Curr. Issues Mol. Biol. 7: 197–212. Online journal at www.cimb.

org

The Evolution of Flea-borne Transmission in

Yersinia pestis

B. Joseph Hinnebusch al., 1999; Hinchcliffe et al., 2003; Chain et al., 2004).
Presumably, the change from the food- and water-borne
Laboratory of Human Bacterial Pathogenesis, Rocky transmission of the Y. pseudotuberculosis ancestor to
Mountain Laboratories, National Institute of Allergy the flea-borne transmission of Y. pestis occurred during
and Infectious Diseases, National Institutes of Health, this evolutionarily short period of time. The monophyletic
Hamilton, MT 59840 USA relationship of these two sister-species implies that the
genetic changes that underlie the ability of Y. pestis to use
Abstract the flea for its transmission vector are relatively few and
Transmission by fleabite is a recent evolutionary adaptation discrete. Therefore, the Y. pseudotuberculosis –Y. pestis
that distinguishes Yersinia pestis, the agent of plague, species complex provides an interesting case study in
from Yersinia pseudotuberculosis and all other enteric the evolution of arthropod-borne transmission. Some of
bacteria. The very close genetic relationship between Y. the genetic changes that led to flea-borne transmission
pestis and Y. pseudotuberculosis indicates that just a few have been identified using the rat flea Xenopsylla cheopis
discrete genetic changes were sufficient to give rise to flea- as model organism, and an evolutionary pathway can
borne transmission. Y. pestis exhibits a distinct infection now be surmised. Reliance on the flea for transmission
phenotype in its flea vector, and a transmissible infection also imposed new selective pressures on Y. pestis that
depends on genes that are specifically required in the help explain the evolution of increased virulence in this
flea, but not the mammal. Transmission factors identified pathogen.
to date suggest that the rapid evolutionary transition of
Y. pestis to flea-borne transmission within the last 1,500 Y. pestis–flea interactions
to 20,000 years involved at least three steps: acquisition There are an estimated 2,500 species and subspecies
of the two Y. pestis-specific plasmids by horizontal gene of fleas that constitute 220 genera and 15 families
transfer; and recruitment of endogenous chromosomal in the insect order Siphonaptera (Lewis, 1998). Of
genes for new functions. Perhaps reflective of the recent these, approximately 80 species, associated with some
adaptation, transmission of Y. pestis by fleas is inefficient, 200 species of wild rodents, have been found to be
and this likely imposed selective pressure favoring the infected with Y. pestis in nature, or to be susceptible to
evolution of increased virulence in this pathogen. experimental infection (Pollitzer, 1954). Accordingly, the
ecology of plague is extremely complex, involving many
Introduction different rodent-flea cycles.
Pathogenic bacteria must overcome several physiological Different species of fleas vary greatly in their
and immunological challenges to successfully infect even ability to transmit Y. pestis, at least under laboratory
a single type of host, such as a mammal. It is remarkable, conditions; some species, such as the common cat flea
then, that bacteria transmitted by blood-feeding Ctenocephalides felis, are incapable of transmission.
arthropods are capable of infecting two very different Wheeler and Douglas (1945) and Burroughs (1947)
hosts during their life cycle: an invertebrate (usually an developed a mathematical model to estimate the vector
insect or tick) and a mammal. As if this were not enough efficiency of different flea species that took into account
of a challenge, it is not sufficient that an arthropod-borne i) infection potential (the percentage of fleas becoming
bacterium successfully infect both vector and host. It infected after feeding on a septicemic animal); ii) vector
must establish a transmissible infection in both; that is, it potential (the percentage of infected fleas which become
must infect the vector in such a way as to be transmitted infective or blocked, i.e., develop a transmissible infection
during a blood meal, and it must infect the mammal in as described below); and iii) the transmission potential
a way that allows uptake by a blood-feeding arthropod. (the average number of successful transmissions per
This feat of evolution has occurred relatively rarely, but flea). Kartman (1957) later added two more factors:
nonetheless arthropod-borne transmission has developed iv) the life span of infective (blocked) fleas; and v) the
independently in a phylogenetically diverse group of field prevalence index (the average number of fleas per
microorganisms, including the rickettsiae, spirochetes in species per rodent or rodent nest). By these measures
the genus Borrelia, and the Gram-negative bacteria. an experimental vector efficiency could be calculated for
Compared to the ancient relationship of rickettsiae different fleas (Wheeler and Douglas, 1945; Burroughs,
and spirochetes with arthropods, the vector relationship 1947; Kartman, 1957; Kartman and Prince, 1956).
between Y. pestis and fleas is new. Population genetics These comparisons have sometimes been intriguing and
and comparative genomics analyses indicate that Y. pestis enigmatic. For example, the rat flea Xenopsylla cheopis
is a clonal variant of Y. pseudotuberculosis that diverged has most frequently been identified as the most efficient
only within the last 1,500 to 20,000 years (Achtman et vector, yet the closely related Xenopsylla astia is a poor
vector (Hirst, 1923). The physiological mechanisms that
account for differences in vector efficiency among different
For correspondence: [email protected]

© Horizon Scientific Press. Offprints from www.cimb.org

198 Hinnebusch
���������������������� �

Table 1. Flea-specific factors which might affect the ability of Y. pestis

to produce a transmissible infection
Flea anatomy and physiology

Midgut
pH, redox potential, osmolarity, etc.
Biochemical composition of host blood
Digestive enzymes, digestive byproducts
Endogenous microbial flora
Frequency of feeding and defecation
Insect immunity components

Proventriculus
Size (volume)
Number, density, length, and shape of spines
Rate of opening and closing during feeding
Hydrodynamic forces generated during feeding
Insect immunity components

Ecology
Ambient temperature
Flea life span after infection

flea species are not known, but some possible factors are
Fig. 1. (A) Digestive tract anatomy of fleas. E = esophagus; PV =
described in the following sections and listed in Table 1.
proventriculus; MG = midgut; HG = hindgut. The muscles that pump
A high degree of vector specificity is characteristic blood into the midgut (PM) and the malpighian tubules (MT) are also
of many arthropod-borne agents. For example, human indicated. (B) Digestive tract dissected from a blocked X. cheopis flea.
malaria is transmitted by anopheline but not culicine Arrows indicate the large aggregate of Y. pestis that fills and blocks the
proventriculus and an independent bacterial aggregate in the midgut.
mosquitoes, different subspecies of Leishmania are The Y. pestis aggregates are surrounded by a dark colored extracellular
transmitted by different sandfly species, and the closely matrix. Bar = 0.25 mm.
related North American species of Borrelia spirochetes
that cause relapsing fever are each transmitted by a
different species of Ornithodoros tick (Sacks and Kamhawi, blood into the midgut and to keep it from leaking back
2001; Barbour and Hayes, 1986). Whether the same co- out. Fleas usually live on or in close association with
evolutionary process is occurring in Y. pestis remains to their hosts and take small but frequent (every few days)
be demonstrated, but Russian researchers have proposed blood meals. Digestion of the blood meal begins quickly,
that, at least for some natural plague cycles, discrete resulting in hemolysis and liquefaction of ingested blood
triads of flea species, rodent, and subspecies or strain of cells by six hours (Vaughan and Azad, 1993). During the
Y. pestis have co-evolved (Anisimov et al., 2004). next two to three days, the blood meal digest is brown-
colored, viscous, and contains many large and small lipid
The flea gut environment droplets, but is eventually processed to a compact dark
Y. pestis infection of the flea is confined to the digestive residue. Fleas defecate partially digested portions of their
tract, which is depicted in Fig. 1. Storage, digestion, and blood meals, which are used as a food source by flea
absorption of the blood meal all occur in the simple midgut larvae. Unlike other blood-feeding arthropods, fleas do
made of a single layer of columnar epithelial cells and not secrete a chitinous peritrophic membrane around the
associated basement membrane. The proventriculus, blood meal.
a valve at the base of the esophagus that guards the Few details are known about flea gut physiology
entrance to the midgut, is central to the transmission and associated environmental conditions in the digestive
mechanism. The interior of the proventriculus is arrayed tract. A probable midgut pH of 6 to 7 has been cited
with densely packed rows of inward-directing spines, (Wigglesworth, 1972), but other basic parameters such
which are coated with an acellular layer of cuticle, the as osmotic pressure and redox potential are unknown.
same material that makes up the insect exoskeleton (Fig. The biochemical composition may initially reflect that of
2). In X. cheopis, there are a total of 264 proventricular hemolyzed blood, but is subject to rapid change due to
spines in the male and 450 in the female (Munshi, 1960). selective absorption of certain nutrients, ions, and water.
The proventricular valve is normally tightly closed by Insect midgut epithelium secretes a variety of digestive
layers of surrounding muscle. During feeding periods, enzymes that are similar to those of vertebrates, including
however, the proventricular muscles rhythmically open trypsin, chymotrypsin, amino- and carboxypeptidases,
and close the valve in concert with a series of three sets cathepsins, lysozymes, glycosidases, and lipases (Terra
of pump muscles located in the flea’s head to propel and Ferreira, 1994). Mammalian blood is composed
 ���������������������� � Y. pestis Transmission Factors 199

