et al. 2013; Mayerle et al. 2013; Wex et al. 2011). Over
the last decades, there is a significant decrease in prevalence of H. pylori infection,
and the prevalence is lowest in childhood (Bureš et al. 2012). The prevalence of the
infection in the adult population ranges from 20 to 60 % and is age dependent, with
the highest prevalence in patients above 50 years.
P. Malfertheiner (*) • M. Selgrad

Department of Gastroenterology, Hepatology and Infectious Diseases, University of
Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany

DOI 10.1007/978-4-431-55936-8_21
492 P. Malfertheiner and M. Selgrad
In spite of these differences, there are common general aspects relevant for the
management of H. pylori infection, which have been updated in European consen-
sus reports in 4–5 year intervals over the last 18 years. The first European consensus
meeting was held in Maastricht in 1996 (Malfertheiner et al. 1997) and provided a
general frame intending to guide and facilitate the management of H. pylori
infection. The European consensus meetings were since held to four different
occasions. Some national societies have adapted and fine-tuned the guidelines to
their specific local needs.
The most current European consensus of H. pylori management is based on the
Maastricht-Florence four consensus report (Malfertheiner et al. 2012), which
addresses the issues of:
(a) Whom to treat
(b) How to test
(c) How to treat
The challenges in Europe are similar to those as in other parts of the world:
(a) Emergent resistance of H. pylori to antibiotics used in conventional eradica-
tion regimen
(b) The gastric cancer burden which is highly variable in different European
countries
21.2 Indications
Indications for treatment of H. pylori-related gastroduodenal pathologies have been

broadened over the past two decades and are summarized in Table 21.1. The
clinical evidence for H. pylori as a pathogen has grown immensely over the last
30 years (Malfertheiner et al. 2014) and public awareness has been spread. There-
fore among different clinical indications, the wish of patient even in the absence of
symptoms or specific risk conditions has been recommended as an indication for
testing and treating the infection. The third edition of the European Maastricht-
Florence consensus report (Malfertheiner et al. 2007) has been the first to recom-
mend H. pylori eradication in some extra-alimentary conditions, which are poten-
tially related to the infection (Malfertheiner et al. 2012) (Table 21.1).
There has been an immense progress in the management of mucosa-associated
lymphoid tissue (MALT)-lymphoma with the recommendation that H. pylori
should always be the first step of treatment, including patients with advanced
gastric MALT lymphoma as well. Eradication is worth trying also in H. pylori-
negative patients with MALT lymphoma (Ruskoné-Fourmestraux et al. 2011).
In young patients with dyspepsia and no alarm symptoms below the age of
45–50 years, a “test and treat” strategy is considered appropriate because of the low
prevalence of gastric malignancies in these age groups in most European countries
(Wee 2013). However, the adoption of this strategy continues to be debated
although clinical trials confirmed the value of this strategy. In countries with easily
21 Management of H. pylori Infection in Europe 493
Table 21.1 Indications for H. pylori treatment

Who should be treated for H. pylori-related diseases?
Gastrointestinal diseases
Peptic ulcer (duodenal – gastric), complicated and non-complicated
Functional dyspepsia
Dyspepsia (noninvasive test based)
Beneficial before starting NSAID treatment
Advisable in NSAID and aspirin users, because of reduced ulcer risk
If patients with ulcers on NSAID, but PPI needs to be continued
In aspirin users with previous history of gastroduodenal ulcer
Chronic atrophic gastritis
Following gastric cancer resection
MALT lymphoma
Extradigestive diseases
Idiopathic thrombocytopenic purpura
Iron deficiency anemia
Vitamin B12 deficiency
Improvement in the bioavailability of certain drugs (i.e., l-thyroxine, l-dopa)
accessible endoscopy, the “scope and test” strategy remains the recommended
strategy for management of all patients with dyspeptic symptoms, which has been
emphasized in the German national guidelines (Fischbach et al. 2009). Arguments
in favor of the “scope and treat” strategy are the definitive diagnosis by direct
endoscopic inspection and by including histological assessment. A further argu-
ment is the low cost of endoscopy in several European countries. Arguments against
the “scope and test” strategy in the young age group are a normal endoscopic
finding in most cases. Complications (i.e., ulcer, neoplasia) associated to the
H. pylori infection are extremely rare in the young age group. Furthermore,
H. pylori eradication would be the treatment of choice even in case of peptic ulcers.
In Europe, treatment of non-complicated duodenal ulcer can be restricted to the
duration of eradication therapy without further proton-pump inhibitor (PPI) therapy
for additional weeks. In gastric ulcer, PPI needs to be continued until healing of the
gastric ulcer is confirmed and malignancy is excluded via a follow-up endoscopy.
In patients with dyspeptic symptoms undergoing upper gastrointestinal endos-
copy either age dependent (older than 45–50 years) or because of the need for
histological assessment, testing for H. pylori infection should routinely be
performed independent of presence or absence of gross morphologic changes of
the gastric mucosa. H. pylori eradication should routinely be offered to patients
with dyspeptic symptoms (functional dyspepsia) with a positive H. pylori test
result. A positive urease test is considered sufficient to start with the eradication
therapy even if additional management would eventually be necessary on the basis
of the histological findings. The histological assessment is mostly performed
according to the Sidney system. In cases of suspected atrophic gastritis, the updated
Sidney-Houston criteria are preferred and gastritis staging systems with special
reference to gastric atrophy (OLGA) (Rugge et al. 2008) or intestinal metaplasia

(OLGIM) (Capelle et al. 2010) are recommended.
The evidence that H. pylori eradication prevents/reduces gastroduodenal ulcers
in patients before long-term nonsteroidal anti-inflammatory drug (NSAID) expo-
sure and prevents the recurrence of ulcer bleeding in patients on low dose aspirin
has led to the implementation of a series of considerate recommendations in the
European consensus (Table 21.1). However, persisting high rates of NSAID and
aspirin-induced upper gastrointestinal bleeding display an indirect evidence for
room of improvement in the translation of these recommendations. There is great
demand for creating more awareness of the potential to prevent ulcers and compli-
cations by eradicating H. pylori in patients exposed to gastrolesive medication.
Secondary prophylaxis by H. pylori eradication in aspirin users with H. pylori
infection and previous ulcer bleeding is recommended based on a positive study
conducted in Asia (Chan et al. 2013).
A similar study is missing in Europe and no study on primary prophylaxis of
aspirin-induced gastroduodenal lesions by H. pylori eradication is available at
present. The ongoing claim H. pylori may be protective for the esophagus and
beneficial for gastroesophageal reflux disease (GERD) and GERD-related compli-
cations have been rejected in the European consensus on the basis of available
evidence (Malfertheiner et al. 2012), but the issue remains debated on a global
level; however there is no objection to H. pylori.
The role of H. pylori in the pathogenesis of selected systemic diseases has been
corroborated by clinical studies in the most recent European consensus (Banić
et al. 2012; Franceschi et al. 2014). For extragastric diseases causally related with
H. pylori (Table 21.1), it is important to emphasize that other possible causes need
to be ruled out before starting the eradication therapy.
It is also clearly stated in the European consensus that for several other
extragastric diseases associated with H. pylori but with insufficient evidence for
causality, more research is required and eradication is not recommended at present.
There are several autoimmune, neuroinflammatory, and metabolic associations
with H. pylori that deserve further investigations. The hygiene theory has been
revisited and H. pylori has been proposed as an ideal “surrogate marker” for poor
hygiene in childhood and to be beneficial in the prevention of atopic diseases.
Clinical evidence is poorly supporting this relationship but experimental studies
provided positive findings and stimulate further clinical research in this area
(Arnold et al. 2012).
21.3 Prevention Strategies of Gastric Cancer
“Search and treat” and “screen and treat” are strategies with great potential in the
prevention of gastric cancer and other H. pylori-related pathologies (Table 21.2).
However at present in Europe, the recommendation of “search and treat” strategy
for prevention is recommended only in first-degree family members of patients with
Table 21.2 European strategies

Strategies adopted in Europe
Variable among countries
Test (noninvasive) and treat – in young patients (<50) with no alarm symptoms
Scope (endoscopy based) and treat – in all conditions requiring precise diagnosis of gastrodu-
odenal pathologies
Search and treat – in selected populations at risk, i.e., family members of first-degree relatives
with gastric cancer
Screen and treat – in study protocols, proposed in high to moderate risk areas for gastric cancer!
Currently not adopted in any European country
Table 21.3 H. pylori eradication for gastric cancer prevention

H. pylori eradication to prevent gastric cancer in individuals with increased risk
To be considered in:
First-degree relatives of family members with gastric cancer
Patients with:
Previous gastric neoplasia already treated by endoscopy or partial gastrectomy
Severe pan-gastritis, corpus-predominant gastritis, severe atrophy
a
Long-term (>1 year) PPI therapy
Strong environmental risk factors for gastric cancer
Fear of gastric cancer
a
PPI so far only associated with the accelerated development of gastric neoplastic condition
gastric cancer. A recent study from Portugal strengthens this recommendation as

20 % of asymptomatic subjects from these families presented with preneoplastic
conditions (i.e., atrophy, intestinal metaplasia) of their stomach (Marcos-Pinto
et al. 2013). Those patients are at an increased risk for the development of gastric
cancer. These subjects require eradication and regular follow-up after successful
eradication. Patients with preneoplastic conditions of the gastric mucosa should in
any case undergo regular endoscopy controls at scheduled intervals (Dinis-Ribeiro
et al. 2012).
A “search and treat” strategy is also recommended in patients on long-term PPI.
In those patients, the long-term PPI use may have a negative effect by accelerating
the development of preneoplastic conditions (i.e., gastric atrophic changes) in the
presence of H. pylori infection.
In the European consensus report, commitment to implement prevention strat-
egies in areas/countries also in Europe with moderate to high prevalence of gastric
cancer in the population has been recommended (Table 21.3) and some initiatives
in this respect have been started. The positive association of H. pylori with colon
neoplasms (Selgrad et al. 2012, 2014a) and the established clinical benefits
obtained with colonoscopy screening for colorectal cancer (De Angelis
et al. 2014) prompted a European-wide initiative to screen for gastric preneopastic
conditions in the context of screening colonoscopy (“Healthy Stomach Initiative –
study proposals” n.d.).
21.4 Diagnostic Tests
The full spectrum of noninvasive and invasive tests is in use in Europe with
different indications and preferences according to the clinical conditions. Among
noninvasive tests, both the 13C-UBT and acid stool antigen test are considered
equivalent. Both tests are used as primary diagnostic tools in the “test and treat”
strategy of dyspeptic patients and they have the primary role for confirmation of the
H. pylori eradication success.
All patients undergoing eradication therapy with non-complicated peptic ulcer,
dyspepsia, and other nonmalignant pathologies should be tested 4 weeks after
eradication by a noninvasive test. Endoscopy-based testing after therapy is
demanded in patients with complicated duodenal ulcer and in all cases of gastric
ulcer, preneoplastic conditions (severe atrophy), and MALT lymphoma and after
surgical subtotal gastric resection or endoscopic resection of gastric neoplasia. If
endoscopy is carried out for clinical reasons after first-line failure of H. pylori
eradication, culture with susceptibility testing of antibiotics used in eradication
regimen should be performed. After second-line failure, therapy should generally
be chosen in the basis of antibiotic susceptibility testing.
Serology is useful in conditions of recent use of antimicrobials and PPI and in
ulcer bleeding. Serology is also appropriate for testing in patients with dyspeptic
symptoms if other noninvasive tests are not available. The use of rapid in office
tests is discouraged because of insufficient accuracy. H. pylori serology combined
with serum pepsinogen I/II and gastrin 17 offers the possibility to identify patients
with advanced preneoplastic conditions (i.e., gastric atrophy) (Agréus et al. 2012).
This test is widely accepted in Asia as a screening tool for patients at risk for gastric
cancer development.
Novel technologies including in situ hybridization methods for clarithromycin
and fluoroquinolone resistance on gastric biopsies are new options if standard
testing on culture is not possible; however they are not generally available yet.
21.5 Treatment
As in other parts of the world, the efficacy of standard triple PPI (clarithromycin,
amoxicillin, and metronidazole) for H. pylori eradication has significantly dropped
in many European countries with eradication rates below 80 %. The increasing
resistance of H. pylori to clarithromycin has been identified as the principal reason
and this is not surprising as the most effective individual antibiotic in eradication
regimen is clarithromycin.
In contrast, resistance to metronidazole although generally higher than for
clarithromycin has much less impact on H. pylori eradication failures. The main
reason for antibiotic resistance represents the occurrence of point mutations of
H. pylori DNA, which can be explained by inappropriate antibiotic use. In Europe
Table 21.4 Antibiotic resistance in Europe

LEV-
Country CLA-R MZ-R R TC-R RIF-R AC-R References
Europe 17.5 % 34.9 % 14.1 % – – – Megraud et al. (2013)
overall
Poland 23.3 % 66.7 % 6.7 % – – – Gościniak et al. (2014)
Belgium 13.3 % 26.1 % – – – 0.8 % Vekens et al. (2013)
Ireland – – 11.7 % 0% 0% – O’Connor et al. (2013)
Italy 35.2 % 59.3 % 22.1 % – – – Saracino et al. (2012)
Germany 6.7 % 29.4 % 14 % – – – Wüppenhorst
et al. (2014)
Germany 7.5 % 32.7 % 11.7 % 0% 0.8 % 0% Selgrad et al. (2013)
CLA-R clarithromycin resistance, MZ-R metronidazole resistance, LEV-R levofloxacin resistance,
TC tetracycline resistance, RIF-R rifabutin resistance, AC-R amoxicillin resistance
clarithromycin resistance rates show a huge difference between Northern and

Southern European countries. Low rates of resistance have been described for the
Scandinavian countries. In Germany, clarithromycin resistance remained compa-
rably low (<10 %), but it has to be noted that there has been a significant increase
over the last decade (Selgrad et al. 2013; Wolle et al. 2002; Wüppenhorst
et al. 2014). In contrast, in Southern Europe (e.g., Italy, Spain, Portugal, and
Greece), clarithromycin resistance is very high and therefore failure of
clarithromycin-based therapies is high. Table 21.4 gives a general overview about
the antibiotic resistance rates in Europe in the years 2012–2014.
Based on the prevalence of clarithromycin resistance, recommendations for first-
line therapy have been revised and no clarithromycin-containing regimen is
recommended for use in areas where clarithromycin resistance of >15 % is
reported. The prevalence of clarithromycin resistance is very different in
European countries and also within countries (Megraud et al. 2013; Selgrad
et al. 2014b). Accordingly different solutions for improving treatment success
have been offered in various European regions. In areas with clarithromycin
resistance >15 (20 %), bismuth-based quadruple is recommended as first line. In
a European multicenter study, bismuth quadruple (O-BMT¼ omeprazole, bismuth,
metronidazole, tetracycline) has been significantly superior to standard triple PPI
containing clarithromycin (Malfertheiner et al. 2011). In some European countries,
bismuth is not available and therefore several non-bismuth-based quadruple,
sequential (ST), concomitant (CT), or even hybrid therapies have been used
successfully (Gatta et al. 2013; Molina-Infante et al. 2013) (Table 21.5).
In clinical studies the eradication rates of complex non-bismuth quadruple
regimens are above 90 % (Molina-Infante et al. 2013), but in routine clinical
practice, the eradication rate drops below 90 % (McNicholl et al. 2014). Antibiotic
resistance testing in the individual patient with the selection of the regimen based
on antibiotic susceptibility is not recommended as first-line treatment. However it
Table 21.5 Current (proposed) treatment regimens and dosages

Standard triple therapy PPI, clarithromycin, amoxicillin, or 7–10 days
metronidazole
Standard dose b.i.d., 500 mg b.i.d., 1,000 mg
b.i.d. (or 500 mg b.i.d.)
Bismuth-containing qua- PPI, tetracycline, metronidazole, bismuth 10 days
druple therapy (Pylera®)
Standard dose b.i.d., 500 mg q.i.d., 125 mg q.i.d.,
standard dose q.i.d.
Sequential therapy Days 1–5, PPI, amoxicillin 10 days
Standard dose b.i.d., 1,000 mg b.i.d.
Days 6–10, PPI, clarithromycin (levofloxacin),
metronidazole
Standard dose b.i.d., 500 mg b.i.d., (250 mg b.i.d.)
500 mg b.i.d.miku
Concomitant therapy PPI, clarithromycin, amoxicillin, or 7–10 days
metronidazole
Standard dose b.i.d., 500 mg b.i.d., 1,000 mg b.i.d.,
500 mg b.i.d
Hybrid therapy Days 1–7, PPI, amoxicillin 14 days
Standard dose b.i.d., 1,000 mg b.i.d
Days 8–14, PPI, amoxicillin clarithromycin,
metronidazole
Standard dose b.i.d., 1,000 mg b.i.d., 500 mg
b.i.d. 500 mg b.i.d.
should be considered in patients undergoing upper gastrointestinal endoscopy for

diagnostic purpose. Second-line regimen should be chosen in consideration of the
first line and can either be O-BMT or based on levofloxacin-containing regimen
(Table 21.6). Levofloxacin has emerged as the antibiotic of choice for second line,
either in the absence of bismuth quadruple or after O-BMT failure. It is worrisome
that also levofloxacin resistance is increasing in many European countries
(Megraud et al. 2013) and therefore in countries with high levofloxacin resistance,
it is advisable to test for levofloxacin resistance before its use. In spite of some
excellent results of levofloxacin in first line (Federico et al. 2012), it should never be
used in first-line therapy without susceptibility testing. There is a general recom-
mendation to perform antibiotic susceptibility tests after second-line failure
(Table 21.6). Rifabutin for third-line combination is a valid option (Gisbert and
Calvet 2012). In this context, it is worth mentioning that for amoxicillin, tetracy-
cline, and rifabutin, resistance rates are none or neglectable in Europe. Several
studies have been performed in Europe to test for the add-on value of probiotics and
for some of them a positive effect has been shown (Szajewska et al. 2010). The
beneficial effect is considered to be obtained by lowering side effects with better
adherence of patients to the eradication therapy (6). The decision of simultaneous
administration of a “valid” probiotic with eradication therapy should be on a case
Table 21.6 H. pylori – therapy algorithm

Regions with a low clarithromycin
resistance Regions with a high clarithromycin resistance
1st line
PPI-clarithromycin-amoxicillin/ Bismuth quadruple therapy
metronidazole
Or If not available:
Bismuth quadruple therapy Quadruple therapy without bismuth (SQT
or CCT)
2nd line
Bismuth quadruple therapy PPI-levofloxacin/amoxicillin
Or
PPI-levofloxacin/amoxicillin
3rd line
Only based on susceptibility testing Only based on susceptibility testing
by case basis taking into account the susceptibility for side effects reported by
patients from the experience with prior antibiotic therapy.
Current recommendations for H. pylori eradication in Europe are based on good to

excellent evidence regarding gastroduodenal pathologies and related symptoms.
Some extragastric diseases are potentially caused by H. pylori infection and deserve
individual consideration. This is an area of important future research activities.
Commitment for gastric cancer prevention strategies is increasingly recognized in
Europe, especially in countries that continue to carry a significant gastric cancer
burden. Bismuth-based quadruple therapy and various non-bismuth-based quadru-
ples are effective alternatives to standard triple PPI therapies in areas of high
antibiotic (i.e., clarithromycin) resistance. Following failure of first-line therapy,
an algorithm of successive therapies is advised.
References
Agréus L, Kuipers EJ, Kupcinskas L, Malfertheiner P, Di Mario F, Leja M, . . . Sung J (2012)

Rationale in diagnosis and screening of atrophic gastritis with stomach-specific plasma bio-
markers. Scand J Gastroenterol 47(2):136–147. doi:10.3109/00365521.2011.645501
Arnold IC, Hitzler I, Müller A (2012) The immunomodulatory properties of Helicobacter pylori
confer protection against allergic and chronic inflammatory disorders. Front Cell Infect
Microbiol 2:10. doi:10.3389/fcimb.2012.00010
Banić M, Franceschi F, Babić Z, Gasbarrini A (2012) Extragastric manifestations of Helicobacter

pylori infection. Helicobacter 17(Suppl 1):49–55. doi:10.1111/j.1523-5378.2012.00983.x
Bastos J, Peleteiro B, Barros R, Alves L, Severo M, de Fátima Pina M, . . . Lunet N (2013)
Sociodemographic determinants of prevalence and incidence of Helicobacter pylori infection
in Portuguese adults. Helicobacter 18(6):413–422. doi:10.1111/hel.12061
Bureš J, Kopáčová M, Koupil I, Seifert B, Skodová Fendrichová M, Spirková J, . . . Tachecı́ I
(2012) Significant decrease in prevalence of Helicobacter pylori in the Czech Republic. World
J Gastroenterol WJG 18(32):4412–4418. doi:10.3748/wjg.v18.i32.4412
Capelle LG, de Vries AC, Haringsma J, Ter Borg F, de Vries RA, Bruno MJ, . . . Kuipers EJ (2010)
The staging of gastritis with the OLGA system by using intestinal metaplasia as an accurate
alternative for atrophic gastritis. Gastrointest Endosc 71(7):1150–1158. doi:10.1016/j.gie.
2009.12.029
Chan FKL, Ching JYL, Suen BY, Tse YK, Wu JCY, Sung JJY (2013) Effects of Helicobacter
pylori infection on long-term risk of peptic ulcer bleeding in low-dose aspirin users. Gastro-
enterology 144(3):528–535. doi:10.1053/j.gastro.2012.12.038
De Angelis R, Sant M, Coleman MP, Francisci S, Baili P, Pierannunzio D, . . . Capocaccia R
(2014) Cancer survival in Europe 1999–2007 by country and age: results of EUROCARE – 5-a
population-based study. Lancet Oncol 15(1):23–34. doi:10.1016/S1470-2045(13)70546-1
Dinis-Ribeiro M, Areia M, de Vries AC, Marcos-Pinto R, Monteiro-Soares M, O’Connor A, . . .
Kuipers EJ (2012) Management of precancerous conditions and lesions in the stomach
(MAPS): guideline from the European Society of Gastrointestinal Endoscopy (ESGE),
European Helicobacter Study Group (EHSG), European Society of Pathology (ESP), and the
Sociedade Portuguesa. Endoscopy 44(1):74–94. doi:10.1055/s-0031-1291491
Federico A, Nardone G, Gravina AG, Iovene MR, Miranda A, Compare D, . . . Romano M (2012)
Efficacy of 5-day levofloxacin-containing concomitant therapy in eradication of Helicobacter
pylori infection. Gastroenterology 143(1):55–61.e1; quize e13–4. doi:10.1053/j.gastro.2012.
03.043
Fischbach W, Malfertheiner P, Hoffmann JC, Bolten W, Bornschein J, G€ otze O, . . . Vieth M
(2009) S3-guideline “helicobacter pylori and gastroduodenal ulcer disease” of the German
society for digestive and metabolic diseases (DGVS) in cooperation with the German society
for hygiene and microbiology, society for pediatric gastroenterology and nutrition. Z
Gastroenterol 47(12):1230–1263. doi:10.1055/s-0028-1109855
Franceschi F, Zuccala G, Roccarina D, Gasbarrini A (2014) Clinical effects of Helicobacter pylori
outside the stomach. Nat Rev Gastroenterol Hepatol 11(4):234–242. doi:10.1038/nrgastro.
2013.243
Gatta L, Vakil N, Vaira D, Scarpignato C (2013). Global eradication rates for Helicobacter pylori
infection: systematic review and meta-analysis of sequential therapy. BMJ (Clin Res Ed) 347:
f4587. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid¼3736972&
tool¼pmcentrez&rendertype¼abstract
Gisbert JP, Calvet X (2012) Review article: rifabutin in the treatment of refractory Helicobacter
pylori infection. Aliment Pharmacol Ther 35(2):209–221. doi:10.1111/j.1365-2036.2011.
04937.x
Gościniak G, Biernat M, Grabińska J, Bińkowska A, Poniewierka E, Iwańczak B (2014) The
antimicrobial susceptibility of Helicobacter pylori strains isolated from children and adults
with primary infection in the Lower Silesia Region, Poland. Pol J Microbiol 63(1):57–61,
Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/25033663
Healthy Stomach Initiative – study proposals (n.d.) Retrieved 23 Feb 2015, from http://www.
hsinitiative.org/study-proposals.html
Malfertheiner P, Mégraud F, O’Morain C, Bell D, Bianchi Porro G, Deltenre M, . . . Rauws E
(1997) Current European concepts in the management of Helicobacter pylori infection – the
Maastricht Consensus Report. The European Helicobacter Pylori Study Group (EHPSG). Eur J
Gastroenterol Hepatol 9(1):1–2. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/
9031888
Malfertheiner P, Megraud F, O’Morain C, Bazzoli F, El-Omar E, Graham D, . . . Kuipers EJ (2007)

Current concepts in the management of Helicobacter pylori infection: the Maastricht III
Consensus report. Gut 56(6):772–781. doi:10.1136/gut.2006.101634
Malfertheiner P, Bazzoli F, Delchier J-C, Celi~nski K, Giguère M, Rivière M, Mégraud F (2011)
Helicobacter pylori eradication with a capsule containing bismuth subcitrate potassium,
metronidazole, and tetracycline given with omeprazole versus clarithromycin-based triple
therapy: a randomised, open-label, non-inferiority, phase 3 trial. Lancet 377(9769):905–913.
doi:10.1016/S0140-6736(11)60020-2
Malfertheiner P, Megraud F, O’Morain CA, Atherton J, Axon ATR, Bazzoli F, . . . Kuipers EJ
(2012) Management of Helicobacter pylori infection – the Maastricht IV/Florence Consensus
report. Gut 61(5):646–664. doi:10.1136/gutjnl-2012-302084
Malfertheiner P, Link A, Selgrad M (2014) Helicobacter pylori: perspectives and time trends. Nat
Rev Gastroenterol Hepatol 11(10):628–638. doi:10.1038/nrgastro.2014.99
Marcos-Pinto R, Dinis-Ribeiro M, Carneiro F, Wen X, Lopes C, Figueiredo C, . . . Areias J (2013)
First-degree relatives of early-onset gastric cancer patients show a high risk for gastric cancer:
phenotype and genotype profile. Virchows Archiv Int J Pathol 463(3):391–399. doi:10.1007/
s00428-013-1458-5
Mayerle J, den Hoed CM, Schurmann C, Stolk L, Homuth G, Peters MJ, . . . Kuipers E (2013)
Identification of genetic loci associated with Helicobacter pylori serologic status. JAMA 309
(18):1912–1920. doi:10.1001/jama.2013.4350
McNicholl AG, Marin AC, Molina-Infante J, Castro M, Barrio J, Ducons J, . . . Gisbert JP (2014)
Randomised clinical trial comparing sequential and concomitant therapies for Helicobacter
pylori eradication in routine clinical practice. Gut 63(2):244–249. doi:10.1136/gutjnl-2013-
304820
Megraud F, Coenen S, Versporten A, Kist M, Lopez-Brea M, Hirschl AM, . . . Glupczynski Y
(2013) Helicobacter pylori resistance to antibiotics in Europe and its relationship to antibiotic
consumption. Gut 62(1):34–42. doi:10.1136/gutjnl-2012-302254
Molina-Infante J, Romano M, Fernandez-Bermejo M, Federico A, Gravina AG, Pozzati L, . . .
Gisbert JP (2013) Optimized nonbismuth quadruple therapies cure most patients with
Helicobacter pylori infection in populations with high rates of antibiotic resistance. Gastroen-
terology 145(1):121–128.e1. doi:10.1053/j.gastro.2013.03.050
O’Connor A, Taneike I, Nami A, Fitzgerald N, Ryan B, Breslin N, . . . O’Morain C (2013)
Helicobacter pylori resistance rates for levofloxacin, tetracycline and rifabutin among Irish
isolates at a reference centre. Ir J Med Sci 182(4):693–695. doi:10.1007/s11845-013-0957-3
Rugge M, Correa P, Di Mario F, El-Omar E, Fiocca R, Geboes K, . . . Vieth M (2008) OLGA
staging for gastritis: a tutorial. Dig Liver Dis 40(8):650–658. doi:10.1016/j.dld.2008.02.030
Ruskoné-Fourmestraux A, Fischbach W, Aleman BMP, Boot H, Du MQ, Megraud F, . . .
Wotherspoon A (2011) EGILS consensus report. Gastric extranodal marginal zone B-cell
lymphoma of MALT. Gut 60(6):747–758. doi:10.1136/gut.2010.224949
Saracino IM, Zullo A, Holton J, Castelli V, Fiorini G, Zaccaro C, . . . Vaira D (2012) High
prevalence of primary antibiotic resistance in Helicobacter pylori isolates in Italy. J
Gastrointest Liver Dis JGLD 21(4):363–635. Retrieved from http://www.ncbi.nlm.nih.gov/
pubmed/23256118
Selgrad M, Bornschein J, Rokkas T, Malfertheiner P (2012) Helicobacter pylori: gastric cancer
and extragastric intestinal malignancies. Helicobacter 17(Suppl 1):30–35. doi:10.1111/j.1523-
5378.2012.00980.x
Selgrad M, Meissle J, Bornschein J, Kandulski A, Langner C, Varbanova M, . . . Malfertheiner P
(2013) Antibiotic susceptibility of Helicobacter pylori in central Germany and its relationship
with the number of eradication therapies. Eur J Gastroenterol Hepatol 25(11):1257–1260.
doi:10.1097/MEG.0b013e3283643491
Selgrad M, Bornschein J, Kandulski A, Hille C, Weigt J, Roessner A, . . . Malfertheiner P (2014a)
Helicobacter pylori but not gastrin is associated with the development of colonic neoplasms.
Int J Cancer J Int Du Cancer 135(5):1127–1131. doi:10.1002/ijc.28758
Selgrad M, Tammer I, Langner C, Bornschein J, Meißle J, Kandulski A, . . . Varbanova M (2014b)

Different antibiotic susceptibility between antrum and corpus of the stomach, a possible reason
for treatment failure of Helicobacter pylori infection. World J Gastroenterol 20(43):16245–51.
doi:10.3748/wjg.v20.i43.16245
Szajewska H, Horvath A, Piwowarczyk A (2010) Meta-analysis: the effects of Saccharomyces
boulardii supplementation on Helicobacter pylori eradication rates and side effects during
treatment. Aliment Pharmacol Ther 32(9):1069–1079. doi:10.1111/j.1365-2036.2010.04457.x
Vekens K, Vandebosch S, De Bel A, Urbain D, Mana F (2013) Primary antimicrobial resistance of
Helicobacter pylori in Belgium. Acta Clin Belg 68(3):183–187. doi:10.2143/ACB.3233
Wee EWL (2013) Evidence-based approach to dyspepsia: from Helicobacter pylori to functional
disease. Postgrad Med 125(4):169–180. doi:10.3810/pgm.2013.07.2688
Wex T, Venerito M, Kreutzer J, G€otze T, Kandulski A, Malfertheiner P (2011) Serological
prevalence of Helicobacter pylori infection in Saxony-Anhalt, Germany, in 2010. Clin Vaccine
Immunol CVI 18(12):2109–2112. doi:10.1128/CVI.05308-11
Wolle K, Leodolter A, Malfertheiner P, K€onig W (2002) Antibiotic susceptibility of Helicobacter
pylori in Germany: stable primary resistance from 1995 to 2000. J Med Microbiol 51
(8):705–709, Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12171304
Wüppenhorst N, Draeger S, Stüger HP, Hobmaier B, Vorreiter J, Kist M, Glocker E-O (2014)
Prospective multicentre study on antimicrobial resistance of Helicobacter pylori in Germany. J
Antimicrob Chemother 69(11):3127–3133. doi:10.1093/jac/dku243
Chapter 22
Population-Based Strategies for Helicobacter
pylori-Associated Disease Management:
Latin American Perspective
Javier Torres, Pelayo Correa, Rolando Herrero, M. Blanca Piazuelo,

and Catterina Ferreccio
Abstract In 2012 almost 65 % of all cancer deaths occurred in less developed

regions of the world, including Latin America, and this frequency is predicted to
increase in the coming years. We aim to offer an outlook of the burden that gastric
cancer represents for the region, the challenges faced, actions taken by some
countries, and regional efforts to deal with the problem. Latin America is a region
with contrasting gastric cancer mortality rates, with areas presenting among the
highest in the world (Central America and the Andean countries) and areas with the
lowest mortality rates (Paraguay and Argentina). In Colombia, a chemoprevention
trial was carried out in a high-risk region to investigate whether anti-Helicobacter
pylori therapy, vitamin supplements, or both prevented the progression of gastric
premalignant lesions. All three interventions resulted in significant regression of
precancerous lesions. A clinical trial in seven Latin American regions proved the
feasibility of H. pylori eradication in community-based programs using less expen-
sive and locally available generic drugs. A multidisciplinary Latin American team
worked on a consensus to deal with the problem, and they stated that “the potential
benefit of eradicating H. pylori in primary prevention of gastric cancer is highly
suggested. However, there is insufficient evidence to justify large-scale implemen-
tation in the general population.” In 2013 IARC gathered worldwide experts to
make recommendations on H. pylori treatment for gastric cancer prevention. The
group recommended the inclusion of gastric cancer prevention in national cancer
J. Torres (*)
Instituto Mexicano del Seguro Social, Unidad de Investigaci
on en Enfermedades Infecciosas,
Cd. Mexico, Mexico
P. Correa • M.B. Piazuelo
Division of Gastroenterology, Vanderbilt University Medical Center, Nashville, TN, USA
R. Herrero
IARC, Lyon, France
C. Ferreccio
Departamento de Salud Pública/Escuela de Medicina, Universidad Pontificia Cat
olica de
Chile. ACCDIS//FONDAP, Santiago, Chile

DOI 10.1007/978-4-431-55936-8_22
504 J. Torres et al.
programs, including controlled interventions for H. pylori eradication, particularly

in high-incidence areas like Latin America.
Keywords Latin America • Gastric cancer • Helicobacter pylori • Primary

prevention • Eradication treatment • Population based
22.1 Introduction
22.1.1 Global Burden of Cancer and the Current Situation

in Latin America
The International Agency for Research on Cancer (IARC) from the World Health
Organization released in December 2013 its last world report on cancer epidemi-
ology, where they inform that the most commonly diagnosed cancers worldwide
were those of the lung (1.8 million, 13.0 % of the total), breast (1.7 million, 11.9 %),
and colorectum (1.4 million, 9.7 %) (Ferlay et al. 2013). They also reported that the
most common causes of cancer death were cancers of the lung (1.6 million, 19.4 %
of the total), liver (0.8 million, 9.1 %), and stomach (0.7 million, 8.8 %). The global
burden of cancer is projected to rise, and IARC estimates predict a substantive
increase to 19.3 million new cancer cases per year by 2025, due to growth and aging
of the global population (Ferlay et al. 2013). Of relevance for this chapter is the
observation that in 2012 more than half of all cancers (56.8 %) and cancer deaths
(64.9 %) occurred in less developed regions of the world, and these proportions will
increase further in the coming years. Latin America is among these developing
regions where cancer burden is projected to increase significantly and utmost
attention should be given by countries in the area. In this chapter we aimed to
offer a general outlook of the burden that gastric cancer represents for the region,
the challenges faced to address the issue, and examples of actions taken by some of
the countries as well as regional efforts to deal with the problem.
Projections for Latin America and the Caribbean estimate that in 2030 1.7
million cases of cancer will be diagnosed, and more than a million deaths will
occur annually. As indicated above, aging of the population is among the factors
responsible for this expected rise, and this is exemplified by the estimate that by
2020 more than 100 million people older than 60 years will be living in Latin
America and the Caribbean (WHO 2012). A recent report did a critical and
thorough analyses on the causes associated with increasing burden and mortality
due to cancer in the region (Goss et al. 2013), from which selected points are
summarized next. In Latin America, low screening rates, delayed referrals, and
failure to seek medical help when symptoms develop contribute to advanced
disease at presentation and hence to increased mortality for breast, cervical, and
gastric cancer. For example, in the USA 60 % of breast cancers are diagnosed in the
earliest stages, whereas in Brazil only 20 % and in Mexico only 10 % are diagnosed
at an early stage. The problem is further complicated if we consider that of
22 Population-Based Strategies for Helicobacter pylori-Associated. . . 505
590 million inhabitants in Latin America, an estimated 54 % (almost 320 million)

do not have health-care coverage. The causes for this include language barriers,
unemployment, underemployment, geographic isolation, low education levels, and
health illiteracy. In addition, it is estimated that some 400 different indigenous
groups live in Latin America, representing 10 % of the population or about
60 million people (Chomitz et al. 2005). This is relevant because rural and remote
populations are especially vulnerable to adverse cancer outcomes, since they often
reside in areas where oncologists and experts in cancer care are not available and
local health centers cannot provide specialized cancer prevention, screening ser-
vices, treatment, or survivor care. Furthermore, the transition to a lifestyle that
mirrors developed countries is increasing obesity, and concomitant cancer risk is
becoming a greater disease burden than infectious diseases in Latin America (Goss
et al. 2013). The above information summarizes the complexity of the situation in
Latin America that challenges health authorities, politicians, and scientists to work
on the search for solutions. Some of the actions taken mostly by the scientific and
medical community are described in the following sections.
22.2 Helicobacter pylori Infection and Gastric Cancer

in the Region
Helicobacter pylori infection is highly prevalent in Latin America where in most

countries prevalence in adults is around 80 %, (Porras et al. 2013; Pest et al. 1999)
although lower frequencies are reported in a few countries like Paraguay and
Argentina (around 60 % in adults) (Flores-Luna et al. 2013). In spite of the high
prevalence of H. pylori in most countries, Latin America is a region with
contrasting gastric cancer mortality rates between countries, with areas presenting
among the highest mortality rates in the world (particularly in Central America and
the Andean countries), but also other areas among the lowest in mortality rates (like
Paraguay and Argentina) (Fig. 22.1). It has been noted that the highest burden of
gastric cancer mortality in the region is concentrated along the Pacific Rim,
following the Andes Cordillera, from Venezuela to Chile, and the Sierra Madre
and Cordillera in Central America (Torres et al. 2013).
What is relevant is that even within these countries, there are marked contrasts in
mortality, with the highest mortality rates occurring in communities in the moun-
tains and the lowest in the coastal regions. These differences are illustrated by
studies in Colombia, where the Andean Pasto region presents age-standardized
incidence rate of 150, whereas the coastal Tumaco area, only 100 miles away, has
an age-standardized incidence rate of 6, representing a 25-fold difference in inci-
dence rates (Correa et al. 1976). Maps comparing the distribution of the Andean
cordillera and the distribution of gastric cancer mortality rates in Colombia clearly
illustrate the consistent association of higher mortality rates in districts in the
mountainous regions (Fig. 22.2).
Cumulave
POPULATION Numbers Crude Rate ASR (W)
risk
Guatemala 2139 14.1 21.4 2.35
Ecuador 2262 15.2 15.5 1.69
Honduras 819 10.4 15.1 1.69
Chile 3371 19.3 13.8 1.59
El Salvador 864 13.8 13.6 1.52
Peru 3684 12.4 13.1 1.44
Costa Rica 612 12.8 12.0 1.33
Colombia 4981 10.5 11.2 1.25
Nicaragua 427 7.2 10.1 1.17
Panama 315 8.7 8.5 0.95
Uruguay 514 15.2 8.4 0.92
Venezuela 2186 7.3 8.0 0.91
France, Guadeloupe 68 14.6 7.7 0.86
Brazil 16077 8.1 7.4 0.86
Hai 510 5.0 7.3 0.75
France, Marnique 67 16.4 7.1 0.73
Jamaica 229 8.3 7.1 0.80
Bolivia 513 5.0 6.9 0.77
Dominican Republic 605 5.9 6.2 0.71
French Guyana 11 4.5 5.8 0.72
Argenna 3273 8.0 5.7 0.65
Bahamas 21 6.0 5.6 0.71
Paraguay 297 4.4 5.5 0.62
Mexico 6281 5.4 5.5 0.62
Belize 11 3.4 4.9 0.56
Barbados 26 9.5 4.8 0.44
Cuba 916 8.1 4.6 0.52
Suriname 23 4.3 4.2 0.42
Guyana 23 3.0 4.0 0.44
Trinidad and Tobago 53 3.9 3.4 0.32
Puerto Rico 172 4.6 2.5 0.30
Fig. 22.1 Gastric cancer mortality (ASR) in Latin America as reported by Globocan 2012 (Ferlay
et al. 2013). Central America and the Andean countries in the Pacific Rim present among the
highest mortality rates in the world
Fig. 22.2 The distribution of gastric cancer mortality in Colombia follows the location of the
Andes Cordillera. (a) Map showing the distribution of the Andes Cordillera; (b) colormap showing
gastric cancer mortality per districts in Colombia (Torres et al. 2013)
A clear correlation with altitude has been documented in Central America, and
in Costa Rica age-standardized incidence rates of 50 were observed in males of
communities located in the “Cordillera Volcánica Central,” whereas in males of
communities in coastal regions, age-standardized incidence rates of 10 have been
reported (Torres et al. 2013); similar observations were found in Honduras and
Venezuela. The epidemiology of gastric cancer in Chile presents important differ-
ences with the other Andean countries, whereas age-standardized incidence rates in
the country are also among the highest in the world, the rates per counties do not
follow altitude, and highest rates are observed in districts in the center and south of
the country, in regions where prevalence of H. pylori infection in young people is
higher (Torres et al. 2013). We need to better understand this rather complex
geographic diversity of gastric cancer burden in the area for a more rational design
of regional programs for the control of gastric cancer.
22.3 Lesson from Studies in Colombia
The creation of the Cali Cancer Registry in southeastern Colombia in 1962 led to
the identification of very high rates of gastric cancer in the Nari~no region of the
Andes Mountains (Correa et al. 1970, 1975a, b). In a study conducted from 1972 to
1974, the incidence rate for the high-altitude area of Nari~no was estimated to be
150 per 100,000 persons, the rate for the coastal and low-altitude valley areas of
Nari~no was 40 per 100,000 persons, while the incidence rate in Cartagena, on the
Atlantic coast, was 6 per 100,000 (Correa et al. 1976). Alongside with these
observations, multiple studies were carried out in the 1960s and 1970s that involved
systematic evaluation of numerous gastric specimens from necropsies and gastric
biopsies from Colombian subjects (Cuello et al. 1976; Correa et al. 1990a, b). These
studies led Correa and colleagues to propose the sequence of histologic lesions that
currently define the gastric precancerous process: non-atrophic gastritis, multifocal
non-metaplastic atrophic gastritis, intestinal metaplasia, and dysplasia (Correa
1992; Correa et al. 1975b). After H. pylori was first documented as a causal agent
of gastritis in 1983 (Marshall and Warren 1983), it was recognized that the
precancerous process starts with the colonization of the gastric mucosa by this
bacterium.
A double-blind randomized chemoprevention trial was carried out in the high-
risk region of Nari~no between 1991 and 1996 (Correa et al. 2000a, b). The purpose
was to investigate the role of anti-H. pylori therapy and vitamin supplements in
preventing the progression of gastric premalignant lesions. Adult volunteers with
gastric precancerous lesions documented by histology (multifocal atrophic gastritis
with or without intestinal metaplasia or dysplasia) were randomly assigned to
receive anti-H. pylori therapy for 2 weeks (amoxicillin, metronidazole, and bismuth
subsalicylate) and/or supplementation for 6 years with beta-carotene and/or
ascorbic acid or their corresponding placebos in a three-way factorial design.
Subjects assigned to the anti-H. pylori treatment arms, who tested positive for
H. pylori at 3 years of follow-up, were retreated for 14 days (amoxicillin,

