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Current Protein and Peptide Science, 2012, 13, 739-755 739
Interactions in Bacterial Biofilm Development: A Structural Perspective
James A. Garnett and Steve Matthews*
Centre for Structural Biology, Department of Life Sciences, Imperial College London, South Kensington, London SW7
2AZ, UK
Abstract: A community-based life style is the normal mode of growth and survival for many bacterial species. These cel-
lular accretions or biofilms are initiated upon recognition of solid phases by cell surface exposed adhesive moieties. Fur-
ther cell-cell interactions, cell signalling and bacterial replication leads to the establishment of dense populations encapsu-
lated in a mainly self-produced extracellular matrix; this comprises a complex mixture of macromolecules. These fascinat-
ing architectures protect the inhabitants from radiation damage, dehydration, pH fluctuations and antimicrobial com-
pounds. As such they can cause bacterial persistence in disease and problems in industrial applications. In this review we
discuss the current understandings of these initial biofilm-forming processes based on structural data. We also briefly de-
scribe latter biofilm maturation and dispersal events, which although lack high-resolution insights, are the present focus
for many structural biologists working in this field. Finally we give an overview of modern techniques aimed at prevent-
ing and disrupting problem biofilms.
Keywords: Biofilm, adhesion, dispersin, structural biology.
1. INTRODUCTION plex sessile form, planktonic and biofilm phenotypes have

evolved together [12].
The ability of a biological cell to sense and respond to its
local environment underlies the existence of every living The biofilm life-cycle involves several stages (Fig. 1) [5].
organism. Furthermore, communication between sibling and If we start with a single mobile bacterium, we can consider
rival cells has led to the evolution of multicellular, higher the first stage of this process to be cellular adhesion to a
ordered life. For many years it was believed that bacteria solid surface [13]. This initial event is highly reversible, al-
inhabit the planet exclusively in a planktonic form, as free- though upon cues from the environment the production of
living cells, but it is now widely accepted that most microor- additional adhesion molecules can be initiated and attach-
ganisms reside primarily in biofilms [1-4]. A biofilm is a ment becomes more tenacious [13,14]. These cells begin to
complex agglomeration of genetically similar or distinct cells divide and signals from neighbouring bacteria lead to
adhered to a solid surface and to one another, encased in a changes in phenotype and the display of further proteina-
scaffold of self-produced extracellular polymeric substances ceous molecules on their surface, instigating pro-
tein/polysaccharide-mediated inter-bacterial interactions.
(EPS). In their mature form they often appear as pyramid or
Further sensing of the local environment leads to the secre-
mushroom-like structures, which are embedded with cavities
tion of a sticky matrix of extracellular polymeric substances
and channels allowing the sequestration of water together
(EPS); comprising polysaccharides, proteins, nucleic acids
with the exchange of nutrients and waste [5].
and lipids [14]. These components allow the three dimen-
Bacterial biofilms are ubiquitous in the environment and sional architecture of the mature biofilm to form, whilst also
can be found on almost any hydrated non-shedding surface; increasing adhesion between bacteria and with the surface
including rivers, stagnant pools, man-made materials and support. At this stage inter-cellular communication leads to
biological matter. Whilst many of these communities can be synergy within the community [12,15], with the diffusion of
used for the synthesis of valuable products and the treatment oxygen/nutrients inwards and waste/signals outwards. The
of waste, others can cause problems in industrial applications heterogeneous distribution of cells within the biofilm is in
and/or persistent virulence in humans and other animals. part orchestrated by dispersins [16]. These reagents act as
surfactants or enzymes that disrupt interbacterial interac-
These bacterial aggregates provide protection from a wide
tions, producing water filled channels and lead to the active
range of environmental factors such as fluctuating pH [6], and passive dispersal of cells from the biofilm mass. This
exposure to UV light [7], dehydration [8] and antimicrobial cycle is generally accepted as a developmental process,
agents [9,10]. Biofilms have been identified as far back as where a hierarchy of genetic events controls the transition in
3.2-billion years ago [11] and it has been suggested that response to environmental cues such as quorum sensing;
rather than the free living cells developing into a more com- although this is still very much a working model and further
validation is required [17].