The midgut epithelium of mosquitoes and the blood

sucking fly Stomoxys calcitrans has been shown to be an
immune-competent tissue, and the presence of bacteria
in the blood meal of these insects induces the secretion of
antimicrobial peptides into the gut lumen (Dimopoulos et
al., 1997; Lehane et al., 1997). Whether this occurs in fleas
is unknown. At any rate, Y. pestis appears to be inherently
resistant to the flea immune response (Hinnebusch et al.,
1996; and unpublished data).

Biological transmission of Y. pestis by fleas: the

proventricular blockage model
The general mechanism of Y. pestis transmission by
fleas was described by Bacot and Martin (1914). They
observed that in infected X. cheopis and Ceratophyllus
fasciatus fleas, solid masses of Y. pestis could fill the
lumen of the proventriculus and obstruct the flow of blood
(Fig. 1B). These fleas made persistent, vigorous attempts
to feed, but could not pump blood past the blocked
proventriculus. Instead, the esophagus became distended
with blood which was then partially refluxed back into the
bite site due to contraction and relaxation of the cibarial
and pharyngeal pumps. This phenomenon correlated
with efficient transmission. Based on their observations,
Bacot and Martin proposed the proventricular blockage-
regurgitation mechanism for plague transmission.
According to this model, blockage of the proventriculus by
a mass of Y. pestis precedes transmission, and Y. pestis
is conveyed to the bite site by regurgitation when blocked
fleas attempted to feed. Bacot (1915) later amended this
model by proposing that fleas with only partial obstruction
of the proventriculus were actually better transmitters.
Partially blocked fleas can still pump blood into the
midgut through an open channel in the proventriculus.
Fig. 2. Proventricular spines of Xenopsylla cheopis. Side (A) and
front (B) views of an uninfected proventriculus viewed by fluorescence The bacterial growth prevents complete closing of the
microscopy. The proventricular spines are covered with cuticle, which is valve, however, so that blood mixed with Y. pestis from
autofluorescent. (C) Scanning electron microscopy of the interior of an the midgut is able to flow back out the proventriculus into
uninfected proventriculus. Bar = 5 μm.
the bite site. This can happen because during flea feeding
the pumping action is not continuous, but stops for short
principally of protein and lipid, and lipids are a major intervals. Transmission as a result of partial blockage was
energy source for hematophagous arthropods. Lipids are an important addendum to the model because complete
relatively insoluble in water, and the mechanism of their proventricular blockage does not readily develop in some
solubilization and absorption in fleas is unknown. flea species that are good vectors of plague (Burroughs,
It is in this active digestive milieu that Y. pestis lives 1947; Pollitzer, 1954). According to the Bacot model,
in the flea. These conditions must be relatively hostile complete blockage of the proventriculus is not necessary
and refractory to colonization, because fleas have rather for efficient transmission; partial interference with its
limited normal digestive tract flora, and few pathogens are valvular function is sufficient.
transmitted by fleas (Savalev et al., 1978; Beard et al.,
1990). Besides Y. pestis, flea-borne pathogens include Mechanical transmission of Y. pestis by fleas. Although
Bartonella henselae, the agent of cat-scratch disease several investigators have established that biological
and bacillary angiomatosis, and Rickettsia typhi and transmission (requiring Y. pestis growth in the digestive
Rickettsia felis, members of the typhus group (Chomel tract to produce a proventricular infection) is the only
et al., 1996; Azad et al., 1997). The Gram-negative reliable means of transmission (Burroughs, 1947; Pollitzer,
bacterium Francisella tularensis is also associated with 1954), there is evidence that mechanical transmission
fleas, although the importance of flea vectors in the overall may also play a role in the ecology of plague. For
ecology of tularemia is unclear (Hopla, 1974). Fleas have mechanical transmission, infection of the vector is not
also been implicated in transmission of the poxvirus that necessary. It is only necessary that septicemia levels
causes myxomatosis in rabbits (Chapple and Lewis, are high and that Y. pestis survive on the blood-stained
1965). Vaughan and Azad (1993) have hypothesized mouthparts of fleas between consecutive feedings. For
that the rapid digestive process of fleas and lice is not example, X. cheopis and the wild rodent flea Malaraeus
conducive to the development of eukaryotic parasites, but telchinum allowed to feed en masse on uninfected mice
that it can be better tolerated by prokaryotes. one day after feeding on a highly septicemic mouse
200 Hinnebusch