clarithromycin, and either omeprazole or lansoprazole). A total of 630 subjects
completed the 6-year intervention and underwent new endoscopic procedure with
gastric biopsy sampling. All three basic interventions resulted in statistically sig-
nificant increases in the rates of regression of precancerous lesions. Relative risks of
regression in subjects with non-metaplastic atrophic gastritis at enrollment were 4.8
(95 % CI ¼ 1.6–14.2) for anti-H. pylori treatment, 5.1 (95 % CI ¼ 1.7–15) for beta-
carotene treatment, and 5.0 (95 % CI ¼ 1.6–14.2) for ascorbic acid. Corresponding
relative risks of regression in subjects with intestinal metaplasia were 3.1 (95 %
CI ¼ 1.0–9.3), 3.4 (95 % CI ¼ 1.1–9.8), and 3.3 (95 % CI ¼ 1.1–9.8). No additive
effect for any of the combinations was detected. At the end of the 6-year trial,
eradication of the infection was achieved in 75 % of treated subjects. Anti-H. pylori
therapy was then offered to the infected subjects that had not received it. Results
from the 12 years (n ¼ 456) (Mera et al. 2005) and 16 years (n ¼ 456; unpublished
data) of follow-up showed that significant regression of lesions was observed
among subjects free of H. pylori infection during 3–6 years, but the effect of
vitamin intervention was lost. Furthermore, the highest beneficial effect of anti-
H. pylori therapy was observed in subjects that started with the least advanced
lesions, mainly regressing from non-metaplastic atrophic gastritis to non-atrophic
gastritis. The rate of H. pylori reinfection/recrudescence in this cohort has hovered
around 5 new infections per 100 person-years of exposure, while the rate of
spontaneous clearance has been significantly increasing from 2 per 100 person-
years at the beginning of the trial to 7 per 100 person-years in the 12–16 years of
follow-up (our unpublished data).
Characterization of H. pylori cagA and vacA genotypes in a subset of subjects
(n ¼ 165) of this cohort showed that failure to completely eradicate H. pylori from
the stomach resulted in some patients being colonized by less virulent strains
(Correa et al. 2000b). This finding suggests that treatment may be more effective
in eliminating more virulent strains and less virulent strains are less responsive to
treatment.
The reasons for the variation in gastric cancer incidence rates in Colombia were
unknown for a long time, but recent studies have offered possible explanations.
Among the possible explanations are differences in dietary habits, host genetic
susceptibility, and host-bacterium interactions. Inhabitants of the high-altitude
Andes Mountains are predominantly “mestizos,” mixture of aboriginal Amerin-
dians and European migrants. In contrast, residents of the Pacific coast, only about
100 miles apart, are predominantly of African ancestry. This phenomenon in
Colombia is similar to the fact described by the African enigma. Similarly high
prevalence rates (>90 %) of H. pylori infection are observed in the adult population
in both high- and low-risk gastric cancer areas, but differences in virulence of the
H. pylori-infecting strains might partially account for differences in cancer risk. A
study showed higher prevalence of infection with more virulent H. pylori strains in
subjects living in the high-risk area for gastric cancer than in those from the
low-risk coastal area (Bravo et al. 2002). The corresponding prevalence rates of
H. pylori genotypes from high- and low-risk areas were cagA-positive, 90.4 %
vs. 81.1 %; vacA s1 genotype, 93.2 % vs. 83.7 %; and vacA m1 genotype, 83.3 %
vs. 70.2 %.
In addition to the mentioned virulence determinants, the phylogeographic origin
of the H. pylori strains seems to play a role in the presence and severity of gastric
precancerous lesions in Colombia. Multilocus sequence typing of seven housekeep-
ing genes was used to identify the ancestral origin of 64 cagA-positive H. pylori
isolates from these two populations (de Sablet et al. 2011). All H. pylori isolates
from the habitants of the mountains showed a predominantly European
phylogeographic origin. In contrast, two thirds of the H. pylori isolates from
African descendants living on the coast displayed predominantly African origin
and one third displayed European origin. Overall, subjects carrying H. pylori strains
of European origin showed more advanced gastric precancerous lesions and greater
oxidative damage in the gastric mucosa than subjects infected with strains of
African origin.
A recent study involving subjects (n ¼ 126, 40 years or older) from both Colom-
bian regions (mountain and coast of Nari~no) showed that interactions between the
host and H. pylori ancestries completely accounted for the difference in the severity
of gastric lesions (Kodaman et al. 2014). In particular, African H. pylori ancestry
was relatively benign in humans of African ancestry but was deleterious in indi-
viduals with substantial Amerindian ancestry. Thus, coevolution likely modulated
disease risk, and the disruption of coevolved human and H. pylori genomes may
explain the high incidence of gastric disease in the mountain population.
Another factor potentially related with carcinogenesis is oxidative stress related
to polyamines, which are generated by the rate-limiting enzyme ornithine decar-
boxylase (ODC). During H. pylori infection, the enzyme spermine oxidase
(SMOX) is induced, which generates hydrogen peroxide from the catabolism of
the polyamine spermine. When cocultured with gastric epithelial cells, H. pylori
clinical strains from the high-risk region induced more SMOX expression and
oxidative DNA damage and less apoptosis than low-risk strains. In Mongolian
gerbils, a H. pylori strain from the high-risk region induced more SMOX, DNA
damage, dysplasia, and adenocarcinoma than a strain from the low-risk region.
Treatment of gerbils, with either an inhibitor of ODC or an inhibitor of SMOX,
reduced gastric dysplasia and carcinoma, as well as apoptosis-resistant cells with
DNA damage. These data indicate that aberrant activation of polyamine-driven
oxidative stress is a marker of gastric cancer risk and a target for chemoprevention
(Chaturvedi et al. 2015).
22.4 Actions Taken in Chile
As described above, Chile is a high-risk area for gastric cancer in Latin America,
with gastric cancer risk increasing from north to south of the country (Ferreccio
et al. 2007). This population experienced a downward trend of 43 % in the gastric
cancer mortality rates from 1960 to 1980 (from 32 to 20 per 100,000 population);
nevertheless after the 1980s, the rates leveled off and remained around 20 per
100,000 in 2009 (http://www.deis.cl/defunciones-y-mortalidad-por-causas/), still
representing the first cancer killer in the country. Unfortunately, nothing has been
done in the area of primary prevention to reverse gastric cancer mortality in Chile.
However, with respect to secondary prevention of gastric cancer, Chile is the only
Latin American country with a national program, the AUGE program. This pro-
gram guarantees endoscopic examination, including H. pylori detection, biopsy,
and treatment for symptomatic adults aged 40 years and above. A pilot study of this
program was performed from 1996 to 2006 in a county of Santiago, the capital city,
with a population of 223,708. In this 10-year period, 10,284 individuals were
screened, and 190 gastric cancer cases were identified, of which only 32.1 %
were at early stages. The specific 5-year survival rate in this group was 40 %
(Galleguillos 2006), which is significantly higher than the previously reported
population-based survival of 8 % in an unscreened population of Chile (Heise
et al. 2009). Based on these promising results, in 2006 the Ministry of Health
initiated a nationwide gastric cancer detection program, which was “opportunistic,”
focused only on symptomatic individuals (including only patients on demand); it
guaranteed endoscopic examination and H. pylori treatment, if indicated (http://
www.supersalud.gob.cl/difusion/572/articles-651_guia_clinica.pdf). Unfortu-
nately, until now, this program has presented no real impact on reducing national
gastric cancer mortality, most probably due to its very low population coverage
(18 % in 2009) (http://epi.minsal.cl/wp-content/uploads/2012/07/Informe-ENS-
2009-2010.-CAP-5_FINALv1julioccepi.pdf); because of these results, the program
is under review and other more feasible strategies are being considered. It is
currently agreed that the most cost-effective intervention would be H. pylori erad-
ication in high-risk populations, accompanied with a more efficient early detection
scheme, which may include biomarker-based screening before stomach endoscopy,
and this approach is one of the strategies under consideration.
22.5 Regional Efforts to Analyze Evidences and Reach

Consensus
Under the sponsorship of the Chilean Society of Gastroenterology (http://

sociedadgastro.cl), the consensus organizing committee derived from the V Amer-
ican Workshop for the study of H. pylori assembled a multidisciplinary group of
gastroenterologists, epidemiologists, and basic scientists with expertise in various
aspects of H. pylori infection and associated diseases. A modified three-round
Delphi method was used to reach consensus on relevant topics. Consensus state-
ments to deal with the high burden of gastric cancer in the region included (Rollan
et al. 2014):
I. The potential benefit of eradicating H. pylori in primary prevention of gastric
cancer is highly suggested. However, there is insufficient evidence to justify
large-scale implementation in the general population. Further studies should be

performed on high-risk populations in Latin America to confirm the expected
benefit and to evaluate potential adverse effects.
II. The eradication of H. pylori infection is recommended as a routine measure to
prevent recurrence in gastric cancer patients receiving either subtotal surgical
gastrectomy or endoscopic resection.
III. In patients with gastric premalignant lesions, the eradication of H. pylori
infection halts the progression of chronic atrophic gastritis and probably that
of intestinal metaplasia. Although the evidence is still limited, current data
favors the eradication of H. pylori infection in these patients.
IV. High-risk gastric premalignant conditions, such as severe or extensive chronic
active gastritis, intestinal metaplasia, or dysplasia, require periodic follow-up.
Endoscopic examination is recommended every 2–3 years for patients with
moderate to severe chronic active gastritis or intestinal metaplasia, annually for
those with low-grade dysplasia, and every 3–6 months for those with high-
grade dysplasia and no focal lesion on endoscopy.
Recommendations for the region included the following: (a) further investigate
implementation issues and health outcomes of H. pylori eradication for primary
prevention of gastric cancer in high-risk populations, and (b) in order to improve the
population health, more information and demonstration projects are urgently
needed to identify effective and safe primary prevention strategies targeted to the
general population of high-risk areas.
In 1994 Brazil experts formed the Brazilian Helicobacter Study Nucleus, now
part of the Brazilian Federation of Gastroenterology. The group has held three
consensus conferences on H. pylori infection, with the third held in April 2012
(Coelho et al. 2013). Delegates included gastroenterologists, pathologists, epide-
miologists, and pediatricians, and the meeting sought to reexamine the role of
H. pylori in dyspepsia, gastric cancer, and extra-digestive diseases, as well as
address diagnosis and therapeutic options for treating and retreating infection.
Results were detailed in 30 statements and a section of proposals for actions to
reduce H. pylori prevalence, as well as suggestion for action to be implemented by
government agencies to reduce infection in Brazil. Two important statements for
gastric cancer were the following: (1) The optimal timing of H. pylori eradication to
prevent gastric cancer is before the appearance of preneoplastic conditions (atro-
phic gastritis and intestinal metaplasia). (2) In Brazil, surveying and treating the
population as a measure to prevent gastric cancer is not indicated. This last
statement might be due to the fact that Brazil with a reported age-standardized
rate of 7.4 (see Fig. 22.1) is not among the Latin American countries with the
highest age-standardized rates (Ferlay et al. 2013).
22.6 Is Eradication of H. pylori Feasible in Communities

of Latin America?
H. pylori is considered as the main risk for distal gastric cancer, and studies have
suggested that eradication of the infection in regions where this cancer is burden-
some may reduce incidence and mortality rate (Ma et al. 2012; Wong et al. 2004).
Few studies have addressed the question of feasibility of H. pylori eradication at
community level, and most of them have been performed in Asia and Europe. We
performed a randomized clinical trial to compare effectiveness of empiric 14-day
triple, 5-day concomitant, and 10-day sequential therapies for H. pylori eradication
in six Latin American countries, Chile, Colombia, Costa Rica, Honduras, Mexico,
and Nicaragua (Greenberg et al. 2011). The study also aimed to provide insight into
the feasibility of community-based programs of H. pylori eradication in the region.
The work included a random sample of 1,469 individuals recruited from the general
population in urban and rural communities. Based on the assumption that large
programs for H. pylori eradication should require inexpensive antibiotic regimens
that are effective when used in the communities, we chose to work with generic
drugs locally available. The rate of eradication at 6 weeks with the 14-day standard
triple therapy was 82.2 %, which was 8.6 % (95 % CI ¼ 2.6–14.5) higher than
concomitant therapy and 5.6 % (95 % CI ¼ 0.04–11.6) higher than sequential
therapy. At 1 year the rates of eradication were 80.4 %, 79.8 %, and 77.8 % for the
standard triple, the concomitant, and the sequential therapies. These results showed
that in contrast to studies in other parts of the world, in Latin America standard
triple therapy reaches eradication rates similar to sequential and a little higher than
concomitant therapies. The study also proves the feasibility of H. pylori eradication
in community-based programs of countries where gastric cancer is burdensome,
using less expensive and locally available generic drugs. The long-term effective-
ness of H. pylori eradication programs for reducing gastric cancer will depend on
the coverage, the acceptability by the population, the compliance with the treatment
schedule, and recurrence rate, whether due to treatment failure or to reinfection.
This latter aspect has been particularly worrisome in high-prevalence countries like
in Latin America (Leal-Herrera et al. 2003; Soto et al. 2003). In our study, the risk
of H. pylori recurrence 1 year after treatment was 11.5 %, resulting in a 1-year
effectiveness among all enrolled participants (independently of their compliance)
of 72.7 % (Morgan et al. 2013). Recurrence was significantly associated with study
site, nonadherence to initial therapy, and children in the household.
A recent report described a longitudinal study conducted in two remote rural
villages of Bolivia (Sivapalasingam et al. 2014) to evaluate annual recurrence rates
after mass eradication treatment with a triple antibiotic therapy (lansoprazole,
amoxicillin, and clarithromycin). The study included 1,153 individuals, children,
and adults, who were tested for H. pylori infection using the 13C urea breath test
(UBT), and overall cure rates were similar in all age groups (92–98 %). Impor-
tantly, annual recurrence rate was as high as 20 % in children <5 years of age, but
8 % in adults. The study concluded that one-time population-based H. pylori screen
and treat, as strategy to reduce gastric cancer, might be feasible in adults but not in
children in regions with high prevalence of H. pylori infection.
A recent systematic review estimated region and country-specific prevalence of
H. pylori antibiotic resistance in Latin America and found a high resistance to first-
line anti-H. pylori antibiotics in the area (Camargo et al. 2014). The report stresses
the need for appropriate surveillance programs, improved antimicrobial regulation,
and increased public awareness in the region. The above studies are relevant in the
design of public health programs for primary prevention of gastric cancer in high-
incidence countries of Latin America.
22.7 Recommendations for Latin America by the IARC

Expert’s Group
In December of 2013, a working group organized by IARC gathered scientific

experts from around the world to discuss the evidence and controversies and to
make recommendations on H. pylori treatment for gastric cancer prevention (IARC
2014). The high number of cases occurring worldwide, particularly in Asia, Latin
America, and Eastern Europe, in addition to its high lethality, makes it the third
cause of cancer death, with an incidence of nearly one million cases per year. The
demographic transition, resulting in aging populations, will result in stable or
increasing burden of disease, despite general trends of declining incidence rates
for the next several decades. The main etiologic agent of the disease, mainly
non-cardia gastric cancer, has been identified, with approximately 90 % of the
cases caused by chronic infection with H. pylori in conjunction with poorly
identified cofactors. The precancerous process has been characterized in detail for
the epithelial subtype, although many questions remain about the specific carcino-
genic mechanisms. The working group considered the fact that in most high-
incidence areas, infection with H. pylori is very common in adults, with a preva-
lence of about 60–80 %, and noted the extreme regional differences in gastric
cancer incidence between and within countries. Treatment of H. pylori infection is
feasible with properly tailored combinations of antibiotics and antacids, and several
trials, mainly in China, have demonstrated, with some limitations, that treatment of
the infection can reduce the incidence of gastric cancer approximately 30–40 %. In
addition, cost-benefit analyses mainly in developed countries indicate that
population-based treatment programs would be cost-effective. However, the work-
ing group noted that, with very few exceptions, population-based H. pylori treat-
ment programs are not been established as public health interventions (IARC 2014).
The reasons for this inaction have to do with the false notion that gastric cancer is
disappearing in most places, that mass eradication of large fractions of the popu-
lation may not be feasible or effective, and that the absence of H. pylori infection
could have negative effects, including antibiotic resistance, increases in adenocar-
cinoma of the esophagus, and obesity, emphasizing the uncertainty and limitations
of the available data to guide evidence-based interventions. Ongoing research is

likely to provide additional valuable information, but the working group considered
that inaction is no longer acceptable in view of the large future burden of disease
and enormous cost to society represented by gastric cancer. The group
recommended the inclusion of gastric cancer prevention in national cancer control
programs, particularly in high-incidence areas, including interventions for H. pylori
treatment in the context of demonstration projects or trials generating the necessary
data on effectiveness, acceptability, and potential adverse effects.
It was also discussed the role of biomarkers in the strategies to reduce gastric
cancer in populations of Latin America. In contrast to extensive studies in Asia
probing the utility of pepsinogen test to identify individuals with chronic atrophic
gastritis and increased risk to develop gastric cancer (Miki 2006), in Latin America
the limited number of studies has shown poor results with this test (Ley et al. 2001).
There are a number of studies on the correlation of polymorphisms in genes
associated with inflammation and risk for gastric cancer, but they are still far
from being representative of the Latin American region and should be extended
before further consideration. It should be emphasized that Latin America is a region
with limited resources and any test suggested for massive screening should be
noninvasive, simple, and cheap. In this context, we should consider that
(a) H. pylori infection is the most important risk factor to develop gastric cancer,
and longitudinal studies have shown that gastric cancer developed only in those
with a basal infection; (b) precancerous gastric lesions associated with H. pylori are
usually presented in adults (most commonly after the 40 years of age); (c) Latin
America, and in particular Central America, and the Andean countries present
among the highest age-standardized incidence rates for gastric cancer worldwide;
and (d) within these Latin American countries with high mortality rates, there are
districts or countries with even higher mortality rates. These arguments could be
used to propose a strategy to screen and treat individuals infected with H. pylori in
high-risk communities as follows (Fig. 22.3).
In summary, the main issues addressed in this chapter include the following:
(I) Central America and the Andean countries in the Pacific Rim present among
the highest gastric cancer mortality rates in the world. Still, in Latin America low
screening rates, delayed referrals, and failure to seek medical help when symptoms
develop contribute to advanced disease at presentation and hence to increased
mortality for gastric cancer. (II) In Colombia, a chemoprevention trial in a high-
risk region for gastric cancer demonstrated that anti-H. pylori therapy, vitamin
supplements, or both prevented the progression of gastric premalignant lesions.
Test and treat population-based studies in the region have documented an annual
H. pylori recurrence rate of around 10 % in adults. (III) Although the potential
benefit of eradicating H. pylori in primary prevention of gastric cancer is highly
Identify population at risk at the level of country,

district or state, select age group (>40 years?)
Census to identify
Test for Hp by UBT or family history of GC
serology
Family history+: Hp serology,
endoscopy and treatment; and
follow up
Hp- Hp+
Eradication
treatment
Confirm eradication 1
year after by UBT
Hp+ Hp-
Retreat
Fig. 22.3 Suggested minimal screening for identification of patients at risk for gastric cancer in
the Latin American region and follow-up strategies. Hp H. pylori, UBT urea breath test, GC gastric
cancer
suggested, there is insufficient evidence to justify large-scale implementation in the

general population. (IV) In countries with low age-standardized incidence rates like
Brazil, Paraguay, Argentina, or Mexico, mass eradication of H. pylori is not
recommended. (V) And recently IARC gathered world experts to discuss the
evidence and controversies and to make recommendations on H. pylori treatment
for gastric cancer prevention. The group recommended the inclusion of gastric
cancer prevention in national cancer control programs, particularly in high-
incidence areas, including interventions for H. pylori treatment in the context of
demonstration projects or trials generating the necessary data on effectiveness,
acceptability, and potential adverse effects.
References
Bravo LE, van Doom LJ, Realpe JL, Correa P (2002) Virulence-associated genotypes of
Helicobacter pylori: do they explain the African enigma? Am J Gastroenterol 97
(11):2839–2842
Camargo MC, Garcia A, Riquelme A et al (2014) The problem of Helicobacter pylori resistance to
antibiotics: a systematic review in Latin america. Am J Gastroenterol 109(4):485–495
Chaturvedi R, de Sablet T, Asim M et al (2015) Increased helicobacter pylori-associated gastric

cancer risk in the Andean region of Colombia is mediated by spermine oxidase. Oncogene 34
(26):3429–3440
Chomitz KM, Buys P, Thomas TS (2005) Quantifying the rural-urban gradient in Latin America
and the Caribbean. World Bank Policy Research working paper 3634; http://dx.doi.org/10.
1596/1813-9450-3634. Accessed 13 Feb 2013
Coelho LG, Maguinilk I, Zaterka S, et al (2013) 3 Consenso Brasileiro para Estudo do
Helicobacter pylori. Arq Gastroenterol. 50(2) (URL http://www.ncbi.nlm.nih.gov/pubmed/
23748591) pii:S0004-28032013005000113. doi:10.1590/S0004-28032013005000001
Correa P (1992) Human gastric carcinogenesis: a multistep and multifactorial process – first
American cancer society award lecture on cancer epidemiology and prevention. Cancer Res
52(24):6735–6740
Correa P, Cuello C, Duque E (1970) Carcinoma and intestinal metaplasia of the stomach in
Colombian migrants. J Natl Cancer Inst 44(2):297–306
Correa P, Bolanos O, Garcia F et al (1975a) The cancer registry of Cali. Colombia. Epidemiologic
studies of gastric cancer. Recent Results Cancer Res 50:155–169
Correa P, Haenszel W, Cuello C, Tannenbaum S, Archer MA (1975b) Model for gastric cancer
epidemiology. Lancet 2(7924):58–60
Correa P, Cuello C, Duque E et al (1976) Gastric cancer in Colombia. III. Natural history of
precursor lesions. J Natl Cancer Inst 57:1027–1035
Correa P, Haenszel W, Cuello C et al (1990a) Gastric precancerous process in a high risk
population: cross-sectional studies. Cancer Res 50(15):4731–4736
Correa P, Haenszel W, Cuello C et al (1990b) Gastric precancerous process in a high risk
population: cohort follow-up. Cancer Res 50(15):4737–4740
Correa P, Fontham ET, Bravo JC et al (2000a) Chemoprevention of gastric dysplasia: randomized
trial of antioxidant supplements and anti-Helicobacter pylori therapy. J Natl Cancer Inst 92
(23):1881–1888
Correa P, van Doorn LJ, Bravo JC et al (2000b) Unsuccessful treatment results in survival of less
virulent genotypes of Helicobacter pylori in Colombian patients. Am J Gastroenterol 95
(2):564–566
Cuello C, Correa P, Haenszel W et al (1976) Gastric cancer in Colombia. I. Cancer risk and suspect
environmental agents. J Natl Cancer Inst 57(5):1015–1020
de Sablet T, Piazuelo MB, Shaffer CL et al (2011) Phylogeographic origin of Helicobacter pylori
is a determinant of gastric cancer risk. Gut 60(9):1189–1195
Ferlay J, Soerjomataram I, Ervik M, et al (2013) GLOBOCAN 2012 v1.0, cancer incidence and
mortality worldwide. IARC Cancer Base No. 11 [Internet]. Lyon, France. Available from
http://globocan.iarc.fr
Ferreccio C, Rollán A, Harris PR et al (2007) Gastric cancer is related to early Helicobacter pylori
infection in a high-prevalence country. Cancer Epidemiol Biomarkers Prev 16(4):662–667
Flores-Luna L, Camorlinga-Ponce M, Hernandez-Suarez G (2013) The utility of serologic tests as
biomarkers for Helicobacter pylori-associated precancerous lesions and gastric cancer varies
between Latin American countries. Cancer Causes Control 24:241–248. doi:10.1007/s10552-
012-0106-8
Galleguillos B (2006) Evaluacion de 10 A~nos de un Programa de Detecci on Precoz de Cáncer
Gástrico: La Florida – Chile, 1. Master of Epidemiology Thesis, Facultad de Medicina
Pontificia Universidad Catolica de Chile 2013
Goss PE, Lee BL, Badovinac-Crnjevic T et al (2013) Planning cancer control in Latin America and
the Caribbean. Lancet Oncol 14:391–436
Greenberg ER, Anderson GL, Morgan DR et al (2011) 14-day triple, 5-day concomitant, and
10-day sequential therapies for Helicobacter pylori infection in seven Latin American sites: a
randomised trial. Lancet 378(9790):507–514. doi:10.1016/S0140-6736(11)60825-8
Heise K, Bertran E, Andia ME, Ferreccio C (2009) Incidence and survival of stomach cancer in a
high-risk population of Chile. World J Gastroenterol 21(15):1854–1862
IARC Helicobacter pylori Working Group (2014) Helicobacter pylori eradication as a strategy for
preventing gastric cancer. International Agency for Research on Cancer, Lyon, (IARC Work-
ing Group Reports, No. 8). Available from: http://www.iarc.fr/en/publications/pdfsonline/wrk/
wrk8/index.php
Kodaman N, Pazos A, Schneider BG et al (2014) Human and Helicobacter pylori coevolution
shapes the risk of gastric disease. Proc Natl Acad Sci U S A 111(4):1455–1460
Leal-Herrera Y, Torres J, Monath TP et al (2003) High rates of recurrence and of transient
reinfections of Helicobacter pylori in a population with high prevalence of infection. Am J
Gastroenterol 98(11):2395–2402
Ley C, Mohar A, Guarner J et al (2001) Screening markers for chronic atrophic gastritis in
Chiapas, Mexico. Cancer Epidemiol Biomarkers Prev 10(2):107–112. PMID:11219766
Ma JL, Zhang L, Brown LM et al (2012) Fifteen-year effects of Helicobacter pylori, garlic, and
vitamin treatments on gastric cancer incidence and mortality. J Natl Cancer Inst 104
(6):488–492
Marshall BJ, Warren JR (1983) Unidentified curved bacillus on gastric epithelium in active
chronic gastritis. Lancet Oncol 1(8336):1273–1275
Mera R, Fontham ET, Bravo LE et al (2005) Long term follow up of patients treated for
Helicobacter pylori infection. Gut 54(11):1536–1540
Miki K (2006) Gastric cancer screening using the serum pepsinogen test method. Gastric Cancer 9
(4):245–253, http://dx.doi.org/10.1007/s10120-006-0397-0
Morgan DR, Torres J, Sexton R et al (2013) Risk of recurrent Helicobacter pylori infection 1 year
after initial eradication therapy in 7 Latin American communities. JAMA 309(6):578–586
Pest PS, Corti R, Pedrana R et al (1999) Seroprevalence of Helicobacter pylori infection in the
republic of Argentina: influence of age, sex, socioeconomic level, geographical area, and
health infrastructure. Multicenter study by the Club Argentino del Estomago y Duodeno.
Acta Gastroenterol Latinoam 29(5):297–305
Porras C, Nodora J, Sexton R et al (2013) Epidemiology of Helicobacter pylori infection in six
Latin American countries (SWOG Trial S0701). Cancer Causes Control 24(2):209–215.
doi:10.1007/s10552-012-0117-5
Rollan A, Arab JA, Camargo MC, et al (2014) Management of Helicobacter pylori infection in
Latin America: a delphi-based consensus. World J Gastroenterol (in press)
Sivapalasingam S, Rajasingham A, Macyt JT, et al (2014) Recurrence of Helicobacter pylori
Infection in Bolivian children and adults after a population-based “Screen and Treat” strategy.
Helicobacter (in press). doi:10.1111/hel.12137
Soto G, Bautista CT, Roth DE, Gastrointestinal Physiology Working Group in Peru et al (2003)
Helicobacter pylori reinfection is common in Peruvian adults after antibiotic eradication
therapy. J Infect Dis 188(9):1263–1275
Torres J, Correa P, Ferreccio C et al (2013) Gastric cancer incidence and mortality is associated
with altitude in the mountainous regions of Pacific Latin America. Cancer Causes Control
24:249–256. doi:10.1007/s10552-012-0114-8
WHO, PAHO (2012) Health in the Americas, edn. Regional outlook and country profiles.
Washington, DC http://www.paho.org/healthintheamericas/
Wong BC, Lam SK, Wong WM, China Gastric Cancer Study Group et al (2004) Helicobacter
pylori eradication to prevent gastric cancer in a high-risk regi on of China: a randomized
controlled trial. JAMA 291(2):187–194
Chapter 23
Population-Based Strategies for Helicobacter
pylori-Associated Disease Management:
Asian Perspective
Muhammad Miftahussurur and Yoshio Yamaoka
Abstract Asia has the largest population of any continent and the highest inci-
dence of gastric cancer in the world, making it very important in the context of
Helicobacter pylori infection. Several new guidelines in East Asian countries
include expanded indications for H. pylori eradication. Importantly, the Japanese
national health insurance system now covers expenses for all H. pylori-infected
subjects up to second-line treatment. According to current guidelines, standard
triple therapy containing a proton pump inhibitor (PPI) and two antibiotics,
clarithromycin and amoxicillin/metronidazole, is still the preferred first-line regi-
men for treatment of H. pylori infection. However, in following years, the efficacy
of legacy triple regimens has been seriously challenged, and they are becoming
ineffective. Moreover, some regions in Asia show patterns of emerging antimicro-
bial resistance. Therefore, clarithromycin-containing triple therapy should be aban-
doned, as it is no longer effective unless local clarithromycin resistance is low or
culture confirms susceptibility to clarithromycin. More effective clarithromycin-
based regimens are now replacing standard triple therapies as empirical first-line
treatments on the basis of the understanding of the local prevalence of H. pylori
antimicrobial resistance. These include the bismuth and non-bismuth quadruple,
sequential, and dual-concomitant (hybrid) regimens.
Keywords Helicobacter pylori • Asia • Antibiotic resistance • Proton pump

inhibitor (PPI) • Clarithromycin • Amoxicillin • Metronidazole
M. Miftahussurur
Faculty of Medicine, Institute of Tropical Disease, Airlangga University, Surabaya, Indonesia
Faculty of Medicine, Department of Environmental and Preventive Medicine, Oita University,
1-1 Idaigaoka, Hasama-machi, Yufu, Oita 879-5593, Japan
Y. Yamaoka (*)
Faculty of Medicine, Department of Environmental and Preventive Medicine, Oita University,
1-1 Idaigaoka, Hasama-machi, Yufu, Oita 879-5593, Japan
Department of Medicine-Gastroenterology, Baylor College of Medicine, Houston, TX 77030,
USA

DOI 10.1007/978-4-431-55936-8_23
520 M. Miftahussurur and Y. Yamaoka
Abbreviations
cagA Cytotoxin-associated gene
DU Duodenal ulcer
IM Intestinal metaplasia
MALT Mucosa-associated lymphoid tissue
PPI Proton pump inhibitor
PUD Peptic ulcer disease
23.1 Introduction
Helicobacter pylori infection is regarded as a high risk factor for severe gastritis-
associated diseases, including peptic ulcers and gastric cancer (Suerbaum and
Michetti 2002). Asia is a very important continent in the context of H. pylori
infection. It has the largest population of any continent (4.4 billion people) and
the highest incidence of gastric cancer in the world, with an age-standardized
incidence rate of 15.8/100,000 (available from the International Agency for
Research on Cancer; GLOBOCAN2012, http://globocan.iarc.fr/). The population
of India is approximately 1.2 billion people; if H. pylori prevalence was 60 %, then
more than 726 million individuals in India would be infected with H. pylori.
Furthermore, the estimated prevalence of duodenal ulcer (DU) in India is 3 %,
meaning that at least 18 million people could need anti-H. pylori therapy (approx-
imately 50 000 per day if treated over 1 year) (Thirumurthi and Graham 2012). Asia
is subdivided into high-risk, intermediate-risk, and low-risk regions by
age-standardized incidence rate for gastric cancer (Ferlay et al. 2010). High-risk
areas include East Asian countries such as China, Japan, and Korea, where the
age-standardized incidence rate is greater than 20 per 100,000. Intermediate-risk
countries (age-standardized incidence rate 11–20/100,000) include Malaysia, Sin-
gapore, and Taiwan, while low-risk areas (age-standardized incidence rate <10/
100,000) include India, Thailand, and Indonesia.
In Asia, there is geographic variation in the prevalence rates of H. pylori
infection. Generally, developing countries have a higher prevalence than developed
countries (Fock and Ang 2010). Interestingly, the incidence rate of gastric adeno-
carcinoma in Asia tends to mirror the prevalence rate of H. pylori infection. Among
East Asian countries, China has the highest seroprevalence rate (58.1 %) (Wang and
Wang 2003) and Japan the lowest (39.3 %) (Fujisawa et al. 1999), while seroprev-
alence rates in South Korea and Taiwan are similar (59.6 % and 54.5 %, respec-
tively). In Southeast Asian countries, reported seroprevalence rates are 35.9 %,
31.0 %, and 57.0 % in Malaysia, Singapore, and Thailand, respectively (Fock and
Ang 2010). High H. pylori seroprevalence rates have also been reported in Bhutan
(70.2 %) (Vilaichone et al. 2013b) and Vietnam (74.6 %) (Hoang et al. 2005).
Interestingly, Indonesia has a low prevalence rate of H. pylori infection
(Miftahussurur et al. 2014). Using five different methods, the prevalence rate in
Indonesia was measured as only 11.5 % (Miftahussurur et al. 2015), similar to that
of Australia, which has an overall seroprevalence rate of 15.1 % (Moujaber
et al. 2008). In Western Asia, prevalence rates among different countries are
similar. Almost 90 % of the adult population is infected with H. pylori in Iran
(Malekzadeh et al. 2004; Massarrat et al. 1995). Prevalence has been reported to be
about 82 % in Jordan (Bani-Hani and Hammouri 2001) and 78.4 % among
industrial workers and 64.3 % among referent workers in the United Arab Emirates
(Bener et al. 2002). In Turkey and Kuwait (Novis et al. 1998), the overall preva-
lence of H. pylori infection is 81 % and 84 %, respectively. In Southern Central
Asia, the prevalence of H. pylori infection was reported to be almost identical
among two ethnic groups (79 % and 80 % in Russians and Kazakhs, respectively) in
Kazakhstan (Nurgalieva et al. 2002). However, a high prevalence of H. pylori
infection is not always associated with a high incidence of gastric cancer. For
example, despite the high infection rate in South Asian countries (79 %, 45 %,
and 60.2 % in India, Pakistan, and Bangladesh, respectively), the incidence of
gastric cancer in the region is low, which is known as an “Asian enigma” (Miwa
et al. 2002). Eradicating H. pylori not only heals peptic ulcers but also prevents their
recurrence and reduces the risk of development of gastric cancer (Malfertheiner
et al. 2012). Furthermore, other H. pylori-associated disorders such as mucosa-
associated lymphoid tissue (MALT) lymphoma, chronic atrophic gastritis, and
intestinal metaplasia (IM) regress after treatment with antibiotics (Sugiyama
et al. 2002; Vannella et al. 2011). It is therefore suggested that H. pylori should
be eradicated in Asia.
23.2 Indications for the Treatment of H. pylori Infection

in Asia
While European and US guidelines for the management of H. pylori infection have
been available for many years, there were no such guidelines for the Asia-Pacific
region until 1997, when guidelines were developed in Singapore (Lam and Talley
1998). These guidelines recommended that all gastric ulcer (GU) and DU patients
infected with H. pylori should be treated for H. pylori, regardless of whether the
ulcer is active or in remission. Patients requiring long-term nonsteroidal anti-
inflammatory drug therapy who have a current or recent history of dyspepsia,
patients with early gastric cancer or low-grade gastric MALT lymphoma, and
patients with a family history of gastric cancer should be treated (Lam and Talley
1998). The second guidelines in 2008 expanded the indications for treatment of
H. pylori infection (Fock et al. 2009). In addition to the previous indications,
H. pylori eradication was also indicated for H. pylori-infected patients with func-
tional dyspepsia, in those receiving long-term maintenance proton pump inhibitors
(PPIs) for gastroesophageal reflux disease, and in cases of unexplained iron-
deficiency anemia or idiopathic thrombocytopenic purpura (Table 23.1). In
Table 23.1 Comparison indication of treatment of H. pylori infection by four guidelines in Asia
Second Asia-Pacific
Consensus 2009 Japan 2013 China 2013 South Korea 2013
Indications (grade of Approved by the Strongly Strongly
recommendation) Japanese national recommended recommended
health insurance
system
Peptic ulcer disease (A) PUD Peptic ulcer Peptic ulcer
(regardless of active- disease
ness or
complications)
MALT lymphoma (A) After resection Gastric MALT Low-grade gastric
of early gastric lymphoma MALT lymphoma
cancer
Atrophic gastritis (B) Gastric MALT Recommended After resection of
lymphoma early gastric cancer
After gastric cancer Idiopathic Chronic gastritis Recommended
resection (B) thrombocytopenic with dyspepsia
purpura
Patients who have first- H. pylori- Chronic gastritis Chronic atrophic
degree relatives of patients related gastritis with mucosal atro- gastritis or IM
with gastric cancer (B) phy/erosion
Patients’ wishes (after Early gastric can- Family history of
full consultation with their cer resected endo- gastric cancer
physician) (A) scopically or by
subtotal gastrectomy
Nonulcer dyspepsia (A) Long-term use of Functional
PPI dyspepsia
To reduce the risk of Family history of Long-term aspi-
peptic ulcer and upper gastric cancer rin/NSAID medica-
gastrointestinal bleeding tion with history of
in NSAID-naive users (A) peptic ulcer disease
Before starting long- Planning to take Idiopathic throm-
term aspirin therapy for long-term NSAID bocytopenic purpura
patients at high risk for (including low-dose
ulcers and ulcer-related aspirin)
complications (B)
Patients receiving long- Iron-deficiency
term low-dose aspirin anemia of unknown
therapy and who have a causes
past history of upper gas-
trointestinal bleeding and
perforation (B)
GERD patients requir- Idiopathic throm-
ing long-term proton bocytopenic purpura
pump inhibitor (B)
As a strategy for gastric Other H. pylori-
cancer prevention in com- related diseases
munities with high inci- (lymphocytic gastri-
dence of gastric cancer (A) tis, gastric hyper-
plastic polyps,
Ménétrier disease,
etc.)
(continued)