*Address correspondence to this author at the Centre for Structural Biology,
Department of Life Sciences, Imperial College London, South Kensington,
Biofilms are a very attractive area of research as they
London SW7 2AZ, UK; Tel/Fax: +44 (0)20 7594 5315/+44 (0) 207 594 effect many industrial processes, are a major cause of antibi-
3057; Email: [email protected] otic resistance and harbour the potential for novel technolo-
1875-5550/12 $58.00+.00 © 2012 Bentham Science Publishers

740 Current Protein and Peptide Science, 2012, Vol. 13, No. 8 Garnett and Matthews
Fig. (1). Schematic representation of the biofilm life cycle. (1) Free swimming bacteria (2) adhere to a surface using cell surface displayed
adhesin molecules. (3) Bacteria begin to divide and the expression of further macromolecules allows them to stick together in small micro-
colonies. (4) As these colonies grow they begin to secrete a complex mixture of carbohydrates, protein and lipids that encapsulates the bacte-
ria. This biofilm matrix (fuzzy outline) provides protection and stability for the maturing biofilm. (5) When the biofilm reaches maturity, a
number of factors will have developed a heterogeneous arrangement of cells and molecules within the biofilm, and given rise to solvent filled
cavities and channels. This can lead to dispersal of cells from the cellular mass. (6) Upon signal from the environment (waste build up or
demand for nutrients, for example), molecules are released that cause cell lysis and matrix dissemination. Many planktonic cells are now
released and can find a new habitat.
gies and materials. In this review we will be concentrating on 2.1. MSCRAMMs

the architectural protein components of these bacterial accre-
tions and how they mediate persistent host-pathogen interac- Staphylococcus aureus is a regular commensal of the
tions. We will then give a brief discussion of therapeutic skin and anterior nares of animals and humans [23]. Whilst
targets with the aim of dismantling and/or removing prob- harmless in these environments, S. aureus may become a
lematic biofilms. dangerous pathogen if it crosses the epithelial barrier where
it can infect almost any organ and cause abscesses, pneumo-
2. INITIAL SURFACE ADHESION nia, endocarditis, sepsis and infections associated with medi-
cal implants [24]. A number of surface components of S.
The initial attachment of prokaryotes to abiotic and bio- aureus have been implicated in biofilm formation. The
logical surfaces is by far the most understood process of exopolysaccharide biofilm matrix is composed of a polymer
biofilm development. The bacterial surface is decorated with of poly-N-acetyl--(1-6)-glucosamine, termed polysaccha-
an arsenal of diverse and often multifunctional macromole- ride intercellular adhesin (PIA) or poly-N-acetylglucosamine
cules. Adhesive structures are projected away from the cell (PNAG) and mediates intercellular adhesion events [25,26].
surface with sufficient distance to sample the surrounding S. aureus biofilms can also form independently of PIA and
environment and recognize appropriate interactions. At the the composition of this proteinaceous matrix includes SasG
time of this review, a search of the protein data bank (PDB: [27], Protein A (Spa) [28,29], fibronectin binding proteins
www.pdb.org) revealed 140 ‘adhesin’ structures from bacte- (FnBPs) [30,31] and biofilm associated protein (Bap) [32].
ria, many of which are unique proteins. In this section a SasG can bind desquamated nasal epithelial cells [31] and
number of different fibres from a range of bacteria will be promotes inter-bacterial aggregation [33]. Spa binds immu-
discussed, although the focus will be on those structures that noglobulin’s which enables S. aureus to evade in-
contribute to a well-defined understanding of surface at- nate/adaptive immune responses [34] and can promote mul-
tachment. For example, whilst type IV pili (T4P) are very ticellular adherence [28]. FnBPs have been shown to mediate
important factors leading to surface attachment in many bac- intercellular biofilm interactions [30] [31], recognize fi-
teria, no receptor complexes are available and so they will bronectin and fibrinogen [35,36] and can also promote the
not be discussed here. In addition, although the chaperone- invasion of epithelial cells [37]. Bap is involved in the initial
usher (CU) assembled type I and P pili are important lectins attachment to inert surfaces/interbacterial interactions [32]
from Escherichia coli, a wealth of both structural and bio- and also prevents cellular internalization of S. aureus
chemical information has already been amassed and a num- through binding the GP96 host receptor, which interferes
ber of excellent articles and reviews are already available with the FnBP mediated invasion pathway [38].
[18-22], so again these will not be described.