consistently transmitted the disease (Burroughs, 1947). Ymt purified from Y. pestis is toxic (Hinnebusch et al.,
Since that time interval is too short for proventricular 2000; and unpublished data). The explanation for this is
infection to develop, transmission presumably occurred not known, but Walker (1967) suggested that synergism
by mechanical transference of bacteria on contaminated between Ymt, endotoxin, and possibly other Y. pestis
mouthparts. The phenomenon of mechanical or mass factors was responsible for murine toxicity.
transmission provides a potential mechanism for fleas Sequence analysis showed that the Y. pestis ymt
that do not develop proventricular blockage readily, such gene mapped to the 100-kb pFra plasmid and encodes
as M. telchinum and the human flea Pulex irritans, to a 61-kDa protein that is a member of a newly described
transmit Y. pestis during epidemics. Human to human family of phospholipase D enzymes found in all kingdoms
transmission via P. irritans has been hypothesized to have of life: animals, plants, fungi, and eukaryotic viruses
contributed to the plague pandemics of medieval Europe as well as bacteria (Cherepanov et al., 1991; Ponting
(Beaucournu, 1999). Because mechanical transmission and Kerr, 1996). All members of this PLD family have
does not rely on specific interactions with the vector, it will two copies of a signature HKD (HxKx4Dx6GG/S) motif,
not be considered further here. which come together to form the catalytic site for binding
and hydrolysis of the phosphodiester bond (Stuckey
Y. pestis transmission factors and Dixon, 1999). The Y. pestis Ymt has classic PLD
A central hypothesis, now substantiated by experimental activity, as shown by its ability to cleave the polar head
evidence, is that bacteria that cycle between a mammal group from phosphatidylcholine, phosphatidylethanol
and an arthropod express distinct subsets of genes in amine, and other phospholipids. Ymt is also capable of
their two hosts. Genes specifically required to infect the transphosphatidlyation of phospholipid with an alcohol
vertebrate host are referred to as virulence factors, and acceptor, a second characteristic PLD reaction (Rudolph
the analogous genes required to produce a transmissible et al., 1999). Because of this proven biochemistry, it has
infection in the arthropod vector have been termed been proposed that the Y. pestis ymt gene should be
transmission factors (Hinnebusch et al., 1996; Paskewitz, renamed pldA (Carniel, 2003).
1997). Many virulence factor genes of Y. pestis that are Despite the known toxic effects of murine toxin, a ymt
required to infect and cause disease in the mammal have deletion mutant of Y. pestis was essentially fully virulent
been identified and studied. In contrast, the genetic factors for mice (Drozdov et al., 1995; Du et al., 1995; Hinnebusch
required in the insect host have been relatively neglected. et al., 2000). Thus, Ymt is not required for morbidity or
Nevertheless, some of the genetic factors of Y. pestis that mortality, even in mice, but only adds insult to injury. This
are specifically involved in flea-borne transmission have likely reflects the fact that Ymt is not a classic exotoxin,
been identified (Table 2). but is a cytoplasmic enzyme that is only released upon
bacterial cell death and lysis. The full virulence of Ymt– Y.
The Yersinia murine toxin: a phospholipase D required pestis, and other results showing that ymt expression is
for flea gut colonization downregulated at 37°C (Du et al., 1995), suggested that
The Yersinia murine toxin (Ymt) was described in the 1950s the principle biological function of this PLD is not as a
as a protein fraction of Y. pestis that was toxic to mice and virulence factor.
rats (Ajl et al., 1955), so it has universally been considered A role for Ymt in transmission was first indicated by a
to be a virulence factor. Brown and Montie (1977) presented study evaluating the fate of plasmid-cured Y. pestis strains
evidence that Ymt is a β-adrenergic receptor antagonist, in the flea. Whereas the 9.5-kb pPst and the 70-kb pYV
blocking epinephrine-induced mobilization of glucose and virulence plasmid were not required for normal infection
fatty acids. In mice, Ymt causes circulatory failure due to and blockage of X. cheopis, strains lacking the 100-kb
vascular collapse, resulting in death in ten hours with an pFra plasmid failed to block the fleas (Hinnebusch et al.,
LD50 of 0.2 to 3.7 μg (Schär and Meyer, 1956). Ymt is not 1998a). Complementation of the pFra– strains with the
toxic to guinea pigs, rabbits, dogs, or primates even in ymt gene alone fully restored normal ability to infect and
enormous doses, however (Montie and Ajl, 1970). Murine block fleas. Specific Ymt– Y. pestis mutants were used for
toxin was described as a cell-associated protein that is further analysis (Hinnebusch et al., 2002b). Within hours
only released upon bacterial death, and, correspondingly, of being taken up in a blood meal by a flea, Ymt– Y. pestis
its effects are seen only in the late stages of septicemic assumed an aberrant, spheroplast-like cell morphology,
murine plague, when the animal is already succumbing and then rapidly disappeared from the flea midgut within
to the disease (Montie and Ajl, 1970). Interestingly, the first day after infection. Rarely, the Ymt– bacteria
recombinant Ymt protein produced in and purified from established an initial foothold in the proventriculus, which
Escherichia coli is nontoxic for mice, whereas native is part of the foregut and physically separated from

Table 2. Y. pestis genes important for flea-borne transmission

Present in:
Gene Llocation Y. pestis Y. pstb Function in Y. pestis and role in transmission
ymt pFra plasmid +– Phospholipase D, survival in flea midgut
hmsHFSR, T Chromosome ++ Extracellular matrix synthesis, biofilm formation, infection and
blockage of the proventriculus
pla pPst plasmid +– Plasminogen activator, dissemination from fleabite site
 Y. pestis Transmission Factors 201

the midgut by the stomodeal valve except during the Gram-negative outer membrane. Attempts to mimic the
few minutes per week that the flea is actively feeding. flea gut environment by culturing the bacteria in triturated
Secluded in the proventriculus, the mutants could grow flea gut contents; in whole or sonicated mouse blood
normally and eventually cause blockage. Because the containing proteases, lipase, and lysozyme; or under
proventriculus is rarely the primary site of infection, but is osmotic and oncotic pressure, oxidative stress, or low pH
usually seeded secondarily from a prior midgut infection, likewise failed to reveal any difference between mutant
the Ymt– mutant infected < 5% and blocked < 0.5% of and wild type strains (B. J. Hinnebusch, unpublished). In
fleas, compared to the normal infection and blockage sum, no phenotypic difference between Ymt– and Ymt+ Y.
rates of 50–60% and 25–45%, respectively (Hinnebusch pestis has been detected outside of the flea gut.
et al., 2002b). Remarkably, introduction of the ymt Alternatively, according to the antidote model, the
gene into Y. pseudotuberculosis and E. coli significantly toxic agent in the flea gut would interact with both Ymt–
enhanced their ability to colonize the flea midgut also. and Ymt+ Y. pestis, but its effects then be neutralized by
Thus, Ymt may have a similar substrate and mechanism Ymt. In many bacteria, exposure to harmful environments
of action in both Yersinia and E. coli. Members of the induces autolytic pathways that result in self-digestion
PLD family of enzymes to which Ymt belongs are found of the bacterial cell wall by endogenous peptidoglycan
in many different cell types and can have many different hydrolases (Lewis, 2000). The environmental stimuli,
functions. Serendipitously, the PLD activity of Ymt signal transduction mechanisms, and gene expression
enhances survival of Gram-negative bacteria in the flea pathways leading to this programmed cell death are
midgut. Acquisition of this single gene by Y. pestis would incompletely understood in bacteria. If an agent in the flea
have been a crucial step in the evolution of the flea-borne gut stimulates Y. pestis autolysis and leads to the observed
route of transmission. rapid spheroplast formation, intracellular Ymt activity may
block or redirect a step in the autolytic pathway. Such a
Models for the protective mechanism of Ymt role would be analogous to that of mammalian PLD, which
How might an intracellular PLD protect Y. pestis in the is an intracellular effector in multiple signal transduction
flea midgut? The first option to consider is that Ymt might cascades (Gomez-Cambronero and Keire, 1998).
be secreted or released from lysed bacteria in the flea, Whichever model is correct, the agent in the flea gut
and degrade an external cytotoxic agent in the midgut that is harmful to Ymt– Y. pestis appears to derive from a
environment. Three types of experimental results argue digestive product of blood plasma. Elimination from the
against that: 1) Addition of exogenous Ymt protein to the flea gut during the first 24 hours after infection occurred if
infectious blood meal did not enhance the survival of Ymt– either filtered mouse plasma or whole blood was the source
Y. pestis in the flea gut. 2) Coinfection of fleas with an of the infectious meal fed to the fleas. However, if fleas
equal mixture of Ymt+ and Ymt– Y. pestis did not result in were infected by feeding on an artificial plasma substrate
a coequal infection pattern. If active PLD were secreted, consisting of PBS, pH 7.4, containing 7% bovine serum
one might expect that enzyme from Ymt+ bacteria would albumin, 6 mM glucose, 12 mM sodium bicarbonate,
also protect Ymt– bacteria in the flea gut, resulting in 10 mM MgCl2, 2.5 mM CaCl2, and 1 mM citric acid, the
infections consisting of an equal mixture of both strains. Ymt– Y. pestis survived as well as the Ymt+ parent strain
Instead, in these experiments the Ymt– mutant again during the first 24 hours after infection. Identical results
survived primarily in the proventriculus. In the midgut, were obtained when washed mouse red blood cells were
it persisted only in small clusters that were embedded added to the artificial plasma substrate (Hinnebusch et
within larger aggregates of wild-type bacteria. 3) In al., 2002b). The fact that the artificial plasma meals were
digestive tracts dissected from fleas infected with Y. pestis digested by the fleas further suggests that flea digestive
that synthesized a Ymt-GFP fusion protein, fluorescence enzymes, or the digestive milieu per se, do not directly
localized only to the cytoplasm and was never detected harm the mutant. The mutant also survived as well as
extracellularly (Hinnebusch et al., 2002b). wild type Y. pestis after injection into the flea hemocoel.
An intracellular PLD conceivably could protect Y. These results implicate a blood plasma digestive product
pestis in the flea gut either by modifying an endogenous as the cytotoxic agent, but the native substrate of the Y.
membrane component to make the bacteria impervious to pestis PLD and its protective mechanism remain to be
the cytotoxic agent (prophylaxis model), or by neutralizing discovered.
the agent, directly or indirectly, after it interacts with the
bacteria (antidote model). In the prophylaxis model, the The hms genes and the biofilm model of proventricular
outer membrane of Ymt– Y. pestis would be differentially blockage
affected in the flea gut. Loss of outer membrane integrity One of the first temperature-dependent phenotypes
could lead to the observed spheroplasty, because described for Y. pestis was the formation of densely
lysozymes are commonly secreted by insect midgut pigmented colonies when incubated at 28°C or less on
epithelium (Terra and Ferreira, 1994). Evidence for this media containing hemin or the structurally analogous
model was sought by analyzing the outer membrane dye Congo red (Jackson and Burrows, 1956a; Surgalla
composition of Ymt+ and Ymt– Y. pestis. Quantitative and Beesley, 1969). The phenotype is not due to the
comparisons of membrane phospholipids and production of a pigment by Y. pestis, but rather to the avid
phosphodiester-linked substitutions of lipid A revealed no adsorption of the exogenous hemin or Congo red to the
differences. The mutant was also no more susceptible outer membrane (Perry et al., 1993). Despite the fact that
than the parent Y. pestis to polymixin B, SDS and EDTA, the phenotype was not expressed at 37°C, pigmentation
cationic detergents, and other agents that target the correlated with virulence. Spontaneous nonpigmented
202 Hinnebusch