Second Asia-Pacific
Consensus 2009 Japan 2013 China 2013 South Korea 2013
Unexplained iron- Requested by indi-
deficiency anemia or idio- vidual patient
pathic thrombocytopenic
purpura (C)
(Fock et al. 2009; Kim et al. 2013a; Asaka 2013; Chinese Society of Gastroenterology et al. 2013)
addition, a population “test-and-treat” strategy for H. pylori infection in communi-

ties with high incidence of gastric cancer was considered to be an effective strategy
for gastric cancer prevention. It was recommended that H. pylori infection should
be tested for and eradicated prior to long-term aspirin or nonsteroidal anti-
inflammatory drug therapy in patients at high risk for ulcers and ulcer-related
complications (Fock et al. 2009).
The new revised 2013 guidelines from China, Japan, and South Korea include
expanded indications for H. pylori eradication (Table 23.1). In Japan, the first
guidelines for management of H. pylori infection were developed in 2000. GU
and DU were the only diseases for which H. pylori eradication therapy was
recommended by these guidelines. The second guidelines in 2003 advanced the
field greatly. Patients with MALT lymphoma were recommended to receive erad-
ication therapy in addition to those with active GU and/or DU. In addition, the
following three clinical outcomes were upgraded to advisable indications: early
gastric cancer after endoscopic mucosal resection, atrophic gastritis, and gastric
hyperplastic polyp. The guidelines were revised substantially again in January
2009. Most importantly, in the new guidelines all “H. pylori infection” subjects
were put into the “level A” group for eradication therapy (strongly recommended
based on strong evidence). Therefore, all H. pylori infections should be considered
as an indication for eradication irrespective of the background diseases (Shiota
et al. 2010). In 2013, the Ministry of Health, Labour and Welfare of Japan
announced that from February 2013, the Japanese national health insurance system
would begin covering expenses from H. pylori eradication in all infected subjects.
The current indications consist of five categories: (1) peptic ulcer disease (PUD),
(2) after resection of early gastric cancer, (3) gastric MALT lymphoma, (4) idio-
pathic thrombocytopenic purpura, and (5) H. pylori-related gastritis. These
expanded indications are intended not only for the prevention of H. pylori-related
diseases such as gastric cancer but also for the prevention of dissemination. Since
infection seems to disseminate from parents to a child during the childhood period,
dissemination of H. pylori can be prevented by eradicating all infected adults
(Asaka 2013).
The Fourth National Consensus Conference on the management of H. pylori
infection in China expanded the indications for H. pylori eradication to include two
strongly recommended diseases (PUD and gastric MALT lymphoma) and ten
recommended diseases. Most notably, the H. pylori-related diseases lymphocytic

gastritis, Ménétrier disease, and gastric hyperplastic polyps were recommended for
eradication. Chronic gastritis with mucosal atrophy/erosion was also indicated for
eradication but not IM. The reason for this is based on the concept that dysplasia is
frequently accompanied by atrophy and/or IM. Atrophy and metaplasia can occur
after repetitive erosions. Although the optimal period for preventing gastric cancer
by H. pylori eradication is prior to the development of atrophy and IM, eradication
during this period can still ameliorate the inflammatory response, slow or stop the
progression of atrophy, and it is even possible to partially reverse the atrophy. It is
difficult, however, to reverse IM (Wang et al. 2011). Two existing areas of
controversy were mentioned. Although a H. pylori “test-and-treat” strategy has
been recommended by several other consensus guidelines, in China, the cost of
endoscopic examination is low, and the prevalence of upper gastric cancer is high.
Therefore, the introduction of a “test-and-treat” strategy has a high risk of missing
the detection of gastric cancer (Li et al. 2005). The consensus panel also believes
that H. pylori eradication might increase the risk of developing gastroesophageal
reflux disease in Eastern countries, due to the presumed higher incidence of gastric
body dominant gastritis in this region than in the Western countries (Chinese
Society of Gastroenterology et al. 2013).
The latest version of the South Korean guideline consists of 11 statements for the
indication of H. pylori eradication, four statements for diagnosis, and four state-
ments for treatment (Kim et al. 2013a). Highly recommended indications for
H. pylori eradication are (1) PUD including scar, (2) low-grade gastric MALT
lymphoma, and (3) after resection of early gastric cancer. Although the level of
evidence is lower than for these indications, H. pylori eradication may also be
considered for the prevention of gastric cancer in subjects with (4) chronic atrophic
gastritis or IM and (5) a family history of gastric cancer. South Korean studies have
demonstrated the importance of a family history of gastric cancer in determining
when H. pylori eradication is indicated, especially in patients aged 40 years or
younger (Kim et al. 2013a; Lee 2014). Compared to the second Asia-Pacific and
World Gastroenterology Organisation global guidelines, indications in three guide-
lines from East Asian countries are more expansive and aggressive, including
younger populations with acute gastric lesions, who will show markedly greater
improvements than older populations with chronic gastric lesions. As East Asia is a
high-risk region, H. pylori infection would be better eradicated at a reversible stage
before the development of a precancerous condition and prevent many infected East
Asians from going untreated. Such differences in the indications for H. pylori
treatment among countries might be due to differences in the approvals granted
by governments and/or national health insurance systems in each country (Lee
2014).
23.3 Antibiotic Resistance in Asia
Triple therapy regimens that include one PPI and two antimicrobial agents such as
clarithromycin, metronidazole, amoxicillin, levofloxacin, ciprofloxacin, and tetra-
cycline have been widely used to eradicate H. pylori (Malfertheiner et al. 2007;
Fock et al. 2009; Chey et al. 2007). However, prevalence of antibiotic resistance is
now increasing worldwide and varies by the geographic area; it is generally higher
in developing countries than in developed regions (Megraud 2004). Antibiotic
resistance is the most common factor causing treatment failure, the likelihood of
which is further increase by poor compliance or if the patient is a smoker (Jenks
2002; Qasim and O’Morain 2002; Suzuki et al. 2006a). In addition, the antibiotic
resistance rate often parallels the antibiotic consumption rate in the population
(Megraud 1998; Broutet et al. 2003). Table 23.2 summarizes antibiotic resistance
rates from 16 countries and four regions in Asia.
Clarithromycin resistance has been shown to be associated with any one of three
well-known point mutations in the 23S rRNA gene of H. pylori; these three
mutations are responsible for more than 90 % of clarithromycin resistance cases
in developed countries (Megraud and Lehours 2007). Interestingly, the point
mutations inducing clarithromycin resistance in Asian countries differ from those
in Europe and North America (Ierardi et al. 2013). Additional mutations such as
T2183C and A2223G have been frequently found to be the cause of observed
clarithromycin resistance, while the A2143G mutation, which has a much stronger
impact than A2142G, and A2142C (De Francesco et al. 2006), which are respon-
sible for 90 % of cases of primary clarithromycin resistance in H. pylori strains
isolated in Western countries (Oleastro et al. 2003), accounted only for 23 % of
resistant strains in Asia (Oleastro et al. 2003). In East Asian countries, high levels of
clarithromycin resistance have been recorded. For example, in Japan the most
recent estimate of resistance rate is 32.4 % (Kato and Fujimura 2010), and annual
surveillance for 5 years conducted between 2002 and 2006 showed that the mean
nationwide clarithromycin resistance rates had increased from 18.9 % (2002) to
27.2 % (2006) (Kobayashi et al. 2007). In China, resistance rates ranged between
21.5 and 23.8 % from 2000 to 2009 (Su et al. 2013; Gao et al. 2010), and the overall
resistance increased annually from 14.8 to 65.4 % (Gao et al. 2010). Increasing
rates of clarithromycin primary resistance have also been reported in South Korea
(17.2–23.7 %) from 2003 to 2012 (Lee et al. 2013). However, the primary resis-
tance rate of clarithromycin in Taiwan is only 8.3 %. In South Asia, India (58.8 %)
has a higher resistance rate than Pakistan (36 %) (Khan et al. 2012; Pandya
et al. 2014; Poon et al. 2009). In Western Asia, resistance rates have been increasing
over the last 20 years. In Iran, clarithromycin resistance has increased from 1.4 % in
1997 to 26.5 % in 2013 (Fakheri et al. 2014; Safaralizadeh et al. 2006). Turkey and
Bahrain also have high rates of clarithromycin resistance (21.3 and 32.5 %) (Ozbey
et al. 2013; Bindayna 2001). Interestingly, while resistance rates in Vietnam and
Indonesia are considered high (33 % and 27.8 %, respectively) (Binh et al. 2013), in
Thailand and Singapore the resistance rates are very low (3.7 % and 6 %,
526
Table 23.2 Antibiotic resistance rates from 16 countries and four regions in Asia
Clarithromycin Metronidazole Levofloxacin Tetracycline Amoxicillin
Reference Country Year Patients Methods (%) (%) (%) (%) (%) Others
Eastern Asia
Kobayashi Japan 2002–2003 1069 Agar dilu- 18.9 4.9 – – 15.2 –
et al. (2007) 2003–2004 1381 tion method 21.1 5.3 – – 21.4 –
2004–2005 1257 27.7 3.3 – – 16.3
Gao China- 2000–2009 290 Epsilometer 23.8 56.6 36.9 1.0 0.3 Moxifloxacin
et al. (2010) Beijing test (41.2 %)
Su Southeast 2010–2012 17,731 Agar dilu- 21.5 95.4 20.6 – 0.1 Furazolidone
et al. (2013) China tion method (0.1 %), genta-
micin (0.1 %)
Wang China- NM 83 Agar dilu- 10.8 49.4 – – –
et al. (2000) Hong tion method
Kong
Poon Taiwan 1998–2004 218 Epsilometer 8.3 31.7 – – 0.0 1998–2004
et al. (2009) test
Lee South 2003–2005 70 Agar dilu- 22.9 34.3 5.7 18.6 7.1 Azithromycin
et al. (2013) Korea tion method (25.7 %),
moxifloxacin
(5.7 %)
2006–2008 201 25.5 26.0 27.4 32.8 9.5 Azithromycin
(27.4 %),
moxifloxacin
(27.9 %)
2009–2012 162 37.0 35.8 34.6 35.2 18.5 Azithromycin
(34.0 %),
moxifloxacin
(34.6 %)
M. Miftahussurur and Y. Yamaoka
23
Western Asia
Abadi Iran 2009 197 Disk diffu- 45.2 65.5 37.1 – 23.9 Ciprofloxacin
et al. (2011) sion method (34.5 %), fura-
zolidone
(61.4 %)
Ozbey Turkey 2009–2010 61 Disk diffu- 21.3 42.6 3.3 0.0 0.0
et al. (2013) sion method
Eltahawy Saudi 2002 223 Disk diffu- 4.0 80.0 – 0.4 1.3
(2002) Arabia sion method
Bindayna Bahrain 1998–1999 83 Epsilometer 32.5 57.0 – 0.0 0.0
(2001) test
Southern Asia
Pandya India 2008–2011 80 Disk diffu- 58.8 83.8 72.5 53.8 72.5 Ciprofloxacin
et al. (2014) sion method (50 %)
Khan Pakistan 2005–2008 178 Not 36.0 89.0 – 12.0 37.0 Ofloxacin
et al. (2012) mentioned (18.5 %)
South Eastern Asia
Kumala and Indonesia 2006 72 Disk diffu- 27.8 100.0 1.4 – 19.4 Ciprofloxacin
Rani (2006) sion method (6.9 %),
moxifloxacin
(1.4 %),
ofloxacin (6.9 %)
Vilaichone Thailand 2004–2012 400 Epsilometer 3.7 36.0 7.2 1.7 5.2 Ciprofloxacin
et al. (2013a) test (7.7 %)
Population-Based Strategies for Helicobacter pylori-Associated. . .
Hua Singapore 1995–1998 282 Disk diffu- 6.0 46.0 – – –

et al. (2000) sion method
Ahmad Malaysia 2004–2007 187 Epsilometer 2.1 36.4 1.0 0.0 0.0 Ciprofloxacin
et al. (2011) test (0.0 %)
Vilaichone Bhutan 2010 111 Epsilometer 0.0 82.9 2.7 0.0 0.0 Ciprofloxacin
et al. (2013c) test (2.7 %)
Binh Vietnam 2008 103 Epsilometer 33.0 69.9 18.4 5.8 0.0
et al. (2013) test
527
respectively) (Vilaichone et al. 2013a; Hua et al. 2000). Moreover, in Bhutan and
Malaysia, no H. pylori strains showed resistance to clarithromycin (Vilaichone
et al. 2013c; Goh and Navaratnam 2011). This suggested that Southeast Asia is
the region of Asia with the lowest clarithromycin resistance rates.
Metronidazole is another agent frequently included in regimens to eradicate
H. pylori. Therefore, the presence of metronidazole resistance may also affect
therapeutic outcomes. The mechanisms of metronidazole resistance are complex
but are largely associated with inactivating mutations of the rdxA and frxA genes,
which encode reductases required for the activation of metronidazole (Gerrits
et al. 2004). However, development of metronidazole resistance can occur inde-
pendently of these mutations, suggesting alternative, as yet unknown, resistance
mechanisms exist (Bereswill et al. 2003). In East Asian countries, China has the
highest prevalence of metronidazole resistance (56.6–95.4 %) (Su et al. 2013; Gao
et al. 2010). Prevalence of resistance in Hong Kong is 49.4 % (Wang et al. 2000),
while the 10-year prevalence of resistance in South Korea is 34.3–35.8 % (Lee
et al. 2013). However, contrary to the general phenomenon whereby prevalence of
clarithromycin resistance tends to be much lower than that of metronidazole, the
metronidazole resistance rate in Japan is only 3.3–4.9 %, as recorded by annual
surveillance for 5 years (Kobayashi et al. 2007). In Southeast Asia, only Thailand
and Malaysia (Vilaichone et al. 2013a; Ahmad et al. 2011) have metronidazole
resistance rates below 40 %. Rates of resistance to metronidazole were found to be
in 46 % in Singapore and 69.9 % in Vietnam (Hua et al. 2000; Binh et al. 2013).
Bhutan (82.9 %) and Indonesia (100 %) have the highest prevalence of metronida-
zole resistance in this region (Vilaichone et al. 2013c; Kumala and Rani 2006).
High prevalence of metronidazole resistance was also reported in Western and
Southern Asia. Resistance rates in Iran, Turkey, Bahrain, and Saudi Arabia have
been reported as 65.5 %, 42.6 %, 57.0 %, and 50.0 %, respectively (Abadi
et al. 2011; Ozbey et al. 2013; Eltahawy 2002; Bindayna 2001). High resistance
rates are also reported in Pakistan and India (89 % and 83.8 %, respectively) (Khan
et al. 2012; Pandya et al. 2014).
Loss of penicillin-binding protein is known to be associated with amoxicillin
resistance (De Francesco et al. 2006). However, research into rates of amoxicillin
resistance is limited. Although most studies estimate rates of resistance to amoxi-
cillin as <1 % in China, Taiwan, Turkey, Bahrain, Malaysia, Bhutan, and Vietnam,
the resistance rate in Japan is >10 % (Kobayashi et al. 2007). Increasing amoxi-
cillin primary resistance rates have also been reported in South Korea (7.1–18.5 %)
(Lee et al. 2013). Both countries in Southern Asia also have high resistance rates to
amoxicillin (72.5 % and 37.0 % for India and Pakistan) (Pandya et al. 2014; Khan
et al. 2012). In Southeast and Western Asia, only Indonesia and Iran have reported
high resistance rates of 19.4 % and 23.9 %, respectively (Abadi et al. 2011; Kumala
and Rani 2006).
Fluoroquinolones, especially levofloxacin-based triple therapy, achieve good
H. pylori eradication rates. As with other bacteria, resistance of H. pylori to
fluoroquinolones is due to point mutations in the quinolone resistance determining
regions of gyrA (Megraud 1998). Rates of resistance to fluoroquinolones also
mirror the level of use of these kinds of drugs. In Asia, fluoroquinolone resistance
rates differ among countries. The resistance is higher in the Southeast coastal region
of China (20.6 %) and Beijing (36.9 %). A high rate (41.2 %) of primary
moxifloxacin resistance was also reported in Beijing (Su et al. 2013; Gao
et al. 2010). Moreover, the primary levofloxacin and moxifloxacin resistance rate
in South Korea rose from 5.7 % in 2003–2005 to 34.6 % in 2009–2012 (Lee
et al. 2013). In Western and Southern Asia, Turkey was found to have a low
levofloxacin resistance rate (3.3 %) (Ozbey et al. 2013), whereas high levofloxacin
and ciprofloxacin resistance rates (37.1 % and 34.5 %, respectively) were reported
in Iran and India (72.5 % and 50.0 %, respectively) (Abadi et al. 2011; Pandya
et al. 2014). Although resistance rates of 18.4 % have been reported in Vietnam
(Binh et al. 2013), levofloxacin resistance rates in Southeast Asia are otherwise low.
Results of a nationwide survey in Thailand found rates of ciprofloxacin and
levofloxacin resistance to be 7.7 % and 7.2 %, respectively (Vilaichone
et al. 2013a). Levofloxacin and ciprofloxacin resistance rates are reported to be
1 % and 0 % in Malaysia (Ahmad et al. 2011), 1.4 and 6.9 % in Indonesia (Kumala
and Rani 2006), and both of 2.7 % in Bhutan (Vilaichone et al. 2013c), respectively.
The tetracycline resistance mechanism has been characterized as a change in
three contiguous nucleotides in the 16S rRNA gene (AGA 926-928RTTC). Resis-
tance to tetracycline is very low, or even absent, in most countries (Megraud 1998).
Indeed, resistance to tetracycline has been shown to be infrequent in Beijing,
occurring in 1 of 49 (2.0 %) cases in 2006–2007, 0 of 63 cases in 2008, and 1 of
52 (1.9 %) cases in 2009 (Gao et al. 2010). In contrast, higher values were found in
South Korea: these increased from 18.6 % in 2003–2005 to 35.2 % in 2009–2012
(Lee et al. 2013). Resistance rates in Saudi Arabia, Thailand, and Vietnam have
been reported to be 0.4 %, 1.7 %, and 5.8 %, respectively. However, in South Asia
(India and Pakistan), tetracycline resistance rates are high (Eltahawy 2002;
Vilaichone et al. 2013a; Binh et al. 2013). Tetracycline resistance is absent in
Turkey, Bahrain, Malaysia, and Bhutan (Vilaichone et al. 2013c; Ozbey et al. 2013;
Bindayna 2001; Ahmad et al. 2011).
23.4 The Relationship Between Virulence Factors

and H. pylori Antibiotic Resistance in Asia
In addition to host factors and diet, virulence factors of H. pylori have been
demonstrated to be predictors of gastric atrophy, IM, and severe clinical outcomes
(Yamaoka 2010). Several studies have reported a relationship between H. pylori
antibiotic resistance patterns and virulence factor genotypes in Asia. In 184 patients
in Turkey treated with clarithromycin-based triple therapy for H. pylori eradication,
the eradication rate was higher in cytotoxin-associated gene (cagA)-positive
patients (87.4 %) than in those who were cagA-negative (71.9 %). The same
study found that TNF-α levels were higher in the cagA-positive than in the cagA-
negative group. This result is concordant with a meta-analysis by Suzuki

et al. (2006b), which reported pooled successful H. pylori eradication rates of
84 % for cagA-positive and 73 % for cagA-negative cases. The summary risk
ratio for eradication failure in cagA-negative relative to cagA-positive cases was
2.0 (95 % CI, 1.6–2.4; P < 0.01). The relationship of cagA with the success and
failure of H. pylori eradication therapy is clarified by enhanced gastric mucosal
inflammation. A close correlation between cagA positivity and severe gastric
inflammation has been confirmed (van der Hulst et al. 1997). Patients with severe
inflammatory cell infiltration in the antral mucosa have significantly higher cure
rates than those with milder inflammation. As gastric inflammation increases
mucosal blood flow, the increased blood flow may facilitate the diffusion of
antibiotics (Maeda et al. 1999). Another study found that vacuolating cytotoxin
gene (vacA) m1 allele was disproportionately represented among patients with
eradication failures (68 %) than in those with successful eradication (39 %) but
found no significant association of vacA s1 with eradication failure. However, there
was no relationship between clarithromycin resistance and cagA or vacA status in a
Japanese population where all cases were cagA-positive and vacA s1m1 (Shiota
et al. 2012).
Lu et al. (2005) were first to describe a novel virulence factor, the duodenal
ulcer-promoting gene (dupA), which is located in the plasticity region of the
H. pylori genome. Furthermore, they reported that infections with dupA-positive
strains increased the risk for DU but were protective against gastric atrophy, IM,
and gastric cancer in Japanese, Korean, and Colombian subjects. A more recent
study showed that gastric acid output is significantly higher in dupA-positive than in
dupA-negative patients (Imagawa et al. 2010); serum gastrin levels were also higher
in the dupA-positive group than in the dupA-negative group, suggesting that gastric
acid secretion might be higher in the dupA-positive group than in the dupA-negative
group (Shiota et al. 2012). In an in vitro study, Lu et al. reported that the presence of
dupA was associated with increased resistance to low pH (Lu et al. 2005). Strains
that are dupA-positive may induce a high level of gastrin and high gastric acid
secretion. The high gastric acid secretion in the dupA-positive group might have
been related to the lower rate of eradication, although the prevalence of dupA was
not different between DU and gastritis cases in this study (Shiota et al. 2012). If
dupA positivity is associated with high acid output, its prevalence should be higher
in DU than in gastritis. Other mechanisms may correlate with the low rate of
eradication in patients with dupA-positive H. pylori infection. Therefore, further
study is necessary to identify the mechanism of the lower rate of eradication in
dupA-positive groups.
23.5 Treatment Regimens for H. pylori Eradication in Asia
Various combinations of PPIs and antimicrobial agents have been designed to treat
H. pylori infection. These regimens include triple therapy, bismuth- and non-
bismuth-containing quadruple therapy, sequential therapy, and hybrid therapy. As
a general rule, clinicians should prescribe therapeutic regimens that have a 90 %
or preferably 95 % eradication rate locally (Rimbara et al. 2011). If no available
regimen can achieve 90 % eradication, clinicians should use the most effective
regimens available locally.
Guidelines for the management of H. pylori infection are still evolving and vary
according to the geographic area. First-line, alternative first-line, second-line, or
even third-line therapies have been proposed. Recent guidelines proposed for the
Asia-Pacific region, World Gastroenterology Organisation global guidelines for
developing countries, and guidelines for three countries in East Asia are summa-
rized in Table 23.3 (Fock et al. 2009; Kim et al. 2013a; Asaka 2013; Chinese
Society of Gastroenterology et al. 2013). However, some regimens are confined to
very small geographic districts, therefore not encouraging the development of
therapeutic guidelines that could be valid worldwide. Therefore, geographic pat-
terns of antibiotic resistance must be considered. According to current guidelines,
standard triple therapy containing a PPI and two antibiotics, clarithromycin and
amoxicillin/metronidazole, is still the first-line regimen for treatment of H. pylori
infection (Fock et al. 2009; Kim et al. 2013a; Asaka 2013; Chinese Society of
Gastroenterology et al. 2013). However, in recent years, the efficacy of legacy triple
regimens has been seriously challenged, and eradication rates lower than 70 % are
now reported in many countries (Papastergiou et al. 2014). In East Asia, the revised
2013 version of the Japanese guideline recommends a lower dose of antibiotics for
a shorter duration (7 days) than guidelines from China or South Korea. No 14-day
treatments or bismuth-based regimens are recommended as first- or second-line
treatments in Japan. The first-line therapy approved by the Japanese health insur-
ance system is clarithromycin-containing triple therapy. Therefore, many Japanese
physicians currently prescribe clarithromycin-containing triple therapy according
to the national health insurance system, even with the knowledge that this regimen
is not effective in areas with a high prevalence of clarithromycin-resistant strains.
Although the Japanese health insurance system has not approved a metronidazole-
containing regimen as a first-line eradication regimen yet, it would be a better future
first-line therapy in Japan than clarithromycin-containing triple therapy. When
H. pylori eradication fails in patients undergoing clarithromycin-based triple ther-
apy, metronidazole-based triple therapy can be used as a second-line eradication
regimen. This second-line therapy was reported to be highly successful, with an
eradication rate of more than 90 % in Japan (Murakami et al. 2008; Shimoyama
Table 23.3 Treatment regimens for H. pylori eradication in Asia

Guidelines First-line treatment Second-line treatment
Second Asia- Standard PPI-based triple therapy: Quadruple therapy: 7–14 days
Pacific Consen- 7–14 days
sus 2009 PPI, amoxicillin 1 g, PPI twice daily, bismuth 240 mg
clarithromycin 500 mg twice daily twice daily, metronidazole 400 mg
twice daily or three times daily, tet-
racycline 500 mg four times daily
PPI, metronidazole 400 mg, Levofloxacin-based triple ther-
clarithromycin 500 mg twice daily apy: 10-day PPI, levofloxacin
PPI, amoxicillin 1 g, metronida- 250 mg (or 500 mg), amoxicillin 1 g
zole 400 mg twice daily twice daily
Quadruple therapy: 7–14 days Rifabutin-based triple therapy:
7–10-day PPI, rifabutin 150 mg,
amoxicillin 1 g twice daily
PPI twice daily, bismuth 240 mg
twice daily, metronidazole 400 mg
twice daily or three times daily, tet-
racycline 500 mg four times daily
Global guide- Triple therapy: 7 days Quadruple therapy
lines for devel- PPI + amoxicillin + clarithromycin PPI + bismuth + tetracycline +
oping countries /furazolidone, twice daily metronidazole for 10–14 days
Quadruple therapy (clarithromycin PPI + furazolidone + tetracycline
resistance > 20 %): 7–10 days + bismuth for 10 days
PPI twice daily + bismuth + tetra- Triple therapy
cycline + metronidazole all four
times daily
Quadruple therapy (no known PPI + furazolidone + levofloxacin
clarithromycin resistance, bismuth for 10 days
unavailable): 14 days
PPI + clarithromycin + metronida- PPI + amoxicillin +
zole + amoxicillin clarithromycin for 7 days
Sequential therapy: 10 days PPI + amoxicillin + levofloxacin
PPI + amoxicillin for 5 days for 10 days
followed by PPI + clarithromycin
and a nitroimidazole (tinidazole) for
5 days
Japan 2013 Triple therapy: 7 days Triple therapy: 7 days
Amoxicillin 750 mg, Amoxicillin 750 mg, metronida-
clarithromycin 200 mg (or 400 mg), zole 250 mg, and PPI twice daily
and PPI twice daily
China 2013 Triple therapy: 7–14 days Quadruple therapy: 10–14 days
Amoxicillin 1 g (or metronidazole Bismuth 220 mg, tetracycline
400 mg), clarithromycin 500 mg, and 750 mg, metronidazole 400 mg
PPI twice daily twice, and PPI twice daily for 10 or
14 days
Korea 2013 Triple therapy: 7–14 days Quadruple therapy: 7–14 days
Amoxicillin 1 g, clarithromycin Bismuth 120 mg four times, tet-
500 mg, and PPI twice daily racycline 500 mg four times, metro-
nidazole 500 mg thrice, and PPI
twice daily for 7–14 days
et al. 2004). Using the metronidazole breakpoint of 8 μg/ml established by the

European Study Group, resistance rates did not change from 2002–2003 to
2004–2005 (4.9 %and 3.3 %, respectively) (Kobayashi et al. 2007). In Asia, only
Japan, Thailand, and Malaysia have populations with <40 % metronidazole resis-
tance (Table 23.2), and the Maastricht III Consensus Report stated that the use of
the PPI + clarithromycin + metronidazole regimen is preferable for these countries.
On the contrary, a regimen including metronidazole is not suitable and should not
be chosen as first-line treatment therapy in most other Asian countries. Amoxicillin-
containing combinations are recommended over those containing metronidazole,
due to metronidazole resistance rates. Alternatively, PPI + clarithromycin + amox-
icillin treatment is the recommended first-choice treatment in populations with less
than 15–20 % clarithromycin resistance (Malfertheiner et al. 2007). Therefore, this
treatment combination is preferred in almost all Southeast Asian countries
(Thailand, Singapore, Malaysia, and Bhutan).
Bismuth quadruple therapy is not completely novel but rather represents an
enhanced evolution of the older regimen comprising a bismuth salt, tetracycline,
and metronidazole (Papastergiou et al. 2014). This regimen is now designated as a
preferred first-line empirical treatment, achieving >90 % eradication in the pres-
ence of clarithromycin resistance and >85 % in regions with a high rate of
metronidazole resistance (Fischbach et al. 2004). Increased efficacy against
metronidazole-resistant strains, which offsets the ability of standard therapies to
overcome clarithromycin resistance, is likely the key for the improved performance
with bismuth quadruple therapy. It is also a valid, and cost-effective, rescue option
after failure of clarithromycin-based regimens. Second Asia-Pacific consensus and
global guidelines for developing countries recommend bismuth quadruple therapy
as an alternative first-line treatment or as a second-line treatment. As second-line
treatment, bismuth quadruple therapy combined with high-dose metronidazole
(2000 mg/day) resulted in 90.8 % per-protocol efficacy rates in a Taiwanese
study (Kuo et al. 2013). Additionally, efficacy rates of 93.1 % were obtained
using standard-dose metronidazole (1600 mg/day) in Shanghai, China, in a setting
of high metronidazole resistance (Liang et al. 2013). Interestingly, re-treatment
with bismuth quadruple therapy was also acceptable as a third-line option (66.7 %
by intention-to-treat analysis and 75.0 % by per-protocol analysis) after second-line
eradication failures with the same regimen in Korea (Lee et al. 2011). Although
bismuth quadruple therapy has been officially substituted for standard triple therapy
in high clarithromycin resistance areas, due to its side effects, bismuth is no longer
available in many countries, including Japan, Malaysia, Indonesia, and Australia.
Therefore, sequential treatment or a non-bismuth quadruple therapy (concomitant)
treatment is recommended as the alternative first-line treatment in areas of high
clarithromycin resistance (Yang et al. 2014; Yanai et al. 2012). Unfortunately, out
of a total of nine bismuth quadruple therapy regimens tested in Iran, with varying
(7-, 10-, and 14-day) durations, none had acceptable eradication rates. Similarly,
only one of nine studies conducted in Turkey has reported a 90.1 % per-protocol
eradication rate (Fakheri et al. 2014).
A novel non-bismuth quadruple therapy combination has also been developed.
This was originally developed in an attempt to decrease the duration of treatment
for H. pylori infection. In studies performed in the late 1990s, data from Europe and
Japan suggested that a short course of 3–5 days using three antibiotics and a PPI
could achieve reasonable eradication rates. A meta-analysis of 2070 patients
including 14 studies from Asia revealed a mean H. pylori intention-to-treat cure
rate of 88 % for non-bismuth quadruple therapy (PPI + clarithromycin + amoxicillin
+ nitroimidazole) (Gisbert and Calvet 2012). Two studies in Japan using PPI +
clarithromycin + amoxicillin + metronidazole as quadruple therapy reported
intention-to-treat cure rates of 92.5 and 94.5 % (Nagahara et al. 2000, 2001). On
the another hand, two studies in South Korea reported intention-to-treat cure rates
of only 81.1 % for the PPI + clarithromycin + amoxicillin + metronidazole regimen.
The rate was increased by using levofloxacin-based non-bismuth quadruple therapy
(intention-to-treat cure rate 87.5–91.4 %) for 5 and 7 days (Kim et al. 2013b; Kwon
et al. 2000). However, we must remember the importance of levofloxacin-based
therapy as a rescue treatment after failure with other regimens.
Although it consists two dosing periods, sequential therapy is a quadruple
therapy consisting of one PPI and three antibiotics. Hypothetically, amoxicillin
during the first 5 days of therapy would weaken the bacterial cell wall, which
prevents the formation of the channels that prevent clarithromycin from entering
the bacterium and hence confer resistance to the antibiotic. Then, in the second
phase of therapy, clarithromycin and a nitroimidazole are added for a further 5 days.
PPI is continued throughout the treatment. Global guidelines recommend sequential
therapy as an alternative first-line treatment. A meta-analysis of 46 randomized
controlled trials, including several countries in Asia (nine in China, seven in South
Korea, and three in Taiwan), found that sequential therapy was superior to 7 days of
triple therapy and marginally superior to 10 days of triple therapy, but not superior
to 14 days of triple therapy, bismuth quadruple therapy, or non-bismuth quadruple
therapy (Gatta et al. 2013). Although several studies have demonstrated the efficacy
of sequential therapy, most of them did not perform susceptibility testing. A study
conducted a multicenter randomized controlled trial to compare the efficacy of
sequential therapy for 10 and 14 days with that of 14 days of triple therapy as first-
line treatment (Liou et al. 2011). In addition to antibiotic resistance, they examined
host cytochrome P 2C19 polymorphisms and bacterial virulence factors such as
cagA and vacA. They found that the successful eradication rate was significantly
higher following 14 days of sequential therapy than 14 days of triple therapy. In
addition, 14 days of sequential therapy was more effective against either
clarithromycin-sensitive or clarithromycin-resistant strains of H. pylori than
10 days of sequential therapy. Cytochrome P 2C19 polymorphisms and bacterial
virulence factors did not influence the rate of successful eradication, but the
eradication rates of each treatment were affected by clarithromycin resistance.
Even after 14 days of sequential therapy, successful eradication was achieved in

only 71 % of patients with clarithromycin-resistant H. pylori strains. This finding
suggests that antibiotics other than clarithromycin, rather than sequential therapy,
are better for eradication in this population.
Dual-concomitant (hybrid) therapy is another novel regimen, consisting of dual
therapy with a PPI and amoxicillin over the first 7 days, followed by a concomitant
quadruple therapy containing a PPI plus amoxicillin, clarithromycin, and metroni-
dazole over the second 7 days. This regimen seems to be effective in areas with dual
resistance to metronidazole and clarithromycin. A study by Hsu et al. reported a
99 % per-protocol eradication rate with hybrid therapy (Hsu et al. 2011). In an
Iranian study, 420 patients were randomized to receive either hybrid or sequential
therapy. The eradication rates with hybrid therapy were 92.9 % and 89.5 % on
per-protocol and intention-to-treat analysis, respectively, while sequential regimen
had 79.9 % per-protocol eradication and 76.7 % intention-to-treat eradication rates
(Fakheri et al. 2014).
In conclusion, eradicating H. pylori not only heals peptic ulcers but also prevents
their recurrence, reduces the risk of development of gastric cancer, may prevent the
spread of infection, and reduces future costs arising from treatment of subsequent
H. pylori-associated diseases. Several new guidelines in East Asian countries
contain expanded indications for H. pylori eradication. According to current guide-
lines, standard triple therapy containing a PPI and two antibiotics, clarithromycin
and amoxicillin/metronidazole, is still the preferred first-line regimen for treatment
of H. pylori infection. However, in recent years, the efficacy of legacy triple
regimens has been seriously challenged, and their rates of effectiveness have fallen.
Moreover, some regions in Asia are exhibiting emerging patterns of antimicrobial
resistance. Clarithromycin-containing triple therapy should be abandoned, as it is
no longer effective unless local clarithromycin resistance is low or culture confirms
susceptibility to clarithromycin. More effective clarithromycin-based regimens are
now replacing standard triple therapies as empirical first-line treatments, on the
basis of local rates of H. pylori antimicrobial resistance (Table 23.4). These
regimens include bismuth and non-bismuth quadruple, sequential, and dual-con-
comitant (hybrid) regimens.
Table 23.4 Resistance region and possibility regimens for H. pylori eradication in Asia
536
First- and second-line therapy Rescue therapy

Resistance Country Clarithromycin- Metronidazole- Bismuth- Non-bismuth Sequential Hybrid Levofloxacin- Rifabutin-
region type based triple based triple based quadruple therapy therapy based triple based tri-
therapy therapy quadruple “concomitant” therapy ple
therapy therapy therapy
Low four anti- Taiwan, √ √ √ √ √ √ √ √
biotic resistance Thailand,
Malaysia
High Japan √ √ √ √ √ √ √
clarithromycin
resistance
(>20 %)
High metroni- China-Hong √ √ √ √ √ √ √
dazole resis- Kong, Saudi
tance (>40 %) Arabia, Sin-
gapore,
Bhutan
High Turkey, √ √ √ √ √
clarithromycin Bahrain,
and metronida- Vietnam
zole resistance
M. Miftahussurur and Y. Yamaoka
23
High South Korea √ √ √ √ √ √ √

clarithromycin
and levofloxacin
resistance
High China-Bei- √ √ √ √
clarithromycin, jing and
metronidazole, Southeast
and levofloxacin China
resistance
High Indonesia √ √ √ √
clarithromycin,
metronidazole,
and amoxicillin
resistance
High Iran, India, √ √
clarithromycin, Pakistan
metronidazole,
amoxicillin, and
levofloxacin
(ciprofloxacin)
resistance
Population-Based Strategies for Helicobacter pylori-Associated. . .
537
References
Abadi AT, Taghvaei T, Mobarez AM et al (2011) Frequency of antibiotic resistance in

Helicobacter pylori strains isolated from the northern population of Iran. J Microbiol
49:987–993
Ahmad N, Zakaria WR, Mohamed R (2011) Analysis of antibiotic susceptibility patterns of
Helicobacter pylori isolates from Malaysia. Helicobacter 16:47–51
Asaka M (2013) A new approach for elimination of gastric cancer deaths in Japan. Int J Cancer
132:1272–1276
Bani-Hani KE, Hammouri SM (2001) Prevalence of Helicobacter pylori in Northern Jordan.
Endoscopy based study. Saudi Med J 22:843–847
Bener A, Uduman SA, Ameen A et al (2002) Prevalence of Helicobacter pylori infection among
low socio-economic workers. J Commun Dis 34:179–184
Bereswill S, Krainick C, Stahler F et al (2003) Analysis of the rdxA gene in high-level metroni-
dazole-resistant clinical isolates confirms a limited use of rdxA mutations as a marker for
prediction of metronidazole resistance in Helicobacter pylori. FEMS Immunol Med Microbiol
36:193–198
Bindayna KM (2001) Antibiotic susceptibilities of Helicobacter pylori. Saudi Med J 22:53–57
Binh TT, Shiota S, Nguyen LT et al (2013) The incidence of primary antibiotic resistance of
Helicobacter pylori in Vietnam. J Clin Gastroenterol 47:233–238
Broutet N, Tchamgoue S, Pereira E et al (2003) Risk factors for failure of Helicobacter pylori
therapy-results of an individual data analysis of 2751 patients. Aliment Pharmacol Ther
17:99–109
Chey WD, Wong BC, G. Practice Parameters Committee of the American College of (2007)
American College of Gastroenterology guideline on the management of Helicobacter pylori
infection. Am J Gastroenterol 102:1808–1825
Chinese Society of Gastroenterology CSGOHP, Liu WZ, Xie Y, Cheng H et al (2013) Fourth
Chinese National Consensus report on the management of Helicobacter pylori infection. J Dig
Dis 14:211–221
De Francesco V, Margiotta M, Zullo A et al (2006) Clarithromycin-resistant genotypes and
eradication of Helicobacter pylori. Ann Intern Med 144:94–100
Eltahawy AT (2002) Prevalence of primary Helicobacter pylori resistance to several antimicro-
bials in a Saudi Teaching Hospital. Med Princ Pract 11:65–68
Fakheri H, Bari Z, Aarabi M et al (2014) Helicobacter pylori eradication in West Asia: a review.
World J Gastroenterol 20:10355–10367
Ferlay J, Shin HR, Bray F et al (2010) Estimates of worldwide burden of cancer in 2008:
GLOBOCAN 2008. Int J Cancer 127:2893–2917
Fischbach LA, Van Zanten S, Dickason J (2004) Meta-analysis: the efficacy, adverse events, and
adherence related to first-line anti-Helicobacter pylori quadruple therapies. Aliment Pharmacol
Ther 20:1071–1082
Fock KM, Ang TL (2010) Epidemiology of Helicobacter pylori infection and gastric cancer in
Asia. J Gastroenterol Hepatol 25:479–486
Fock KM, Katelaris P, Sugano K et al (2009) Second Asia-Pacific Consensus guidelines for
Helicobacter pylori infection. J Gastroenterol Hepatol 24:1587–1600
Fujisawa T, Kumagai T, Akamatsu T et al (1999) Changes in seroepidemiological pattern of
Helicobacter pylori and hepatitis A virus over the last 20 years in Japan. Am J Gastroenterol
94:2094–2099
Gao W, Cheng H, Hu F et al (2010) The evolution of Helicobacter pylori antibiotics resistance
over 10 years in Beijing, China. Helicobacter 15:460–466
Gatta L, Vakil N, Vaira D et al (2013) Global eradication rates for Helicobacter pylori infection:
systematic review and meta-analysis of sequential therapy. BMJ 347:f4587
Gerrits MM, Van der Wouden EJ, Bax DA et al (2004) Role of the rdxA and frxA genes in oxygen-
dependent metronidazole resistance of Helicobacter pylori. J Med Microbiol 53:1123–1128
Gisbert JP, Calvet X (2012) Update on non-bismuth quadruple (concomitant) therapy for eradi-
cation of Helicobacter pylori. Clin Exp Gastroenterol 5:23–34
Goh KL, Navaratnam P (2011) High Helicobacter pylori resistance to metronidazole but zero or
low resistance to clarithromycin, levofloxacin, and other antibiotics in Malaysia. Helicobacter
16:241–245
Hoang TT, Bengtsson C, Phung DC et al (2005) Seroprevalence of Helicobacter pylori infection in
urban and rural Vietnam. Clin Diagn Lab Immunol 12:81–85
Hsu PI, Wu DC, Wu JY et al (2011) Modified sequential Helicobacter pylori therapy: proton pump
inhibitor and amoxicillin for 14 days with clarithromycin and metronidazole added as a
quadruple (hybrid) therapy for the final 7 days. Helicobacter 16:139–145
Hua JS, Bow H, Zheng PY et al (2000) Prevalence of primary Helicobacter pylori resistance to
metronidazole and clarithromycin in Singapore. World J Gastroenterol 6:119–121
Ierardi E, Giorgio F, Losurdo G et al (2013) How antibiotic resistances could change Helicobacter
pylori treatment: a matter of geography? World J Gastroenterol 19:8168–8180
Imagawa S, Ito M, Yoshihara M et al (2010) Helicobacter pylori dupA and gastric acid secretion
are negatively associated with gastric cancer development. J Med Microbiol 59:1484–1489
Jenks PJ (2002) Causes of failure of eradication of Helicobacter pylori. BMJ 325:3–4
Kato S, Fujimura S (2010) Primary antimicrobial resistance of Helicobacter pylori in children
during the past 9 years. Pediatr Int 52:187–190
Khan A, Farooqui A, Manzoor H et al (2012) Antibiotic resistance and cagA gene correlation: a
looming crisis of Helicobacter pylori. World J Gastroenterol 18:2245–2252
Kim SG, Jung HK, Lee HL et al (2013a) Guidelines for the diagnosis and treatment of
Helicobacter pylori infection in Korea, 2013 revised edition. Korean J Gastroenterol 62:3–26
Kim SY, Lee SW, Hyun JJ et al (2013b) Comparative study of Helicobacter pylori eradication
rates with 5-day quadruple “concomitant” therapy and 7-day standard triple therapy. J Clin
Kobayashi I, Murakami K, Kato M et al (2007) Changing antimicrobial susceptibility epidemiol-
ogy of Helicobacter pylori strains in Japan between 2002 and 2005. J Clin Microbiol
45:4006–4010
Kumala W, Rani A (2006) Patterns of Helicobacter pylori isolate resistance to fluoroquinolones,
amoxicillin, clarithromycin and metronidazoles. Southeast Asian J Trop Med Public Health
37:970–974
Kuo CH, Hsu PI, Kuo FC et al (2013) Comparison of 10 day bismuth quadruple therapy with high-
dose metronidazole or levofloxacin for second-line Helicobacter pylori therapy: a randomized
controlled trial. J Antimicrob Chemother 68:222–228
Kwon DH, El-Zaatari FA, Kato M et al (2000) Analysis of rdxA and involvement of additional
genes encoding NAD(P)H flavin oxidoreductase (FrxA) and ferredoxin-like protein (FdxB) in
metronidazole resistance of Helicobacter pylori. Antimicrob Agents Chemother 44:2133–2142
Lam SK, Talley NJ (1998) Report of the 1997 Asia Pacific Consensus Conference on the
management of Helicobacter pylori infection. J Gastroenterol Hepatol 13:1–12
Lee SY (2014) Current progress toward eradicating Helicobacter pylori in East Asian countries:
differences in the 2013 revised guidelines between China, Japan, and South Korea. World J
Lee SK, Lee SW, Park JY et al (2011) Effectiveness and safety of repeated quadruple therapy in
Helicobacter pylori infection after failure of second-line quadruple therapy: repeated quadru-
ple therapy as a third-line therapy. Helicobacter 16:410–414
Lee JW, Kim N, Kim JM et al (2013) Prevalence of primary and secondary antimicrobial
resistance of Helicobacter pylori in Korea from 2003 through 2012. Helicobacter 18:206–214
Li XB, Liu WZ, Ge ZZ et al (2005) Helicobacter pylori “test-and-treat” strategy is not suitable for
the management of patients with uninvestigated dyspepsia in Shanghai. Scand J Gastroenterol
40:1028–1031
Liang X, Xu X, Zheng Q et al (2013) Efficacy of bismuth-containing quadruple therapies for