mutants had greatly reduced virulence for mice after nutritionally has also been disproven (Hinnebusch et al.,
peripheral routes of infection unless iron salts were 1996; Lillard et al., 1999). In contrast, nonpigmented Y.
injected simultaneously (Jackson and Burrows, 1956b). pestis strains lacking a functional hmsHFRS locus, or
Further study of nonpigmented Y. pestis strains showed the entire 102-kb Pgm locus, were completely unable
that they did not grow in iron-chelated media in vitro, failed to produce proventricular blockage in X. cheopis fleas,
to synthesize several iron-regulated proteins, and did not although they survived in and established a chronic
interact with pesticin, the bacteriocin encoded on the pPst infection of the midgut at the same rate as the isogenic
plasmid (Brubaker, 1969; Sikkema and Brubaker, 1987, pigmented Y. pestis. The ability of Pgm– Y. pestis to infect
1989). and block the proventriculus could be completely restored
The reason for the wide range of physiological effects by reintroducing the hmsHFRS genes alone, indicating
associated with loss of pigmentation gradually emerged that the HPI and other genes in the 102-kb Pgm locus are
from a series of molecular genetics analyses. Robert Perry not required in the flea (Hinnebusch et al., 1996). Earlier,
and colleagues used transposon mutagenesis to identify working with genetically undefined strains, Bibikova (1977)
a 9.1-kb chromosomal locus required for the pigmentation correlated the pigmentation phenotype with the ability to
phenotype (Lillard et al., 1997). Nucleotide sequence of this cause proventricular blockage in X. cheopis; and Kutyrev
region, termed the hemin storage (hms) locus, revealed et al. (1992) reported that a nonpigmented but pesticin
a 4-gene operon, hmsHFRS. Two unlinked genes, hmsT sensitive and virulent Y. pestis strain failed to survive in
and hmsP, were later found to be essential for the normal the vole flea Nosopsyllus laeviceps. Whether physiological
pigmentation phenotype (Hare and McDonough, 1999; differences between the two flea species account for the
Jones et al., 1999; Kirillina et al., 2004). Concurrently, ability of nonpigmented Y. pestis to colonize X. cheopis
investigations by several groups into the iron-regulated but not N. laeviceps is unknown.
proteins synthesized by pigmented Y. pestis culminated
in the characterization of the Yersinia high-pathogenicity Role of the hms genes: Production of a Y. pestis biofilm
island (HPI), which encodes a siderophore-based iron required for proventricular blockage. Ironically, given its
acquisition system (for recent reviews see Lesic and close phylogenetic relationship with enteric pathogens,
Carniel, 2004; Perry and Fetherston, 2004). A key unifying Y. pestis does not penetrate or even adhere to the flea
discovery was reported by Fetherston et al. (1992) showing midgut epithelium, but remains confined to the lumen of
that most nonpigmented Y. pestis mutants resulted from the digestive tract. Because Y. pestis is not invasive in the
spontaneous deletion of a 102-kb segment of the Y. pestis flea, it is at constant risk of being eliminated by peristalsis
chromosome that was termed the pigmentation (Pgm) and excretion in the feces. In fact, approximately half of X.
locus. The 102-kb Pgm locus contains not only the hms cheopis fleas spontaneously rid themselves of infection
genes, but also the Yersinia HPI. It is flanked by IS100 in this way even if they feed on highly septicemic blood
elements, and homologous recombination between these (Pollitzer, 1954; Hinnebusch et al., 1996). Success or failure
extensive direct repeat sequences likely accounts for in stable colonization of the flea gut depends on the ability
the high spontaneous deletion rate (10–5 to 10–3) of the of the bacteria to produce aggregates that are too large
entire 102-kb segment (Hare et al., 1999; Fetherston and to be excreted (Fig. 1B). Both Hms+ and Hms– Y. pestis
Perry, 1994). Thus, elimination of this large locus by a are able to do this, and so achieve comparable infection
single deletion event results not only in the nonpigmented rates in X. cheopis. However, transmission to a new host
(Hms–) phenotype, but also in decreased virulence due to further requires that Y. pestis, which is nonmotile, move
concomitant loss of the HPI. Not all nonpigmented mutants against the direction of blood flow when the flea feeds.
result from the loss of the entire 102-kb segment, however. As described above, this is accomplished by infecting the
The existence of certain nonpigmented hmsHFSR- proventriculus, interfering with its valvular action in such
negative, HPI-positive Y. pestis strains indicated that the a way as to generate backflow of blood into the bite site.
hmsHFRS locus could be autonomously deleted and that The hms genes are required for proventricular infection,
pigmentation and iron acquisition phenotypes are clearly and recent evidence suggests that they synthesize an
separable (Iteman et al., 1993; Buchrieser et al., 1998). extracellular matrix required for biofilm formation.
The incidental linkage of the hmsHFRS locus and the HPI HmsH and HmsF were characterized as surface-
within the same deletion-prone segment accounts for the exposed outer membrane proteins (HmsF also contains
long-held consideration of pigmentation as a virulence a lipid attachment site typical of a lipoprotein), and HmsR,
determinant, reinforced by referring to the entire 102-kb HmsR, and HmsT contain transmembrane domains
segment as the Pgm locus. However, the connection and appear to be inner membrane proteins (Parkhill et
between pigmentation per se and virulence turns out to be al., 2001; Pendrak and Perry, 1993; Perry et al., 2004),
merely “guilt by association” with the HPI. In retrospect, the but the first predictive clue as to the function of the hms
temperature-dependence of the pigmentation phenotype genes came from database searches showing that they
provided an important clue as to its true biological role. are similar to glycosyl transferase and polysaccharide
As noted previously, pigmentation develops only at deacetylase genes in other bacteria that are required to
temperatures less than about 28°C, a temperature that produce extracellular polysaccharides (Fig. 3). Notably,
matches the flea environment. It is not detected at 37°C, the E. coli operon pgaABCD is homologous to Y. pestis
the mammalian body temperature. In fact, nonpigmented hmsHFRS; pgaC and pgaD restore pigmentation to Y.
Y. pestis strains containing specific loss-of-function pestis hmsR and hmsS mutants, respectively, and the
mutation of the hms genes are fully virulent, at least in adjacent ycdT gene is an hmsT homolog (Lillard et al.,
mice, and the hypothesis that hemin storage is important 1997; Jones et al., 1999; Wang et al., 2004). The four
 Y. pestis Transmission Factors 203

Fig. 3. Comparison of the ica, hms, and pga operons of S. epidermidis, Y. pestis, and E. coli, respectively. Numbers indicate the percent amino acid
similarity of the predicted products of Y. pestis hms genes with ica, pga, and ycd gene products. Single asterisks indicate polysaccharide deacetylase
domains, double asterisks indicate glycosyl transferase domains, and GGDEF indicates diguanylate cyclase domains.

pga gene products are predicted to be outer surface their predicted catalytic activities, Kirillina et al. (2004)
proteins that synthesize extracellular poly-β-1,6-N-acetyl- proposed that HmsT and HmsP regulate Hms-dependent
D-glucosamine (PGA) that is required for biofilm formation extracellular polysaccharide production (and therefore
(Wang et al., 2004; Itoh et al., 2005). Similarity was also biofilm formation) by coordinately controlling the level of
detected between hmsR and hmsF and two genes in the the cyclic-di-GMP activator. Transcription of the hmsT
ica (intercellular adhesion) operon of Staphylococcus and the hmsHFRS operons is not affected by growth
epidermidis that is required to synthesize a linear β-1,6-N- temperature; however, protein levels of HmsT, HmsH,
acetyl-D-glucosamine polymer called the polysaccharide and HmsR are much lower in Y. pestis grown at 37°
intercellular adhesin (PIA) (Heilmann et al., 1996; Lillard than at 26°C, which likely accounts for the temperature-
et al., 1999;). PIA is an extracellular polysaccharide that dependence of the pigmentation phenotype (Perry et al.,
leads to bacterial cell-cell aggregation and is required for 2004).
the formation of staphylococcal biofilms. Interestingly, PIA The amino acid sequence comparisons indicate
as well as the extracellular polysaccharide associated that the hms genes encode products that synthesize
with several other bacterial biofilms binds Congo red the extracellular matrix of a biofilm. A bacterial biofilm
(Heilmann and Götz, 1998; Weiner et al., 1999). The ica is a complex, compact community of cells enclosed
operon consists of four genes (icaADBC). HmsR has 39% in an extracellular matrix, often attached to a surface
identity and 58% amino acid sequence similarity to IcaA, (Costerton et al., 1995). Biofilms can form in spite of high
an N-acetylglucosamine transferase that functions to shear forces and rapid currents, and are produced in
polymerize UDP-N-acetylglusosamine units; and HmsF vivo, particularly on implanted medical devices, by many
has 23% identity and 41% similarity to IcaB, a poly (β-1, 6) bacterial pathogens (Costerton et al., 1999). Previous
N-acetylglocosamine deacetylase that removes N-acetyl investigations have shown that the dense aggregates of
groups from the extracellular PIA polymer (Götz, 2002). Y. pestis that develop in the flea midgut and block the
Thus, it is likely that the Y. pestis hms gene products also proventriculus are surrounded by an extracellular matrix,
synthesize a PGA-like extracellular polysaccharide, but fitting the operational definition of a biofilm (Hinnebusch
its chemical structure remains to be determined. et al., 1998a; 2002a; Jarrett et al., 2004). The ability of Y.
The chromosomal hmsT and hmsP genes are pestis to produce an extracellular matrix in the flea, along
unlinked to the hmsHFRS operon and to each other but with the ability to block the proventriculus, depends on the
are required for normal expression of the pigmentation hms genes. The role of the individual hms genes in this in
phenotype and biofilm formation (Hare and McDonough, vivo phenotypes have not been systematically studied, but
1999; Jones et al., 1999; Kirillina et al., 2004). Although mutation of hmsR or hmsT eliminates or greatly reduces
the biochemistry remains to be demonstrated, the the ability of Y. pestis to block fleas (Hinnebusch et al.,
presence of specific domains within HmsT and HmsP 1996; and unpublished data). The hms genes are also
indicate their probable function. HmsT belongs to the required for the ability of Y. pestis to produce an adherent
family of GGDEF domain proteins (Jones et al., 1999) biofilm on the surface of a glass flowcell, and to synthesize
and is predicted to synthesize cyclic-di-GMP, a known an extracellular material observed by scanning electron
effector of extracellular polysaccharide production in other microscopy (Jarrett et al., 2004). Like pigmentation, the in
bacteria (Kirillina et al., 2004; Ross et al., 1987; Ryjenkov vitro biofilm and extracellular material are only produced
et al., 2005). HmsP belongs to the family of EAL domain at low temperatures and not at 37°C. Darby et al. (2002)
proteins and is predicted to have phosphodiesterase and Joshua et al. (2003) have also shown that Y. pestis
activity that hydrolyzes cyclic-di-GMP (Kirillina et al., and Y. pseudotuberculosis produce biofilm-like growth on
2004). Based on the presence of these domains and agar plates that accumulates on the external mouthparts
204 Hinnebusch