clarithromycin-, metronidazole-, and fluoroquinolone-resistant Helicobacter pylori infections
in a prospective study. Clin Gastroenterol Hepatol 11:802–807
Liou JM, Chen CC, Chen MJ et al (2011) Empirical modified sequential therapy containing
levofloxacin and high-dose esomeprazole in second-line therapy for Helicobacter pylori
infection: a multicentre clinical trial. J Antimicrob Chemother 66:1847–1852
Lu H, Hsu PI, Graham DY et al (2005) Duodenal ulcer promoting gene of Helicobacter pylori.
Maeda S, Yoshida H, Ikenoue T et al (1999) Structure of cag pathogenicity island in Japanese
Helicobacter pylori isolates. Gut 44:336–341
Malekzadeh R, Sotoudeh M, Derakhshan MH et al (2004) Prevalence of gastric precancerous
lesions in Ardabil, a high incidence province for gastric adenocarcinoma in the northwest of
Iran. J Clin Pathol 57:37–42
Malfertheiner P, Megraud F, O’Morain C et al (2007) Current concepts in the management of
Helicobacter pylori infection: the Maastricht III consensus report. Gut 56:772–781
Malfertheiner P, Megraud F, O’Morain CA et al (2012) Management of Helicobacter pylori
infection – the Maastricht IV/Florence Consensus report. Gut 61:646–664
Massarrat S, Saberi-Firoozi M, Soleimani A et al (1995) Peptic ulcer disease, irritable bowel
syndrome and constipation in two populations in Iran. Eur J Gastroenterol Hepatol 7:427–433
Megraud F (1998) Epidemiology and mechanism of antibiotic resistance in Helicobacter pylori.
Megraud F (2004) H pylori antibiotic resistance: prevalence, importance, and advances in testing.
Gut 53:1374–1384
Megraud F, Lehours P (2007) Helicobacter pylori detection and antimicrobial susceptibility
testing. Clin Microbiol Rev 20:280–322
Miftahussurur M, Tuda J, Suzuki R et al (2014) Extremely low Helicobacter pylori prevalence in
North Sulawesi, Indonesia and identification of a Maori-tribe type strain: a cross sectional
study. Gut Pathog 6:42
Miftahussurur M, Shiota S, Suzuki R et al (2015) Identification of Helicobacter pylori infection in
symptomatic patients in Surabaya, Indonesia, using five diagnostic tests. Epidemiol Infect
143:986–996
Miwa H, Go MF, Sato N (2002) H. pylori and gastric cancer: the Asian enigma. Am J
Moujaber T, MacIntyre CR, Backhouse J et al (2008) The seroepidemiology of Helicobacter
pylori infection in Australia. Int J Infect Dis 12:500–504
Murakami K, Okimoto T, Kodama et al (2008) Evaluation of three different proton pump
inhibitors with amoxicillin and metronidazole in retreatment for Helicobacter pylori infection.
J Clin Gastroenterol 42:139–142
Nagahara A, Miwa H, Ogawa K et al (2000) Addition of metronidazole to rabeprazole-
amoxicillin-clarithromycin regimen for Helicobacter pylori infection provides an excellent
cure rate with five-day therapy. Helicobacter 5:88–93
Nagahara A, Miwa H, Yamada T et al (2001) Five-day proton pump inhibitor-based quadruple
therapy regimen is more effective than 7-day triple therapy regimen for Helicobacter pylori
Novis BH, Gabay G, Naftali T (1998) Helicobacter pylori: the Middle East scenario. Yale J Biol
Med 71:135–141
Nurgalieva Z, Malaty HM, Graham DY et al (2002) Helicobacter pylori infection in Kazakhstan:
effect of water source and household hygiene. Am J Trop Med Hyg 67:201–206
Oleastro M, Ménard A, Santos A et al (2003) Real-time PCR assay for rapid and accurate detection
of point mutations conferring resistance to clarithromycin in Helicobacter pylori. J Clin
Ozbey G, Dogan Y, Demiroren K (2013) Prevalence of Helicobacter pylori virulence genotypes
among children in Eastern Turkey. World J Gastroenterol 19:6585–6589
Pandya HB, Agravat HH, Patel JS et al (2014) Emerging antimicrobial resistance pattern of
Helicobacter pylori in central Gujarat. Indian J Med Microbiol 32:408–413
Papastergiou V, Georgopoulos SD, Karatapanis S (2014) Treatment of Helicobacter pylori
infection: meeting the challenge of antimicrobial resistance. World J Gastroenterol
20:9898–9911
Poon SK, Lai CH, Chang CS, Lin WY, Chang YC, Wang HJ, Lin PH, Lin HJ, Wang WC (2009)
Prevalence of antimicrobial resistance in Helicobacter pylori isolates in Taiwan in relation to
consumption of antimicrobial agents. Int J Antimicrob Agents 34:162–165
Qasim A, O’Morain CA (2002) Review article: treatment of Helicobacter pylori infection and
factors influencing eradication. Aliment Pharmacol Ther 16(Suppl 1):24–30
Rimbara E, Fischbach LA, Graham DY (2011) Optimal therapy for Helicobacter pylori infections.
Nat Rev Gastroenterol Hepatol 8:79–88
Safaralizadeh R, Siavoshi F, Malekzadeh R et al (2006) Antimicrobial effectiveness of furazoli-
done against metronidazole-resistant strains of Helicobacter pylori. East Mediterr Health J
12:286–293
Shimoyama T, Fukuda S, Mikami T et al (2004) Efficacy of metronidazole for the treatment of
clarithromycin-resistant Helicobacter pylori infection in a Japanese population. J
Shiota S, Murakami K, Fujioka T (2010) Population-based strategies for Helicobacter pylori-
associated disease management: a Japanese perspective. Expert Rev Gastroenterol Hepatol
4:149–156
Shiota S, Nguyen LT, Murakami K (2012) Association of Helicobacter pylori dupA with the
failure of primary eradication. J Clin Gastroenterol 46:297–301
Su P, Li Y, Li H et al (2013) Antibiotic resistance of Helicobacter pylori isolated in the Southeast
Coastal Region of China. Helicobacter 18:274–279
Suerbaum S, Michetti P (2002) Helicobacter pylori infection. N Engl J Med 347:1175–1186
Sugiyama T, Sakaki N, Kozawa H et al (2002) Sensitivity of biopsy site in evaluating regression of
gastric atrophy after Helicobacter pylori eradication treatment. Aliment Pharmacol Ther 16
(Suppl 2):187–190
Suzuki T, Matsuo K, Ito H et al (2006a) Smoking increases the treatment failure for Helicobacter
pylori eradication. Am J Med 119:217–224
Suzuki T, Matsuo K, Sawaki A et al (2006b) Systematic review and meta-analysis: importance of
CagA status for successful eradication of Helicobacter pylori infection. Aliment Pharmacol
Ther 24:273–280
Thirumurthi S, Graham DY (2012) Helicobacter pylori infection in India from a western perspec-
tive. Indian J Med Res 136:549–562
Van der Hulst RW, Van der Ende A, Dekker FW (1997) Effect of Helicobacter pylori eradication
on gastritis in relation to cagA: a prospective 1-year follow-up study. Gastroenterology
113:25–30
Vannella L, Lahner E, Bordi C et al (2011) Reversal of atrophic body gastritis after H. pylori
eradication at long-term follow-up. Dig Liver Dis 43:295–299
Vilaichone RK, Gumnarai P, Ratanachu-Ek T et al (2013a) Nationwide survey of Helicobacter
pylori antibiotic resistance in Thailand. Diagn Microbiol Infect Dis 77:346–349
Vilaichone RK, Mahachai V, Shiota S et al (2013b) Extremely high prevalence of Helicobacter
pylori infection in Bhutan. World J Gastroenterol 19:2806–2810
Vilaichone RK, Yamaoka Y, Shiota S et al (2013c) Antibiotics resistance rate of Helicobacter
pylori in Bhutan. World J Gastroenterol 19:5508–5512
Wang KJ, Wang RT (2003) Meta-analysis on the epidemiology of Helicobacter pylori infection in
China. Zhonghua Liu Xing Bing Xue Za Zhi 24:443–446
Wang WH, Wong BC, Mukhopadhyay AK et al (2000) High prevalence of Helicobacter pylori
infection with dual resistance to metronidazole and clarithromycin in Hong Kong. Aliment
Pharmacol Ther 14:901–910
Wang J, Xu L, Shi R et al (2011) Gastric atrophy and intestinal metaplasia before and after
Helicobacter pylori eradication: a meta-analysis. Digestion 83:253–260
Yamaoka Y (2010) Mechanisms of disease: Helicobacter pylori virulence factors. Nat Rev
Yanai A, Sakamoto K, Akanuma M et al (2012) Non-bismuth quadruple therapy for first-line
Helicobacter pylori eradication: a randomized study in Japan. World J Gastrointest Pharmacol
Ther 3:1–6
Yang JC, Lu CW, Lin CJ (2014) Treatment of Helicobacter pylori infection: current status and
future concepts. World J Gastroenterol 20:5283–5293
Chapter 24
Probiotics as an Alternative Therapy
for Helicobacter pylori-Associated Diseases
Filipa F. Vale, Jorge M.B. Vı́tor, and Monica Oleastro
Abstract Probiotics are live microorganisms which, when administered in ade-

quate amounts, confer a health benefit to the host. Probiotics are not part of the
current treatment therapies prescribed for Helicobacter pylori eradication, but there
are numerous studies, most of them using probiotics as adjuvants to therapy,
showing a reduction of side effects in the greater majority of cases. The probiotics
administered vary hugely in the composition of microorganisms used, as well as the
duration and mode of administration, which renders the comparison difficult.
However, the most used probiotics for H. pylori infection are composed of Lacto-
bacillus, Bifidobacterium, Saccharomyces, and Streptococcus. The mode of action
of these probiotics relies on competition for nutrients and for adhesion to cell
receptors, antimicrobial activity, and modulation of the immune system and
microbiota.
Keywords Helicobacter pylori • Probiotics • Microbiota • Lactobacillus •

Bifidobacterium • Saccharomyces • Streptococcus
F.F. Vale (*)

Laboratoire de Bactériologie, Université de Bordeaux, Bordeaux, France
INSERM U853, Bordeaux, France
Host-Pathogen Interaction Unit, Research Institute for Medicines (iMed-ULIsboa), Faculty of
Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
e-mail: [email protected]; vale.fi[email protected]
J.M.B. Vı́tor
Biochemistry and Human Biology Department, Faculty of Pharmacy, Universidade de Lisboa,
Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
M. Oleastro
Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Av. Padre
Cruz, 1649-016 Lisboa, Portugal

DOI 10.1007/978-4-431-55936-8_24
544 F.F. Vale et al.
24.1 Introduction
Helicobacter pylori, a curved, microaerophilic, Gram-negative bacterium which

colonizes the mucus layer of the gastric epithelium, is the causative agent of chronic
active gastritis and ulcer disease and it is associated with an increased risk for
gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma
(Basso et al. 2010).
Eradication of H. pylori is recommended in patients with (a) peptic ulcer disease
and low-grade MALT lymphoma, (b) gastric precancerous lesions, (c) first-degree
relatives of patients with gastric cancer, and (d) patients with unexplained iron
deficiency anemia (Malfertheiner et al. 2012). Gastric cancer is a major public
health issue and the global burden of gastric cancer is increasing, particularly in
developing countries (Crew and Neugut 2006). Several studies highlight that
H. pylori eradication could reduce the risk of gastric cancer, thus playing a central
role in the prevention of this malignancy (Wong et al. 2004).
H. pylori eradication is currently a challenge in many parts of the world, due to
enormous raise in antibiotic-resistant strains. Therefore, alternative therapeutic
regimens are urgently needed.
Probiotics are living organisms that have a long history of use to promote human
health. In relation to disease, their beneficial effects have been explored as well,
especially in the prevention and treatment of gastrointestinal disorders such as
diarrhea, inflammatory bowel disease, irritable bowel syndrome, and liver disease.
In the context of H. pylori infection, the effect of probiotics is not so straight-
forward, due to specific niche of H. pylori, the human stomach.
In theory, probiotics could be used as a single therapy against H. pylori. Indeed,
lactic acid bacteria and bifidobacteria produce several substances, such as organic
acids, hydrogen peroxide, carbon dioxide, and other bactericidal compounds with
the potential to inhibit H. pylori (Servin 2004). In addition, probiotics could
compete with H. pylori for adhesion receptors and nutrients and strengthen the
mucin barrier of the epithelium, inhibiting the bacteria’s adherence to epithelial
cells (Mattar et al. 2002; Nam et al. 2002). However, to date, no study has
demonstrated complete eradication of H. pylori infection by probiotic treatment
and therefore, their use as a single therapy is currently unviable.
Nevertheless, the use of probiotics as an adjuvant of antibiotic-based therapies,
in order to reduce side effects or even to increase therapy efficacy, is an interesting
approach. Probiotics can modify the immunologic response of the host by
interacting with epithelial cells and modulating the secretion of anti- and
pro-inflammatory cytokines, what could result in a reduction of gastric activity
and inflammation associated with H. pylori infection, as experimentally demon-
strated with animal models (reviewed in (Patel et al. 2014)). These effects could
increase the efficacy of antibiotic therapies and also help the injured gastric
epithelium to restore itself more rapidly. Accordingly, the use of probiotics with
concomitant antibiotic therapy against H. pylori would serve a role in restoring the
homeostasis of microbiota and therefore in reducing side effects. However, the
24 Probiotics as an Alternative Therapy for Helicobacter pylori-Associated Diseases 545
clinical trials using probiotics as a complement to H. pylori eradication treatment

are very diverse in their design and probiotic content, which may explain the
diversity of the effect observed (Canducci et al. 2000; Goldman et al. 2006).
24.2 The Human Gut Microbiota
The definition of gut microbiota corresponds to the collective set of microorgan-

isms that colonize the human gastrointestinal tract. A typical human may carry over
40,000 different bacterial species in the intestinal microbiome (McFarland 2010).
Until recently, newborn babies were considered to be born sterile, but current
evidence points otherwise (Jimenez et al. 2008), showing that Bacilli and other
Firmicutes are the main bacteria groups detected in the meconium while
Proteobacteria dominate in the fecal samples (Moles et al. 2013). Indeed, most
phylotypes belong to the phyla Proteobacteria, Firmicutes, Actinobacteria,
Bacteroidetes, and Fusobacteria (Gotteland et al. 2006). At the microbiome phy-
lum level, humans are similar to one another and to other mammals, whereas at the
genus, species, and strain population levels, the diversity is highly specific for each
individual (Blaser 2010; Costello et al. 2009). Several pathologies are associated
with alterations in host-associated microbial communities, including obesity, mal-
nutrition, and a variety of inflammatory diseases of the skin, mouth, and intestinal
tract. The human body today is viewed as an ecosystem, and human health can be
construed as a product of ecosystem services delivered in part by the microbiota
(Costello et al. 2012).
A recent study showed that the host diet is of paramount importance on gut
microbiota composition and activity, strengthening the ecosystem view. Schnorr
and co-workers (2014) studied the phylogenetic diversity and metabolite produc-
tion of the gut microbiota from 27 hunter-gatherers, the Hadza of Tanzania, and
compared them with 16 urban Italian adults from Bologna, Italy. They observed
that their gut microbiome profiles are quite distinct even at the phylum level.
Firmicutes and Bacteroidetes are the dominant phyla in both Hadza and Italian
gut microbiome; Hadza are characterized by a relatively higher abundance of
Bacteroidetes and a lower abundance of Firmicutes. The two gut microbiome
ecosystems are remarkably different with respect to subdominant phyla (<10 %
relative abundance): Hadza are largely enriched in Proteobacteria and Spirochetes,
which are extremely rare in the Italian, while Actinobacteria, an important sub-
dominant component of the Italian microbiome, are almost completely absent from
the Hadza microbiome. They also conclude that the microbiome, as a diverse and
responsive ecosystem, is adapting continuously as a commensal component of the
host supraorganism; the functional redundancy found in bacterial communities
indicates that microbial activity is the important aspect, rather than species
composition.
The gut microbiome is so complex and diverse that one may have the impression
that it will take a long time before some of this information can be used to treat
certain human pathologies. The normal microbiome remains to be defined but when
known may be useful for its reposition in patients (Ling et al. 2013). Indeed, the
administration of exogenous bacteria has been shown to improve some conditions,
such as Bacteroides fragilis which in a mouse model corrected gut permeability,
altered microbial composition, and improved defects in communicative, stereo-
typic, anxiety-like, and sensorimotor behavior (Hsiao et al. 2013), or the fecal
transplants in the treatment of the Clostridium difficile infection, so far the most
efficient way to remove the pathogen from the gut (van Nood et al. 2013).
The gut microbiota may serve as a virtual endocrine organ, which is corrobo-
rated by their direct role in metabolic pathways which produce and regulate
multiple compounds that reach the circulation and influence the function of distal
organs and systems. For example, the probiotic Lactobacillus rhamnosus PL60
produces conjugated linoleic acid which has been shown to reduce body weight
gain and white adipose tissue without effects on food intake (Clarke et al. 2014).
H. pylori colonize a peculiar ecological niche, the stomach mucin layer, prob-
ably being the principal bacteria present in the stomach of infected patients,
although the presence of a transient gastric flora, to which Lactobacillus strains
belong, cannot be ignored (Gotteland et al. 2006). Moreover, a recent study reports
that Bifidobacteriaceae species present in the oral cavity can colonize the
omeprazole-treated hypochlorhydria stomach to quite a large degree (Mattarelli
et al. 2014). There are conflicting findings regarding the influence of H. pylori in the
stomach microbiota (Wang et al. 2014). The microbiota of the human stomach and
its influence on H. pylori colonization has been characterized showing that H. pylori
does not affect the composition of the gastric community (Bik et al. 2006). How-
ever, a positive H. pylori status has been associated to an increased relative
abundance of non-Helicobacter bacteria from the Proteobacteria, Spirochetes,
and Acidobacteria and with decreased abundance of Actinobacteria, Bacteroidetes,
and Firmicutes (Wang et al. 2014).
The gastric microbiota is highly controlled by pH and may play a role in the
development of gastric cancer. In the presence of lower acid secretion due to gastric
atrophy, bacteria overgrowth is favored in the gastric fluid, enhancing the produc-
tion of carcinogenic N-nitrosamine compounds (Wang et al. 2014). The gastric
microbiota in gastric cancer patients is comprised predominately of Veillonella,
Haemophilus, Streptococci, Lactobacillus, Prevotella, and Neisseria (Dicksved
et al. 2009).
24.3 The Omics Era in Probiotics
The first genome to be completely sequenced was published in Nature in 1976. The
sequencing was carried out by Walter Fiers and his group in Gent, Belgium. It
concerned the bacteriophage MS2, whose genome is a small linear single-stranded
RNA molecule with 3569 bases, which codes for four genes (Fiers et al. 1976).
Twenty-one years later, the first complete microbial genome of Haemophilus
influenzae Rd, with 1,830,137 base pairs, was published (Fleischmann et al. 1995),
and soon after a large number of other bacteria were sequenced, as well as the fruit
fly, baker’s yeast, and the human genome.
All of this sequencing interest stimulated the improvement of the DNA sequenc-
ing method developed by Fred Sanger as well as the search for novel sequencing
technologies, and today, there are several companies selling large and small
sequencing machines, based on several different technologies. They are faster,
less expensive, and more informative than the pioneer Sanger method (Brown
2013; Sanger et al. 1977). These new technologies, known as “next-generation
sequencing technologies” (NGS), are able to surpass the major limitation of micro-
biology: the inability to cultivate most of the earth’s microorganisms (Brown 2013).
The first attempts to understand the diversity of microorganisms in the human
body were carried out using polymerase chain reaction (PCR)-based technologies,
targeting 16S RNA sequences, and soon it was clear that the number of species
present in the human gut was very large (Hayashi et al. 2002; Suau et al. 1999).
Using NGS will probably clarify the (1) identity of the microbes that populate each
host, (2) the role of the microbiota, (3) the response of the host to these microbes,
(4) the forces that maintain equilibrium among the populations, and (5) the unique
characteristics of each individual (Blaser 2010). After a full comprehension of the
microbiota, it will be possible to study how probiotics interfere and module it. The
National Institute of Health (NIH) decided to start a program to finance the study of
the microbiota by examining at least four body sites: the gastrointestinal tract, the
mouth, the vagina, and the skin. The Human Microbiome Project was designed and
its major goal was to demonstrate the feasibility of characterizing the human
microbiome well enough to enable study of its variation and its influence on
disease. Fifteen projects were funded, and more than half were gut related (Peterson
et al. 2009).
24.3.1 Probiotics
The term probiotics means “for life” and was most probably first addressed by Ilya
Mechnikov (1908 Nobel Prize winner, natural scientist/microbiologist at Pasteur
Institute), who proposed the use of diet containing milk fermented by bacilli which
produce large amounts of lactic acid to prevent the multiplication of certain bacteria
that poison the body (reviewed in the Nobel Lectures, (Nobel Foundation 1967)).
Also in France, the pediatrician Henry Tissier suggested the administration of
Bifidobacterium (designated as Y-shaped bacteria abundant in healthy children)
to patients with diarrhea to restore gut flora (Tissier 1906).
Presently, probiotics are defined as “live organisms which administered in
adequate amounts confer a health benefit to the host” (Pineiro and Stanton 2007).
Although probiotics may exert their function by products of theirs metabolism, such
as enzymes or bacteriocins (proteins lethal to bacteria other than the producing
strain) that do not require live cells, dead microorganisms are not probiotics.
Moreover, microbiota microorganisms are not probiotics. Microbiota may be the

source of probiotics, but the term should only be used after strain isolation and
characterization, namely, considering their health effects, dose, and stability
(Sanders 2008). Specificity of probiotic action and the ability to remain viable at
the target site is the most important point rather than the source of the microorgan-
ism (Pineiro and Stanton 2007). In fact, the guidelines established by Food and
Drug Administration (FAO) and World Health Organization (WHO) recommend
(1) the identification to the level of strain of all probiotics in the product, (2) the
depositing of all strains in an international culture collection, (3) naming the strain
according to the International Code of Nomenclature, (4) characterization of strain
safety and function (probiotic effects are strain specific), and (5) validation of
health benefits in human studies, including the dose required to provide the benefit
(FAO and WHO 2001, 2002). The mode of action of probiotics is discussed below,
but their action may prevent pathogenic bacteria infection through stimulation of
the immune response, competition with pathogenic bacteria, prevention of antibi-
otic side effects, and improvement of eradication rates (Lesbros-Pantoflickova
et al. 2007; Lionetti et al. 2010; Miki et al. 2007; Vitor and Vale 2011).
The increasing difficulty in eradicating H. pylori when therapy is recommended
has incited a search of new treatments and an attractive field within H. pylori
research. Probiotics are probably at the top of the list for alternative therapies
against H. pylori, although frequently administrated in conjugation with antibiotics
(Vitor and Vale 2011). According to the Web of Science, the number of publica-
tions addressing probiotics and H. pylori increased especially in the ten last years
(Fig. 24.1). The total number of publications in Thomson Reuters Web of Science
addressing H. pylori and probiotics is 394 (December 2014). The analysis of
Fig. 24.1 shows that the most studied probiotic microorganism is Lactobacillus
followed by Bifidobacterium, Saccharomyces, and Streptococcus. The two
Fig. 24.1 Number of Thomson Reuters Web of Science publications addressing H. pylori and
probiotics from 1997 to 2014. The number of publications is only displayed if 2
microorganisms used the most for studying their impact on H. pylori infection,
Lactobacillus and Bifidobacterium, belong to the genera most present as probiotics
in food (Pineiro and Stanton 2007). The number of publications addressing the use
of probiotics reflects their increasing importance in H. pylori infection, as scientific
evidence continues to accumulate on the properties, functionality, and benefits of
probiotics for the promotion of human health, namely, improving immunological
and digestive functions and alleviating infectious disease (Pineiro and Stanton
2007).
Probiotic studies include not only a wide range of microbial species, adminis-
tered alone or in combination with each other, which complicates the comparison of
studies. However, an observation of the most recent meta-analysis shows that the
majority of the probiotics reveal a consistent, statistically significant increase in
eradication rates and reduced side effects from antibiotic therapy (Molina-Infante
and Gisbert 2013).
The clinical trials included in the meta-analysis of Tong and co-workers (2007)
applying multiple strains from different species (Cao et al. 2005; Cremonini
et al. 2002; Myllyluoma et al. 2005; Sheu et al. 2002) presented an odds ratio
(OR) favoring probiotics, except for the study of Goldman et al. (2006) which only
included children. However, only one of the trials (Sheu et al. 2002) applying a
combination of Lactobacillus and Bifidobacterium was statistically significant.
Furthermore, when the Goldman study was compared with other clinical trials
performed on children (Pacifico et al. 2014), it favored probiotic administration
even though the results were not statistically significant. Pacifico and colleagues
reported only one study applying a mixture of probiotics to children (Ahmad
et al. 2013) was statistically significant (Pacifico et al. 2014).
The effect of probiotics is known to be species specific and the next topics
present work developed specifically according to the bacterial genus.
24.3.2 Lactobacillus
Over 100 species belong to the genus Lactobacillus, which is the largest group
among the Lactobacteriacea. Lactobacilli are nutritionally fastidious and are asso-
ciated with a large variety of plants and animals, including the human gut
microbiota. These lactic acid bacteria, whose primary fermentation end product is
lactic acid, are used extensively for the fermentation of dairy products (Canchaya
et al. 2006). The species used the most frequently in clinical trials are Lactobacillus
acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus
plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus
salivarius, and Lactobacillus sporogenes (Pacifico et al. 2014; Tong et al. 2007;
Wang et al. 2013).
Lactobacillus species is not only the most studied probiotic but also the first
tested against H. pylori. Shortly after the discovery of H. pylori, L. acidophilus was
described in the in vitro inhibition of H. pylori, as was the inhibition by both
L. acidophilus culture supernatant and 1 and 3 % lactic acid (Bhatia et al. 1989). At
this time, the inhibitory effect was related to the extracellular secretory products of
Lactobacillus, since H. pylori was also inhibited by solutions of lactic acid (Bhatia
et al. 1989; Lambert and Hull 1996). Other Lactobacillus species, especially
L. casei, were then also associated with an inhibitory effect on the growth of
H. pylori. Strain specificity concerning an inhibitory effect on H. pylori was also
described, since not all strains tested presented the same effect (Midolo et al. 1995).
There are numerous in vitro studies demonstrating the capacity of inhibition of
H. pylori growth by Lactobacillus spp. (reviewed in (Gotteland et al. 2006; Vitor
and Vale 2011)). Lactobacillus spp. belong to the group of microbiota gut bacteria
which being acid resistant can persist in the stomach longer than other bacteria
(Gotteland et al. 2006). An important additional characteristic of Lactobacillus spp.
is their ability to adhere to gastric cells in vitro (Conway et al. 1987) and in vivo,
both in animals (Declich et al. 2000) and in humans (Valeur et al. 2004), and to
inhibit H. pylori adherence to gastric epithelial cells, which is fundamental for the
establishment of the infection, as demonstrated in vitro using cultured standard
laboratory cell lines such as AGS (Lin et al. 2009; Rokka et al. 2008) and Caco-2
(Myllyluoma et al. 2008).
Valeur and co-workers demonstrated by fluorescence in situ hybridization
(FISH) the colonization of the human stomach, duodenum, and ileum by
L. reuteri after the administration to 19 volunteers of a dietary supplementation
of strain ATCC 55730, a heterofermentative autochthonous bacterium of the
gastrointestinal tract of humans and animals (Valeur et al. 2004). The presence of
probiotics was also highlighted by strain-specific real-time PCR, showing that the
concentrations of probiotics were significantly higher in the probiotic group than in
the placebo group during the probiotic intervention (Karjalainnen et al. 2012;
Myllyluoma et al. 2005), reinforcing the capacity of colonization of the gut by
probiotics.
Recent meta-analyses reinforce the increased eradication rate and reduction of
antibiotic side effects during the (antibiotic) treatment of H. pylori supplemented by
probiotics containing Lactobacillus, as well as Bifidobacterium (Tong et al. 2007;
Wang et al. 2013). Moreover, the most recent meta-analysis considering ten clinical
trials presented an odds ratio (OR) of 0.31 (95 % confidence interval (CI),
0.12–0.79) for the incidence of total side effects in the probiotic supplementation
(Lactobacillus and Bifidobacterium) versus treatment without probiotics, while
eradication rates by intention-to-treat (ITT) analysis presented an OR of 2.07
(95 % CI 1.40–3.06), favoring probiotics. However, current clinical trials, besides
being very diverse in their design, have not included all human populations,
namely, North Americans and black Africans (Wang et al. 2013).
Clinical trials using Lactobacillus or placebo supplementation to antibiotic
therapy present eradication rates favoring probiotics (Tong et al. 2007), especially
in the study by Canducci and colleagues (2000), which showed a statistical signif-
icant result in comparison to other clinical trials (Armuzzi et al. 2001a, b; Sykora
et al. 2005). The comparison of seven clinical trials targeting children (Pacifico
et al. 2014) with one group receiving antibiotic therapy and another receiving the
same therapy plus a probiotic showed a globally beneficial effect of probiotics

concerning the probiotic group, although of the three studies that used only Lacto-
bacillus strains (Lionetti et al. 2006; Sykora et al. 2005; Szajewska et al. 2009), only
one (Sykora et al. 2005) presented a statistically significant result. In a clinical trial,
patients who received a lyophilized and inactivated culture of L. acidophilus
showed an improved eradication rate in comparison to patients just receiving
antibiotic therapy (Canducci et al. 2000), but according to the definition of probi-
otic, this is not a probiotic trial.
Several clinical trials rely on the administration of dairy products. The storage
conditions to preserve the required number of probiotics are extremely important.
For instance, the initial dose of colony-forming units (CFU) (1–1.4 107) of
L. gasseri (LG 21) present in a yogurt decreased by approximately 50 % following
1 week of storage (Sakamoto et al. 2001), which may introduce a bias in the clinical
trials since the probiotic load appears to be very important for a successful
eradication.
24.3.3 Bifidobacterium
The genus Bifidobacterium belongs to the phylum Actinobacteria and is character-

ized by high G+C content Gram-positive bacteria, which are common inhabitants of
the gastrointestinal tract of mammals, birds, and certain cold-blooded animals
(Turroni et al. 2011). Bifidobacteria were first isolated from feces of a breast-fed
infant in 1899 and were quickly associated with a healthy infant gut because of their
predominance in breast-fed infants in comparison to formula-fed ones. The human
milk seems to be a source of living bifidobacteria for the infant gut (Martin
et al. 2009). Due to their morphological and physiological features, similar to
lactobacilli, they were classified as members of the genus Lactobacillus during a
large part of the twentieth century and have been recently recognized as a different
genus. Currently, the genus Bifidobacterium is comprising over 30 species.
Bifidobacteria are important members of the human gut microbiota and play a
beneficial role in maintaining the health of the host (Martin et al. 2009; Turroni
et al. 2011). Together with lactic acid bacteria, bifidobacteria are used in yogurt
production and are known to have probiotic effects (Wang et al. 2004).
Bifidobacteria are known to adhere to epithelial cells and inhibit enteropatho-
genic cell interactions (Bernet et al. 1993). The inhibition of H. pylori by
Bifidobacterium appears to occur via immunomodulatory effects (Shirasawa
et al. 2010; Wang et al. 2013; Zhang et al. 2008). In fact, H. pylori upregulates
the expression of the inflammatory chemokine interleukin (IL)-8 in the gastric
mucosa. A microarray study demonstrated that the expression of IL-8 diminished
in the human scirrhus-type gastric cancer epithelial cell line, GCIY, which had been
preincubated with the probiotic Bifidobacterium bifidum BF-1 and then infected
with H. pylori. Moreover, the action of Bifidobacterium appears to affect the
regulatory mechanism of the nuclear factor kappa-B (NF-κB) signaling pathway,
since H. pylori-induced and B. bifidum-suppressed genes overlap with the genes

regulated by NF-κB (Shirasawa et al. 2010).
The majority of the clinical trials that include Bifidobacteria also include other
probiotic microorganisms. For example, in one trial, Bifidobacterium BF1 and
Streptococcus thermophilus YIT 2021 fermented milk were administrated to a
group of 79 randomized healthy individuals and appeared important for the main-
tenance of stomach health (Miki et al. 2007); others are described in Table 24.1.
Nevertheless, in vitro and in vivo animal studies using only Bifidobacterium strains
(B. bifidum CECT 7366) showed anti-H. pylori effects and partially repaired
damages in the gastric tissue of the BALB/c mouse model (Chenoll et al. 2011).
24.3.4 Saccharomyces
The third most used probiotic is a eukaryotic microorganism. The yeast Saccharo-
myces comprises eight species (Hittinger 2013). Most of the yeast strains used for
alcoholic fermentation are now recognized as Saccharomyces cerevisiae, which is
used for baking, brewing, winemaking, and distillation industries, and it was first
used for the production of fermented beverages in China about 9000 years ago
(Dequin and Casaregola 2011). The probiotic yeast is Saccharomyces boulardii,
which is used worldwide to combat antibiotic side effects. This probiotic yeast was
originally isolated from litchi fruit in Indonesia and described as a separate species
but is now considered to be conspecific with S. cerevisiae. However, the probiotic
strains of S. boulardii present tight clustering both genetically and metabolically
(MacKenzie et al. 2008).
A recent meta-analysis focusing on the efficacy and safety of S. boulardii in
treating several diseases recommends the use of 1000 mg/day for 2 weeks in
supplementation to standard triple therapy to prevent H. pylori symptoms
(McFarland 2010).
S. boulardii is considered to be safe but may cause fungemia, especially in
patients with central venous lines, and this issue should be considered while the
number of antimycotics available is reduced (Vandenplas et al. 2009).
Most clinical trials are consensual in demonstrating that the administration of
S. boulardii diminishes the incidence of side effects (Cindoruk et al. 2007; Duman
et al. 2005; Hurduc et al. 2009; Song et al. 2010) but rarely points to an improve-
ment in the eradication rate (Song et al. 2010).
S. boulardii survives in gastric acid and bile only short time (Vandenplas
et al. 2009), as less than 1 % of the S. boulardii cells are viable 2 h under gastric
conditions (Vanhee et al. 2010). A stable concentration of this probiotic is reached
after three consecutive days of administration and S. boulardii is undetectable
1 week after the end of the treatment (Vandenplas et al. 2009). S. boulardii appears
to alter the structure of H. pylori, as demonstrated in an ultrastructure study in
which the closest bacteria to the yeast showed altered morphology (Vandenplas
et al. 2009). S. boulardii inhibits the NF-κB inflammation pathway via the secretion
Table 24.1 Probiotics tested in blind, placebo-controlled, randomized trials
24
Meta-analysis results
Improved A – Tong et al. (2007)
Patients Improved side B – Wang et al. (2013)
Probiotic enrolled eradication effects Other comments Reference C – Li et al. (2014)
Lactobacillus casei, Lactobacil- 180 No Yes/no 1 108 CFU, twice daily for Shavakhi
lus rhamnosus, Lactobacillus 14 days et al. (2013)
acidophilus, Lactobacillus Less frequent diarrhea/more
bulgaricus, Bifidobacterium frequent abdominal pain
breve, Bifidobacterium longum, 66 Yes Yes 1 109 CFU once daily for Ahmad C – Improved eradication
and Streptococcus thermophilus (children) 4 weeks et al. (2013) and diminished total side
effects and nausea/vomiting
Lactobacillus plantarum, Lacto- 68 No Yes 1 109 CFU to 5 109 CFU Tolone C – Diminished total side
bacillus reuteri, Lactobacillus (children) daily for 7 days et al. (2012) effects and diarrhea
casei subsp. rhamnosus,
Bifidobacterium infantis,
Bifidobacterium longum, Lacto-
bacillus salivarius, Lactobacil-
lus acidophilus, Streptococcus
thermophilus, and Lactobacillus
sporogenes (Probinul –
CaDiGroup)
Saccharomyces boulardii 991 Yes Yes 1.8 109 CFU (Bioflor250, Song
Kuhnil Pharmacy), three times a et al. (2010)
day for 4 weeks. Helped com-
pleting therapy
90 No Yes 1.8 109 CFU (Enterol, Hurduc C – Diminished total side
Probiotics as an Alternative Therapy for Helicobacter pylori-Associated Diseases
(children) Biocodex) once daily for et al. (2009) effects

4 weeks
(continued)
553
554

124 No Yes 500 mg twice daily for 2 weeks Cindoruk
(Reflor, Sanofi–Synthelabo Ilac et al. (2007)
A.S.)
389 No Yes Prevented antibiotic-associated Duman A – Diminished side effects
diarrhea 500 mg twice daily for et al. (2005) (diarrhea)
2 weeks
Lactobacillus GG 83 No No 109 CFU for 7 days Szajewska C – Did not improved
(children) et al. (2009) eradication or side effects
60 No Yes 6 109 (Giflorex®, Errekappa Armuzzi A – Did not improved
Euroterapici) twice daily for et al. (2001a) eradication
14 days Diminished side effects
(diarrhea, taste disturbance,
and nausea)
120 No Yes 6 109 twice daily for 14 days Armuzzi A – Did not improved
et al. (2001b) eradication
Diminished side effects
(diarrhea and taste
disturbance)
Lactobacillus reuteri 40 No Yes 108 CFU (Reuterin, Noos) once Francavilla
daily for 4 weeks. Decreased the et al. (2008)
occurrence of dyspeptic
symptoms
40 No Yes 108 CFU (Reuterin, Noos) once Lionetti
(children) daily for 20 days et al. (2006)
Lactobacillus casei DN-114 001 86 Yes Infrequent 1010 CFU – fermented milk Sykora A – Improved eradication
(children) side (Actimel) for 2 weeks et al. (2005) C – Improved eradication
effects
F.F. Vale et al.
Lactobacillus casei subsp. casei 70 Yes Yes 109 CFU twice daily for 10 days Tursi A – Diminished total side
DG et al. (2004) effects
24
Lactobacillus johnsonii La1 326 Yes –a >107/ml (80 ml Chamyto, Cruchet

(children) Nestlé-Chile SA) for 4 weeks et al. (2003)
Lactobacillus gasseri OLL2716 29 Yes –a 1–1.4 107 CFU/g (90 g yogurt) Sakamoto
twice daily for 16 weeks et al. (2001)
Bacillus subtilis and Streptococ- 352 Yes Yes 8 weeks Park
cus faecium et al. (2007)
Bacillus clausii (Enterogermina, 120 No Yes 2 109 spores Nista A – Eradication not
Sanofi–Synthelabo) et al. (2004) significant
Once daily for 3 weeks Diminished side effects
(diarrhea, epigastric pain,
and nausea)
Bifidobacterium animalis and 65 No No 107/ml (200 ml yogurt) once Goldman A – Did not improve
Lactobacillus casei (children) daily for 3 months et al. (2006) eradication
B – Did not improve
eradication
C – Did not improve
eradication
Lactobacillus and 138 Yes Yes 5 109/ml (200 ml AB-Yogurt, Sheu A – Improved eradication
Bifidobacterium President Corp) for 4 weeks et al. (2006) B – Improved eradication
160 Yes Yes Restored the depletion of Sheu A – Improved eradication
Bifidobacterium in stools after et al. (2002) B – Improved eradication
triple therapy. 5 109/ml
(200 ml AB-Yogurt, President
Corp) twice daily for 5 weeks
(continued)
555
556

Lactobacillus acidophilus LB 254 Yes –a LA 109 heat killed (Lacteol Gotteland
and Saccharomyces boulardii (children) Forte, Laboratory of et al. (2005)
Dr. Boucard), SB (Perenteryl,
Merck Quı́mica Chilena)
250 mg for 8 weeks
S. boulardii-eradicated
H. pylori in 12 % of the colo-
nized children, and
L. acidophilus in 6 %
Lactobacillus plantarum, Lacto- 206 Yes Yes 1–2 109 (Lf100, Dicofarm®; de Bortoli B – Improved eradication,
bacillus reuteri, L. casei, Lacto- and Probinul, CaDiGroup®) et al. (2007) diminished side effects
bacillus salivarius, twice daily for 7 days
Lactobacillus acidophilus, 31 No No LR 108 CFU (Reuflor), others Scaccianoce B – Eradication and side
Bifidobacterium infantis, 1–5 109 CFU (Probinul) twice et al. (2008) effects did not improve
Bifidobacterium longum, Strep- daily for 14 weeks
tococcus thermophilus
Lactobacillus acidophilus La5 70 Yes –a 107 CFU (AB-Yogurt) twice Wang
or Bifidobacterium lactis Bb12 daily for 6 weeks et al. (2004)
Lactobacillus acidophilus 337 No No LA, LC >105 CFU/ml, BL >106 Yoon B – Eradication and side
HY2177, Lactobacillus casei CFU/ml, ST >108 CFU/ml et al. (2011) effects did not improve
HY2743, Bifidobacterium (150 ml Will yogurt) once daily
longum HY8001, and Strepto- for 4 weeks
coccus thermophilus B-1 347 Yes No Will yogurt once daily for Kim
3 weeks et al. (2008)
Lactobacillus rhamnosus GG, 58 – Yes 109 CFU/ml twice daily for Myllyluoma
Lactobacillus rhamnosus 1 week and once daily for et al. (2007)
LC705, Propionibacterium 3 weeks. Probiotic combination
F.F. Vale et al.
freudenreichii ssp. shermanii JS, resulted in only minor changes

and Bifidobacterium breve Bb99 in the microbiota
24
L. rhamnosus GG, L. rhamnosus 47 No Yes 1 109 CFU/ml Myllyluoma A – Eradication and side
LC705, Bifidobacterium breve Twice daily during 7 days and et al. (2005) effects not significant
Bb99, and Propionibacterium once daily for 3 weeks
freudenreichii ssp. shermanii JS
Lactobacillus GG, S. boulardii, 85 No Yes 5–6 109 twice daily 14 days Cremonini A – Eradication not
Lactobacillus acidophilus, and (Giflorex, Errekappa et al. (2002) significant
Bifidobacterium lactis Euroterapici; Codex, Diminished side effects
SmithKline Beecham; Ferzym, (diarrhea and taste
Specchiasol) disturbance)
a
Probiotics administered alone (without antibiotic therapy)
557
of a soluble, heat-stable, small (<1 kDa) anti-inflammatory factor. This anti-

inflammatory factor blocks NF-κB activation and NF-κB-mediated IL-8 gene
expression, which may be responsible for the anti-inflammatory action of
S. boulardii (Sougioultzis et al. 2006).
24.3.5 Streptococcus
Streptococcus spp. are Gram-positive anaerobic aerotolerant bacteria and the gen-
era Streptococcus and Lactobacillus belong to the same order, Lactobacillales. The
most used Streptococcus species is Streptococcus thermophilus, which is also
considered as a lactic acid bacterium, since it is found in milk-derived products.
The clinical trials that included S. thermophilus used combined probiotics, together
with Lactobacillus and Bifidobacterium (Shavakhi et al. 2013; Ahmad et al. 2013;
de Bortoli et al. 2007; Scaccianoce et al. 2008; Kim et al. 2008; Yoon et al. 2011).
The results obtained with these clinical trials are rather diverse, although the
majority point to a decrease in side effects (Table 24.1). Streptococcal virulence-
related genes (associated with pathogenic streptococci) are either inactivated or
absent in sequenced genomes of S. thermophilus (Prajapati et al. 2013).
S. thermophilus isolates produce bacteriocins, which are excreted antimicrobial
peptides with the ability to inhibit the growth of other bacteria (Gul et al. 2012).
Sterilized and neutralized fluid supernatants of S. thermophilus (and Lactobacillus
spp.) were shown to inhibit H. pylori in vitro, which suggests the presence of
bacteriocin-like substances (Aslim et al. 2011). The other tested probiotic Strepto-
coccus is Streptococcus faecalis, administrated in combination with Bacillus
subtilis (Park et al. 2007). S. faecalis is a microbiota bacterium of the human
gastrointestinal tract. This isolated study showed an increased eradication rate
and decrease in side effects (Table 24.1).
24.3.6 Propionibacterium
The Propionibacterium genus, as well as the Bifidobacterium genus, belongs to the