of Caenorhabditis elegans nematodes placed on them, borne bubonic plague epidemics terminate abruptly with
and that this phenotype is hms-dependent. the onset of hot, dry weather (Cavanaugh and Marshall,
Taken together, the genetic, in vitro, and in vivo 1972). Cavanaugh (1971) hypothesized that Pla activity
observations strongly suggest that Y. pestis forms could explain both phenomena. Pla synthesis is not
an hms-dependent biofilm to infect the hydrophobic, temperature-dependent, but its plasminogen activator
acellular surface of the flea’s proventricular spines, and in ability that leads to fibrinolysis is much greater at 37°C than
this way overcomes the rhythmic, pulsating action of the at temperatures below 28°C (McDonough and Falkow,
proventricular valve and the inward flow of blood during 1989). In fact, Pla has an opposite procoagulant activity at
feeding that would otherwise counteract transmission by low temperatures, although this fibrin clot-forming ability
washing the bacteria backwards into the midgut. Given is weak and is detected only in rabbit plasma and not in
the homology between the Y. pestis hms genes and the mouse, rat, guinea pig, squirrel, or human plasma (Jawetz
staphylococcal ica genes, it seems likely that the function and Meyer, 1944; Beesley et al., 1967). Nevertheless, it
of the hms gene products is to synthesize an extracellular was hypothesized that this low-temperature activity of
polysaccharide required for biofilm development. The Pla formed what was presumed to be the fibrin matrix
composition of the extracellular matrix that surrounds the of the blocking mass of Y. pestis in the flea. The clot-
Y. pestis biofilm in the flea is unknown, but appears to dissolving plasminogen activator function was invoked to
contain flea midgut-derived lipid components as well as explain why blockage does not develop in fleas at higher
hms-dependent components (Jarrett et al., 2004). The temperatures. McDonough et al. (1993) later reported that
hms genes do not appear to be required in the mammal; Pla+ Y. pestis caused greater mortality in fleas than an
thus, their primary biological function is to enable flea- isogenic Pla- mutant, and attributed this to an increased
borne transmission (Hinnebusch et al., 1996; Lillard et blockage rate. Blockage was not directly monitored in that
al., 1999). Transmission of Leishmania parasites also study, however, and the mortality occurred only four days
depends on a foregut-blocking phenomenon in the sandfly after infection, well before blockage would be expected
vector (Stierhof et al., 1999), but Y. pestis is unique to occur.
among bacteria characterized to date in using a biofilm When the Cavanaugh hypothesis was put to the
mechanism to enable arthropod-borne transmission. In test, it was found that Pla is not required for normal
retrospect, the first hint that the hms genes pertained to proventricular blockage to develop in the flea (Hinnebusch
biofilm formation was the observation in the original paper et al., 1998a). Hms+ Y. pestis strains lacking pPst were
by Jackson and Burrows (1956a) that cells in pigmented able to infect and block X. cheopis fleas as well as the
colonies resist resuspension and remain bound together wild-type parent strain. Both Pla+ and Pla– strains failed to
in densely packed masses. Surgalla (1960) also observed block fleas kept at 30°C, even though midgut colonization
that another aspect of the pigmentation phenotype is rates were little affected by temperature; in other words,
the production of a substance in liquid cultures at room the identical in vivo phenotype as seen for Hms– Y. pestis.
temperature that promotes autoaggregation and pellicle Thus, the inability of Y. pestis to block fleas kept at 30°C
formation on the sides of the culture vessel – typical of can be fully explained by temperature dependence of
biofilm formation. the Hms phenotype. Furthermore, the presumption that
flea-blocking masses of Y. pestis are embedded in a
The Y. pestis plasminogen activator and fibrin matrix is inconsistent with the fact that the matrix
dissemination following flea-borne transmission is not degraded by proteases or the fibrinolytic enzyme
Like murine toxin and the Hms pigmentation phenotype, plasmin. Therefore, the hms-dependent biofilm model of
the biological functions attributed to the Y. pestis proventricular blockage better fits the available data than
plasminogen activator (Pla) have undergone revision. the Pla-based fibrin clot model.
The pla gene is on the 9.5-kb Y. pestis plasmid referred to
as pPCP1, pPst or pPla (Sodeinde and Goguen, 1988). Insect pathogen-related genes in Y. pestis and Y.
It encodes a surface protease associated with increased pseudotuberculosis
tissue invasiveness and systemic spread of the bacteria Like most bacterial genomes, the Y. pestis genome
(Korhonen et al., 2004). Pla is considered to be an essential contains several loci that appear to have been introduced
factor for the flea-borne route of transmission because it by lateral transfer from unrelated organisms. Among
greatly enhances dissemination following subcutaneous these are several homologs of known insecticidal toxin
injection, which is assumed to mimic transmission by complex (Tc) genes of bacterial pathogens of insects,
fleas (Sodeinde et al., 1992). The requirement for Pla for and a homolog of a baculovirus enhancin protease gene
dissemination from peripheral infection sites may not be required for insect pathogenesis, which conceivably could
universally true for all Y. pestis strains or for all animals, influence Y. pestis interaction with the flea (Parkhill et al.,
however (Samoilova et al., 1996; Welkos et al., 1997). 2001). In beginning efforts to assess the role of these
A prominent role for Pla in proventricular blockage of genes, fleas were infected with Y. pestis strains containing
the flea has been proposed previously. The extracellular specific mutations in the baculovirus enhancin homolog
matrix that embeds the blocking masses of Y. pestis and tcaA, a homolog of one of the Tc genes. Both mutants
in the flea has often been assumed to be a fibrin clot infected and blocked X. cheopis fleas normally, indicating
derived from the flea’s blood meal. It was also known that these two genes are not important for interaction with
that proventricular blockage does not develop normally the flea (B. J. Hinnebusch and R. D. Perry, unpublished).
in fleas kept at elevated temperatures, which helps Many of the insect pathogen-related genes are also present
explain striking epidemiological observations that flea- in Y. pseudotuberculosis; therefore, their acquisition
 Y. pestis Transmission Factors 205

appears to predate the divergence of Y. pestis. When primary lesion at the fleabite site and disseminates first
outside the host in soil and water, Y. pseudotuberculosis to the local draining lymph node, seems to better fit an
would be expected to come into contact with and even intradermal transmission model.
be ingested by insects and other invertebrates, and the Flea saliva is also secreted into the bite site. The
insecticidal toxins may help the bacteria survive those saliva of all blood-feeding arthropod vectors contains
encounters. Because Y. pestis transmission depends on anticoagulants, and may contain other factors that
chronic infection of the flea gut, overt toxicity would be influence the outcome of transmission. For example,
counterproductive. Thus, it seems likely that insect toxicity a component of sandfly saliva greatly enhances the
would be lost or moderated in Y. pestis. infectivity of Leishmania (Titus and Ribeiro, 1988). Flea
saliva is known to contain the anticoagulant apyrase,
Y. pestis at the host–vector interface an enzyme which acts to inhibit platelet and neutrophil
Successful transmission of an arthropod-borne agent aggregation (Ribeiro et al., 1990), but this is the only
and subsequent infection depends on a complex co- component that has been identified to date.
evolved interaction between pathogen, vector, and host
that has not been well-characterized for any arthropod- The transmission phenotype of Y. pestis
borne disease. Plague is initiated during the brief The phenotype of Y. pestis as it exits the flea and enters
encounter between an infectious flea and a vertebrate the mammal is clearly different from in vitro growth
host. For practical reasons, intradermal or subcutaneous phenotypes. As described in previous sections, Y. pestis
inoculation by needle and syringe of in vitro-grown Y. growth in the flea resembles a biofilm and is associated
pestis is routinely used for pathogenesis studies in animal with an extracellular membrane. The infectious units
models in lieu of flea-borne transmission. While this is a transmitted by the flea may consist not only of individual
reasonable challenge method which may be adequate for Y. pestis, but small clumps of bacteria derived from the
most purposes, certain aspects of the flea-bacteria-host periphery of the proventriculus-blocking mass. If pieces
transmission interface are unique, and have unknown of the biofilm are regurgitated by fleas, the bacteria
effects on the host-pathogen interaction and the initiation within them may be protected from the initial encounter
of disease. with the host innate immune response, because bacteria
embedded in a biofilm have been shown to be more
Feeding mechanism of fleas and the resistant to uptake or killing by phagocytes (Donlan and
microenvironment of the transmission site Costerton, 2002). Because known antiphagocytic factors
Two basic strategies have been described for the manner such as the F1 capsule and the Type III secretion system
in which blood-feeding arthropods acquire a blood meal are not produced by Y. pestis at the low temperature
(Lavoipierre, 1965). Vessel feeders, such as triatomine of the flea gut (Straley and Perry, 1995; Perry and
bugs, penetrate the skin and cannulate a superficial blood Fetherston, 1997), the extracellular matrix associated with
vessel with their mouthparts before they begin to feed. growth in the flea may provide initial protection until the
In contrast, pool feeders, such as ticks and tsetse flies, known antiphagocytic virulence factors are synthesized.
lacerate blood vessels with their mouthparts as they probe, Secondarily, regurgitated aggregates that are larger than
and feed from the resulting extravascular hemorrhage. the diameter of the intradermal blood vessels would
Fleas, like mosquitoes, were originally classified as preclude direct intravenous transmission.
vessel feeders, but this may be an oversimplification. The In nature, Y. pestis in a particular phenotype is
flea mouthparts include a pair of thin serrated laciniae that transmitted along with flea saliva into an intradermal
act as cutting blades to perforate the dermis. Alternating, microenvironment. Details of the flea-bacteria-host
rapid contractions and thrusts of the left and right laciniae interface during and after transmission have not been
pierce the skin, and this pneumatic drill-like cutting motion characterized, and cannot be satisfactorily mimicked by
continues as the mouthparts move vertically and laterally transmission using a needle and syringe. Consequently,
in the dermal tissue during probing (Wenk, 1980), an aspects of host-parasite interactions specific to the unique
activity that can cause hemorrhage. When blood is context of the fleabite site are unknown and merit future
located, the laciniae and epipharynx come together to investigation.
form a feeding channel, and feeding ensues. Whether
the tip of the mouthparts is inserted into a vessel or in Evolution of arthropod-borne transmission
an extravascular pool of blood has obvious implications Y. pestis provides a fascinating case study of how a
for plague pathogenesis. If intravascular feeding occurs, bacterial pathogen can evolve a vector-borne route of
Y. pestis might be regurgitated directly into the blood transmission. Given the short evolutionary timeframe in
stream (i.v. transmission). If extravascular feeding occurs, which it occurred, the change from an enteric, food- and
intradermal transmission is the appropriate model (the water-borne pathogen to systemic, insect-borne pathogen
flea mouthparts are not long enough to penetrate into the was too abrupt to result from the slow evolutionary process
subcutaneous tissue). The most careful observations of of random mutation of individual genes leading to natural
flea feeding (Deoras and Prasad, 1967; Lavoipierre and selection. Instead, more rapid evolutionary processes
Hamachi, 1961) suggest that fleas can suck extravascular were responsible, such as horizontal gene transfer and
blood that leaks from a capillary, but prefer to feed directly the fine-tuning of existing genetic pathways to perform
from a blood vessel. Whether blocked fleas show the same new functions.
discretion, however, is unknown. The usual progression Carniel (2003) has proposed a sequential evolutionary
of bubonic plague, in which Y. pestis can produce a scenario for the switch to vector-borne transmission in the
206 Hinnebusch

yersiniae. Because the ymt (pldA) gene enhances survival would have strongly favored this. Some consideration of
in the flea digestive tract, a likely first step was acquisition the dynamics of the Y. pestis-flea relationship serve to
of the 100-kb pFra plasmid by horizontal transfer to reinforce this point (Fig. 4). First, flea-borne transmission
generate a Y. pseudotuberculosis (pFra) or pre-pestis is actually quite inefficient, which may reflect the fact that
1 clone. Y. pseudotuberculosis can cause septicemia in Y. pestis has only recently adapted to its insect host. The
rodents, so it probably was taken up by fleas periodically. number of Y. pestis needed to infect 50% of susceptible
This would have been a dead end, until the hypothetical mammals (the ID50, often referred to as the minimum
pre-pestis 1 clone acquired the PLD activity encoded infectious dose) is the same as the 50% lethal dose (LD50)
on the pFra replicon, which allowed it to survive in and – less than 10 (Perry and Fetherston, 1997). In contrast,
colonize the flea midgut. the ID50 of Y. pestis for X. cheopis is about 5,000 bacteria
A second necessary step in the evolution of flea-borne (Lorange et al., 2005). Fleas take small blood meals
transmission took advantage of a pre-existing biofilm- (0.1–0.3 μl), so Y. pestis must achieve a level of >107 per
forming capacity, which involves the hms genes. Y. pestis milliliter in the peripheral blood in order to have a 50%
makes an hms-mediated biofilm to produce an obstructing chance of infecting its vector. Bacteremias of 108 to 109 per
infection in the proventricular valve, which is required for milliliter are routinely present in moribund white laboratory
efficient transmission. The hms genes are present in Y. mice (Douglas and Wheeler, 1943). The concept of a very
pseudotuberculosis, but, curiously, most isolates that have high threshold level of bacteremia, below which infection
been tested do not exhibit the pigmentation phenotype of feeding fleas does not occur or is rare, is supported
(Brubaker, 1991). Some Y. pseudotuberculosis strains by the observations of several investigators (Douglas and
do show the Congo red-binding pigmentation phenotype Wheeler, 1943; Pollitzer, 1954; Kartman and Quan, 1964).
in vitro, but all Y. pseudotuberculosis that have been Thus, Y. pestis does not infect the flea very efficiently in
tested, whether pigmented or not, are unable to block the first place, and this would have been strong selective
the proventriculus of X. cheopis (B. J. Hinnebusch, pressure favoring more invasive, and consequently, more
unpublished). Thus, a separate, as yet undiscovered virulent strains able to produce the severe bacteremia
genetic change may have occurred in pre-pestis 1 to that typifies plague.
extend its biofilm-forming capacity to include the flea gut A second weak link in the Y. pestis life cycle is that,
environment. The presumptive change likely affected the even after successful infection of the vector, subsequent
outer membrane in such a way as to enhance aggregate transmission is not very efficient. Not all infected fleas
formation on the hydrophobic proventricular spines in the develop transmissible proventricular infections – for X.
context of the flea digestive tract milieu. cheopis, the rate is only about 50%, and this rate can be
A third important step in the evolution of flea- much lower for other flea species (Wheeler and Douglas,
borne transmission occurred when the progenitor clone 1945; Burroughs, 1947; Pollitzer, 1954). Furthermore, it
acquired the small plasmid containing the pla gene, which is well established that the bite of a blocked flea does not
is thought to enable Y. pestis to disseminate from the always result in disease. Past studies report that individual
fleabite site after transmission. The clone containing both blocked X. cheopis fleas that feed on a susceptible host
of the new Y. pestis-specific plasmids has been referred transmit plague only about 50% of the time (Burroughs,
to as pre-pestis 2 by Carniel (2003). Given the ecology of 1947). Very few studies have addressed the number of Y.
the Y. pseudotuberculosis ancestor, horizontal transfer of pestis transmitted by a single infectious fleabite. Burroughs
pFra and pPst could have occurred in a mammal, a flea, (1947) triturated and plated mouse-skin
���������������������� � biopsies taken
or the environment. Of course, it is likewise impossible to from the site where a blocked flea had been allowed to
know with certainty the order in which the plasmids were
transferred, or the plasmid donors, although molecular
biology analyses may provide some clues. For example,
the 100-kb pFra shares major sequence identity with
a Salmonella Typhi plasmid, suggesting that the Y.
pseudotuberculosis ancestor acquired what became pFra
from a Salmonella donor (Prentice et al., 2001). This
horizontal transfer to generate the Y. pseudotuberculosis
(pFra) clone may have occurred in the digestive tract of a
rodent, since both bacteria are enteric pathogens. On the
other hand, plasmid transfer by conjugation occurs readily
in mixed bacterial biofilms, both in the environment and in
the flea gut (Hinnebusch et al., 2002a).