Actinobacteria class. Propionibacterium are Gram-positive, high G+C%,
mesophilic, and aerotolerant bacteria, with pleomorphic rods. The main end prod-
uct of fermentation is named propionic acid. The Propionibacterium genus is
currently comprised of 14 species, divided in two groups, dairy and cutaneous
propionibacteria. Four typical dairy species were described early: Propioni-
bacterium freudenreichii, Propionibacterium acidipropionici, Propionibacterium
jensenii, and Propionibacterium thoenii. P. freudenreichii, a food grade bacterium
consumed both in cheeses and in probiotic preparations, first described more than a
century ago in Swiss Emmental cheese (Thierry et al. 2011), has been used in
combination with other probiotics to adjuvant H. pylori therapy. P. freudenreichii
was in the past divided into two subspecies: P. freudenreichii subsp. freudenreichii
which is unable to ferment lactose and shows nitrate reductase activity and
P. freudenreichii subsp. shermanii which exhibits the opposite properties.
P. freudenreichii consumption modulates the gut microbiota, enhancing the
bifidobacterial population and decreasing the Clostridium and Bacteroides
populations (Saraoui et al. 2013). In vitro studies showed that P. freudenreichii
subsp. shermanii inhibits H. pylori adhesion to epithelial cells and, in combination
with other probiotics, inhibits H. pylori-induced cell membrane leakage and
H. pylori-induced IL-8 release (Myllyluoma et al. 2008). P. freudenreichii ssp.
shermanii JS has been used in combination with other probiotics (Myllyluoma
et al. 2005, 2007), showing an improvement of side effects, which were not
confirmed by further meta-analysis studies (Tong et al. 2007).
24.3.7 Other Probiotic Microorganisms
Clinical trials presented in Table 24.1 also show two isolated studies that used
Bacillus species. Park and co-workers used Bacillus subtilis in combination with
S. faecium (see above) (Park et al. 2007) and Nista and colleagues used Bacillus
clausii spores alone (Nista et al. 2004), both showing reduction of antibiotic side
effects. Bacillus are Gram-positive bacteria capable of forming spores. Bacillus
clausii spores can survive the gastric pH, activate and reach the intestinal tract
where they germinate to vegetative forms (Urdaci et al. 2004). B. clausii is a
probiotic used for viral diarrhea in children and for antibiotic-related side effects
that can release antimicrobial substances. B. clausii spores and cells can adhere to
the bowel wall and colonize the mucosa (Nista et al. 2004).
The list of other probiotics tested in vitro on H. pylori cultures is much more
extensive. The in vitro test of the effect of 32 microorganisms against H. pylori
clinical isolates was determined by a diffusion method, showing that several species
presented an inhibitory action against H. pylori. These were both Gram-positive
and Gram-negative microorganisms, such as Staphylococcus spp. (Staphylococcus
auricularis, Staphylococcus epidermis, Staphylococcus hominis, and Staphylococ-
cus aureus), Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloa-
cae, and Stenotrophomonas maltophilia (Lopez-Brea et al. 2008). Excluding the
pathogenic microbes, the other microorganisms could be a starting point to explore
other probiotics to be used with H. pylori infection.
24.4 Characterization of Probiotics Studies
A probiotic study typically starts with in vitro tests, where a potential probiotic
microorganism is tested by a diffusion method or something similar on an H. pylori
culture. Microorganisms presenting an inhibitory effect against H. pylori are then
further characterized. To determine if the inhibitory action is dependent on a

secreted antimicrobial metabolite, culture supernatants of the probiotic may be
obtained, concentrated, and tested again by the agar diffusion method (Lopez-
Brea et al. 2008). Inhibition liquid culture assay may alternatively be used to screen
potential anti-H. pylori activity of probiotics (Hsieh et al. 2012). Also of importance
is the study of adherence to mucus and/or human epithelial cells and cell lines. The
use of gastric epithelial cells cocultured with H. pylori and the probiotic allows
further characterization, for instance, of changes in the adhesion of H. pylori to
these cells or alteration of cytokine secretion by the epithelial cells (Hsieh
et al. 2012; Myllyluoma et al. 2008; Shirasawa et al. 2010). Of course, before
proceeding to animal trials, certain tests should be performed, namely, the resis-
tance to acidity, bile acid resistance, and the ability to reduce pathogen adhesion to
surfaces (FAO and WHO 2002). Animal models permit further study of the gastric
acid regulation, the histopathological analysis, the bacterial load and motility, and
immunologic response, among others (Hsieh et al. 2012; Isobe et al. 2012). The
own microbiota of the animal model is not currently considered but may interfere
with the effect of the probiotics administered. The final step is the use of clinical
trials (Table 24.1). The randomized, double-blind placebo-controlled human trials
available are still few, some involving a small numbers of patients, and often
difficult to compare because of differences in probiotic strains employed, doses,
and formulation (Bergonzelli et al. 2005).
24.5 Probiotics and Immune System Stimulation
The beneficial effect of probiotics on human health was originally attributed to

improvements in the intestinal microbial balance, but there is now growing evi-
dence that probiotics can also provide benefits by modulating immune functions. A
few examples of probiotic impact at different levels of the immune system will be
given.
Probiotics can act at different levels as immunomodulators, by influencing
specially the adaptive immunity, both the systemic and the mucosal types
(reviewed in (Kemgang et al. 2014)).
The mucosal immunity is mediated by intraepithelial lymphocytes and other
immunomodulatory cells of the mucosal lamina propria, such as cytokine-
producing cells, phagocytic cells, goblet cells, and IgA-secreting cells.
Several studies support the effect of probiotic lactobacilli in IgA secretion in
intestinal fluid and in the number of IgA-secreting cells. For example, two studies
using mice models showed the ability of L. casei Zhang (Ya et al. 2008) and
Lactobacillus crispatus KT-11 (Tobita et al. 2010) to increase the resistance of
gastrointestinal mucosa to infections by inducing a significant increase in the
concentration of secretory IgA in intestinal fluid, in a dose-dependent manner
compared with the controls. Another study showed that yogurt supplemented with
L. acidophilus and Bifidobacterium spp. administered to mice stimulated enhanced

mucosal and systemic anticholera toxin IgA (Sanders and Klaenhammer 2001).
The most extensively studied effect of immunomodulation by probiotics is
cytokine secretion from intestinal epithelial cells, which is strongly dependent on
the probiotic strain, with differences being observed even among strains of the same
species.
Several probiotic strains can prevent degradation of the NF-κB inhibitor, there-
fore preventing the expression of pro-inflammatory cytokines like IL-8 by the
intestinal epithelial cells (Thomas and Versalovic 2010). Zhang et al. (2005),
using an epithelial cell model, demonstrated the effect of both viable and heat-
killed Lactobacillus GG in the decrease in degradation of the NF-κB inhibitor and
subsequent inhibition of NF-κB translocation into the nucleus, resulting in
decreased IL-8 production (Zhang et al. 2005). Another study showed that
pretreatment of epithelial cells with L. casei DN-114 001 decreased Shigella
flexneri-induced NF-κB activation due to inhibition of NF-κB inhibitor degradation
(Tien et al. 2006).
Toll-like receptors (TLR) are proteins involved in the prokaryotic macromolec-
ular motifs recognition. TLR stimulation by probiotics has been extensively studied
in vivo and in vitro with different types of immune cells, and this effect appears to
be strain specific. An example of this effect is the probiotic Lactobacillus paracasei
ssp. paracasei DC412 stimulation of TLR2- (peptidoglycan recognition) and
TL4-mediated (lipopolyssaccharide recognition) signaling events that lead to secre-
tion of several cytokines (Kourelis et al. 2010). These strong pro-inflammatory
responses after TLR stimulation are important host-defense mechanisms against
dangerous exogenous and endogenous factors. Another example of the immuno-
modulatory effect of probiotics at the mucosal level is their action on mucins and
antimicrobial peptide production. Recently, Tobita and co-workers demonstrated
that L. crispatus K-11 increased mucin 13 and defensin alpha genes expression in
Peyer’s patches by twofold compared with control, ensuring constant protection
against the attack of digestive fluid, microorganisms, pollutants, and toxins (Tobita
et al. 2010).
Via the lymph nodes, immune cells can migrate from the lamina propria into the
circulation system, therefore influencing the systemic immune system involving the
production of IgA, IgG antibodies, and a wide range of cytokines (Th1, Th2, and
regulatory cytokines). It was recently demonstrated that Lactobacillus plantarum,
L. salivarius, and Lactococcus lactis attenuate Th2 response and increase the
production of regulatory cytokines in healthy mice in a strain-dependent manner
(Smelt et al. 2012).
Another study, using seven probiotic strains from Lactobacillus and
Bifidobacterium genera, administered individually in the form of two capsules/
day for 21 days containing 1 1010 CFU/capsule to 83 healthy volunteers aged
18–62 years, also receiving an oral cholera vaccine, showed an effect of some of the
tested strains on specific humoral responses. During early response (day 0–21),
serum IgG significantly increased in subjects consuming B. lactis Bl-04 and
L. acidophilus La-14 compared with controls. These results suggest that specific
strains of probiotics may thus act as adjuvants to the humoral immune response
following oral vaccination (Paineau et al. 2008).
24.6 Mechanism of Action
The mode of action of probiotics is not completely understood, but they probably
replace normal microflora following antibiotic therapy until recovery is achieved
(McFarland 2010). The use of probiotics to directly inhibit H. pylori does not seem
viable in the acidic stomach. However, probiotics can help the microbiome of the
patients during and after antibiotic therapy.
The specificity of action rather than the source of the microorganism is the most
important (Pineiro and Stanton 2007).
In a general way, probiotics’ action occurs via competition for adhesion sites and
nutrients. This impairs the colonization of the pathogen and increases the produc-
tion of antimicrobial compounds like acids or bacteriocins which inhibit pathogen
growth and reduce inflammation and tissue destruction, enhancing the host immune
response and thus promoting indirectly pathogen elimination (Rastogi et al. 2011).
Most of the described mechanisms are related to the immune system modulation
described above.
There are a few examples in which the mechanism of action is more detailed and
they are presented below. The active compounds identified in Propionibacterium
are 1,4-dihydroxy-2-naphtoic acid (DHNA), which is the penultimate intermediate
in the biosynthesis pathway of vitamin K2 (Isawa et al. 2002), and conjugated
linoleic acids (CLAs), which have anticarcinogenic properties (Thierry et al. 2011).
The L. casei strain Shirota YIT9029 presented in vitro and in vivo antagonism of
H. pylori and Salmonella, inhibiting the swimming motility of these bacteria. The
probiotic action on Salmonella was reversible. However, this probiotic produced an
irreversible inhibition of the swimming motility of H. pylori accompanied by the
presence of coccoid morphologies and loss of FlaA and FlaB flagellin expression
(Le et al. 2013). L. gasseri OLL2716 strain also induces the coccoid conversion of
H. pylori (Fujimura et al. 2012).
24.7 Safety and Delivery Systems of Probiotics
Probiotics are living microorganisms and therefore may represent a risk for human
health. They are all nonpathogenic; however, since probiotics interfere with com-
mensal microflora, as well as have immunomodulatory effects, they can result in
opportunistic outcomes in the host due to bacteremia and fungemia. The main
observed adverse effects of probiotics are sepsis, fungemia, and gastrointestinal
ischemia. Generally, critically ill patients in intensive care units, critically sick
children, postoperative and hospitalized patients, and patients with immune-
compromised complexity are the most at-risk populations (reviewed in (Didari

et al. 2014; Redman et al. 2014)).
Another important aspect of probiotic safety is the antibiotic resistance charac-
teristics. Indeed, they should not carry transmissible antibiotic resistance genes that
could potentially be transferred by horizontal gene transfer to pathogenic bacteria.
For example, Lactobacilli, one of the most common probiotics, display a wide
range of antibiotic resistances naturally (Charteris et al. 1998), but in most cases,
antibiotic resistance is not of the transmissible type. In addition, plasmid-linked
antibiotic resistances are not very common among lactobacilli, but they can occur
(Rinckel and Savage 1990), and their safety implications should be taken into
consideration. Resistance transfer from commensal microbiota to probiotics is
also an issue of concern.
Many systems have been developed for the delivery of probiotics to the gastro-
intestinal system including both conventional pharmaceutical systems and
nonconventional commercial products. Probiotic delivery systems vary greatly in
efficacy to exert health benefits for a patient. The degree of health benefits provided
by these probiotic formulations varies in their ability to deliver viable, functional
bacteria in large enough numbers (effectiveness), to provide protection against the
harsh effects of the gastric environment and intestinal bile (in vivo protection), and
to survive formulation processes (viability) (reviewed in (Govender et al. 2014)).
Nonconventional probiotic formulations include mostly cheeses, yogurts,
creams, chocolates, milk, and meat. Due to their easy availability and convenience,
they are good delivery systems that, in addition, can be beneficial to the patient,
when effective. Some of these products have the ability to deliver viable probiotic
bacterial cells to the human intestine but this ability differs greatly. This difference
is a result of various reasons ranging from formulation processes and viability of
dosed bacteria, as well as variability in the ability of different species of bacteria to
survive physiological conditions. For example, Lactobacillus spp. are more viable
in gastric conditions compared to other probiotic species, making it the most ideal
probiotic for delivering systems that do not provide gastric protection
(Maragkoudakis et al. 2006). In the last decades, a considerable amount of research
has been consecrated to improving commercial food-based probiotics delivery
systems.
Conventional pharmaceutical products tend to be more effective regarding
protection of the probiotic bacteria from the human gastrointestinal tract. Indeed,
providing probiotic living cells with a physical barrier against adverse environmen-
tal conditions is an approach which is currently of considerable interest. In addition,
these systems have been characterized much more compared to commercial food-
based carrier systems. Examples of pharmaceutical formulations for the delivery of
probiotics currently include beads, capsules, and tablets (Schrezenmeir and de
2001; Solanki et al. 2013).
24.8 Screening of New Probiotic Bacteria, Production,

and Available Products
The classical procedures and cultured media that have been used so far for bacte-
rially based drug discovery are unable to increase the number of bacteria available
for to assay. These limitations and the frequent rediscovery of known compounds
led the pharmaceutical companies to abandoned screening for new antibiotics. A
novel approach to screening is needed. A good example of a new and efficient
methodology for finding new antibiotics was recently described: sequencing soil
microbiomes and using bioinformatics methods to identify gene clusters predicted
to encode for metabolites that are evolutionarily related to families of natural
products with known bioactivities (Charlop-Powers et al. 2014). A similar approach
could be envisaged for probiotics selection. The Human Microbiome Project is
financing several projects that hopefully will bring new probiotics.
The starting point in screening new probiotic bacteria are in vitro tests as
mentioned above. Of course, there are characteristics which are necessary for the
probiotics, namely, viability at the target site, acid (and bile) tolerance, antimicro-
bial production, and adherence ability to human intestinal cells (Pineiro and Stanton
2007). The mechanism of action should be studied. In vivo tests start with animal
models which, if effective, are then translated into clinical trials. These include
randomized double-blind placebo-controlled human trials which should be under-
taken to establish the efficacy of the probiotic product. Eradication rate and
secondary effects are the most used indicators to evaluate the efficacy of probiotics
for which a statistically significant result is necessary.
Probiotics are often used in fermented foods (Table 24.2) and fermentation
metabolic products appear in the food product, including acetic acid, lactic acid,
and possibly bacteriocins, resulting in a decrease of the product’s pH. Such changes
may affect the stability of probiotic bacteria and may alter the probiotic’s functional
properties. Moreover, long-term industrial use of the starter culture for production
purposes may influence viability and functional properties, as well as storage. Thus,
it is important to constantly control the properties of the probiotics (Tuomola
et al. 2001).
The European Medicines Agency does not coordinate the evaluation of
probiotics; these are evaluated and approved at a national level by the regulatory
agencies of each country. The Food and Drug Administration (FDA) approves the
probiotics available in the USA. The complete list of currently available probiotics
is very difficult to obtain, due to the constant changes, variability of compositions,
and designations given in each country. Table 24.2 presents a nonexhaustive list of
generally recognized as safe (GRAS) microorganisms added to food from the FDA.
There is a high variation in the composition of microorganisms of each product.
Table 24.2 FDA generally recognized as safe (GRAS) microorganisms added to food
Date of
Request by Species Intended use closure
Avitop, Bacteroides xylanisolvens DSM Fermented low-fat and skim Dec
Germany 23964 milk and milk products. Heat- 16, 2013
inactivated cells
Cargill, Inc., Bifidobacterium animalis subsp. Foods Sep
USA lactis Bf-6 29, 2011
Danisco, Inc., Bifidobacterium animalis subsp. Ready-to-eat breakfast Apr
USA lactis strains HN019, Bi-07, cereals, bars, cheeses, milk 10, 2013
Bl-04, and B420 drinks and milk products,
bottled water and teas, fruit
juices, fruit nectars, fruit
“ades” and fruit drinks,
chewing gum, and
confections
Morinaga Milk Bifidobacterium breve M-16V Baked goods, breakfast Sept
Industry Co., cereals, fruit juices and nec- 27, 2013
Ltd., Japan tars, fruit ices, vegetable
juices, milk-based drinks and
powders, dairy product ana-
logs, frozen dairy desserts,
processed cheese, imitation
cheese, cheese spreads,
butter-type products, snack
foods, gelatin, pudding, fill-
ings, meal replacements,
snack bars, nut and peanut
spreads, hard and soft
candies, cocoa-type powder,
and condiment sauces
454 Morinaga Bifidobacterium breve M-16V Nonexempt powdered term Sept
Milk Industry infant formulas (milk or soy 27, 2013
Co., Ltd., based) and exempt powdered
Japan term infant formula
containing partially hydro-
lyzed milk or soy proteins
Danone Trad- Bifidobacterium breve M-16V Exempt term powdered Sept
ing B.V., the amino acid-based formulas 30, 2013
Netherlands
Nestlé, USA Bifidobacterium lactis Bb12 and Milk-based infant formula Mar
Streptococcus thermophilus Th4 that is intended for consump- 19, 2002
tion by infants 4 months and
older
Morinaga Milk Bifidobacterium longum BB536 Breads/baked goods, cereals, July
Industry Co., dairy products/dairy-based 8, 2009
Ltd., Japan foods and dairy substitutes,
fruit products, candy,
chewing gum, cocoa powder,
condiment sauces, flavored
beverage syrups, fruit-
(continued)

Date of
flavored powder beverage
mixes, gelatin desserts,
gravies, margarine, peanut
and other nut butter/spreads,
snack foods, and in milk-
based powdered infant
formula
Danisco, Inc., Lactobacillus acidophilus NCFM In certain dairy products, Apr
USA functional beverages, nutri- 19, 2011
tional powders, juices, bars,
ready-to-eat breakfast
cereals, chewing gum, and
confections
Nutrition Phys- Lactobacillus acidophilus, Lacto- Antimicrobial to control Dec
iology Corp., bacillus lactis, and Pediococcus pathogenic bacteria in meat 7, 2005
USA acidilactici and poultry products
Yakult Honsha Lactobacillus casei Shirota Ingredient in fermented dairy Dec
Co., Ltd., products 10, 2012
Japan
Mead Johnson Lactobacillus casei subsp. Ingredient in term infant May
& Company, rhamnosus GG formula 29, 2008
USA
BioGaia AB, Lactobacillus reuteri DSM 17938 Ingredient in processed Nov
Sweden cheeses, yogurt, ice cream, 18, 2008
fruit juices, fruit drinks,
processed vegetables,
processed vegetable drinks,
beverage bases, energy bars,
energy drinks, and chewing
gum
Nestlé Nutri- Lactobacillus reuteri DSM 17938 Ingredient in powdered Mar
tion, USA whey-based term infant 26, 2012
formula
Micropharma Lactobacillus reuteri NCIMB Beverages and beverage Feb
Ltd., Canada 30242 bases, breakfast cereals, 12, 2013
cheeses, dairy product ana-
logs, fats and oils, frozen
dairy desserts, grain products
and pastas, milk products,
processed fruits and fruit
juices, and sugar substitutes
Fonterra Lactobacillus rhamnosus HN001 Various foods, including cer- Nov
Co-operative tain beverages and beverage 1, 2009
Group, bases (excluding soft drinks);
New Zealand cheeses; milk drinks; milk
products; meal replacements;
energy bars; ready-to-eat
(continued)

Date of
cereals; fruit juices, nectars,
ades, and drinks; confections;
chewing gum; and hard
candies
Fonterra Lactobacillus rhamnosus HN001 Milk-based powdered term Aug
Co-operative produced in a milk-based medium infant formula that is 31, 2009
Group, intended for consumption
New Zealand from the time of birth, as well
as in milk-based powdered
follow-on formula
PURAC, the Cultured dairy sources, sugars, Antimicrobial agents in a Mar
Netherlands wheat, malt, and fruit- and variety of food categories 26, 2012
vegetable-based sources
fermented by Streptococcus
thermophilus, Bacillus
coagulans, Lactobacillus aci-
dophilus, Lactobacillus paracasei
subsp. paracasei, Lactobacillus
plantarum, Lactobacillus sakei,
Lactobacillus bulgaricus, and
Propionibacterium freudenreichii
subsp. shermanii or mixtures of
these strains
Meiji Co., Ltd., Heat-killed Propionibacterium Beverages and beverage Dec
Japan freudenreichii ET-3 culture bases, breakfast cereals, 26, 2012
(powder) cheeses, coffee and tea, fats
and oils, frozen dairy desserts
and mixes, gelatins, pud-
dings, and fillings, grain
products and pastas, milk
products, processed fruits and
fruit juices, processed vege-
tables and vegetable juices,
and soft candy
Taking into consideration the increasing knowledge of the human gut microbiota
and the fact that probiotics are generally regarded as safe, reducing greatly the
secondary effects produced by current therapies applied, probiotics appear to be
here to stay. The administration of probiotics alone does not eradicate H. pylori but
consistently diminish the side effects when added as an adjuvant to the therapy.
Indirectly, probiotics can contribute to the eradication of H. pylori. The mecha-
nisms of action are not fully understood but appear to be based on three pillars:
competition, antimicrobial activity, and modulation of the immune system and
microbiota. The new tools to study the human microbiome will probably give new
probiotics. Quality assurance protocols should be constantly applied to guarantee
the safety of probiotics.
References
Ahmad K, Fatemeh F, Mehri N, Maryam S (2013) Probiotics for the treatment of pediatric
Helicobacter pylori infection: a randomized double blind clinical trial. Iran J Pediatr 23:79–84
Armuzzi A, Cremonini F, Bartolozzi F, Canducci F, Candelli M, Ojetti V et al (2001a) The effect
of oral administration of Lactobacillus GG on antibiotic-associated gastrointestinal side-effects
during Helicobacter pylori eradication therapy. Aliment Pharmacol Ther 15:163–169
Armuzzi A, Cremonini F, Ojetti V, Bartolozzi F, Canducci F, Candelli M et al (2001b) Effect of
Lactobacillus GG supplementation on antibiotic-associated gastrointestinal side effects during
Helicobacter pylori eradication therapy: a pilot study. Digestion 63:1–7
Aslim B, Onbasili D, Yuksekdag ZN (2011) Determination of lactic acid production and antag-
onistic activity against Helicobacter pylori of L. delbrueckii subsp bulgaricus and
S. thermophilus strains. Kafkas Univ Vet Fak Derg 17:609–614
Basso D, Plebani M, Kusters JG (2010) Pathogenesis of Helicobacter pylori infection.
Helicobacter 15(Suppl 1):14–20
Bergonzelli GE, Blum S, Brussow H, Corthesy-Theulaz I (2005) Probiotics as a treatment strategy
for gastrointestinal diseases? Digestion 72:57–68
Bernet MF, Brassart D, Neeser JR, Servin AL (1993) Adhesion of human bifidobacterial strains to
cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.
Appl Environ Microbiol 59:4121–4128
Bhatia SJ, Kochar N, Abraham P, Nair NG, Mehta AP (1989) Lactobacillus acidophilus inhibits
growth of Campylobacter pylori in vitro. J Clin Microbiol 27:2328–2330
Bik EM, Eckburg PB, Gill SR, Nelson KE, Purdom EA, Francois F et al (2006) Molecular analysis
of the bacterial microbiota in the human stomach. Proc Natl Acad Sci U S A 103:732–737
Blaser MJ (2010) Harnessing the power of the human microbiome. Proc Natl Acad Sci U S A
107:6125–6126
Brown SM (2013) Introduction to DNA sequencing. In: Brown SM (ed) Next-generation DNA
sequencing informatics. CSHL Press, New York
Canchaya C, Claesson MJ, Fitzgerald GF, van Sinderen D, O’Toole PW (2006) Diversity of the
genus Lactobacillus revealed by comparative genomics of five species. Microbiology
152:3185–3196
Canducci F, Armuzzi A, Cremonini F, Cammarota G, Bartolozzi F, Pola P et al (2000) A
lyophilized and inactivated culture of Lactobacillus acidophilus increases Helicobacter pylori
eradication rates. Aliment Pharmacol Ther 14:1625–1629
Cao YJ, Qu CM, Yuan Q, Wang S, Liang S, Yang X (2005) Control of intestinal flora alteration
induced by eradication therapy of Helicobacter pylori infection in the elders. Chin J
Charlop-Powers Z, Owen JG, Reddy BV, Ternei MA, Brady SF (2014) Chemical-biogeographic
survey of secondary metabolism in soil. Proc Natl Acad Sci U S A 111:3757–3762
Charteris WP, Kelly PM, Morelli L, Collins JK (1998) Antibiotic susceptibility of potentially
probiotic Lactobacillus species. J Food Prot 61:1636–1643
Chenoll E, Casinos B, Bataller E, Astals P, Echevarria J, Iglesias JR et al (2011) Novel probiotic
Bifidobacterium bifidum CECT 7366 strain active against the pathogenic bacterium
Helicobacter pylori. Appl Environ Microbiol 77:1335–1343
Cindoruk M, Erkan G, Karakan T, Dursun A, Unal S (2007) Efficacy and safety of Saccharomyces
boulardii in the 14-day triple anti-Helicobacter pylori therapy: a prospective randomized
placebo-controlled double-blind study. Helicobacter 12:309–316
Clarke G, Stilling RM, Kennedy PJ, Stanton C, Cryan JF, Dinan TG (2014) Gut microbiota: the
neglected endocrine organ. Mol Endocrinol 28:1221–1238
Conway PL, Gorbach SL, Goldin BR (1987) Survival of lactic acid bacteria in the human stomach
and adhesion to intestinal cells. J Dairy Sci 70:1–12
Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R (2009) Bacterial community
variation in human body habitats across space and time. Science 326:1694–1697
Costello EK, Stagaman K, Dethlefsen L, Bohannan BJ, Relman DA (2012) The application of
ecological theory toward an understanding of the human microbiome. Science 336:1255–1262
Cremonini F, Di CS, Covino M, Armuzzi A, Gabrielli M, Santarelli L et al (2002) Effect of
different probiotic preparations on anti-Helicobacter pylori therapy-related side effects: a
parallel group, triple blind, placebo-controlled study. Am J Gastroenterol 97:2744–2749
Crew KD, Neugut AI (2006) Epidemiology of gastric cancer. World J Gastroenterol 12:354–362
Cruchet S, Obregon MC, Salazar G, Diaz E, Gotteland M (2003) Effect of the ingestion of a dietary
product containing Lactobacillus johnsonii La1 on Helicobacter pylori colonization in chil-
dren. Nutrition 19:716–721
de Bortoli N, Leonardi G, Ciancia E, Merlo A, Bellini M, Costa F et al (2007) Helicobacter pylori
eradication: a randomized prospective study of triple therapy versus triple therapy plus
lactoferrin and probiotics. Am J Gastroenterol 102:951–956
Declich P, Ambrosiani L, Grassini R, Tavani E, Bellone S, Bortoli A et al (2000) Fundic gland
polyps: a still elusive entity on the eve of the year 2000. Pol J Pathol 51:3–8
Dequin S, Casaregola S (2011) The genomes of fermentative Saccharomyces. C R Biol
334:687–693
Dicksved J, Lindberg M, Rosenquist M, Enroth H, Jansson JK, Engstrand L (2009) Molecular
characterization of the stomach microbiota in patients with gastric cancer and in controls. J
Didari T, Solki S, Mozaffari S, Nikfar S, Abdollahi M (2014) A systematic review of the safety of
probiotics. Expert Opin Drug Saf 13:227–239
Duman DG, Bor S, Ozutemiz O, Sahin T, Oguz D, Istan F et al (2005) Efficacy and safety of
Saccharomyces boulardii in prevention of antibiotic-associated diarrhoea due to Helicobacter
pylori eradication. Eur J Gastroenterol Hepatol 17:1357–1361
FAO and WHO (2001) Report on joint FAO/WHO expert consultation on evaluation of health and
nutritional properties of probiotics in food including powder milk with live lactic acid bacteria.
Ref Type: Report
FAO and WHO (2002) Report of a joint FAO/WHO working group on drafting guidelines for the
evaluation of probiotics in food. Ref Type: Report
Fiers W, Contreras R, Duerinck F, Haegeman G, Iserentant D, Merregaert J et al (1976) Complete
nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the
replicase gene. Nature 260:500–507
Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR et al (1995)
Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science
269:496–512
Francavilla R, Lionetti E, Castellaneta SP, Magista AM, Maurogiovanni G, Bucci N et al (2008)
Inhibition of Helicobacter pylori infection in humans by Lactobacillus reuteri ATCC 55730
and effect on eradication therapy: a pilot study. Helicobacter 13:127–134
Fujimura S, Watanabe A, Kimura K, Kaji M (2012) Probiotic mechanism of Lactobacillus gasseri
OLL2716 strain against Helicobacter pylori. J Clin Microbiol 50:1134–1136
Goldman CG, Barrado DA, Balcarce N, Rua EC, Oshiro M, Calcagno ML et al (2006) Effect of a
probiotic food as an adjuvant to triple therapy for eradication of Helicobacter pylori infection
in children. Nutrition 22:984–988
Gotteland M, Poliak L, Cruchet S, Brunser O (2005) Effect of regular ingestion of Saccharomyces

boulardii plus inulin or Lactobacillus acidophilus LB in children colonized by Helicobacter
pylori. Acta Paediatr 94:1747–1751
Gotteland M, Brunser O, Cruchet S (2006) Systematic review: are probiotics useful in controlling
gastric colonization by Helicobacter pylori? Aliment Pharmacol Ther 23:1077–1086
Govender M, Choonara YE, Kumar P, du Toit LC, van Vuuren S, Pillay V (2014) A review of the
advancements in probiotic delivery: conventional vs. non-conventional formulations for intes-
tinal flora supplementation. AAPS PharmSciTech 15:29–43
Gul S, Masud T, Maqsood S, Latif A, Irshad I, ul Haque I (2012) Streptococcus thermophilus
bacteriocin, from production to their application: an overview. Afr J Microbiol Res 6:859–866
Hayashi H, Sakamoto M, Benno Y (2002) Phylogenetic analysis of the human gut microbiota
using 16S rDNA clone libraries and strictly anaerobic culture-based methods. Microbiol
Immunol 46:535–548
Hittinger CT (2013) Saccharomyces diversity and evolution: a budding model genus. Trends
Genet 29:309–317
Hsiao EY, McBride SW, Hsien S, Sharon G, Hyde ER, McCue T et al (2013) Microbiota modulate
behavioral and physiological abnormalities associated with neurodevelopmental disorders.
Cell 155:1451–1463
Hsieh PS, Tsai YC, Chen YC, Teh SF, Ou CM, King VA (2012) Eradication of Helicobacter pylori
infection by the probiotic strains Lactobacillus johnsonii MH-68 and L. salivarius ssp.
salicinius AP-32. Helicobacter 17:466–477
Hurduc V, Plesca D, Dragomir D, Sajin M, Vandenplas Y (2009) A randomized, open trial
evaluating the effect of Saccharomyces boulardii on the eradication rate of Helicobacter pylori
infection in children. Acta Paediatr 98:127–131
Isawa K, Hojo K, Yoda N, Kamiyama T, Makino S, Saito M et al (2002) Isolation and identifi-
cation of a new bifidogenic growth stimulator produced by Propionibacterium freudenreichii
ET-3. Biosci Biotechnol Biochem 66:679–681
Isobe H, Nishiyama A, Takano T, Higuchi W, Nakagawa S, Taneike I et al (2012) Reduction of
overall Helicobacter pylori colonization levels in the stomach of Mongolian gerbil by Lacto-
bacillus johnsonii La1 (LC1) and its in vitro activities against H. pylori motility and adherence.
Biosci Biotechnol Biochem 76:850–852
Jimenez E, Marin ML, Martin R, Odriozola JM, Olivares M, Xaus J et al (2008) Is meconium from
healthy newborns actually sterile? Res Microbiol 159:187–193
Karjalainnen H, Ahlroos T, Myllyluoma E, Tynkkynen S (2012) Real-time PCR assays for strain-
specific quantification of probiotic strains in human faecal samples. Int Dairy J 27:58–64
Kemgang TS, Kapila S, Shanmugam VP, Kapila R (2014) Cross-talk between probiotic
lactobacilli and host immune system. J Appl Microbiol 117:303–319
Kim MN, Kim N, Lee SH, Park YS, Hwang JH, Kim JW et al (2008) The effects of probiotics on
PPI-triple therapy for Helicobacter pylori eradication. Helicobacter 13:261–268
Kourelis A, Zinonos I, Kakagianni M, Christidou A, Christoglou N, Yiannaki E et al (2010)
Validation of the dorsal air pouch model to predict and examine immunostimulatory responses
in the gut. J Appl Microbiol 108:274–284
Lambert J, Hull R (1996) Upper gastrointestinal tract disease and probiotics. Asia Pac J Clin Nutr
5:31–35
Le MV, Fayol-Messaoudi D, Servin AL (2013) Compound(s) secreted by Lactobacillus casei
strain Shirota YIT9029 irreversibly and reversibly impair the swimming motility of
Helicobacter pylori and Salmonella enterica serovar Typhimurium, respectively. Microbiol-
ogy 159:1956–1971
Lesbros-Pantoflickova D, Corthesy-Theulaz I, Blum AL (2007) Helicobacter pylori and
probiotics. J Nutr 137:812S–818S
Li S, Huang XL, Sui JZ, Chen SY, Xie YT, Deng Y et al (2014) Meta-analysis of randomized
controlled trials on the efficacy of probiotics in Helicobacter pylori eradication therapy in
children. Eur J Pediatr 173:153–161
Lin WH, Lin CK, Sheu SJ, Hwang CF, Ye WT, Hwang WZ, Tsen HY (2009) Antagonistic activity
of spent culture supernatants of lactic acid bacteria against Helicobacter pylori growth and
infection in human gastric epithelial AGS cells. J Food Sci 74:M225–M230
Ling Z, Liu X, Luo Y, Yuan L, Nelson KE, Wang Y et al (2013) Pyrosequencing analysis of the
human microbiota of healthy Chinese undergraduates. BMC Genomics 14:390
Lionetti E, Miniello VL, Castellaneta SP, Magista AM, de Canio A, Maurogiovanni G et al (2006)
Lactobacillus reuteri therapy to reduce side-effects during anti-Helicobacter pylori treatment
in children: a randomized placebo controlled trial. Aliment Pharmacol Ther 24:1461–1468
Lionetti E, Indrio F, Pavone L, Borrelli G, Cavallo L, Francavilla R (2010) Role of probiotics in
pediatric patients with Helicobacter pylori infection: a comprehensive review of the literature.
Lopez-Brea M, Alarcon T, Domingo D, az-Reganon J (2008) Inhibitory effect of Gram-negative
and Gram-positive microorganisms against Helicobacter pylori clinical isolates. J Antimicrob
Chemother 61:139–142
MacKenzie DA, Defernez M, Dunn WB, Brown M, Fuller LJ, de Herrera SR et al (2008)
Relatedness of medically important strains of Saccharomyces cerevisiae as revealed by
phylogenetics and metabolomics. Yeast 25:501–512
Malfertheiner P, Megraud F, O’Morain CA, Atherton J, Axon AT, Bazzoli F et al (2012) Man-
agement of Helicobacter pylori infection – the Maastricht IV/Florence consensus report. Gut
61:646–664
Maragkoudakis PA, Zoumpopoulou G, Miaris C, Kalantzopoulos G, Pot B, Tsakalidou E (2006)
Probiotic potential of Lactobacillus strains isolated from dairy products. Int Dairy J
16:189–199
Martin R, Jimenez E, Heilig H, Fernandez L, Marin ML, Zoetendal EG, Rodriguez JM (2009)
Isolation of bifidobacteria from breast milk and assessment of the bifidobacterial population by
PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR. Appl Environ
Mattar AF, Teitelbaum DH, Drongowski RA, Yongyi F, Harmon CM, Coran AG (2002) Probiotics
up-regulate MUC-2 mucin gene expression in a Caco-2 cell-culture model. Pediatr Surg Int
18:586–590
Mattarelli P, Brandi G, Calabrese C, Fornari F, Prati GM, Biavati B, Sgorbati B (2014) Occurrence
of Bifidobacteriaceae in human hypochlorhydria stomach. Microb Ecol Health Dis 25:
10.3402/mehd.v25.21379
McFarland LV (2010) Systematic review and meta-analysis of Saccharomyces boulardii in adult
patients. World J Gastroenterol 16:2202–2222
Midolo PD, Lambert JR, Hull R, Luo F, Grayson ML (1995) In vitro inhibition of Helicobacter
pylori NCTC 11637 by organic acids and lactic acid bacteria. J Appl Bacteriol 79:475–479
Miki K, Urita Y, Ishikawa F, Iino T, Shibahara-Sone H, Akahoshi R et al (2007) Effect of
Bifidobacterium bifidum fermented milk on Helicobacter pylori and serum pepsinogen levels
in humans. J Dairy Sci 90:2630–2640
Moles L, Gomez M, Heilig H, Bustos G, Fuentes S, de Vos W et al (2013) Bacterial diversity in
meconium of preterm neonates and evolution of their fecal microbiota during the first month of
life. PLoS One 8:e66986
Molina-Infante J, Gisbert JP (2013) Probiotics for Helicobacter pylori eradication therapy: not
ready for prime time. Rev Esp Enferm Dig 105:441–444
Myllyluoma E, Veijola L, Ahlroos T, Tynkkynen S, Kankuri E, Vapaatalo H et al (2005) Probiotic
supplementation improves tolerance to Helicobacter pylori eradication therapy – a placebo-
controlled, double-blind randomized pilot study. Aliment Pharmacol Ther 21:1263–1272
Myllyluoma E, Ahlroos T, Veijola L, Rautelin H, Tynkkynen S, Korpela R (2007) Effects of anti-
Helicobacter pylori treatment and probiotic supplementation on intestinal microbiota. Int J
Antimicrob Agents 29:66–72
Myllyluoma E, Ahonen AM, Korpela R, Vapaatalo H, Kankuri E (2008) Effects of multispecies

probiotic combination on Helicobacter pylori infection in vitro. Clin Vaccine Immunol
15:1472–1482
Nam H, Ha M, Bae O, Lee Y (2002) Effect of Weissella confusa strain PL9001 on the adherence
and growth of Helicobacter pylori. Appl Environ Microbiol 68:4642–4645
Nista EC, Candelli M, Cremonini F, Cazzato IA, Zocco MA, Franceschi F et al (2004) Bacillus
clausii therapy to reduce side-effects of anti-Helicobacter pylori treatment: randomized,
double-blind, placebo controlled trial. Aliment Pharmacol Ther 20:1181–1188
Nobel Foundation (1967) Nobel lectures. Physiology or medicine 1901–1921. Elsevier Publishing
Company, Amsterdam
Pacifico L, Osborn JF, Bonci E, Romaggioli S, Baldini R, Chiesa C (2014) Probiotics for the
treatment of Helicobacter pylori infection in children. World J Gastroenterol 20:673–683
Paineau D, Carcano D, Leyer G, Darquy S, Alyanakian MA, Simoneau G et al (2008) Effects of
seven potential probiotic strains on specific immune responses in healthy adults: a double-
blind, randomized, controlled trial. FEMS Immunol Med Microbiol 53:107–113
Park SK, Park DI, Choi JS, Kang MS, Park JH, Kim HJ et al (2007) The effect of probiotics on
Helicobacter pylori eradication. Hepatogastroenterology 54:2032–2036
Patel A, Shah N, Prajapati JB (2014) Clinical appliance of probiotics in the treatment of
Helicobacter pylori infection-a brief review. J Microbiol Immunol Infect 47:429–437
Peterson J, Garges S, Giovanni M, McInnes P, Wang L, Schloss JA et al (2009) The NIH human
microbiome project. Genome Res 19:2317–2323
Pineiro M, Stanton C (2007) Probiotic bacteria: legislative framework – requirements to evidence
basis. J Nutr 137:850S–853S
Prajapati JB, Nathani NM, Patel AK, Senan S, Joshi CG (2013) Genomic analysis of dairy starter
culture Streptococcus thermophilus MTCC 5461. J Microbiol Biotechnol 23:459–466
Rastogi P, Saini H, Dixit J, Singhal R (2011) Probiotics and oral health. Natl J Maxillofac Surg
2:6–9
Redman MG, Ward EJ, Phillips RS (2014) The efficacy and safety of probiotics in people with
cancer: a systematic review. Ann Oncol 25:1919–1929
Rinckel LA, Savage DC (1990) Characterization of plasmids and plasmid-borne macrolide
resistance from Lactobacillus sp. strain 100-33. Plasmid 23:119–125
Rokka S, Myllykangas S, Joutsjoki V (2008) Effect of specific colostral antibodies and selected
lactobacilli on the adhesion of Helicobacter pylori on AGS cells and the Helicobacter-induced
IL-8 production. Scand J Immunol 68:280–286
Sakamoto I, Igarashi M, Kimura K, Takagi A, Miwa T, Koga Y (2001) Suppressive effect of
Lactobacillus gasseri OLL 2716 (LG21) on Helicobacter pylori infection in humans. J
Antimicrob Chemother 47:709–710
Sanders ME (2008) Probiotics: definition, sources, selection, and uses. Clin Infect Dis 46(Suppl 2):
S58–S61
Sanders ME, Klaenhammer TR (2001) Invited review: the scientific basis of Lactobacillus
acidophilus NCFM functionality as a probiotic. J Dairy Sci 84:319–331
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc
Saraoui T, Parayre S, Guernec G, Loux V, Montfort J, Le CA et al (2013) A unique in vivo
experimental approach reveals metabolic adaptation of the probiotic Propionibacterium
freudenreichii to the colon environment. BMC Genomics 14:911
Scaccianoce G, Zullo A, Hassan C, Gentili F, Cristofari F, Cardinale V et al (2008) Triple therapies
plus different probiotics for Helicobacter pylori eradication. Eur Rev Med Pharmacol Sci
12:251–256
Schnorr SL, Candela M, Rampelli S, Centanni M, Consolandi C, Basaglia G et al (2014) Gut
microbiome of the Hadza hunter-gatherers. Nat Commun 5:3654
Schrezenmeir J, de Vrese M (2001) Probiotics, prebiotics, and synbiotics – approaching a
definition. Am J Clin Nutr 73:361S–364S
Servin AL (2004) Antagonistic activities of lactobacilli and bifidobacteria against microbial