Coevolution of flea-borne transmission and

increased virulence in Y. pestis
The evolutionary path that led to flea-borne transmission
also led to Y. pestis becoming one of the most virulent
and feared pathogens of human history. It is probably
no accident that increased virulence coevolved with Fig. 4. Dynamics of flea-borne transmission of plague. The high ID50 of Y.
vector-borne transmission. In fact, reliance on the flea pestis for fleas is compensated for by the ability to produce a high-density
septicemia in the rodent. The inefficient transmission by blocked fleas is
for transmission imposed new selective pressures that compensated for by a low ID50 (LD50) from a peripheral inoculation site.
 Y. pestis Transmission Factors 207

attempt to feed. Only one of thirty samples was positive, in their mammalian hosts, but infections in the arthropod
which contained 88 Y. pestis colony-forming units (CFU). vector are rarely symptomatic. As a rule it would be
This number was multiplied by a factor to compensate counterproductive to impair the health of the vector they
for the low plating efficiency from other skin samples into rely on for transmission. For example, malaria parasites
which known numbers of Y. pestis had been injected, do not cause morbidity in the mosquitoes that transmit
to calculate an estimate of 11,000 to 24,000 bacteria them to new hosts. Y. pestis breaks this rule – it causes
transmitted by the flea. However, this estimate was derived an overwhelming, fatal infection in the flea as well as the
indirectly from a single positive sample, which seems less mammal. A single blocked flea contains greater than 106
than satisfactory. We recently reexamined this topic by Y. pestis on average, and proventricular blockage leads
allowing individual blocked X. cheopis fleas to feed on a to dehydration and death by starvation within a few days
mouse or an artificial feeding device and quantifying the (Hinnebusch et al., 1998b; Kartman and Prince, 1956;
number of Y. pestis transmitted. Only 45% of the fleabites Pollitzer, 1954)). During this time, however, a blocked
resulted in transmission, and the median number of Y. flea will make persistent, frequent attempts to feed. A
pestis transmitted was less than 100 (Lorange et al., normal flea probes the skin and feeds to repletion within
2005). This rather erratic transmission, frequently involving a few minutes. A blocked flea, in contrast, will try to take a
only a small number of bacteria, would have favored the blood meal in one location for a period of several minutes,
selection of more invasive clones (such as the ancestral then withdraw, move to another location, and try again.
pPla+ clone), able to successfully disseminate from the As it gradually starves, this process is repeated many
fleabite site even from a small initial dose. times. Finally, the dehydrated blocked flea often remains
Because reliance on the flea vector to complete with its mouthparts embedded in the skin for an hour or
the Y. pestis life cycle is contingent upon the ability to more, expending its last bit of energy in a futile attempt
produce a high-density bacteremia and to invade from a to satisfy its appetite. This anomalous feeding behavior
small infectious dose in the skin, the evolution of vector- provides multiple opportunities for transmission to occur
borne transmission and of increased virulence would before the flea dies, somewhat analogous to the manner
have been mutually reinforcing. Parasite evolution is often in which the altered behavior of a rabid dog enhances the
assumed to evolve towards commensalism, or a state of transmission of rabies virus.
peaceful coexistence with the host. Actually, the degree
of virulence that is evolved by a successful parasite is Conclusions
functionally coupled with transmissibility (Ewald, 1983). Y. pestis exhibits a distinct life stage in its flea vector. The
That is, pathogens will tend to evolve to a level of virulence barriers to infection and the environmental conditions it
that optimizes their chance of successful transmission. faces in the flea digestive tract are quite different from
Paul Ewald (1983) has argued that parasites transmitted those encountered in the mammalian host. Consequently,
by blood-feeding arthropods pay little cost for harming the development of a transmissible infection requires a
their hosts, and may actually benefit by being virulent. distinct subset of genes. None of the known mammalian
The extensive reproduction and hematogenous spread virulence factors that have been tested (the F1 capsule,
associated with severe disease increases the probability pla, the type III secretion system encoded on the pYV
that a blood-feeding vector would acquire an infectious virulence plasmid, and the HPI) are required in X. cheopis
dose; and host immobilization and morbidity would not (Hinnebusch et al., 1996; 1998). Conversely, the two
hinder, and may even enhance, the ability of a vector to genetic loci (ymt and hms) that have been shown to be
find the host and feed to repletion. required for the flea-specific phenotype are not required
The risk for a pathogen as virulent as Y. pestis is for virulence in the mammal. Thus, a distinction can be
killing the host too quickly for transmission to occur. Y. made between Y. pestis genes required for pathogenesis
pestis may have gotten away with the risky strategy of in the mammal (virulence factors) and the genes
being rapidly fatal because most mammals maintain a required to produce a transmissible infection in the flea
more or less permanent flea population in their fur and in (transmission factors).
their burrows. It is not unusual for a single rodent to carry When a flea takes up Y. pestis in a blood meal, the
5 to 80 fleas, with more than 100 fleas present in the nest bacteria experience a drop in temperature from 37°C to
or burrow, and it is simply a fact of life for most rodents the ambient temperature of the flea. This temperature
to experience daily fleabites throughout their lives (Traub, shift appears to be an important environmental signal
1972). Thus, Y. pestis does not have to rely on a chance for the bacteria to regulate gene expression appropriate
encounter with a flying vector like a mosquito. Even if there to the invertebrate or vertebrate host. The expression of
are only a few hours between the development of high- many Y. pestis virulence factors is upregulated at 37°C
density septicemia and death, there is good probability compared to room temperature (Straley and Perry, 1995;
that one or more resident fleas will take an infectious blood Perry and Fetherston, 1997), whereas the upregulation of
meal during that short window of time. Because fleas do the Hms phenotype and the ymt gene at room temperature
not readily leave their host mammal, killing the host may compared to 37°C was a predictive initial clue that their
actually be important to Y. pestis transmission. Brubaker biological role might occur in the flea. With the advent of
(2000) has made the point that death of the host compels DNA microarray and proteomics technologies, the global
the resident infected fleas to seek a new, healthy host – a effect of the temperature shift from mammal to flea on
necessary step to complete the transmission cycle. Y. pestis gene expression can be analyzed, which may
Arthropod-transmitted pathogens may be indifferent identify new candidate transmission factors (Han et al.,
to or even benefit from producing morbidity and mortality 2004; Motin et al., 2004).
208 Hinnebusch

In this review, I have focused on recent work using Anisimov, A.P. 1999. [Factors providing the blocking
X. cheopis as the animal model and genetically defined activity of Yersinia pestis]. Mol. Gen. Mikrobiol. Virusol.
strains of Y. pestis KIM (biovar Medievalis) and 195/P (Russ.) 4: 11–15.
(biovar Orientalis). Earlier work, particularly by Russian Anisimov, A.P., Lindler, L.E., and Pier, G.B. 2004.
investigators, led to often contradictory conclusions, Intraspecific diversity of Yersinia pestis. Clin. Microbiol.
with some studies suggesting a role in flea infection or Rev. 17: 434–464.
blockage for the F1 capsule and Pla virulence factors, and Azad, A.F., Radulovic, S., Higgins, J.A., Noden, B.H., and
others finding no role (Kutyrev et al., 1992; McDonough Troyer, J.M. 1997. Flea-borne rickettsioses: ecologic
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I thank Clayton Jarrett, Roberto Rebeil, and Florent Buchrieser, C., Prentice, M., and Carniel, E. 1998. The 102-
Sebbane for their contributions to the work described in kilobase unstable region of Yersinia pestis comprises
this review and for discussions about it. Robert Perry and a high-pathogenicity island linked to a pigmentation
Andrey Anisimov kindly provided preprints of in press segment which undergoes internal rearrangement. J.
manuscripts. Research in my laboratory is supported in Bacteriol. 180: 2321–2329.
part by a New Scholars in Global Infectious Diseases Burroughs, A.L. 1947. Sylvatic plague studies. The
award from the Ellison Medical Foundation. vector efficiency of nine species of fleas compared with
Xenopsylla cheopis. J. Hygiene. 45: 371–396.
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