pathogens. FEMS Microbiol Rev 28:405–440
Shavakhi A, Tabesh E, Yaghoutkar A, Hashemi H, Tabesh F, Khodadoostan M et al (2013) The
effects of multistrain probiotic compound on bismuth-containing quadruple therapy for
Helicobacter pylori infection: a randomized placebo-controlled triple-blind study.
Sheu BS, Wu JJ, Lo CY, Wu HW, Chen JH, Lin YS, Lin MD (2002) Impact of supplement with
Lactobacillus- and Bifidobacterium-containing yogurt on triple therapy for Helicobacter pylori
eradication. Aliment Pharmacol Ther 16:1669–1675
Sheu BS, Cheng HC, Kao AW, Wang ST, Yang YJ, Yang HB, Wu JJ (2006) Pretreatment with
Lactobacillus- and Bifidobacterium-containing yogurt can improve the efficacy of quadruple
therapy in eradicating residual Helicobacter pylori infection after failed triple therapy. Am J
Clin Nutr 83:864–869
Shirasawa Y, Shibahara-Sone H, Iino T, Ishikawa F (2010) Bifidobacterium bifidum BF-1 sup-
presses Helicobacter pylori-induced genes in human epithelial cells. J Dairy Sci 93:4526–4534
Smelt MJ, de Haan BJ, Bron PA, van Swam I, Meijerink M, Wells JM et al (2012) L. plantarum,
L. salivarius, and L. lactis attenuate Th2 responses and increase Treg frequencies in healthy
mice in a strain dependent manner. PLoS One 7:e47244
Solanki HK, Pawar DD, Shah DA, Prajapati VD, Jani GK, Mulla AM, Thakar PM (2013)
Development of microencapsulation delivery system for long-term preservation of probiotics
as biotherapeutics agent. Biomed Res Int 2013:620719
Song MJ, Park DI, Park JH, Kim HJ, Cho YK, Sohn CI et al (2010) The effect of probiotics and
mucoprotective agents on PPI-based triple therapy for eradication of Helicobacter pylori.
Sougioultzis S, Simeonidis S, Bhaskar KR, Chen X, Anton PM, Keates S et al (2006) Saccharo-
myces boulardii produces a soluble anti-inflammatory factor that inhibits NF-kappaB-mediated
IL-8 gene expression. Biochem Biophys Res Commun 343:69–76
Suau A, Bonnet R, Sutren M, Godon JJ, Gibson GR, Collins MD, Dore J (1999) Direct analysis of
genes encoding 16S rRNA from complex communities reveals many novel molecular species
within the human gut. Appl Environ Microbiol 65:4799–4807
Sykora J, Valeckova K, Amlerova J, Siala K, Dedek P, Watkins S et al (2005) Effects of a specially
designed fermented milk product containing probiotic Lactobacillus casei DN-114 001 and the
eradication of H. pylori in children: a prospective randomized double-blind study. J Clin
Szajewska H, Albrecht P, Topczewska-Cabanek A (2009) Randomized, double-blind, placebo-
controlled trial: effect of Lactobacillus GG supplementation on Helicobacter pylori eradication
rates and side effects during treatment in children. J Pediatr Gastroenterol Nutr 48:431–436
Thierry A, Deutsch SM, Falentin H, Dalmasso M, Cousin FJ, Jan G (2011) New insights into
physiology and metabolism of Propionibacterium freudenreichii. Int J Food Microbiol
149:19–27
Thomas CM, Versalovic J (2010) Probiotics-host communication: modulation of signaling path-
ways in the intestine. Gut Microbes 1:148–163
Tien MT, Girardin SE, Regnault B, Le BL, Dillies MA, Coppee JY et al (2006) Anti-inflammatory
effect of Lactobacillus casei on Shigella-infected human intestinal epithelial cells. J Immunol
176:1228–1237
Tissier H (1906) Traitement des infections intestinales par la méthode de la flore bactérienne de
l’intestin. C R Soc Biol 60:361
Tobita K, Yanaka H, Otani H (2010) Lactobacillus crispatus KT-11 enhances intestinal immune
functions in C3H/HeN mice. J Nutr Sci Vitaminol (Tokyo) 56:441–445
Tolone S, Pellino V, Vitaliti G, Lanzafame A, Tolone C (2012) Evaluation of Helicobacter pylori
eradication in pediatric patients by triple therapy plus lactoferrin and probiotics compared to
triple therapy alone. Ital J Pediatr 38:63
Tong JL, Ran ZH, Shen J, Zhang CX, Xiao SD (2007) Meta-analysis: the effect of supplementa-
tion with probiotics on eradication rates and adverse events during Helicobacter pylori
eradication therapy. Aliment Pharmacol Ther 25:155–168
Tuomola E, Crittenden R, Playne M, Isolauri E, Salminen S (2001) Quality assurance criteria for
probiotic bacteria. Am J Clin Nutr 73:393S–398S
Turroni F, van Sinderen D, Ventura M (2011) Genomics and ecological overview of the genus
Bifidobacterium. Int J Food Microbiol 149:37–44
Tursi A, Brandimarte G, Giorgetti GM, Modeo ME (2004) Effect of Lactobacillus casei supple-
mentation on the effectiveness and tolerability of a new second-line 10-day quadruple therapy
after failure of a first attempt to cure Helicobacter pylori infection. Med Sci Monit 10:CR662–
CR666
Urdaci MC, Bressollier P, Pinchuk I (2004) Bacillus clausii probiotic strains: antimicrobial and
immunomodulatory activities. J Clin Gastroenterol 38:S86–S90
Valeur N, Engel P, Carbajal N, Connolly E, Ladefoged K (2004) Colonization and immunomo-
dulation by Lactobacillus reuteri ATCC 55730 in the human gastrointestinal tract. Appl
Environ Microbiol 70:1176–1181
van Nood E, Vrieze A, Nieuwdorp M, Fuentes S, Zoetendal EG, de Vos WM et al (2013) Duodenal
infusion of donor feces for recurrent Clostridium difficile. N Engl J Med 368:407–415
Vandenplas Y, Brunser O, Szajewska H (2009) Saccharomyces boulardii in childhood. Eur J
Pediatr 168:253–265
Vanhee LM, Goeme F, Nelis HJ, Coenye T (2010) Quality control of fifteen probiotic products
containing Saccharomyces boulardii. J Appl Microbiol 109:1745–1752
Vitor JM, Vale FF (2011) Alternative therapies for Helicobacter pylori: probiotics and
phytomedicine. FEMS Immunol Med Microbiol 63:153–164
Wang KY, Li SN, Liu CS, Perng DS, Su YC, Wu DC et al (2004) Effects of ingesting Lactoba-
cillus- and Bifidobacterium-containing yogurt in subjects with colonized Helicobacter pylori.
Am J Clin Nutr 80:737–741
Wang ZH, Gao QY, Fang JY (2013) Meta-analysis of the efficacy and safety of Lactobacillus-
containing and Bifidobacterium-containing probiotic compound preparation in Helicobacter
pylori eradication therapy. J Clin Gastroenterol 47:25–32
Wang LL, Yu XJ, Zhan SH, Jia SJ, Tian ZB, Dong QJ (2014) Participation of microbiota in the
development of gastric cancer. World J Gastroenterol 20:4948–4952
Wong BC, Lam SK, Wong WM, Chen JS, Zheng TT, Feng RE et al (2004) Helicobacter pylori
eradication to prevent gastric cancer in a high-risk region of China: a randomized controlled
trial. JAMA 291:187–194
Ya T, Zhang Q, Chu F, Merritt J, Bilige M, Sun T et al (2008) Immunological evaluation of
Lactobacillus casei Zhang: a newly isolated strain from koumiss in Inner Mongolia, China.
BMC Immunol 9:68
Yoon H, Kim N, Kim JY, Park SY, Park JH, Jung HC, Song IS (2011) Effects of multistrain
probiotic-containing yogurt on second-line triple therapy for Helicobacter pylori infection. J
Zhang L, Li N, Caicedo R, Neu J (2005) Alive and dead Lactobacillus rhamnosus GG decrease
tumor necrosis factor-alpha-induced interleukin-8 production in Caco-2 cells. J Nutr
135:1752–1756
Zhang L, Su P, Henriksson A, O’Rourke J, Mitchell H (2008) Investigation of the immunomod-
ulatory effects of Lactobacillus casei and Bifidobacterium lactis on Helicobacter pylori
infection. Helicobacter 13:183–190
Chapter 25
Vaccination Against Helicobacter pylori
Infection
Sukanya Raghavan and Marianne Quiding-Järbrink
Abstract Helicobacter pylori is well adapted to colonize the human gastric

mucosa and induces a relatively mild but persistent inflammation and activation
of adaptive B- and T-cell responses. A subset of infected individuals experience
symptoms or develop ulcer disease or gastric adenocarcinoma that might be
treatable with antibiotics. At the same time, the resistance to antibiotics is rapidly
increasing among H. pylori isolates, and access to an efficient vaccine would
improve treatment options considerably. Still, the complex pathogenesis and
many different virulence factors of the bacterium have made vaccine development
a challenging task. In this review, we discuss the possibilities of constructing a
future vaccine against H. pylori based on the choice of antigens and mucosal
adjuvants and describe our knowledge of protective immune responses that may
be necessary to generate for eradication of H. pylori. In addition, the preclinical and
clinical testing of available vaccine candidates is reviewed. The immunological
response to H. pylori infection is multifaceted, and inflammatory responses are
mounted side by side with a prominent regulatory response. The potential problems
caused by the immune response, comprising both tolerance induction by the
infection and the risk of developing post-immunization gastritis, are also discussed.
Keywords Helicobacter pylori • Vaccine • Adjuvant • Cytokine • T cell •

Gastritis • Regulatory T cell • Clinical trial
25.1 Introduction
Helicobacter pylori infection has a major impact from a public health-care per-
spective since an estimated half of the world’s population chronically harbor this
bacterium in the stomach. A birth cohort study in a rural area of Bangladesh showed
that H. pylori infection was already acquired by 60 % of the children within the first
2 years of their life (Bhuiyan et al. 2009). The highest prevalence of the infection in
S. Raghavan • M. Quiding-Järbrink (*)

Department of Microbiology and Immunology, Institute of Biomedicine, University of
Gothenburg, Box 435, 405 30 G€oteborg, Sweden
e-mail: [email protected]; [email protected]

DOI 10.1007/978-4-431-55936-8_25
576 S. Raghavan and M. Quiding-Järbrink
children is in low- and middle-income countries and can be related back to the low
socioeconomic status of parents (Malaty et al. 1996a, b). The infection is mainly
acquired via close contact with family members, between mother and child
(Yamaoka et al. 2000) or between siblings (Goodman and Correa 2000). Although
the fecal-oral route of transmission has been suggested as for other enteric infec-
tions, H. pylori may also be spread via the oral-oral route during outbreak of Vibrio
cholerae or enterotoxigenic Escherichia coli (ETEC) infections (Janzon
et al. 2009).
Approximately 1 in 10 H. pylori-infected individuals will develop symptoms,
including PUD, and, in the worst case, approximately 1 in 100, gastric cancer.
Whether chronic infection will lead to symptoms (or not) is dependent both on the
host and bacterial genetics (Ernst and Gold 2000; Lochhead and El-Omar 2007). As
discussed in detail in Chap. 12, there is also a vast body of epidemiological and
experimental evidence to suggest that chronic infection with H. pylori during early
childhood can protect from asthma and allergy later in life (Kosunen et al. 2002;
Blaser et al. 2008). In infected individuals, activation of innate and adaptive
immune system leads to the recruitment to the stomach of a wide range of cell
types, including dendritic cells, macrophages, neutrophils, mast cells, and T and B
cells (Robinson et al. 2007). H. pylori colonization also induces a strong systemic
antibody response with a rise in H. pylori-specific IgA and IgG which has been
exploited for diagnostic purposes. The immune cells recruited to the stomach
secrete a range of pro-inflammatory cytokines such as interleukin-1 (IL-1), IL-6,
tumor necrosis factor (TNF), interferon-γ (IFN-γ), and IL-17. In spite of the robust
immune response evident in the stomach, the chronic lifelong infection does not
protect against a second encounter with the bacteria, and spontaneous eradication of
the bacteria is rare. This has led to a high prevalence of the infection which can be
up to 90 % of the adult population in low- and middle-income countries where
H. pylori infection is endemic.
25.2 The Need for a Vaccine
In most individuals, the infection will persist for life unless they seek antibiotic
treatment. The current H. pylori eradication treatment schemes with antibiotics
combined with proton pump inhibitors have been improved in recent years and are
discussed in detail in Chap. 20. Although the treatment leads to eradication of the
infection, in many cases depending on the country, it still poses several difficulties
related to side effects and development of antibiotic resistance. Epidemiological
studies have reported a reinfection rate of 20–30 %/year after antibiotic treatment in
low- and middle-income countries which is accompanied by the return of peptic
ulcer disease (PUD) and the risk for gastric cancer (Parsonnet 2003). Thus, there is
an urgent need to develop novel treatment strategies not dependent on the use of
antibiotics and, ideally, a highly efficacious vaccine, based on our understanding of
H. pylori bacteria and its interplay with the human host. Candidate prophylactic and
25 Vaccination Against Helicobacter pylori Infection 577
therapeutic vaccines are already in clinical trials and have been projected to be cost-
effective in reducing the prevalence of the infection and incidence of gastric cancer
in the USA and Japan (Rupnow et al. 2000, 2001). Since the prevalence of the
infection in developed countries is decreasing naturally without intervention, a
vaccination scheme running for 10 years has been predicted to eradicate the
infection from the population (Rupnow et al. 2001). Furthermore, the vaccine
need not be highly efficacious or have wide coverage. In low- and middle-income
countries, however, the predicted scenario is different; the high prevalence rate
means that it would require not only a longer time (>10 years of continuous
vaccination) but also a wider vaccine reach for it to effectively reduce the preva-
lence of H. pylori infection in the population and symptoms related to chronic
infection (Rupnow et al. 2001).
It is noteworthy that the oral Dukoral cholera vaccine was found to provide not
only direct protection to vaccine recipients in Bangladesh but also conferred
significant herd immunity to non-vaccinated individuals (Ali et al. 2005). Since
the mode of transmission of H. pylori seems to be similar to that of V. cholerae as
evidenced by the fact that new infections with H. pylori in young children occur
during the same biannual peaks as diarrheal diseases (Janzon et al. 2009), it would
be interesting to evaluate whether a H. pylori vaccine would also afford herd
immunity which might reduce the need for the vaccine to have a wide reach in
the population as discussed above. It is also important to consider the target group
for vaccination against H. pylori infection in low- and middle-income countries.
Considering the evidence that suggests that childhood H. pylori infection might be
protective against allergy and asthma (Blaser et al. 2008), it might be worth
vaccinating only symptomatic adults in conjunction with antibiotic therapy. Such
a treatment may be considered as a prophylactic vaccine in the traditional sense
because the vaccine can be given to adults that have cleared their infection with
antibiotics and protection against reinfection can be followed.
25.3 Protective Immune Mechanisms for Eradication

of H. pylori
The complex pathogenicity and large variability of H. pylori are well described in
Chaps. 1 and 9 of this book and thus make vaccine development a major challenge.
To design an effective vaccine, we need to identify the protective immune mech-
anisms that will eventually lead to bacterial eradication, keeping in mind that they
are most probably different, quantitatively or qualitatively, from what the infection
as such gives rise to. Most of this work will, by necessity, be performed in small
rodent models, but will eventually have to be verified in humans or nonhuman
primate models.
The definition of protection also deserves some clarification. Most vaccination
regimens in mice lead to a significant one- or two-log decrease in bacterial burden
in the gastric tissue, but rarely to complete eradication of the bacterium. However,
most studies have examined bacterial numbers a few weeks after challenge, and
there are data suggesting that waiting for a longer time will actually lead to a
complete vaccine-induced eradication of the infection (Garhart et al. 2002). In
humans, the situation is even more complicated by the patchy colonization pattern
in the stomach, which makes it hard to determine the overall bacterial density even
when multiple sampling sites are examined. It is thus hard to evaluate the effect of
model vaccines if complete eradication is not achieved. In this chapter, we use the
word “protection” to indicate a significant reduction in the gastric colony-forming
units.
25.3.1 T-Cell-Mediated Protection
When research on anti-Helicobacter vaccines was first initiated, it was generally

believed that a secretory IgA response would suffice to prevent infection, based on
the extracellular lifestyle of the bacterium and the success of other oral vaccines
promoting the barrier function. However, early studies in mice clearly showed that
CD4+ T helper cells, but not CD8+ cytotoxic T cells or antibodies, were crucial for
vaccine-induced protection (Ermak et al. 1998; Blanchard et al. 1999; Pappo
et al. 1999). In addition, there is a prominent gastric IgA response to the infection
(Luzza et al. 1995; Mattsson et al. 1998b), without any apparent effect on bacterial
colonization. Much attention has since been devoted to elucidating the function,
especially cytokine production, of protective T helper cell subsets.
In summary, several studies using knockout animals or antibodies to neutralize
cytokine signaling indicate that both Th1- and Th17-type responses are important
for vaccine-induced protection against H. pylori in mice. The importance of Th1
cells for protection after oral immunization with H. pylori sonicate and cholera
toxin (CT) adjuvant was shown by Akhiani and coworkers (Akhiani et al. 2002), in
IFN-γ/ mice, and later confirmed by Sayi and coworkers (Sayi et al. 2009). In
other studies, however, no effect of IFN-γ on vaccine-induced protection could be
detected (Sawai et al. 1999; Garhart et al. 2002; Flach et al. 2011a). However, it was
consistently shown that IL-12 p40 subunit was important for protection (Akhiani
et al. 2002; Garhart et al. 2003; Hitzler et al. 2011; Ding et al. 2013). This later
raised the question whether Th17 responses supported by IL-23 (a cytokine sharing
the p40 subunit with IL-12) were actually the ones mediating the documented
protection. Studies using IL-12 p35/ mice lacking IL-12 showed varying effects
of the IL-12 defect on protection, which was dependent on the genetic background
of the host (Panthel et al. 2003; Hitzler et al. 2011). When IL-23 p19-deficient
animals lacking IL-23 became available, it was shown that also IL-23 was needed to
mount a fully protective immune response following vaccination, as p19/ mice
display some but not as good protection as wild-type mice (Hitzler et al. 2011; Ding
et al. 2013).
IL-23 is a potent driver of Th17 responses, and both Th1 and Th17 responses
appear to contribute to the H. pylori-induced chronic gastritis, as outlined in
Chap. 12. Thus, the results using IL-12- and IL-23-deficient animals raised the
question about the relative importance of Th1 and Th17 responses also in protective
immunity after vaccination. The first studies of IL-17 in protective immunity after
immunization used neutralizing antibodies during the effector phase of the immune
response and showed that IL-17 was necessary for protection (Velin et al. 2009;
Flach et al. 2011a). However, when IL-17A- or IL-17 receptor A (IL-17RA)-
deficient animals were prophylactically immunized, they were protected as well
as the wild-type mice (DeLyria et al. 2011). As the different studies cited above
have used partly different model systems and immunization schedules, it is difficult
to make absolute statements about the need for either Th1 or Th17 cells to achieve
protective immunity, but it seems reasonable to assume that both Th1 and Th17
responses arise following immunization and that they both contribute to bacterial
elimination. In some experimental systems, they may both be needed for successful
immunization, while in others they may compensate for each other and the effect
may not be seen due to redundancy.
Furthermore, it was also shown in the H. felis model that expression of the
mucosal homing receptor integrin α4β7 was necessary for vaccine-induced protec-
tion in mice following prophylactic immunization (Michetti et al. 2000). Integrin
α4β7 binds to mucosal addressin cell adhesion molecule (MAdCAM-1) which is
expressed at high levels by the vascular endothelium in gastrointestinal mucosae.
These findings are also supported by studies showing local T-cell activation in the
gastric mucosa following immunization with various vaccine preparations, but less
in systemic lymphoid tissues (Sayi et al. 2009; Becher et al. 2010; Flach
et al. 2011a; Hitzler et al. 2011). Thus, we can conclude that CD4+ T helper cells
with the ability to migrate to mucosal effector sites and secrete either Th1 or Th17
cytokines, or both, are crucial for vaccine-induced protection. In this work, we must
also remember that the pathology created by Helicobacter infection also appears to
be mediated by T cells.
Mice lacking conventional T cells harbor large bacterial numbers, but are free
from inflammation and epithelial damage (Roth et al. 1999; Hitzler et al. 2012), and
in virtually all immunization studies, gastritis is correlated to lower bacterial
burden. Therefore, much work has been devoted to trying to separate inflammation
driving and protective immune responses, to avoid the so-called post-immunization
gastritis (discussed later). However, a thorough study following immunized and
infected mice for a year showed that although a strong post-immunization gastritis
developed during the first weeks after infection in the prophylactically immunized
animals, this response was reduced with time and had virtually disappeared (as did
the bacteria) 1 year after infection (Garhart et al. 2002).
25.3.2 Innate Immune Functions Mediating Protection
Presumably, T cells are not directly responsible for bacterial elimination, and some
attempts have been made to define the final effector mechanisms mediating the
clearance of bacteria. In this regard, it is interesting to note that protective immunity
induced by vaccination leads to gastric production of chemokines, small chemo-
tactic proteins, with the ability to recruit not only effector T cells but also mast cells
and neutrophils (DeLyria et al. 2009; Flach et al. 2012). These cells are also
increased in the gastric mucosa in protected animals (Velin et al. 2005; DeLyria
et al. 2009; Flach et al. 2012), and several studies have been performed to inves-
tigate the effect of neutrophils and mast cells on H. pylori clearance. Neutrophils
are capable to translocate across the gastric epithelium into the lumen, where they
can contribute to H. pylori clearance by phagocytosis (Zu et al. 2000). Antibody-
mediated depletion of neutrophils during the effector phase of a vaccine-induced
immune response abrogated the ability to mount a protective response to H. pylori
(DeLyria et al. 2009). Further support for the role of neutrophils during H. pylori
elimination comes from experiments in IL-10/ mice that usually clear H. pylori
spontaneously. However, if neutrophils are depleted from the IL-10-deficient mice,
the bacterial clearance is delayed (Ismail et al. 2003). Thus, neutrophils appear to
contribute to vaccine-induced protection, but their effect may be dependent on the
infection model (see below).
Mast cells can kill H. pylori directly, at least in vitro, and have also been shown
to degranulate in the gastric mucosa of immunized mice (Velin et al. 2005).
However, in models of systemic bacterial infection, mast cells promote bacterial
clearance rather by enhancing the recruitment of neutrophils to the site of infection
(Echtenacher et al. 1996; Malaviya et al. 1996). The potential importance of mast
cells for protection against H. pylori infection has been investigated in two separate
studies. It was shown that mast cell-deficient mice were only partly protected
compared to wild-type mice following immunization and infection with
H. pylori, and they also failed to recruit neutrophils to the gastric mucosa during
the effector phase of the immune response (Ding et al. 2009). Furthermore, in
another experimental system, mast cell-depleted mice were unable to clear H. felis
infection after immunization (Velin et al. 2005). In this model, however, neutrophil
depletion had no effect on protection. Instead, the mast cells needed CD4+ T cells
for their function in anti-Helicobacter immunity (Velin et al. 2005). Recent studies
indicate that a possible mechanism for the effect of mast cells and neutrophils in
Helicobacter vaccination may be via activation of protease-activated receptors
(PARs) on innate immune cells (Wee et al. 2010; Velin et al. 2011; Chionh
et al. 2015). PARs are activated by serine proteases, which can be derived from
both Helicobacter bacteria and endogenously from degranulating mast cells and
neutrophils (Ossovskaya and Bunnett 2004; Kajikawa et al. 2007). The effects of
PAR1 and PAR2 have been studied in Helicobacter infection, and they appear to
balance the host’s pro-inflammatory and tissue-protective responses, thereby
influencing the outcome of infection and vaccination. Activation of PAR2 promotes
pro-inflammatory responses, and PAR2/ mice have a higher gastric Helicobacter

burden than wild-type mice (Wee et al. 2010) and were not as well protected against
H. felis infection following immunization as wild-type mice (Velin et al. 2011). It
was suggested that activation of PAR2 on dendritic cells (DCs) leads to improved
Th17 responses, and activation of PAR2 may thus be part of a positive feedback
loop where neutrophil degranulation leads to improved Th17 responses and more
neutrophil recruitment, altogether contributing to bacterial clearance (Velin
et al. 2011). In contrast, PAR1/ mice have lower colonization than wild-type
mice and display better protection after immunization in a H. pylori challenge
model (Wee et al. 2010; Chionh et al. 2015).
25.4 Mechanistic Studies in Experimental Animals

for Optimization of Immunization Protocols
A large body of evidence in the literature show that protection against H. pylori
infection can be achieved by vaccination either prophylactically or therapeutically
in animal models although prophylactic studies dominate. Based on numerous
vaccination studies in the mouse model of H. pylori infection (summarized in
Tables 25.1 and 25.2), one can come to the conclusion that the choice of adjuvant
and route of immunization plays a crucial role in the induction of the protective
immune responses which will be important to consider when proceeding to clinical
trials.
25.4.1 Choice of Antigen(s)
The feasibility of mucosal immunization was first demonstrated in mice immunized

orally with bacterial lysates or formalin-inactivated whole-cell bacteria together
with CT, enterotoxigenic E. coli heat-labile toxin (LT), or their nontoxic mutants. If
included in a H. pylori vaccine, crude antigens like the lysate preparation of the
bacteria will be difficult if not impossible to pass through regulatory authorities, due
to difficulties in batch-to-batch variation and lack of thorough characterization of
the preparation. The lysate preparation does have the advantage that it contains all
the known immunodominant and protective antigens such as CagA, HpaA, Urease,
VacA, etc. (unpublished observations). However, the lysate preparation also con-
tains a high concentration of pathogen-associated molecular patterns (PAMPs),
such as lipopolysaccharides (LPS), flagellin, and unmethylated cytosine guanine
dinucleotides (CpG DNA), that could potentially trigger an inflammatory reaction
when given together with a strong mucosal adjuvant.
A striking feature of H. pylori LPS is the expression of Lewis (Le) and blood
group antigens in the O-antigen component of the LPS molecule (Moran 2008). The
Table 25.1 Prophylactic vaccination studies against H. pylori infection in the mouse modela
Antigen Adjuvant Route Fold protectionb Reference
Formalin M-cell IG 10-fold Chionh et al. (2009)
whole cell targeting
dmLT IG 10–100-fold Summerton et al. (2010)
Urease LT IG 50–100-fold Kleanthous et al. (1998) and
Weltzin et al. (2000)
IN 10–100-fold Ermak et al. (1998), Kleanthous
et al. (1998), and Park
et al. (2000)
Alum s.c. 5-fold Ermak et al. (1998)
IN 100-fold Park et al. (2000)
LT Rectal 100-fold Kleanthous et al. (1998)
LT s.c. 50-fold Weltzin et al. (2000)
LTB IG No protection
LTB s.c. 50-fold
CT SL 10-fold Flach et al. (2011b)
Salmonella IG 5-fold Gomez-Duarte et al. (1998)
expressing
UreB
HpaA CT SL 10-fold Flach et al. (2011b)
CT IG 10-fold in BALB/c Sutton et al. (2007)
mice. No protection in
B6
Lysate CT IN 100-fold Garhart et al. (2002)
CT IG 10-fold Pappo et al. (1999) and
Raghavan et al. (2010)
CT SL 200-fold Raghavan et al. (2010)
CTA1- IN 10-fold Akhiani et al. (2006)
DD
IFA sc/ip 50-fold Eisenberg et al. (2003)
CFA sc/ip 50-fold Eisenberg et al. (2003)
CpG IG 3-fold Taylor et al. (2008)
dmLT IG 10-fold Sjokvist Ottsjo et al. (2013)
dmLT SL 200-fold Sjokvist Ottsjo et al. (2013)
CpG- IN 3-fold Nystrom-Asklin et al. (2008)
CTB
HpaA and CT SL 40-fold Flach et al. (2011b)
UreB IG 5-fold
dmLT SL 44-fold Sjokvist Ottsjo et al. (2013)
a
Only studies where H. pylori were used as a challenge strain for infection- and colony-forming
units subsequently quantified were included
b
Fold protection was roughly calculated as the decrease in the number of colony-forming units in
the stomach compared to unvaccinated infected mice
Table 25.2 Therapeutic vaccination studies against H. pylori infection in the mouse modela
Fold
Antigen Adjuvant Route protection Reference
Formalin whole cell CT IG 10–100- Raghavan et al. (2002a)
fold
Urease LT IG 100-fold Guy et al. (1999)
QS21 s.c. <1000-
back fold
Salmonella expressing IG 10-fold Liu et al. (2011)
CagA, VacA, and UreB
HpaA CT IG 10-fold Nystrom and Svennerholm
(2007) and Sutton et al. (2007)
Lysate CT IG 5-fold Raghavan et al. (2002b)
Alum ip 2-fold Nystrom et al. (2006)
HpaA and UreB CT IG >100- Nystrom and Svennerholm
fold (2007)
a
Only studies where H. pylori were used as a challenge strain for infection- and colony-forming
units subsequently quantified were included
expression of Lex and Ley antigens is a common property of H. pylori strains since as
many as 80–90 % of isolates from various geographical regions worldwide have
been found to express these antigens when screened using anti-Le antibodies as
probes (Simoons-Smit et al. 1996; Moran 2008). Although controversial, there
could be the possibility that vaccination with H. pylori LPS containing lysate
preparation or whole-cell bacteria together with an adjuvant could induce antibodies
to the bacterial Lex and Ley that could cross-react and bind to the Lex and Ley
structures expressed by the host epithelium and trigger autoimmunity (Negrini
et al. 1996; Appelmelk et al. 1998). If whole-cell bacteria/lysates are to be used as
vaccine in humans, it would be important to perhaps reduce the LPS content and
follow closely the Lex and Ley antibody titers in the vaccinated individuals.
Bacterial vectors for the delivery of antigens to the host have also been exploited
for the delivery of H. pylori antigens to the host particularly urease. For example,
attenuated Salmonella typhi, Lactobacillus ssp., and polio virus have all been used
as vectors to deliver urease antigen (Corthesy et al. 2005; Smythies et al. 2005;
Aebischer et al. 2008). The advantage on this approach is that it avoids the need for
a mucosal adjuvant. Attenuated Salmonella as a vector has been the most exten-
sively studied both for oral and intranasal delivery. The Salmonella vector
expressing urease was based on the licensed typhoid fever vaccine strain Ty21a
which is a chemically induced avirulent mutant of S. enterica serovar Typhi with an
ability to induce broad cellular and humoral immunity at mucosal sites. The results
showed that although the vaccination with Salmonella expressing urease worked
well in mice, when taken to clinical trials, the response to urease in the volunteers
was weak and did not provide protection to infectious challenge (discussed below).
Finally, some of the most promising results on vaccine-induced protection have
been obtained using recombinant antigens from H. pylori either alone or in
combination. Numerous studies have tested the efficacy of urease as a protective

antigen using different adjuvants and routes of immunization (Tables 25.1 and
25.2). The efficacy of prophylactic or therapeutic protection against H. pylori
infection in mice has also been demonstrated for a variety of other native and
recombinant antigens such as shock proteins and native and recombinant VacA,
CagA, NapA, catalase, and HpaA among others (Radcliff et al. 1997; Marchetti
et al. 1998). Importantly, a synergistic effect on vaccine-induced protection was
seen when two antigens, HpaA and UreB, were combined together with CT or
double-mutant heat-labile toxin from E. coli (dmLT) as an adjuvant compared to
immunization with either antigen alone (Nystrom and Svennerholm 2007; Flach
et al. 2011b; Sjokvist Ottsjo et al. 2013). In the future, information derived from the
knowledge of the H. pylori genome that is now easily available will probably lead
to the identification of additional candidate antigens that could be safely included in
a multicomponent vaccine against H. pylori infection.
25.4.2 Choice of Adjuvants
The mucosal route of immunization requires the use of a strong adjuvant because
proteins are poor immunogens when given mucosally. The strongest mucosal
adjuvants are bacterial toxins such as CT and LT; however, they both cause severe
diarrhea limiting their use in humans. Both CT and the heat-labile toxin from E. coli
(LT) belong to the class of AB5 toxins with a characteristic A1 subunit linked to a
pentamer of B subunits via the A2 fragment (Rappuoli et al. 1999) (Fig. 25.1). The
B subunit is responsible for the binding to monosialotetrahexosylganglioside
(GM1) present on all nucleated cells, and the A1 subunit is a ribosyltransferase
promoting adenosine diphosphate ribosylation of stimulatory guanine nucleotide-
binding proteins (G proteins), resulting in increased intracellular levels of cAMP
and enhanced fluid secretion and diarrhea. The ribosyltransferase activity of the A1
subunit has been shown to be responsible also for the adjuvanticity of CT and LT
(Giuliani et al. 1998). Intense efforts in the last 20 years have focused on developing
molecules with reduced toxicity but intact adjuvanticity. Site-directed mutagenesis
was used to replace single or two amino acids within the enzymatic A1 subunit of
LT leading to reduced or eliminated enzymatic activity (LTK63, LTR192G, and
dmLT) (Giuliani et al. 1998; Norton et al. 2011) (Fig. 25.1). Another approach to
decrease the toxicity of CT has been to link the enzymatically active CTA1 subunit
to the cell-binding moiety of Staphylococcus aureus protein A (CTA1-DD) (Agren
et al. 1997). These nontoxic mutant molecules have been tested as mucosal adju-
vants in combination with H. pylori antigens in the mouse model, primates, and
human clinical trials (described below).
Studies on the proof of principle that a H. pylori vaccine can promote immunity
and reduce bacterial load in the stomach of mice were performed initially using CT
as an adjuvant. Prophylactic or therapeutic mucosal vaccination with a range of
H. pylori antigens was found to protect against H. pylori infection (Tables 25.1 and
A Subunit B Subunit
ETEC heat labile toxin (LT)
A1
A2
A1 enzymatically active when cleaved
from A2
Mutant ETEC heat-labile toxin (LTK63)
serine to lysine substitution in position 63 of the A

subunit
Single mutant ETEC heat-labile toxin (LTR192G)
192
glycine to arginine sustitution at position 192 of the A1

subunit
Double mutant ETEC heat-labile toxin (dmLT)
192 211
glycine to arginine substitution at position 192 and

leucine to alanine substitution at position 211
Fig. 25.1 Site-directed mutagenesis to reduce/remove the ADP ribosylase activity of the A1
subunit of the ETEC heat-labile toxin (LT). Several startegies have been employed to reduce the
toxicity of LT while retaining the adjuvanticity of the molecule. The figure shows a schematic
representation of native LT and the different LT variants with mutations in selected sites that have
been evaluated as mucosal adjuvants together with candidate H. pylori antigens
25.2). Most of the animal studies on the protective immune responses after vacci-
nation described earlier have also been carried out using CT. Thus, CT has served as
a “golden standard” for evaluating other nontoxic mucosal adjuvants to be included
in a H. pylori vaccine. Similar to CT, LT has been used as a mucosal adjuvant in
animal studies (Tables 25.1 and 25.2). The nontoxic derivatives of LT, LTK63,
LTR72, and dmLT have also been found to strongly potentiate immune response to
various parenterally and mucosally administered H. pylori antigens making them
promising adjuvants to include in future vaccines (Lu et al. 2010; Summerton
et al. 2010; Norton et al. 2011; Sjokvist Ottsjo et al. 2013). Finally, CTA1-DD
has been shown to function as an adjuvant with a H. pylori vaccine in mice when
administered intranasally but not intragastrically or sublingually (Akhiani
et al. 2006). The choice of the adjuvant and mucosal route of immunization will
be crucial for the safety aspect and induction of appropriate and long-lasting
immune responses against H. pylori antigens as discussed below.
Neutrophils T CD4+ Th cells Treg CD4+ regulatory T cells
mucus
Anmicrobial pepdes
Defensins
IgA
IL-8 MLN
T
Blood vessel
IFNγ
3 Via blood
α4β7 T 2
Treg MAdCAM-1
T
T T T
T Treg
T Treg
Treg
IL-17 4
Fig. 25.2 Interactions between CD4+ T-cell subsets and the gastric epithelium and cytokines
important for vaccine-induced protection. H. pylori reside in the mucous gel layer. (1) Antigens
shed by H. pylori are picked up by dendritic cells which migrate to the draining lymph node to
prime CD4+ T cells and induce regulatory T cells (Treg). IL-8 secretion by the epithelial cells in
response to the infection can directly recruit neutrophils to the site of infection. (2) H. pylori-
specific CD4+ T cells and Treg home to the gastric mucosa via α4β7 interaction with MAdCAM-1
and proliferate locally in the tissue. (3) CD4+ effector T cells secrete IFN-γ and IL-17A in response
to H. pylori infection which may bind to their respective receptors on the epithelium. (4) Finally,
Treg can suppress the activation of effector T cells in the stomach
25.4.3 Vaccination Routes
Since the bacteria are localized extracellularly in the stomach, particular emphasis
has been given to immunization via the intragastric route although other mucosal
routes of immunization as well as the parenteral route and prime-boost regimens
have also been evaluated. Alternatives to the intragastric route of immunization
have been sought since the stomach poses additional challenges on the stability of
the antigen and adjuvants used. Indeed, the intranasal and the sublingual route of
immunization has consistently proved to induce a stronger protection against
H. pylori infection (in the range of 100–200-fold reduction in bacterial load)
compared to the intragastric route which gives a reduction in bacterial load in
order of five to tenfold (Tables 25.1 and 25.2) irrespective of the antigen and
adjuvant used. However, the intranasal route of immunization may not be the route
of choice since studies in humans have indicated that the nasal route of immuniza-
tion is ineffective in stimulating immune responses in the intestine or stomach
(Johansson et al. 2004). In addition, intranasal immunization is associated with a
risk of translocation of GM1-binding adjuvants like CT to the olfactory bulb of the
brain, restricting its applicability in humans (van Ginkel et al. 2000).
The sublingual route of immunization on the other hand has been reported to be
safe at least in mice and particularly ideal when using antigens or adjuvants that
might be sensitive to the harsh environment of the stomach acid (Czerkinsky
et al. 2011). CD4+ T cells primed in the cervical lymph node after sublingual
immunization have been shown to migrate to peripheral lymphoid organs and
further to the mucosal tissues such as the lungs, stomach, intestine, and vagina
and afford protection against H1N1 influenza, H. pylori, and genital papilloma virus
infections (Song et al. 2009; Raghavan et al. 2010; Czerkinsky et al. 2011). The
induction of rather poor immunity after intragastric administration of the H. pylori
vaccine could be either due to the degradation of antigens or due to dilution of the
vaccine in the contents of the gastrointestinal tract, an aspect that must be kept in
mind when designing clinical trials in humans.
Another approach for the delivery of vaccines has been to target it to the M cells
lining the payer patches of the small intestine. M cells selectively absorb the antigen
by endocytosis or pinocytosis and direct it to antigen-presenting cells (macro-
phages, DCs, B lymphocytes). Whole-cell bacteria agglutinated with the Ulex
Europaeus Lectin 1 (UAE1) and administered orally to mice could presumably
bind to M-cell glycocalyx in the small intestine where they can be taken up for
presentation to the immune system. Remarkably, vaccination with lectin-
agglutinated bacteria was as effective as using formalin-inactivated whole-cell
bacteria and CT (Chionh et al. 2009). An advantage of mucosal vaccination or
M-cell targeting of the vaccine is that mucosal priming induces the lymphocytes to
express the α4β7 integrin. As mentioned previously, α4β7+ cells have been shown
to be essential for vaccine-induced protection against Helicobacter infection in
mice indicating the importance of priming at a mucosal site (Michetti et al. 2000).
By, contrast T cells that are primed peripherally typically display the α4β1 integrin
and CC chemokine receptor 4 (CCR4) and so do not migrate to or respond in
mucosal sites. This selective expression of integrin on lymphocytes explains why
mucosal vaccination might be required to protect against mucosal infections and
why peripheral administration of vaccine antigens is often ineffective against
mucosal infections which may be the case for a H. pylori vaccine.
Another approach that has been evaluated for H. pylori vaccines is the mucosal
prime and systemic boost strategy particularly with purified antigens that might be
weakly immunogenic. This strategy would require that the antigen and adjuvant are
safe to administer via the mucosal and the systemic route. Mucosal immunization
alone often requires two to three boosters for an effective immune response and
reduction in the bacterial load in the stomach of mice (Sutton et al. 2000). With the
mucosal prime-systemic boost strategy, on the other hand, mucosally induced T
cells could be effectively expanded with a single systemic boost. The systemic
boost would also be dose sparing both for the antigen and adjuvant. In this regard,
intranasal mucosal priming with Salmonella expressing urease and systemic boost
with purified recombinant urease with alum was shown to be more effective in
reducing the bacterial load in the stomach of mice post-challenge compared to
single mucosal or parenteral immunizations alone (Londono-Arcila et al. 2002). In
another study, using two separate adjuvants, Ermak and coworkers showed that
mucosal intranasal prime with urease and LT and systemic boost with urease and
alum was effective in reducing the bacterial load in the stomach of mice (Ermak
et al. 1998). The mucosal prime and systemic boost strategy although promising
might be difficult to implement in the field as it may increase the cost of the vaccine.
25.5 Studies in Nonhuman Primates and Clinical Trials
25.5.1 Immunization Studies in Nonhuman Primates
The use of nonhuman primates in medical research is inevitably connected with

high costs and difficult ethical considerations. Still, in Helicobacter research, the
model carries several advantages, such as the route of transmission, which is
presumably the same in humans and socially housed Rhesus macaques (Macaca
mulatta), the resulting chronic active gastritis (Dubois et al. 1994), and the ability of
the bacterium to re-infect previously infected individuals treated with antibiotics
(Dubois et al. 1999). A further advantage is the possibility to follow individual
animals with repeated biopsy sampling after treatment or vaccinations attempts.
Several immunization studies using Rhesus macaques to evaluate antigens and
immunization routes were performed in the late 1990s, but in the last decade,
there has been a few vaccination studies using this model that has made it all the
way to publication.
Dubois and coworkers (1998) showed that prophylactic immunization of
socially housed macaques with urease together with E. coli LT as adjuvant induced
significant protection against infection acquired from other monkeys in the colony
and also that the immunized but subsequently infected monkeys had significantly
lower gastritis score than untreated and infected animals. In contrast, a prophylactic
immunization with urease and LT given orally or intramuscularly failed to induce
protection during subsequent experimental challenge with H. pylori (Solnick
et al. 2000).
At the same time, attempts at therapeutic immunization with urease and LT
adjuvant of already infected macaques had no effect on gastric bacterial load in a
separate study (Lee et al. 1999b). Still, when the same animals were treated with
antibiotics to eliminate the infection, vaccinated again, and then rechallenged with
H. pylori, they had significantly lower bacterial carriage than previously
unvaccinated but infected monkeys. In an attempt to define better immunization
schedules, the same group also combined mucosal and parenteral immunizations in
antibiotic-treated animals with previous infection (Lee et al. 1999a). This treatment
reduced bacterial numbers in the vaccinated animals, but did not achieve full
eradication after challenge.
The factors differing between these studies suggest that in order to achieve
successful vaccination actually preventing H. pylori infection, prophylactic immu-
nizations, the natural infection route rather than challenge studies, and a reasonably
large number of animals should be used. If full eradication is not achieved, the
patchy distribution of H. pylori in the gastric mucosa will probably make it difficult
to assess potential differences in bacterial load. Finally, it is encouraging to note
that none of the studies cited above reports any serious post-immunization gastritis.
If anything, gastritis scores appear to be slightly reduced in immunized and
subsequently infected animals compared to animals who were immunologically
naı̈ve when they were infected.
25.5.2 Clinical Trials of H. pylori Vaccines
Urease was also used in the first trials with human volunteers receiving experimen-
tal H. pylori vaccines, and LT was again used as adjuvant. This carries obvious
problems with toxin-induced diarrhea, and native LT will not be used as adjuvant in
a human vaccine, when available. Oral immunization with urease and LT in
asymptomatic but H. pylori-infected volunteers induced specific serum IgA
responses and circulating IgA-secreting cells (Michetti et al. 1999) indicating the
induction of a gastrointestinal immune responses (Brandtzaeg and Johansen 2005).
There was also a modest but significant decrease in the gastric bacterial counts
1 month after immunization, but the volunteers were then treated with antibiotics,
and no further effect on the bacterial counts could be followed. The same group also
evaluated lower doses of LT in uninfected volunteers and found that these generally
reduced the frequencies of responders to oral urease immunization (Banerjee
et al. 2002). Still, a significant increase in CD69+ activated lymphocytes within
the gut-homing α4β7+ population could be documented following immunization.
Another approach, using a mutated LT without toxic effect (LTR192G) combined
with a formalin-inactivated whole-cell vaccine, was also evaluated at the same time
(Kotloff et al. 2001; Losonsky et al. 2003). This formulation induced mucosal IgA
responses in both uninfected and H. pylori-infected volunteers, and a systemic
T-cell response evident as secretion of IFN-γ, but interestingly enough only in the
uninfected volunteers. Still, this vaccine formulation could not achieve bacterial
eradication when given to asymptomatic H. pylori-infected volunteers. As the
bacterial density was not examined, it could not be evaluated if the vaccination
had any effect on bacterial density in the stomach.
In addition to the inactivated vaccine formulations, attempts to use live recom-
binant S. typhi Ty21a strains expressing urease have been partly successful. The
S. typhi Ty21a is the same strain as in the Vivotif™ vaccine against typhoid fever
and has an attractive safety profile. When engineered to express UreA and UreB
from H. pylori, it induces very weak B-cell responses, but significant T-cell
responses to urease following oral delivery in a subset of volunteers, especially if
the vaccinees had been exposed to the carrier Salmonella strain previously
(Bumann et al. 2001; Metzger et al. 2004).
As H. pylori preferentially infects young children living in crowded households
with poor hygiene (Kivi and Tindberg 2006), the use of natural infection to evaluate
the efficacy of vaccine candidates is not a viable starting point, even though the
initial primate studies suggested that this may be a possible way to record protective
immune responses. In order to evaluate the protective effect of any vaccine
candidate in healthy adults, the access to a safe and relevant human challenge
model is therefore invaluable. One such model was developed some 10 years ago
and made use of an antibiotic-sensitive cagPAI- H. pylori strain isolated from a
patient with mild gastritis (Graham et al. 2004). Inoculation with this strain induced
a typical active chronic gastritis, infiltration of CD4+ and CD8+ T cells in the gastric
mucosa, and development of a serum IgM response (Graham et al. 2004;
Nurgalieva et al. 2005). This challenge model was also used in subsequent attempts
to correlate immune responses to vaccine-induced protection, again using the
urease-expressing S. typhi Ty21a as vaccine (Aebischer et al. 2008). The live
vaccine was delivered orally three times every second day, and the volunteers
challenged with H. pylori 1–5 months later. This regimen resulted in the prevention
of infection in some (three out of nine) volunteers in a first round of experiments,
but none in a subsequent series of 12 vaccinees (Aebischer et al. 2008). The
comprehensive analysis of immune responses made it possible to correlate low or
absent H. pylori infection with detectable CD4+ T-cell responses to the vaccinees
antigens. It was shown that presence of circulating CD4+ integrin β7+ T cells
producing IFN-γ or IL-2 following antigen-specific stimulation was much more
common in protected individuals (defined as having no or few remaining H. pylori),
while vaccine-specific B-cell responses could not be detected in any of the
vaccinees (Aebischer et al. 2008). These data strongly indicate that CD4+ gut-
homing (α4β7+) effector T cells may be important for protection in humans as well.
However, the presence of such cells has not yet been evaluated in the gastric
mucosa of vaccinated volunteers.
A second line of studies investigated the route antigen delivery that resulted in
gastric immune responses using already available oral vaccines and the homing
commitments of gastrically activated effector B and T cells. The immunogenic and
well-tolerated oral Dukoral™ cholera vaccine was used for several of these studies.
This is an inactivated whole-cell vaccine substituted with cholera toxin B subunit
(CTB). Initial studies in H. pylori-infected and uninfected volunteers showed that
although the two groups of volunteers displayed similar vaccine-specific B-cell
responses in the duodenum, only the infected volunteers had detectable gastric
B-cell responses to the cholera antigens (Mattsson et al. 1998a). The ability to
induce gastric B-cell immunity in uninfected individuals was shown a few years
later, when another study showed weak gastric reactivity to a higher dose of
LTR192G (25 μg versus 10 μg CTB in Dukoral) also in uninfected volunteers
(Losonsky et al. 2003), but again, duodenal immune responses were considerably
higher. To investigate if gastric B-cell responses could be induced directly in the

gastric mucosa or if they were the result of migration of cells activated at distant
sites, such as Peyer’s patches, another series of experiments with gastric or intes-
tinal delivery of the Dukoral vaccine was performed. These experiments showed
that both gastric and intestinal antigen delivery could induce gastric B-cell
responses, but only in the H. pylori-infected individuals (Quiding-Jarbrink
et al. 2001b). T-cell reactivity was not evaluated in the gastric cell suspensions,
but another study showed that H. pylori-reactive T cells in the circulation of
infected individuals carry the α4β7 mucosal homing receptor, as do specific B
cells induced by gastric antigen delivery (Quiding-Jarbrink et al. 2001a). The
increased migration of effector B cells to the H. pylori-infected gastric mucosa
could be caused by an increased production of the chemokine CCL28, which
specifically recruits IgA+ plasmablasts to mucosal tissues (Kunkel et al. 2003),
and was found to be increased in H. pylori-infected human stomach (Hansson
et al. 2008). Taken together, these studies suggests that induction of adaptive gastric
immune responses can be achieved by intestinal antigen administration and that a
therapeutic vaccine given to already infected individuals should have the possibility
to induce a strong gastric response. However, these studies were all performed with
adult volunteers, and it may well be that young children or infants respond differ-
ently, and this possibility must be taken into account when designing future vaccine
trials.
A different approach to H. pylori vaccination in humans is the use of three well-
characterized recombinant H. pylori antigens (CagA, VacA, and NapA) given
intramuscularly with alum adjuvant (Malfertheiner et al. 2008). As would be
expected, this antigen regimen resulted in strong serum IgG response, and there
was also a substantial IFN-γ production from restimulated circulating T cells.
However, when volunteers receiving this vaccine were challenged with a cagPAI+
H. pylori strain, there was no difference in bacterial clearance in the vaccine
and placebo groups (Malfertheiner et al. 2012).
When human and animal studies are considered together, it appears that an
appropriate T-cell response is a key to protective immune responses against
H. pylori infection. In this regard, it is interesting and encouraging that the
human studies performed so far do not report on any overt vaccine-induced
gastritis, even when a T-cell response to H. pylori has clearly been induced. In
the previous primate studies, mucosal IgA responses were recorded, but at that
time, unfortunately no attempts were made to correlate T-cell immunity to bacterial
burden. Both in the human and primate studies performed so far, a long-time
follow-up of gastric bacterial counts are lacking, to properly evaluate the effect of
vaccine candidates. This would obviously be a time-consuming and a costly
endeavor, but will probably be necessary to evaluate a future promising vaccine
candidate.
25.6 Remaining Obstacles for Successful Vaccination

Against H. pylori
25.6.1 Treg-Mediated Suppression of Effector Functions
In spite of a vigorous immune response generated in the stomach with infiltration of

T and B cells, it is not clear exactly how H. pylori infection persists for many
decades in the infected individuals. One reason that has been suggested is that the
immune system has developed mechanisms to protect the host by ensuring the
development of an immune response in the absence of harmful inflammation and
damage to the host tissue. The mucosal immune system in particular is highly
adapted toward tolerance, the breakdown of which can result in disease (Izcue
et al. 2006). A specific subset of CD4+ T cells co-expressing CD25 called regula-
tory T cells (Tregs) was first described in 1995 to be crucial in the active suppres-
sion of autoimmune inflammation and colitis in healthy individuals (Sakaguchi
et al. 1995; Izcue et al. 2006; Ohkura et al. 2013). These Tregs could be identified,
purified, and characterized by their high expression of CD25, CTLA-4, and later the
transcription factor Foxp3 (FOXP3 in humans). The Tregs also specifically secreted
TGFβ and IL-10 when activated through their T-cell receptor. In subsequent
studies, Tregs have been shown to be readily induced by oral administration of
antigen, but this induction must be avoided if mucosal vaccination is to be
successful.
The role of Tregs in H. pylori infection was first described in the mouse model
where it was shown to dampen the H. pylori-induced immunopathology by reduc-
ing the activation of CD4+ IFN-γ-producing cells (Raghavan et al. 2003; Kaparakis
et al. 2006; Rad et al. 2006; Stuller et al. 2008; Sayi et al. 2009). Indications that
Treg can suppress H. pylori-induced inflammation can perhaps also be deduced
from the fact that both B-cell knockout and NADPH phagocyte oxidase knockout
mice that both have lower frequencies of Treg in vivo compared to wild-type
controls also have much lower bacterial loads and exacerbated inflammation in
the stomach (Blanchard et al. 2003; Akhiani et al. 2004).
Vaccination with H. pylori antigens and a strong mucosal adjuvant to overcome
Treg activity could possibly be a mechanism to enhance immunity. However, the
vaccine-specific response may itself be dampened by the presence of Treg as
evidenced by the presence of Treg in the stomach of vaccinated mice (Becher
et al. 2010) and lack of sterilizing immunity reported in several studies (Tables 25.1
and 25.2). Thus, vaccination combined with Treg depletion has been suggested as
strategy to enhance the protective effect of the vaccine. Currently there is only one
study reporting enhanced vaccine-induced response to H. pylori infection after
depletion of Treg in mice (Hitzler et al. 2011). Surprisingly the bacteria were still
not eradicated from the stomach of Treg-depleted mice indicating that Foxp3neg
regulatory cells such as IL-10-producing T regulatory 1 cells might play a role in
controlling effector T-cell responses in the stomach. Whether vaccination com-
bined with short-term Treg depletion would be a viable option for the treatment of
H. pylori infection remains to be seen, as there are inherent risks of blocking Treg
activity due to their role in preventing autoimmunity and inflammation.
25.6.2 Post-immunization Gastritis
Several studies have characterized the immune cell infiltration in the stomach of
vaccinated mice compared to unimmunized infected mice reporting enhanced
infiltration of CD4+ T cells, B cells, neutrophils, M1 macrophages, DCs, mast
cells, and eosinophils and chemokine and cytokine response post-challenge (Ermak
et al. 1998; Quiding-Jarbrink et al. 2010; Flach et al. 2011a, 2012; Hitzler
et al. 2011) (Fig. 25.2) often referred to as post-immunization gastritis. In this
complicated picture of the inflammatory infiltrate in the stomach of vaccinated
mice, it is a daunting task to decipher the function of the individual cell types and
cytokines in inflammation and protection. As mentioned previously, an increase in
the IFN-γ and IL-17 response in the stomach of vaccinated mice has been shown to
correlate with a decrease in bacterial load in the stomach of individual mice (Flach
et al. 2011a). Recent studies have shown that vaccinated IL-12p35/ and IFN-γ/
mice are protected against H. pylori infection with minimal inflammation with a
consequent increase in IL-17A levels in the stomach (Ding et al. 2013; Sjokvist
Ottsjo et al. 2015), indicating that post-immunization gastritis might be promoted
by IFN-γ, while IL-17A mainly affects the bacterial load in the stomach. These
results were consistent with another study showing that depletion of macrophages
using loaded liposomes reduced vaccination-induced gastritis presumably by
blocking macrophage derived IFN-γ response (Kaparakis et al. 2008) but did not
affect protection (Walduck et al., personal communication). Further studies are
warranted and important because future vaccines should be designed based on the
induction of appropriate immune response with minimal post-immunization
gastritis.
25.6.3 Challenges in Vaccination Against H. pylori

in the Developing World
The main target population for a vaccine against H. pylori infection are symptom-
atic individuals living in low- and middle-income countries and those with the risk
for developing gastric cancer. Thus, one of the biggest challenges for the vaccine
would be to make it available at an affordable price. Another important aspect to
consider is that previous oral live vaccines have shown reduced immunogenicity
when used in low- and middle-income countries compared to industrialized coun-
tries (Richie et al. 2000; Ogra et al. 2011). The reasons for the difference in
immunogenicity although not completely defined have been suggested to be due
to nutrition-related factors including protein-calorie and micronutrient malnutri-

tion. In addition, interference of vaccine take by maternal IgA antibodies during
breastfeeding, presence of intestinal parasitic infection, and possibly also host
genetic factors have been shown to contribute to the reduced efficacy of oral
vaccines in low- and middle-income countries. Indeed zinc supplementation and
temporary withdrawal of breast milk during vaccination in infants has given
promising results when evaluated with the oral cholera vaccine (Ahmed
et al. 2009).
Another aspect that needs to be taken into consideration is that due to high costs
for production of the vaccine according to Good Manufacturing Practices, the
involvement of a pharmaceutical company would be required. Previously several
companies were actively pursuing the development of a H. pylori vaccine, but due
to the poor results later in clinical trials, the interest has declined. With the
development of new and promising mucosal adjuvants, hopefully there will be
renewed interest in the production of an H. pylori vaccine for evaluation in clinical
trials.
As with other bacterial vaccines, either inactivated whole cells or a mixture of

putative protective recombinant antigens should be included in the vaccine against
H. pylori with a strong, safe, and effective mucosal adjuvant that can induce
mucosal homing CD4+ T-cell responses. Vaccination of symptomatic adults in
conjunction with antibiotic therapy might be considered due to the recent data
suggesting that H. pylori infection during childhood may actually protect against
development of asthma and allergies. Indeed, it has been demonstrated in adult
volunteers that it is possible to induce stronger immune responses by vaccination in
the H. pylori-infected mucosa compared to uninfected mucosa; thus, a therapeutic
vaccine would probably have a chance for success (Mattsson et al. 1998a). Fur-
thermore, the evaluation of the safety of the H. pylori mucosal vaccine in future
clinical trials will be of utmost priority. Two recently developed mucosal vaccines
for human use against rotavirus diarrhea and influenza were unfortunately with-
drawn after a short period due to reported adverse reactions, emphasizing the future
challenging task in formulating a H. pylori vaccine which would be safe to use in
children and adults primarily in developing countries.
References
Aebischer T, Bumann D, Epple HJ, Metzger W, Schneider T, Cherepnev G et al (2008) Correla-

tion of T-cell response and bacterial clearance in human volunteers challenged with
Helicobacter pylori revealed by randomised controlled vaccination with Ty21a-based Salmo-

nella vaccines. Gut 57:1065–1072
Agren LC, Ekman L, Lowenadler B, Lycke NY (1997) Genetically engineered nontoxic vaccine
adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit. J
Immunol 158:3936–3946
Ahmed T, Arifuzzaman M, Lebens M, Qadri F, Lundgren A (2009) CD4+ T-cell responses to an
oral inactivated cholera vaccine in young children in a cholera endemic country and the
enhancing effect of zinc supplementation. Vaccine 28:422–429
Akhiani AA, Pappo J, Kabok Z, Schon K, Gao W, Franzen LE, Lycke N (2002) Protection against
Akhiani AA, Schon K, Franzen LE, Pappo J, Lycke N (2004) Helicobacter pylori-specific
antibodies impair the development of gastritis, facilitate bacterial colonization, and counteract
resistance against infection. J Immunol 172:5024–5033
Akhiani AA, Stensson A, Schon K, Lycke N (2006) The nontoxic CTA1-DD adjuvant enhances
protective immunity against Helicobacter pylori infection following mucosal immunization.
Scand J Immunol 63:97–105
Ali M, Emch M, von Seidlein L, Yunus M, Sack DA, Rao M et al (2005) Herd immunity conferred
by killed oral cholera vaccines in Bangladesh: a reanalysis. Lancet 366:44–49
Appelmelk BJ, Faller G, Claeys D, Kirchner T, Vandenbroucke-Grauls CM (1998) Bugs on trial:
the case of Helicobacter pylori and autoimmunity. Immunol Today 19:296–299
Banerjee S, Medina-Fatimi A, Nichols R, Tendler D, Michetti M, Simon J et al (2002) Safety and
efficacy of low dose Escherichia coli enterotoxin adjuvant for urease based oral immunisation
against Helicobacter pylori in healthy volunteers. Gut 51:634–640
Becher D, Deutscher ME, Simpfendorfer KR, Wijburg OL, Pederson JS, Lew AM et al (2010)
Local recall responses in the stomach involving reduced regulation and expanded help mediate
vaccine-induced protection against Helicobacter pylori in mice. Eur J Immunol 40:2778–2790
Bhuiyan TR, Qadri F, Saha A, Svennerholm AM (2009) Infection by Helicobacter pylori in
Bangladeshi children from birth to two years: relation to blood group, nutritional status, and
seasonality. Pediatr Infect Dis J 28:79–85
Blanchard TG, Czinn SJ, Redline RW, Sigmund N, Harriman G, Nedrud JG (1999) Antibody-
independent protective mucosal immunity to gastric Helicobacter infection in mice. Cell
Immunol 191:74–80
Blanchard TG, Yu F, Hsieh CL, Redline RW (2003) Severe inflammation and reduced bacteria
load in murine helicobacter infection caused by lack of phagocyte oxidase activity. J Infect Dis
187:1609–1615
Brandtzaeg P, Johansen FE (2005) Mucosal B cells: phenotypic characteristics, transcriptional
regulation, and homing properties. Immunol Rev 206:32–63
Bumann D, Metzger WG, Mansouri E, Palme O, Wendland M, Hurwitz R et al (2001) Safety and
immunogenicity of live recombinant Salmonella enterica serovar Typhi Ty21a expressing
urease A and B from Helicobacter pylori in human volunteers. Vaccine 20:845–852
Chionh YT, Wee JL, Every AL, Ng GZ, Sutton P (2009) M-cell targeting of whole killed bacteria
induces protective immunity against gastrointestinal pathogens. Infect Immun 77:2962–2970
Chionh YT, Ng GZ, Ong L, Arulmuruganar A, Stent A, Saeed MA et al (2015) Protease-activated
receptor 1 suppresses Helicobacter pylori gastritis via the inhibition of macrophage cytokine
secretion and interferon regulatory factor 5. Mucosal Immunol 8:68–79
Corthesy B, Boris S, Isler P, Grangette C, Mercenier A (2005) Oral immunization of mice with
lactic acid bacteria producing Helicobacter pylori urease B subunit partially protects against
challenge with Helicobacter felis. J Infect Dis 192:1441–1449
Czerkinsky C, Cuburu N, Kweon MN, Anjuere F, Holmgren J (2011) Sublingual vaccination.
Hum Vaccin 7:110–114
DeLyria ES, Redline RW, Blanchard TG (2009) Vaccination of mice against H pylori induces a
strong Th-17 response and immunity that is neutrophil dependent. Gastroenterology
136:247–256
DeLyria ES, Nedrud JG, Ernst PB, Alam MS, Redline RW, Ding H et al (2011) Vaccine-induced
immunity against Helicobacter pylori in the absence of IL-17A. Helicobacter 16:169–178
Ding H, Nedrud JG, Wershil B, Redline RW, Blanchard TG, Czinn SJ (2009) Partial protection
against Helicobacter pylori in the absence of mast cells in mice. Infect Immun 77:5543–5550
Ding H, Nedrud JG, Blanchard TG, Zagorski BM, Li G, Shiu J et al (2013) Th1-mediated
immunity against Helicobacter pylori can compensate for lack of Th17 cells and can protect
mice in the absence of immunization. PLoS One 8:e69384
Dubois A, Fiala N, Heman-Ackah LM, Drazek ES, Tarnawski A, Fishbein WN et al (1994)
Natural gastric infection with Helicobacter pylori in monkeys: a model for spiral bacteria
infection in humans. Gastroenterology 106:1405–1417
Dubois A, Lee CK, Fiala N, Kleanthous H, Mehlman PT, Monath T (1998) Immunization against
natural Helicobacter pylori infection in nonhuman primates. Infect Immun 66:4340–4346
Dubois A, Berg DE, Incecik ET, Fiala N, Heman-Ackah LM, Del Valle J et al (1999) Host
specificity of Helicobacter pylori strains and host responses in experimentally challenged
nonhuman primates. Gastroenterology 116:90–96
Echtenacher B, Mannel DN, Hultner L (1996) Critical protective role of mast cells in a model of
acute septic peritonitis. Nature 381:75–77
Eisenberg JC, Czinn SJ, Garhart CA, Redline RW, Bartholomae WC, Gottwein JM et al (2003)
Protective efficacy of anti-Helicobacter pylori immunity following systemic immunization of
neonatal mice. Infect Immun 71:1820–1827
Ermak TH, Giannasca PJ, Nichols R, Myers GA, Nedrud J, Weltzin R et al (1998) Immunization
of mice with urease vaccine affords protection against Helicobacter pylori infection in the
absence of antibodies and is mediated by MHC class II-restricted responses. J Exp Med
188:2277–2288
Ernst PB, Gold BD (2000) The disease spectrum of Helicobacter pylori: the immunopathogenesis
of gastroduodenal ulcer and gastric cancer. Annu Rev Microbiol 54:615–640
Flach CF, Ostberg AK, Nilsson AT, Malefyt Rde W, Raghavan S (2011a) Proinflammatory
cytokine gene expression in the stomach correlates with vaccine-induced protection against
Helicobacter pylori infection in mice: an important role for interleukin-17 during the effector
phase. Infect Immun 79:879–886
Flach CF, Svensson N, Blomquist M, Ekman A, Raghavan S, Holmgren J (2011b) A truncated
form of HpaA is a promising antigen for use in a vaccine against Helicobacter pylori. Vaccine
29:1235–1241
Flach CF, Mozer M, Sundquist M, Holmgren J, Raghavan S (2012) Mucosal vaccination increases
local chemokine production attracting immune cells to the stomach mucosa of Helicobacter
pylori infected mice. Vaccine 30:1636–1643
Garhart CA, Redline RW, Nedrud JG, Czinn SJ (2002) Clearance of Helicobacter pylori infection
and resolution of postimmunization gastritis in a kinetic study of prophylactically immunized
Garhart CA, Heinzel FP, Czinn SJ, Nedrud JG (2003) Vaccine-induced reduction of Helicobacter
pylori colonization in mice is interleukin-12 dependent but gamma interferon and inducible
nitric oxide synthase independent. Infect Immun 71:910–921
Giuliani MM, Del Giudice G, Giannelli V, Dougan G, Douce G, Rappuoli R, Pizza M (1998)
Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-
labile enterotoxin with partial knockout of ADP-ribosyltransferase activity. J Exp Med
187:1123–1132
Gomez-Duarte OG, Lucas B, Yan ZX, Panthel K, Haas R, Meyer TF (1998) Protection of mice
against gastric colonization by Helicobacter pylori by single oral dose immunization with
attenuated Salmonella typhimurium producing urease subunits A and B. Vaccine 16:460–471
Goodman KJ, Correa P (2000) Transmission of Helicobacter pylori among siblings. Lancet
355:358–362
Graham DY, Opekun AR, Osato MS, El-Zimaity HM, Lee CK, Yamaoka Y et al (2004) Challenge
model for Helicobacter pylori infection in human volunteers. Gut 53:1235–1243
Guy B, Hessler C, Fourage S, Rokbi B, Millet MJ (1999) Comparison between targeted and
untargeted systemic immunizations with adjuvanted urease to cure Helicobacter pylori infec-
tion in mice. Vaccine 17:1130–1135
Hansson M, Hermansson M, Svensson H, Elfvin A, Hansson LE, Johnsson E et al (2008) CCL28 is
increased in human Helicobacter pylori-induced gastritis and mediates recruitment of gastric
immunoglobulin A-secreting cells. Infect Immun 76:3304–3311
Hitzler I, Oertli M, Becher B, Agger EM, Muller A (2011) Dendritic cells prevent rather than
promote immunity conferred by a Helicobacter vaccine using a mycobacterial adjuvant.
Gastroenterology 141:186–196, 196 e181
Hitzler I, Kohler E, Engler DB, Yazgan AS, Muller A (2012) The role of Th cell subsets in the
Immunol 3:142
Ismail HF, Fick P, Zhang J, Lynch RG, Berg DJ (2003) Depletion of neutrophils in IL-10(-/-) mice
delays clearance of gastric Helicobacter infection and decreases the Th1 immune response to
Helicobacter. J Immunol 170:3782–3789
Izcue A, Coombes JL, Powrie F (2006) Regulatory T-cells suppress systemic and mucosal immune
activation to control intestinal inflammation. Immunol Rev 212:256–271
Janzon A, Bhuiyan T, Lundgren A, Qadri F, Svennerholm AM, Sjoling A (2009) Presence of high
numbers of transcriptionally active Helicobacter pylori in vomitus from Bangladeshi patients
suffering from acute gastroenteritis. Helicobacter 14:237–247
Johansson EL, Bergquist C, Edebo A, Johansson C, Svennerholm AM (2004) Comparison of
different routes of vaccination for eliciting antibody responses in the human stomach. Vaccine
22:984–990
Kajikawa H, Yoshida N, Katada K, Hirayama F, Handa O, Kokura S et al (2007) Helicobacter
pylori activates gastric epithelial cells to produce interleukin-8 via protease-activated receptor
2. Digestion 76:248–255
Kaparakis M, Laurie KL, Wijburg O, Pedersen J, Pearse M, van Driel IR et al (2006) CD4+ CD25+
regulatory T cells modulate the T-cell and antibody responses in helicobacter-infected BALB/c
Kaparakis M, Walduck AK, Price JD, Pedersen JS, van Rooijen N, Pearse MJ et al (2008)
Macrophages are mediators of gastritis in acute Helicobacter pylori infection in C57BL/6
Kivi M, Tindberg Y (2006) Helicobacter pylori occurrence and transmission: a family affair?
Scand J Infect Dis 38:407–417
Kleanthous H, Myers GA, Georgakopoulos KM, Tibbitts TJ, Ingrassia JW, Gray HL et al (1998)
Rectal and intranasal immunizations with recombinant urease induce distinct local and serum
immune responses in mice and protect against Helicobacter pylori infection. Infect Immun
66:2879–2886
Kosunen TU, Hook-Nikanne J, Salomaa A, Sarna S, Aromaa A, Haahtela T (2002) Increase of
allergen-specific immunoglobulin E antibodies from 1973 to 1994 in a Finnish population and
a possible relationship to Helicobacter pylori infections. Clin Exp Allergy: J Br Soc Allergy
Clin Immunol 32:373–378
Kotloff KL, Sztein MB, Wasserman SS, Losonsky GA, DiLorenzo SC, Walker RI (2001) Safety
and immunogenicity of oral inactivated whole-cell Helicobacter pylori vaccine with adjuvant
among volunteers with or without subclinical infection. Infect Immun 69:3581–3590
Kunkel EJ, Kim CH, Lazarus NH, Vierra MA, Soler D, Bowman EP, Butcher EC (2003) CCR10
expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting
cells. J Clin Invest 111:1001–1010
Lee CK, Soike K, Giannasca P, Hill J, Weltzin R, Kleanthous H et al (1999a) Immunization of

rhesus monkeys with a mucosal prime, parenteral boost strategy protects against infection with
Helicobacter pylori. Vaccine 17:3072–3082
Lee CK, Soike K, Hill J, Georgakopoulos K, Tibbitts T, Ingrassia J et al (1999b) Immunization
with recombinant Helicobacter pylori urease decreases colonization levels following experi-
mental infection of rhesus monkeys. Vaccine 17:1493–1505
Liu KY, Shi Y, Luo P, Yu S, Chen L, Zhao Z et al (2011) Therapeutic efficacy of oral
immunization with attenuated Salmonella typhimurium expressing Helicobacter pylori
CagA, VacA and UreB fusion proteins in mice model. Vaccine 29:6679–6685
Lochhead P, El-Omar EM (2007) Helicobacter pylori infection and gastric cancer. Best Pract Res
Clin Gastroenterol 21:281–297
Londono-Arcila P, Freeman D, Kleanthous H, O’Dowd AM, Lewis S, Turner AK et al (2002)
Attenuated Salmonella enterica serovar Typhi expressing urease effectively immunizes mice
against Helicobacter pylori challenge as part of a heterologous mucosal priming-parenteral
boosting vaccination regimen. Infect Immun 70:5096–5106
Losonsky GA, Kotloff KL, Walker RI (2003) B cell responses in gastric antrum and duodenum
following oral inactivated Helicobacter pylori whole cell (HWC) vaccine and LT(R192G) in H
pylori seronegative individuals. Vaccine 21:562–565
Lu YJ, Yadav P, Clements JD, Forte S, Srivastava A, Thompson CM et al (2010) Options for
inactivation, adjuvant, and route of topical administration of a killed, unencapsulated pneu-
mococcal whole-cell vaccine. Clin Vaccine Immunol 17:1005–1012
Luzza F, Imeneo M, Maletta M, Monteleone G, Doldo P, Biancone L, Pallone F (1995) Isotypic
analysis of specific antibody response in serum, saliva, gastric and rectal homogenates of
Helicobacter pylori-infected patients. FEMS Immunol Med Microbiol 10:285–288
Malaty HM, Kim JG, Kim SD, Graham DY (1996a) Prevalence of Helicobacter pylori infection in
Korean children: inverse relation to socioeconomic status despite a uniformly high prevalence
in adults. Am J Epidemiol 143:257–262
Malaty HM, Paykov V, Bykova O, Ross A, Graham DP, Anneger JF, Graham DY (1996b)
Helicobacter pylori and socioeconomic factors in Russia. Helicobacter 1:82–87
Malaviya R, Ikeda T, Ross E, Abraham SN (1996) Mast cell modulation of neutrophil influx and
bacterial clearance at sites of infection through TNF-alpha. Nature 381:77–80
Malfertheiner P, Schultze V, Rosenkranz B, Kaufmann SH, Ulrichs T, Novicki D et al (2008)
Safety and immunogenicity of an intramuscular Helicobacter pylori vaccine in noninfected
volunteers: a phase I study. Gastroenterology 135:787–795
Malfertheiner P, Selgrad M, Wex T, Bornschein J, Palla E, Del Giudice G et al (2012) Efficacy of
an investigational recombinant antigen based vaccine against a CagA H. pylori infectious
challenge in healthy volunteers. Gastroenterology 142:S184–S184
Marchetti M, Rossi M, Giannelli V, Giuliani MM, Pizza M, Censini S et al (1998) Protection
against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens
is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant.
Vaccine 16:33–37
Mattsson A, Lonroth H, Quiding-Jarbrink M, Svennerholm AM (1998a) Induction of B cell
responses in the stomach of Helicobacter pylori- infected subjects after oral cholera vaccina-
tion. J Clin Invest 102:51–56
Mattsson A, Quiding-Jarbrink M, Lonroth H, Hamlet A, Ahlstedt I, Svennerholm A (1998b)
Antibody-secreting cells in the stomachs of symptomatic and asymptomatic Helicobacter
pylori-infected subjects. Infect Immun 66:2705–2712
Metzger WG, Mansouri E, Kronawitter M, Diescher S, Soerensen M, Hurwitz R et al (2004)
Impact of vector-priming on the immunogenicity of a live recombinant Salmonella enterica
serovar typhi Ty21a vaccine expressing urease A and B from Helicobacter pylori in human
volunteers. Vaccine 22:2273–2277
Michetti P, Kreiss C, Kotloff KL, Porta N, Blanco JL, Bachmann D et al (1999) Oral immunization
with urease and Escherichia coli heat-labile enterotoxin is safe and immunogenic in
Helicobacter pylori-infected adults. Gastroenterology 116:804–812
Michetti M, Kelly CP, Kraehenbuhl JP, Bouzourene H, Michetti P (2000) Gastric mucosal alpha
(4)beta(7)-integrin-positive CD4 T lymphocytes and immune protection against helicobacter
infection in mice. Gastroenterology 119:109–118
Moran AP (2008) Relevance of fucosylation and Lewis antigen expression in the bacterial
gastroduodenal pathogen Helicobacter pylori. Carbohydr Res 343:1952–1965
Negrini R, Savio A, Poiesi C, Appelmelk BJ, Buffoli F, Paterlini A et al (1996) Antigenic mimicry
between Helicobacter pylori and gastric mucosa in the pathogenesis of body atrophic gastritis.
Norton EB, Lawson LB, Freytag LC, Clements JD (2011) Characterization of a mutant
Escherichia coli heat-labile toxin, LT(R192G/L211A), as a safe and effective oral adjuvant.
Clin Vaccine Immunol: CVI 18:546–551
Nurgalieva ZZ, Conner ME, Opekun AR, Zheng CQ, Elliott SN, Ernst PB et al (2005) B-cell and
T-cell immune responses to experimental Helicobacter pylori infection in humans. Infect
Immun 73:2999–3006
Nystrom J, Svennerholm AM (2007) Oral immunization with HpaA affords therapeutic protective
immunity against H. pylori that is reflected by specific mucosal immune responses. Vaccine
25:2591–2598
Nystrom J, Raghavan S, Svennerholm AM (2006) Mucosal immune responses are related to
reduction of bacterial colonization in the stomach after therapeutic Helicobacter pylori immu-
nization in mice. Microbes Infect/Inst Pasteur 8:442–449
Nystrom-Asklin J, Adamsson J, Harandi AM (2008) The adjuvant effect of CpG oligodeoxynu-
cleotide linked to the non-toxic B subunit of cholera toxin for induction of immunity against
H. pylori in mice. Scand J Immunol 67:431–440
Ogra PL, Okayasu H, Czerkinsky C, Sutter RW (2011) Mucosal immunity to poliovirus. Expert
Rev Vaccines 10:1389–1392
Ohkura N, Kitagawa Y, Sakaguchi S (2013) Development and maintenance of regulatory T cells.
Immunity 38:414–423
Ossovskaya VS, Bunnett NW (2004) Protease-activated receptors: contribution to physiology and
disease. Physiol Rev 84:579–621
Panthel K, Faller G, Haas R (2003) Colonization of C57BL/6J and BALB/c wild-type and
knockout mice with Helicobacter pylori: effect of vaccination and implications for innate
and acquired immunity. Infect Immun 71:794–800
Pappo J, Torrey D, Castriotta L, Savinainen A, Kabok Z, Ibraghimov A (1999) Helicobacter pylori
infection in immunized mice lacking major histocompatibility complex class I and class II
functions. Infect Immun 67:337–341
Park EJ, Chang JH, Kim JS, Yum JS, Chung SI (2000) The mucosal adjuvanticity of two nontoxic
mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes. Exp Mol
Med 32:72–78
Parsonnet J (2003) What is the Helicobacter pylori global reinfection rate? Can J Gastroenterol ¼ J
Can Gastroenterol 17(Suppl B):46B–48B
Quiding-Jarbrink M, Ahlstedt I, Lindholm C, Johansson EL, Lonroth H (2001a) Homing commit-
ment of lymphocytes activated in the human gastric and intestinal mucosa. Gut 49:519–525
Quiding-Jarbrink M, Lonroth H, Ahlstedt I, Holmgren J, Svennerholm AM (2001b) Human gastric
B cell responses can be induced by intestinal immunisation. Gut 49:512–518
Quiding-Jarbrink M, Raghavan S, Sundquist M (2010) Enhanced M1 macrophage polarization in
human helicobacter pylori-associated atrophic gastritis and in vaccinated mice. PLoS One 5:
e15018
Rad R, Brenner L, Bauer S, Schwendy S, Layland L, da Costa CP et al (2006) CD25+/Foxp3+ T
cells regulate gastric inflammation and Helicobacter pylori colonization in vivo. Gastroenter-
ology 131:525–537
Radcliff FJ, Hazell SL, Kolesnikow T, Doidge C, Lee A (1997) Catalase, a novel antigen for
Helicobacter pylori vaccination. Infect Immun 65:4668–4674
Raghavan S, Hjulstrom M, Holmgren J, Svennerholm AM (2002a) Protection against experimen-
tal Helicobacter pylori infection after immunization with inactivated H. pylori whole-cell
vaccines. Infect Immun 70:6383–6388
Raghavan S, Svennerholm AM, Holmgren J (2002b) Effects of oral vaccination and immunomo-
dulation by cholera toxin on experimental Helicobacter pylori infection, reinfection, and
gastritis. Infect Immun 70:4621–4627
Raghavan S, Fredriksson M, Svennerholm AM, Holmgren J, Suri-Payer E (2003) Absence of CD4
+CD25+ regulatory T cells is associated with a loss of regulation leading to increased
pathology in Helicobacter pylori-infected mice. Clin Exp Immunol 132:393–400
Raghavan S, Ostberg AK, Flach CF, Ekman A, Blomquist M, Czerkinsky C, Holmgren J (2010)
Sublingual immunization protects against Helicobacter pylori infection and induces T and B
cell responses in the stomach. Infect Immun 78:4251–4260
Rappuoli R, Pizza M, Douce G, Dougan G (1999) Structure and mucosal adjuvanticity of cholera
and Escherichia coli heat-labile enterotoxins. Immunol Today 20:493–500
Richie EE, Punjabi NH, Sidharta YY, Peetosutan KK, Sukandar MM, Wasserman SS et al (2000)
Efficacy trial of single-dose live oral cholera vaccine CVD 103-HgR in North Jakarta,
Indonesia, a cholera-endemic area. Vaccine 18:2399–2410
Robinson K, Argent RH, Atherton JC (2007) The inflammatory and immune response to
Helicobacter pylori infection. Best Pract Res Clin Gastroenterol 21:237–259
Roth KA, Kapadia SB, Martin SM, Lorenz RG (1999) Cellular immune responses are essential for
the development of Helicobacter felis-associated gastric pathology. J Immunol 163:1490–1497
Rupnow MF, Shachter RD, Owens DK, Parsonnet J (2000) A dynamic transmission model for
predicting trends in Helicobacter pylori and associated diseases in the United States. Emerg
Infect Dis 6:228–237
Rupnow MF, Shachter RD, Owens DK, Parsonnet J (2001) Quantifying the population impact of a
prophylactic Helicobacter pylori vaccine. Vaccine 20:879–885
Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M (1995) Immunologic self-tolerance
maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown
of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol
155:1151–1164
Sawai N, Kita M, Kodama T, Tanahashi T, Yamaoka Y, Tagawa Y et al (1999) Role of gamma
interferon in Helicobacter pylori-induced gastric inflammatory responses in a mouse model.
Simoons-Smit IM, Appelmelk BJ, Verboom T, Negrini R, Penner JL, Aspinall GO et al (1996)
Typing of Helicobacter pylori with monoclonal antibodies against Lewis antigens in lipopoly-
saccharide. J Clin Microbiol 34:2196–2200
Sjokvist Ottsjo L, Flach CF, Clements J, Holmgren J, Raghavan S (2013) A double mutant heat-
labile toxin from Escherichia coli, LT(R192G/L211A), is an effective mucosal adjuvant for
vaccination against Helicobacter pylori infection. Infect Immun 81:1532–1540
Sjokvist Ottsjo L, Flach CF, Nilsson S, Malefyt RDW, Walduck AK, Raghavan S (2015) Defining
the roles of IFN-γ and IL-17A in inflammation and protection against Helicobacter pylori
infection. PLOSone 10:e0131444
Smythies LE, Novak MJ, Waites KB, Lindsey JR, Morrow CD, Smith PD (2005) Poliovirus
replicons encoding the B subunit of Helicobacter pylori urease protect mice against H. pylori
infection. Vaccine 23:901–909
Solnick JV, Canfield DR, Hansen LM, Torabian SZ (2000) Immunization with recombinant
Helicobacter pylori urease in specific-pathogen-free rhesus monkeys (Macaca mulatta). Infect
Immun 68:2560–2565
Song JH, Kim JI, Kwon HJ, Shim DH, Parajuli N, Cuburu N et al (2009) CCR7-CCL19/CCL21-
regulated dendritic cells are responsible for effectiveness of sublingual vaccination. J Immunol
182:6851–6860
Stuller KA, Ding H, Redline RW, Czinn SJ, Blanchard TG (2008) CD25+ T cells induce
Helicobacter pylori-specific CD25- T-cell anergy but are not required to maintain persistent
hyporesponsiveness. Eur J Immunol 38:3426–3435
Summerton NA, Welch RW, Bondoc L, Yang HH, Pleune B, Ramachandran N et al (2010)
Toward the development of a stable, freeze-dried formulation of Helicobacter pylori killed
whole cell vaccine adjuvanted with a novel mutant of Escherichia coli heat-labile toxin.
Vaccine 28:1404–1411
Sutton P, Wilson J, Lee A (2000) Further development of the Helicobacter pylori mouse vacci-
nation model. Vaccine 18:2677–2685
Sutton P, Doidge C, Pinczower G, Wilson J, Harbour S, Swierczak A, Lee A (2007) Effectiveness
of vaccination with recombinant HpaA from Helicobacter pylori is influenced by host genetic
background. FEMS Immunol Med Microbiol 50:213–219
Taylor JM, Ziman ME, Canfield DR, Vajdy M, Solnick JV (2008) Effects of a Th1- versus a
Th2-biased immune response in protection against Helicobacter pylori challenge in mice.
Microb Pathog 44:20–27
van Ginkel FW, Jackson RJ, Yuki Y, McGhee JR (2000) Cutting edge: the mucosal adjuvant
cholera toxin redirects vaccine proteins into olfactory tissues. J Immunol 165:4778–4782
Velin D, Bachmann D, Bouzourene H, Michetti P (2005) Mast cells are critical mediators of
vaccine-induced helicobacter clearance in the mouse model. Gastroenterology 129:142–155
Velin D, Favre L, Bernasconi E, Bachmann D, Pythoud C, Saiji E et al (2009) Interleukin-17 is a
critical mediator of vaccine-induced reduction of Helicobacter infection in the mouse model.
Gastroenterology 136:2237–2246 e2231
Velin D, Narayan S, Bernasconi E, Busso N, Ramelli G, Maillard MH et al (2011) PAR2 promotes
vaccine-induced protection against Helicobacter infection in mice. Gastroenterology
141:1273–1282, 1282 e1271
Wee JL, Chionh YT, Ng GZ, Harbour SN, Allison C, Pagel CN et al (2010) Protease-activated
receptor-1 down-regulates the murine inflammatory and humoral response to Helicobacter
Weltzin R, Guy B, Thomas WD Jr, Giannasca PJ, Monath TP (2000) Parenteral adjuvant activities
of Escherichia coli heat-labile toxin and its B subunit for immunization of mice against gastric
Helicobacter pylori infection. Infect Immun 68:2775–2782
Yamaoka Y, Malaty HM, Osato MS, Graham DY (2000) Conservation of Helicobacter pylori
genotypes in different ethnic groups in Houston, Texas. J Infect Dis 181:2083–2086
Zu Y, Cassai ND, Sidhu GS (2000) Light microscopic and ultrastructural evidence of in vivo
phagocytosis of Helicobacter pylori by neutrophils. Ultrastruct Pathol 24:319–323
Index
A AmiF, 33, 34, 44

Acid adaptation, 32, 33, 46, 47, 51, 202 Ammonia, 30, 32–37, 39, 44, 50, 66, 167
Acidity, 30, 39, 44, 280, 560 metabolism, 30, 32, 39–44
Acid-responsive signalling regulon (ArsRS), Ancestry, 14–16, 345, 508, 509
31, 33, 34, 37, 59, 65, 155, 202 Ancient DNA, 22, 23
Acinetobacter baumannii, 559 Andes Cordillera, 505, 506
Actinobacteria, 283, 545, 546, 551, 558 AnsB, 40, 42, 44
ADAM, 331 Antibiotic resistance, 203, 458–460, 472, 474,
ADAM17, 104 476, 480, 482, 485, 496, 497, 513,
Adenocarcinoma, 58, 64, 94, 251, 252, 254, 525–531, 534, 536, 563, 576
274, 278, 279, 281, 282, 285, 353, 374, Antibiotics, 57, 283, 286, 308, 314, 347, 425,
381, 390, 392, 398, 399, 403–408, 411, 428, 436, 445, 453, 455, 459, 460,
412, 509, 513 472–476, 478, 479, 482–485, 492,
Adhesins 496–499, 512, 513, 521, 525, 530, 531,
AlpA, 61, 62 534, 535, 544, 548–552, 554, 557, 559,
AlpB, 61, 62 562, 564, 576, 577, 588, 589, 594
BabA, 60, 61, 118, 143–159, 166, 286, 408, Antisense RNA, 196, 198, 200–203, 206
409, 448 Antisense transcription, 150, 190, 192, 201,
OipA, 61, 281 202, 208
SabA, 60, 61, 143–159, 166, 409, 448 Antisense transcriptional start site (asTSS),
Adjuvants, 307, 311, 544, 558, 562, 567, 578, 195, 196
581–589, 591, 592, 594 Apoptosis, 63, 64, 66, 71, 73–76, 93, 95, 99,
African, 7, 12, 14, 15, 17–20, 22, 23, 221, 226, 126, 128–130, 166–168, 170, 173, 175,
346, 372, 404, 446, 508, 509, 550 247, 308, 326, 330, 350, 429, 430,
Agrobacterium tumefaciens, 91 435, 509
AGS, 44, 66, 75, 91, 97, 100, 101, 104, 167, Aptamer-tagged RNA, 206
168, 172, 179, 247, 550 Arginase, 34, 44, 45, 73
Aliphatic, 34, 44 ArsRS. See Acid-responsive signalling regulon
Allergic asthma, 313–315 (ArsRS)
α-carbonic anhydrase, 34–36 Artificial antisense RNA (asRNA), 200–203
α-Pix, 99–101 A second out-of-Africa migration, 21–22
Amerindian, 221, 223, 226, 509 Asia, 7, 11, 14, 16, 17, 20, 21, 23, 179, 221,
Amidase, 33, 34, 43, 44 228, 368, 376, 379, 391, 403, 479, 480,
Amidotransferase, 43 494, 496, 512–514, 520–536
AmiE, 33, 34, 44 Asparaginase, 41, 44–45

DOI 10.1007/978-4-431-55936-8
604 Index
Asparagine, 39, 40, 42, 176 280, 281, 284, 300, 301, 303, 407, 411,
Aspartyl-tRNA synthetase, 43 448, 590, 591
Aspirin, 118, 367, 371, 493, 494, 522, 523 cagA, 62, 63, 70, 89–105, 118, 301
Asthma, 72, 168, 170, 173, 175, 300, 313, 314, cagL, 63, 92, 93, 104, 105
316, 379, 381, 451, 455, 576, 577, 594 T4SS, 62, 66, 90–94, 97, 102–105, 118,
Asymptomatic carrier, 71, 304, 306, 316 180, 276, 281, 282, 300, 301, 407, 411
Atopic dermatitis, 314 Cag pathogenicity island, 62–63, 90, 91, 118,
Atopy, 455 151, 240, 275, 300, 407
Atrophic gastritis, 118, 276, 305, 307, 350, CagS, 93
373, 387–390, 392–394, 404, 450, 493, CagZ, 93
507, 508, 511, 514, 521–524 Carbon dioxide, 34, 37, 49, 58, 544
Autoimmune disease, 429, 431, 453 Carbonic anhydrase, 34, 35, 37–39
Autophagy, 64, 70, 73, 76, 124–126, 130 Carbon storage regulator (CsrA), 204, 205
Autotransporter, 63, 65–66, 114, 115 CARMA, 433, 434
Carnivores, 251
Caspase, 302, 429
B Cats, 114, 235, 236, 238–242, 253, 274, 284
Bacillus clausii, 555, 559 cDNA, 190–195, 198, 209, 280, 331, 333
Bacillus subtilis, 169, 555, 558, 559 libraries, 190–194
Bacteriocin, 547, 558, 562, 564 Cell permeability, 127
Bacteroides fragilis, 546 Cell polarity, 63, 95, 98, 101
BALB/c mice, 241, 242, 246, 582 Cell-translocating serine/threonine kinase
Bantu migrations, 8, 9 (CtkA), 175
B-cell lymphoma (BCL), 74, 168, 432 Chemotaxis, 32, 37, 59, 60, 129, 169, 170, 174,
B-cells, 129, 303, 306, 308, 424, 425, 427–429, 199, 201, 245, 281
431, 433–435, 590–593 Chemotaxis receptor TlpB, 60, 199, 201
Bifidobacteria, 544, 551, 552 Childhood infection, 30, 306, 341, 405, 444,
Bifidobacterium, 283, 547–553, 555–558, 448, 454, 460, 523, 576, 577, 594
561, 565 Chile, 370, 444, 448, 452, 505, 507, 509–510,
B. bifidum, 551 512, 555
Bismuth, 472–476, 478, 481–486, 497, 498, China, 7, 11, 228, 370, 377, 444, 449, 473, 480,
507, 531–533, 535, 536 482–484, 513, 520, 522–526, 528, 529,
Bismuth quadruple therapy, 476, 478, 531–534, 537, 552
481–485, 497–499, 533–536 Cholera toxin adjuvant, 307, 311
Blood group antigen-binding (BabA) Chromosomal translocations, 424, 429
ABO blood group antigen receptors, 145 Chronic inflammation, 58, 105, 357, 424, 427
binding to gastric mucins, 241 Ciprofloxacin, 482, 525, 527, 529, 537
gastric disease, 150–151 Cis-encoded, 197–199, 201–203
genomic location, 147 Clarithromycin, 238, 458–460, 472, 473, 475,
identification of BabA, 145–147 476, 478–485, 496–499, 508, 512, 525,
homologous recombination, 148, 158 526, 528–537
outer membrane vesicles, 157 resistance, 458–460, 473, 478–480, 485,
regulation of BabA expression, 149–150 496, 497, 499, 525, 528, 530, 532–536
slipped-strand mispairing, 148, 149, 153 Clinical manifestations
Brazil, 373, 377, 452, 456, 457, 504, 511, 515 asthma, 451, 455
beneficial effects, 455–456
chronic idiopathic thrombocytopenic
C purpura, 453–454
C57BL/6, 60, 241, 246, 277, 305, 413 extra gastric diseases, 451–455
CagAPY, 94, 95, 97, 100 ghrelin, 454
CagD, 93 growth retardation, 454–455
CagL, 63, 92, 93, 102, 104, 105, 177, 300 peptic ulcer disease, 449–450
CagPAI, 61, 62, 66, 70, 89–105, 118, 151, 180, recurrent abdominal pain, 450–451
240, 243, 245, 247, 250, 251, 275, 276, sideropenic anaemia, 452–453
Index 605
Clinical trials, 238, 283, 453, 454, 492, 512, Differential RNA-seq, 190, 192, 194
545, 549–552, 558, 559, 564, 577, 583, Diffuse large B-cell lymphoma (DLBCL), 424,
584, 587–591, 594 425, 427, 430, 431, 436
CLIP-seq, 206 DNA duplication associated with inversion
Coalescence, 17–22 (DDAI), 224–226
Co-immunoprecipitation of RNA binding DNA glycosylase, 228
proteins, 191, 205 DNA methylation, 226, 228–229, 326
Colombia, 7, 155, 454, 505–509, 512, 514 DNA methyltransferases, 218, 226, 228
Colony-forming units, 286, 412, 551, 578, 582, DNA repeats, 92
583 Dogs, 235, 236, 238–242, 253, 274
Commensal, 4, 310, 413, 545, 562, 563 Dolphins, 236, 249, 250
Concomitant, 48, 242, 286, 476, 478–481, 483, Dosages, 460, 498
485, 497, 498, 505, 512, 533, 535, dRNA-seq, 190, 192–203, 208, 209
536, 544 Dual-concomitant, 535
COX-/PGE, 127–129, 168, 280, 326–332, Dukoral™ vaccine, 590
335, 336 Duodenal ulcer-promoting gene A (dupA), 68,
Crohn’s disease (CD), 376, 378, 379 179–180, 530
Cross-linking immunoprecipitation
sequencing, 206
Cytokines, 65, 72, 129, 150, 166, 170–173, E
175, 246, 277, 279–281, 303–308, East Asia migrations, 11, 228
310–313, 316, 329–333, 335, 340, 342, East Asian, 11, 20, 63, 221, 222, 224, 226, 288,
344, 348, 349, 353–354, 366, 368, 376, 391, 520, 524, 525, 528, 535
378, 427, 432, 448, 449, 544, 560, 561, E-cadherin, 67, 99, 101, 166, 174, 177, 178
576, 578, 579, 586, 593 Endosomal compartments, 121–125
Cytoskeleton, 127, 177, 254 Enigma, 508, 521
Cytotoxin-associated gene product (CagA), Enterobacter cloacae, 559
62–64, 66, 70, 71, 73–75, 89–105, 117, Enterohepatic helicobacters, 48, 234, 236,
118, 130, 150, 165, 166, 172, 174, 252–254
177–180, 194, 221–223, 280–282, 301, Enterotoxigenic E. coli heat labile
343, 366, 368, 374, 380, 407, 408, 410, toxin (LT), 581
411, 430, 449, 508, 509, 529, 530, 534, Eosinophilia, 314
581, 583, 584, 591 Epidemiology
age of acquisition, 444
cohort phenomenon, 444
D immigrants, 479
Deep sequencing, 190, 193 incidence, 444–445
Defensin, 561 prevalence, 444–445
Dendritic cells (DCs), 61, 68, 72, 104, 128, transient infection, 287
129, 166, 168, 175, 276, 302–304, Epidermal growth factor receptor (EGFR), 97,
309–315, 378, 379, 576, 581, 586, 587, 103, 104, 301, 331, 332, 335, 350
593 Epithelial-mesenchymal transition (EMT), 68,
maturation, 61, 72, 129, 311, 312 100, 101, 172, 175
Diagnostic tests EPIYA, 63, 94, 95, 100, 221–223, 407, 449
invasive tests, 456 motifs, 63, 94, 95, 221, 223, 407, 449
PCR from stools, 458 Eradication, 157, 242, 308, 314, 327, 352, 357,
serology, 457–458 366, 369–374, 376, 378–381, 397, 410,
stool antigen test, 458 425, 428, 430, 436, 450, 453, 455, 459,
urea breath test, 457 460, 472–477, 479–481, 483–485,
Diet, 251, 282, 288, 406, 409–411, 413, 452, 492–499, 508, 510–513, 515, 521, 523,
529, 545, 547 524, 528–536, 544, 545, 548–558, 564,
Diagnostic tests, 457 567, 576, 577, 581, 589
606 Index
Esophagitis, 237, 313, 372, 373, 396 313, 315, 316, 327, 329, 330, 335, 342,
Esophagus 344, 351, 380, 381, 389, 391, 393, 396,
adenocarcinoma, 513 397, 427, 428, 449, 454, 456, 493, 495,
Barrett’s, 313, 366, 374 507, 509, 530, 544, 551, 579, 580, 586,
European strategies, 495 589–591
Excludon, 202 Gastric pathologies, 72, 177, 238–241, 391, 413
Gastrin, 104, 241, 244, 278, 280, 367, 368, 375,
393, 394, 398, 410, 496, 530
F Gastritis, 3, 58, 72, 90, 98, 118, 145, 155, 158,
Ferrets, 236, 250–251, 274 168, 172, 235, 237, 239, 242, 244, 246,
Fibronectin, 104, 166, 174, 177, 178 248, 250, 252, 275–277, 279–282,
fineSTRUCTURE, 221 285–288, 303, 305, 307, 329, 331, 332,
First-line, 459, 460, 476–482, 485, 496–499, 335, 340, 347, 350, 354, 355, 366, 367,
513, 531–535 373–375, 378, 380, 381, 387–394,
FlaA, 59, 69, 70, 205, 251, 281, 282, 411, 562 397–399, 404, 410, 411, 449, 450, 452,
FlaB, 59, 205, 251, 281, 282, 411, 562 454–456, 472, 493, 495, 507, 508,
Flagella, 59, 60, 65, 251, 275, 281, 302 511, 514, 520–524, 530, 544, 579,
Fluoroquinolone, 474, 475, 482–484, 496, 588–591, 593
528, 529 Gastroesophageal reflux disease (GERD), 366,
Formamidase, 33, 44 372–374, 381, 447, 458, 494, 521
FoxP, 72, 304, 310, 311, 378, 592 Genetic diversity, 5, 12–15, 20, 58, 68, 407
FrpB4, 46 Genetic polymorphisms, 346, 347, 355, 356
Functional dyspepsia, 366, 374–377, 381, Genome sequencing, 22, 196, 245
493, 522 Genome wide association studies (GWAS),
Fur, 33, 249 341, 345–347, 349–351
Furazolidone, 472, 482–484, 486, 526, Genomics, 22–23, 240–242, 245–247
527, 532 Gerbil, 33, 47, 60, 61, 74, 94, 105, 131, 132,
Fusobacteria, 545 148–151, 201, 236, 241, 246, 247, 274,
279, 283, 286, 289, 301, 408–411, 509
Ghrelin, 375, 454
G Global H. pylori phylogeny, 18–19
Gamma-glutamyl transpeptidase, 41, 44, Glutamine, 39–42, 48, 66, 71, 167, 168, 247,
66–67, 71, 72, 151, 166–168, 175, 245, 310
247, 309–311 Global burden, 504–505, 544
Gan mice, 327–331, 333–335 Glutamine synthetase (GlnA), 39–42
Gastric adenocarcinoma, 58, 64, 94, 251, 274, Glutamyl-tRNA synthetase, 40, 43
278, 279, 281, 282, 285, 353, 392, Glutathione, 66, 125, 126, 167, 247, 310,
403–407, 411, 520 334, 352
Gastric cancer, 30, 68, 74, 75, 90, 94, 98, 104, Glycogen synthase kinase (GSK), 99, 101
105, 113, 117, 118, 144, 150, 155, 158, Grb2, 95–100
172, 179, 241, 276, 278–283, 288, 301, Guidelines, 247, 279, 371, 456, 460, 472, 483,
303, 305, 307, 308, 313, 326, 327, 484, 492, 493, 521–524, 531–535, 548
329–331, 333, 336, 340, 347–351, 357,
374, 387, 391, 396, 398, 399, 403–413,
424, 452, 455, 460, 472, 492–496, 499, H
504–515, 520–524, 530, 535, 544, 546, Halitosis, 378–379
551, 576, 577, 593 Helicobacter felis, 236–242, 252, 274, 275,
Gastric disease, 76, 94, 114, 117, 118, 130, 277, 278, 289, 410, 411, 579–581
150–151, 248, 316, 340, 343, 509 Helicobacter heilmannii, 235–243, 245,
Gastric mucosa, 58, 61, 66–68, 73–76, 90, 118, 246, 286, 287, 428
130, 131, 144, 146, 152, 155, 157, 158, Helicobacter mustelae, 236, 250, 251, 274
201, 233, 239–241, 246, 250, 251, Helicobacter outer membrane protein Q
277–280, 282, 300, 301, 304, 305, 311, (HopQ), 166, 174, 178–180
Index 607
Helicobacter outer membrane protein Z Hypochlorhydria, 130, 241, 242, 250, 279, 340,
(HopZ), 166, 174, 178–180 347, 348, 366, 372, 380, 392, 394, 452,
Helicobacter pylori (H. pylori), 3–23, 29–51, 454, 473, 546
57–76, 90–92, 94–99, 101–105,
113–132, 144–153, 155–159, 165–180,
189–209, 217–229, 233–255, 273–289, I
299–316, 326, 327, 335, 340–349, 351, Idiopathic thrombocytopenic purpura,
352, 354–357, 365–381, 387–399, 379–380, 493, 521, 522
403–413, 423–436, 443–460, 471–486, Illegitimate recombination, 218, 223, 224
491–499, 503–515, 519–537, 543–568, Immune response, 45, 58, 66, 68, 69, 71–72,
575–594 76, 92, 98, 102, 128, 157, 170, 175, 242,
Helicobacter pylori human association, 246, 275, 276, 287, 300–306, 311, 316,
19, 20 329, 330, 335, 355, 376, 378, 424, 449,
Helicobacter pylori out of Africa, 12, 19, 21 456, 460, 548, 562, 576, 578–581, 585,
Helicobacter suis, 167, 235–238, 243–248, 587, 589–594
286, 287 Immune systems, 58, 68–70, 90, 168–171, 175,
Helicobacter pylori eradication, 327, 352, 190, 300, 301, 306, 316, 343, 354, 381,
369–374, 376, 378, 379, 381, 425, 428, 426, 560–562, 567, 576, 587, 592
430, 436, 455, 472, 477, 492–496, 499, Immune tolerance, 72, 303, 304, 306, 310, 311,
511–513, 515, 521, 523, 524, 528–537, 313–316
544, 545, 567, 576 Immunoglobulinantibody test, 428
Heme oxygenase (HO-1), 97, 98 Immunomodulating autotransporter (ImaA),
Herd immunity, 577 65, 66
Hfq, 198, 204, 206, 208, 209 Immunomodulation, 300, 561
High and low clarithromycin resistance, 480, Immunomodulators, 560
499, 533, 536 Immunopathology, 303–306, 308, 592
High temperature requirement A (HtrA), 67, Indications, 252, 254, 255, 451, 456, 492, 494,
166, 174–178, 180 496, 521, 523, 524, 535, 592
High-throughput sequencing, 190 Indigenous groups, 505
Histology, 132, 237, 398, 404, 426, 448, 449, Inducible nitric oxide synthase (iNOS), 45, 73,
456, 507 278, 280
Histone, 103 Infection, 22, 30, 32, 45, 57–62, 65, 68, 72, 73,
Homeostasis, 30, 36, 37, 46, 47, 330, 411, 412, 75, 240–248, 305, 341–347, 351, 410,
544 411, 413, 521–524
Homologous recombination, 13, 17, 148, 158, Inflammasome, 303
218–221, 223, 224, 228, 229 Inflammatory bowel disease (IBD), 253, 254,
Honduras, 507, 512 303, 313–315, 354, 376–378, 451,
Host genetics, 304, 340–352, 354, 357, 406, 456, 544
508 Innate immune responses, 69, 102, 300–304,
Housekeeping gene, 4, 11, 12, 16–19, 22, 23, 329, 330, 335
284 Insulin-gastrin (INS-GAS), 241, 242, 278, 410,
House-keeping RNAs, 198, 200 412, 413
Hp0169, 166, 174, 176 Integrin α5β1, 63, 66, 104, 105, 300
Hpn, 48 Intention-to-treat, 480, 533–535, 550
HspA, 47–48 Interferon-γ (IFN-γ), 71, 246, 277, 280, 305,
HtrA. See High temperature requirement A 307, 313, 376, 449, 576, 578, 586,
(HtrA) 589–593
Human DNA, 6, 7, 11–12, 22 Intergenic regions (IGR), 191, 199, 208
Hummingbird phenotype, 63 Interleukin (IL), 128, 241, 246, 253, 300,
Hybrid, 15, 16, 21, 207, 221, 478, 479, 481, 302–305, 307, 309, 310, 312–315,
497, 498, 531, 535, 536 329–332, 340, 342, 344, 348, 349, 378,
Hydrogenase, 46–50 407, 410, 411, 430, 432, 448, 449, 452,
608 Index
455, 551, 558, 559, 561, 576, 578–580, Lymfomagenesis, 246, 424, 428, 430–433, 435
586, 590, 592, 593 Lymphocytes, 68, 71, 72, 128, 237, 242, 246,
Interleukin-10 (IL-10), 61, 246, 253, 277, 280, 247, 276, 287, 309, 426, 427, 432, 560,
302–305, 310–313, 315, 348, 378, 448, 587, 589
455, 580, 592
Internal TSS (iTSS), 195, 197
International Agency for Research on Cancer M
(IARC), 58, 504, 513–515 Maastricht-Florence consensus report., 371,
Intestinal metaplasia, 118, 237, 242, 252, 276, 374, 380, 492
282, 288, 305, 390, 392, 398, 404, 408, Macrophage apoptosis, 102
456, 494, 495, 507, 508, 511, 521 Macrophages, 45, 68–70, 73, 74, 97, 98, 102,
Inversion, 218, 224–226 129, 171, 172, 327, 329–333, 335, 343,
Iron, 8, 33, 44, 46, 73, 105, 130, 170, 205, 241, 344, 576, 587, 593
242, 251, 282, 380, 409–411, 451–453, MALT, 235, 237, 242, 246, 251, 254, 340,
493, 521, 522, 544 351–356, 423–436, 492, 493, 496,
Iron deficiency anemia (IDA), 380, 452, 453, 521–524, 544
493, 521, 523, 544 MALT lymphoma, 237, 242, 246, 247, 251,
Isolation-by-distance (IBD), 12 254, 340, 351–356, 424–433, 435, 436,
493, 521–524, 544
Mitogen activated protein kinases (MAPK), 62,
J 96, 103, 127, 170, 179
Junctions, 95, 96, 99, 100, 124, 174, 177, 178, Marginal zone, 351, 423, 424
277, 404 Marine mammals, 249–250
Mast cells, 129, 169, 313, 375, 576, 580, 593
Maturation, 33, 37, 38, 46, 48–50, 61, 70, 72,
K 75, 125, 129, 191, 200, 202, 204, 207,
Klebsiella pneumoniae, 559 311, 312, 379, 431
Mechanism of action, 562, 564
Membrane channels, 64, 74, 115, 118, 119,
L 124, 126, 132
Lack of H. pylori among Pygmies, 22 Metal, 32–34, 37, 45–50, 190, 191, 223
Lactic acid bacteria, 544, 549, 551 metallochaperone, 47, 49, 50
Lactobacillus, 413, 546, 548–551, 553–558, Metaplasia, 65, 118, 132, 179, 237, 242, 252,
560–563, 566, 567, 583 276, 277, 279, 282, 288, 305, 308, 314,
L. acidophilus, 549–551, 553, 556, 557, 327, 390, 392, 397, 398, 404, 408, 494,
561, 566, 567 495, 507, 508, 511, 524
L. bulgaricus, 549, 553, 567 Methylome, 218, 226–229
L. casei, 549, 550, 553–556, 560–562, 566 Metronidazole, 238, 459, 460, 472–476,
L. platarum, 549, 553, 556, 561, 567 478–485, 496–499, 507, 525, 526, 528,
L. reuterii, 549, 550, 554, 556, 566 531–537
L. rhamnosus, 546, 549, 553, 556, 557, 566, México, 473, 504, 512, 515
567 Microbiome, 229, 283, 381, 406, 412–413,
L. salivarius, 549, 553, 556, 561 453, 459, 545–547, 562, 564, 568
L. sporogens, 549, 553 Microbiota, 248, 278, 283, 306, 316, 406, 412,
Latin America, 479–481, 504–506, 509, 413, 544–551, 556, 558–560, 563,
511–515 567, 568
Levofloxacin, 474, 475, 483, 484, 497–499, microRNA, MIR30B, 75
525, 526, 528, 529, 532, 534, 536, 537 MicroRNAs (miRNA), 74, 75, 102, 332,
Lewis blood group antigens, 145 335, 375
Linkage model, 15–17 miR-, 246, 335
Lipopolysaccharide (LPS), 69, 72, 129, 301, Mitochondria, 74, 119, 121–123, 126, 127
302, 311, 332, 343, 354, 355, 432, 449, Multilocus sequence typing (MLST), 4, 5, 22,
581, 583 218, 219, 221, 509
Index 609
Modification enzyme, 226 Nucleotide oligomerization domain (NOD)

Molybdenum, 222, 223 NOD1, 62, 90
Mongolian gerbils, 61, 74, 94, 105, 148–151, NOD2, 104, 355
201, 241, 246, 247, 274, 279–283,
409, 509
Motility, 32, 33, 59, 60, 65, 69, 70, 97, 100, O
104, 155, 196, 204, 205, 208, 245, 251, OMVs. See Outer membrane vesicles (OMVs)
281, 300, 375, 560, 562 Operon, 33, 37, 49, 62, 195, 197, 199, 202,
Mucins, 144, 147, 155–157, 241, 350, 351, 204, 209
445, 544, 546, 561 Optimal, 46, 128, 154, 433, 444, 472, 475–477,
Mucosal immune system, 316, 592 482, 484, 485, 511, 524
Mucosal vaccination, 584, 587, 592 Optimization, 41, 476–478, 484, 485, 581–588
Mucosal-prime and systemic boost strategy, 587 Orphan TSS (oTSS), 195
Multi-locus sequence typing (MLST), 4, 5, 22, Outer inflammatory protein A (OipA), 61, 281
218, 219, 221, 509 Outer membrane protein (OMP), 33, 60, 157,
Mutations, 4–6, 12, 13, 17–19, 58, 101, 103, 178, 223, 449
116, 148, 178, 190, 209, 218, 223, 278, Outer membrane vesicles (OMVs), 62, 157
288, 302, 304, 330, 343, 356, 388, 392, Ovalbumin (OVA), 314, 455
428, 456, 473, 474, 496, 525, 528, 585 Oxidative stress, 67, 73, 126, 190, 205, 245,
430, 509
N
Neutrophil activating protein A (NapA), 73, P
166, 169–171, 174–176, 178, 180, 379, P, 118, 127, 192–194, 198, 207, 238, 301,
584, 591 306–316, 331–335, 341, 342, 367, 373,
Natural host, 21, 236, 237, 241, 255 375, 379–380, 397, 407, 408, 474, 521,
ncRNA, 190, 191 530, 534, 558
Neuropeptide-Y (NPY), 375 P53, 74, 75, 98, 99, 103, 278, 388
Neutrophils, 61, 68, 69, 129, 153, 155, 156, Par1b, 99, 101
166, 169, 170, 276, 287, 313, 346, 349, Parasites, 316
424, 427, 576, 580, 586, 593 Parietal cells, 129, 130, 240, 242, 244, 247,
NF-κB. See Nuclear factor kappa B (NF-κB) 248, 287, 393, 395, 404, 412
Nickel, 30, 33, 34, 37, 44–51, 59, 251 PARs. See Protease-activated receptors
Nickel response regulator, 59 (PARS)
NikR, 33, 37, 46–48, 59 Pars esophagea, 244, 245
Nilo-Saharan migrations, 9 Pathogen associated molecular pattern
Nitric oxide (NO), 45, 72 (PAMPs), 300–303, 312, 343, 581
NixA, 47 Pathogenesis, 30, 59, 62–68, 70, 76, 90, 94, 95,
Non-bismuth quadruple therapy, 478, 482, 102, 105, 114, 130, 144, 159, 166,
533, 534 168–170, 172, 175–178, 180, 235,
non-coding RNA, 189–209 240–242, 245–247, 251, 255, 275, 282,
Nonhuman primates, 235, 243, 245, 248, 274, 289, 303, 340, 342, 347, 349–357, 394,
276, 283–289, 301, 588–591 395, 397, 407, 410, 411, 424–435, 444,
Nonsteroidal anti-inflammatory drug 448–449, 494
(NSAIDs), 326, 347, 366–369, 371, pathogenic factors, 448
450, 493, 494, 521–523 Th1 response, 246
NOX complex, 333–335 T regulatory cells, 448
Noxo, 333–334 Peptic ulcer
Nuclear factor kappa B (NF-κB), 62, 69, bleeding, 369
98–101, 103, 129, 170–172, 179, 280, disease, 113, 117, 144, 150, 158, 251, 300,
300, 301, 334, 335, 343, 344, 350, 355, 304, 305, 308, 340, 366–368, 381, 449,
412, 424, 429, 431–436, 449, 551, 552, 455, 522, 523, 544, 576
558, 561 duodenal, 367–371
610 Index
Peptic ulcer (cont.) 460, 472, 474–479, 481, 483–485, 493,

gastric, 366, 368, 369 495–499, 522, 525, 531–535, 576
perforation, 370
Peptidoglycan, 59, 62, 63, 66, 91, 102, 103,
205, 561 Q
Periplasmic pH, 35, 36, 38, 39 Quadruple therapy, 478, 480, 484, 485, 499,
Per-protocol (PP), 459, 472, 533–535 532, 535
Persistence, 30, 51, 70, 71, 74, 130, 132, 168,
173, 304, 308, 309, 311, 316, 369, 449
Persistent infection, 58, 61, 92, 113, 150, 286, R
300, 305, 308, 311, 313, 315, 346 RAG, 277
PGE, 327 Reactive oxygen species (ROS), 72, 75, 247,
Phagocytosis, 68, 70–71, 102, 153, 156, 332–335, 352, 412, 424, 427, 530
346, 580 REBASE, 226
pH exposure, 30, 35, 39 Recent migrations, 7
Phosphoproteomics, 103 Recombination, 3–23, 58, 148, 149, 158,
pH sensing, 34 218–224, 228, 229, 286, 424
pH-tactic behaviour, 60 Recurrence rate, 369, 512, 514
Phylogenetic tree, 218, 220, 222, 234 Regulation, 37, 38, 41, 50, 60, 61, 72, 149–150,
Phylogeny, 18–22, 218–219, 278 153–155, 157, 166, 168, 172, 190, 196,
Piglet, 39, 59, 66, 131, 167, 244, 245, 274, 275 198, 199, 201, 203, 204, 209, 307, 334,
Pigs, 235, 238, 243–247, 253, 274, 344 368, 378, 381, 560
PMSS1, 276 Regulatory T-cells, 71, 277, 300, 310–313,
Population structure, 3–23, 218–221 315, 378, 379, 448, 586
Population trees, 14–15 Rescue, 38, 105, 200, 472, 482, 486, 533, 534
Post-immunization gastritis, 579, 589, 593 Resistance, 30, 71, 126, 203, 238, 341, 435,
PPI. See Proton pump inhibitor (PPI) 456, 472, 492, 513, 525–529, 560, 576
Prehistoric migrations, 8–11 Restriction enzyme, 226, 228
Prevalence, 146, 235, 238, 239, 243–244, 250, Restriction-modification (RM), 224, 226, 228
253, 284, 306, 366, 370, 372, 374, 376, RGD motif, 93, 104
380, 388, 391, 395, 396, 428, 444–446, Ribonuclease, 202, 206–208
448, 449, 453–455, 473, 476, 480, 483, Ribonucleoprotein complex, 43, 204
485, 491, 492, 495, 497, 505, 507, 508, Riboregulation, 150, 190, 198, 199, 204,
511, 513, 520, 521, 524, 525, 528, 530, 208, 209
531, 575–577 Rifabutin, 473, 474, 482, 483, 485, 498
Prevention, 168, 170, 175, 255, 371, 494–495, RIP-seq, 206
499, 505, 510, 511, 513–515, 522–524, Risk factors
544, 548, 590 breast feeding, 445
Primary (pTSS), 195, 196 genetic factors, 446
Probiotics, 498, 543–568 socioeconomic status, 445
Pro-inflammatory signalling, 62, 98, 102, 303 RUNX3, 98, 100
Promoter motif, 195, 196 RNA-binding proteins (RBPs), 204
Propionibacterium, 558–559, 562 RNA chaperone, 198, 204, 206–208
P. acidipropionici, 558 RNA-immunoprecipitation sequencing,
P. freudenreichii, 556–559, 567 191, 205
P. jensenii, 558 RNA-protein interaction, 204, 206
P. thoenii, 558 RNase E, 206, 207
Protease, 67, 174, 176, 177, 331, 580 RNase III, 202, 206, 207
Protease-activated receptors (PARS), 580 RNase J, 206, 207
Protective immunity, 307, 313, 579, 580 RNA sequencing (RNA-seq), 190–194, 197,
Proteobacteria, 545, 546 198, 201, 202, 205, 206, 208, 209
Proton buffering, 34 Rodents, 236, 237, 240–242, 253, 279, 281,
Proton pump inhibitor (PPI), 238, 369, 370, 284, 301, 316, 407, 410, 577
373–374, 391, 396, 399, 451, 453, 459, ROS. See Reactive oxygen species (ROS)
Index 611
S Storage, 34, 46–48, 204, 551, 564

Saccharomyces, 548, 552 Streptococcus, 548, 558
S. boulardii, 552, 553, 556–558 S. faecalis, 558
S. cerevisiae, 552 S. thermophilus, 552, 553, 556, 558,
Safety and Delivery systems of probiotics, 565, 567
562–563 Stress resistance proteins
Sahul migrations, 10, 20 AhpC, 73
Salt, 282, 409–410, 476, 478, 481, 483, 484, Bcp, 73
533 katA, 73
San and hpAfrica, 21 MdaB, 73
SCID, 277 NapA, 73
Secondary TSS (sTSS), 195, 196 SodB, 73
Second-line, 482, 485, 496, 498, 531–533, Tpx, 73
536, 590 STRUCTURE, 6, 18, 219, 221
Secretory IgA, 445, 560, 578 Sublingual route of immunization, 586, 587
Sequential therapy, 459, 478–480, 498, 512, Suboperon, 195, 197
531, 532, 534–536 Superlineages, 18, 19, 21
SH2 domain, 63, 95, 97, 100 Sydney Strain 1 (SS1), 274–276
Shine-Dalgarno motif, 196 Synteny, 224
Sialic acid-binding (SabA) Systemic diseases, 367, 494
binding to gastric mucins, 156
gastroduodenal diseases, 155–156
H. pylori hemagglutinin, 153 T
identification of SabA, 152–153 T4SS, 62, 63, 66–68, 71, 90–94, 97, 102–105,
outer membrane vesicles, 157 118, 130, 150, 165, 171, 180, 276, 281,
pH-dependent SabA regulation, 155 282, 286, 300, 301, 311, 407, 411
regulation of sabA expression, 155 T4SS pilus, 66, 90, 92, 93, 300
sialyl-Lewis x and the sialyl-Lewis a T84 cells, 98
antigen receptors, 152 TAK, 100, 101
Signaling, 90, 94, 99, 101, 105, 120, 127, 128, Tandem affinity purification (TAP), 41,
150, 170, 177, 180, 221, 280, 301–303, 193, 194
305, 308, 326, 329–331, 333, 335, Target recognition domain (TRD), 226,
448, 561 228, 229
Signal transduction, 63, 90, 95, 101, 105, 126, T-cell inhibition, 168, 356
127, 129, 130, 171, 172, 176, 178, 343 T cells, 45, 67, 68, 70, 71, 117, 122, 128, 129,
Site-specific recombination, 218, 223 131, 166, 168, 173, 275, 277, 300,
Small ORFs, 192, 203 304–313, 315, 355, 356, 378, 379, 427,
Small Rho family GTPase, 97 433, 448, 449, 578–580, 586, 587,
small RNA (sRNA), 60, 150, 190–192, 196, 590–594
198–201, 203–209 Terminator exonuclease (TEX), 192–194
Somatostatin, 244, 280, 366, 375, 394 Th, 242, 379
Spasmolytic polypeptide-expressing Th response, 304
metaplasia (SPEM), 242, 277, 278 The Austronesian expansion, 20
Spiral morphology, 59 Therapy algorithm, 499
Staphylococcus Tipα, 67, 68, 166, 171–172, 175, 176, 178, 180
S. aureus, 206, 559, 584 Toll-like receptors (TLR), 104, 301–303, 311,
S. epidermis, 559 329, 330, 332, 343–345, 351, 354–355,
S. hominis, 559 446, 561
Stem cell, 327, 330, 331, 333, 406, 411, 412 TNFR1 interacting endonuclease A, 74
Stenotrophomonas maltophila, 559 Tobacco acid pyrophosphatase (TAP), 41,
Stomach, 3, 30, 58–60, 90, 113, 144, 176, 190, 193, 194
218, 233, 279, 300, 327, 348, 387, 404, Toll like receptors (TLRs), 69, 302, 303, 329,
424, 447, 473, 495, 504, 544, 575 330, 332, 343–345, 354, 561
612 Index
Toxin-antitoxin system, 203 Type IV secretion system (T4SS) t, 62, 63,

Transamidosome, 43 66–68, 71, 90–94, 97, 100, 102–105,
Transcriptional regulation, 33 118, 130, 150, 165, 171, 180, 245, 276,
Transcriptional response, 33, 34 281, 282, 286, 288, 300, 301, 311,
Transcriptional start sites (TSS), 149, 190, 407, 411
192–198, 202
Trans-encoded, 198–201, 203, 208
Transforming growth factor-β, 101, 304, 448 U
Translation, 39, 42, 43, 196, 198–201, 203, Uptake, 32, 36, 38, 41–42, 46, 47, 102,
205, 206, 432, 494 119–123, 130, 380
Transmission, 61, 238, 239, 249, 250, 284, 288, asparagine, glutamine, 41–42
341, 445–448, 576, 577, 588 Urea, 4, 30, 32–39, 41, 44, 45, 49, 58, 59, 73,
day care centres, 446, 447 127, 194, 237, 240, 251, 286, 306, 381,
diarrheal diseases, 447, 577 410, 428, 457, 512, 515, 589
intrafamilial, 446, 447 Urease, 30, 32–39, 41, 45–50, 58, 59, 70, 73,
molecular fingerprinting, 447 199, 202–204, 233, 245, 249–252, 254,
siblings, 446, 447, 576 275, 286, 288, 306, 428, 456, 493,
vehicle, 447 581–584, 588–590
water, 238 regulation, 38
Transport, 36, 37, 41–44, 46–47, 51, 122, 169, maturation, 33, 37, 46, 48–50
195, 204, 223, 245, 281 regulation, 38
Transporter, 33, 40, 42, 46, 47, 121, 197 UreI
Treatment, 128, 192–194, 238, 242, 247, 255, channel, 35, 36
308, 311, 316, 326, 330, 331, 350, 369, maturation, 49
371–373, 376, 377, 380, 405, 425, 430, USA, 7, 155, 285, 313, 314, 367, 369, 370, 373,
436, 444, 450, 453, 454, 458–460, 376, 379, 393, 404, 444, 447, 473, 504,
472–476, 480–485, 492, 493, 496–499, 564–566, 577
505, 507–510, 512, 513, 515, 521–525,
531–535, 544–546, 550, 552, 576, 577,
588, 589, 592 V
bismuth-based triple therapies, 459 VacA-like protein C (VlpC), 65
clarithromycin resistance, 459, 460, 473, FaaA, 65
478–480, 497, 532–534 Vaccine, 306, 307, 313, 561, 576–581, 583,
current guidelines, 460, 531 584, 586–594
sequential therapy, 459, 479–480, 498, 531, Vacuolating cytotoxin A (VacA), 114–132,
532, 534, 535 151, 157, 240, 243, 245, 247, 251,
Treatment regimens, 460, 482, 498, 531–535 308–311, 366, 368, 408, 410, 448, 581,
Treg cells, 72, 129, 131, 277, 278, 304–306, 583, 584, 591
310–316, 379, 448, 449, 455, 586, allelic diversity, 116–118
592–593 apoptosis, 64, 74, 130, 166, 308
Triple, 238, 250, 450, 451, 459, 460, 472, 473, atrophic gastritis, 118, 508
476, 479, 480, 482–485, 496–499, 512, autophagy, 70, 75, 124–126, 130
525, 528, 529, 531–536, 552, 555 binding to host cells, 64
tRNA-dependent, 40, 42, 43 carcinoids, 393
Tumor-associated macrophages (TAMs), cell death, 74, 123, 124, 126
329, 330 colonic adenocarcinoma, 64, 251, 408, 509
Tumor necrosis factor alpha (TNF-α), 67, 90, colonic hyperplastic polyps, 388
171, 172, 277, 280, 305, 311, 312, 329, duodenal adenomas, 397
331–334, 340, 342, 343, 353–355, 432, effects on cell permeability, 127
449, 529 effects on cytoskeleton, 127
Tumor necrosis factor-α inducing protein effects on dendritic cells, 168
(Tipα), 67, 68, 166, 171–172, 175, 176, effects on endosomal compartments, 121,
178, 180 122, 124
Index 613
effects on lymphocytes, 128–129 Wnt signaling, 327, 328, 331, 332, 334, 335
effects on macrophages, 129
effects on mast cells, 129
effects on neutrophils, 129, 166 Z
effects on parietal cells, 129–130 Zoonotic transmission
effects on signal transduction, 127 adoptive transfer, 277, 307
Elster’s polyps, 388, 395 C57BL/6 mice, 60, 94, 241, 246, 297, 305 413
eosinophilic granulomas, 396 CagA, 280–282
fundic gland polyps, 395, 396 cagPAI, 243, 245, 247, 250–251, 275–276
gastric adenomas, 391 CagY, 276, 280, 286
gastric hyperplastic polyps, 388–389 diet, 282
gastric inflammatory fibroid polyps, 397 gastric adenocarcinoma, 252
gastric neuroendocrine tumors, 393–394 gastric cancer, 241
Helicobacter, 114, 240 gastritis, 252
Helicobacter pylori, 63–65, 90, 114, 240, gerbil, 236
275, 308, 311, 366, 530 H. felis, 236, 238, 239
interaction with mitochondria, 122 H. heilmannii, 236, 238, 239
intracellular trafficking, 119–122 H. mustelae 236
membrane channels, 118–119 H. suis, 236, 238
raised intraepithelial neoplasia, 391 IFNγ, 246
role in gastroduodenal disease, 131–132 IL-, 10, 246
secretion, 114–115 INS-GAS, 241–242
structural properties, 64, 116 iron, 241–242
transcription, 114, 116 metaplasia, 252
vacuolation, 123–125, 130 microbiota, 248
Vacuolation, 114, 116, 119, 123–125, 130, 308 mouse, 236, 237
Venezuela, 146, 505, 507 non-human primate, 245, 248
VirB (CagY), 276, 280, 286 piglet, 244–245
VirB proteins, 91 PMSS1, 276
VirB10, 92, 93 RAG-/-, 277
Virulence factors, 529–530 salt, 282
Vivotif™ vaccine, 589 SCID, 277
SPEM, 242
SS1, 274–276
W T4SS, 276, 281–282
Western and Central Asian migrations, 10, 11 Th1 cells, 246
Wild felines, 242 treg cells, 277–278

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What is the book about?

What is the book about?

What topics are covered in the book?

What topics are covered in the book?

Steffen

Helicobacter pylori Research

ISBN 978-4-431-55934-4 ISBN 978-4-431-55936-8 (eBook)

Library of Congress Control Number: 2016935851

© Springer Japan 2016

Printed on acid-free paper

This Springer imprint is published by Springer Nature

Helicobacter pylori, the Gastric Bacterium Which Still

It is exciting to participate in this new “state-of-the-art” book about Helicobacter

genome is an advantage coupled with the massive numbers of organisms competing

The Helicobacter Research Laboratory Barry Marshall

Part I Bacteriology and Molecular Biology

10 Non-Helicobacter pylori Helicobacter Infections in Humans

Part II Immune Responses and Host Factors

Part III Helicobacter pylori-Associated Diseases

Part IV Treatment of Helicobacter pylori

23 Population-Based Strategies for Helicobacter pylori-Associated

Keywords Helicobacter pylori • Coevolution • Population • Recombination •

© Springer Japan 2016 3

1.2 The Coevolution of Helicobacter pylori and Humans

1.2.1 H. pylori’s Housekeeping Genes

1.2.2 Geographic Clustering of Housekeeping Gene

and hpEastAsia into hspEAsia, hspAmerind, and hspMaori. While numbers of

1.2.2.1 Recent Human Population Movements

1.2.2.2 Prehistoric Human Migrations

When subpopulation-level structure was analyzed, geographic patterns also

Bantu speakers: hpAfrica1

Nilo-Saharan speakers: hpNEAfrica

Australians and New Guineans: hpSahul

Central and Southeast Asians: hpAsia2

Native Americans, Han Chinese, and the Austronesians: hpEastAsia

Among subpopulations of hpEastAsia, hspAmerind was isolated among diverse

1.2.3 Formal Comparisons with Human DNA Data

1.2.4 Recombination and Its Effect on Evolutionary

Strong geographic structuring of population assignment clusters and clear parallels

Fig. 1.4 Determining interpopulation relatedness in a highly recombining genome. (a)

in the substitution model. Due to the excess of homoplasies introduced by recom-

1.2.4.1 Population Trees

Given that recombination is usually geographically restricted, one way of mini-

1.2.4.2 The Linkage Model: The Concept of Ancestral Populations

1.2.5 Enter Coalescence

Inference of population demographic parameters such as migration rates and

1.2.5.1 The Global H. pylori Phylogeny

Until the implementation of coalescent models, the phylogenetic relationships

1.2.5.2 Age of the H. pylori Human Association

of a continuous hominoid-Helicobacter association spanning several million years,

1.2.5.3 Sahul Was Colonized Only Once

1.2.5.4 The Austronesian Expansion

1.2.5.5 San Hunter-Gatherers Are the Original Hosts of hpAfrica2

1.2.5.6 A Second More Recent Out-of-Africa Migration

hpEurope is a hybrid population comprising nucleotides from two ancestral

1.2.5.7 Pygmy Hunter-Gatherers Contracted H. pylori Recently from

Nell and coworkers investigated the population demographics of H. pylori trans-

1.2.6 Outlook: Genomics, Aboriginal Populations,

While the population, phylogeographic and evolutionary, research outlined in this

Achtman M, Azuma T, Berg DE et al (1999) Recombination and clonal groupings within