Interactions in Bacterial Biofilm Development Current Protein and Peptide Science, 2012, Vol. 13, No. 8 741
Spa and FnBPs belong to a large family of proteins called -sheet complementation with the G strand of the N3 domain
MSCRAMMs (microbial surface components recognizing and buries ~1800 Å2 of surface area. Ganesh et al. [47] have
adhesive matrix molecules) [39] that enable many patho- also shown that a mutant of ClfA that contains a disulphide
genic Gram-positive bacteria to interact with the eukaryotic bridge that covalently locks the C-terminal ‘latch’ across the
extracellular matrix (ECM). The ECM is a biologically ac- N2 domain binds to fibrinogen with equivalent affinities to a
tive material that encapsulates eukaryotic cells and contains wild-type construct. This suggests that ClfA does not require
a mixture of macromolecules including collagen, fibronectin an ‘open’ conformation to bind its ligand. This is possibly a
and fibrinogen, and functions in both cellular structure and consequence of ClfA recognizing just the last few residues of
physiology. A number of MSCRAMMs are involved in the the fibrinogen -chain that may be threaded into the binding
first step of S. aureus biofilm formation via attachment to the site.
ECM. These include the fibrinogen binding proteins: clump-
ing factor A (ClfA) and ClfB (~95 kDa) [40-43]; and the The A-region of FnBPA/B can also bind the C-terminal
fibronectin binding proteins: FnBPA and FnBPB (~100 kDa) region of the fibrinogen -chain and likely recognizes this
[35,36,44]. MRCRAMMs allow staphylococci to adhere to a ligand in a similar fashion to ClfA/B [47,56]. The B-region
range of cell lines and because FnBPs also bind host serum of FnBPs is intrinsically unstructured and contains 11
proteins that coat medical implants they can also mediate (FnBPA) or 10 (FnBPB) fibronectin binding repeats
adherence to these devices [40,42,45]. (FnBRs) that interact with consecutive fibronectin type I
(F1) region in the N-terminal domain of fibronectin [57,58].
MSCRAMMs are modular in nature and contain an N- Furthermore, in FnBPA, FnBR-1, -4, -5, -10 and -11 bind
terminal signal sequence followed by an extensive re- with dissociation constants in the nM range, whilst the others
peat/non-repeat region and a C-terminal region for cell wall bind with lower affinities [57]. Jennifer Pott’s group de-
anchoring [46] (Fig. 2A). The repeat/non-repeat region can scribed the NMR structure of a peptide from the Streptococ-
be further divided into an A-region and B-region. The A- cus dysgalactiae FnBP in complex with the fibronectin mod-
region contains ligand binding domains (N1-N3) whilst the ule pair 1F12F1 (pdb: 1O9A) and showed how these class of
role of the B-region is to project the adhesive domains away proteins interact with fibronectin through a tandem -zipper
from the bacterial surface, although they also contain un- mechanism [59]. More recently her group has also presented
known functions. In FnBPs, the B-region in addition har- the crystal structure of peptides corresponding to FnBR-1
bours the fibronectin binding activity. (residues 508-546) and FnBR-5 (residues 639-672) of
The B-region of ClfA/B is composed of mainly serine FnBPA in complex with 2F13F14F15F1 (pdb: 3CAL, 2RL0,
and aspartate residues (R-region) and it has been shown that 2RKY, 2RKZ) [58]. Each FnBR binds to four consecutive
residues 229-545 of ClfA (N2, N3 domains) are sufficient to F1 domains, where they form an antiparallel -strand along
retain the binding activity for the C-terminal region of the strand E of the triple-stranded (strands CDE) -sheet (Fig.
fibrinogen -chain (Kd 657 nM measured by isothermal calo- 2C), again as a tandem -zipper. Although these interactions
rimetry) [47]. The N1 domain is cleaved by the S. aureus are solely mediated by -sheet interactions, there is ~4300
metalloprotease autolysin [48] and recombinant ClfA/B ex- Å2 of surface area buried in each of the FnBR/2F13F14F15F1
pressed in E. coli have unstructured N1 domains that are complexes. It is likely that the interaction with fibronectin is
degraded by endogenous proteases prior to purification [49]. multivalent, binding six to nine fibronectin copies per
The crystal structure of apo-ClfA (pdb: 1N67) was solved in FnBPA/B molecule [60,61] and taking this into account, the
Sthanam Narayana’s group and consists of residues 221-559 burial of surface area increases to between 26000 to 36000
[50] with the N2 and N3 domain connected by a short linker Å2 [58].
(Fig 2B). Each domain is dominated by -sheet secondary
structure that folds into a variation of the IgG fold. This 2.2. Serine-Rich Repeat Containing Fimbriae
DEv-IgG fold, named so because two additional strands (D’ Fimbriae-associated protein 1 (Fap1) was identified by
and D’’) are inserted between the D and E strands of the Paula Fives-Taylor’s group whilst studying novel adhesive
standard IgG fold, was first identified in the collagen binding proteins from the Gram-positive Streptococcus parasanguis
MSCRAMM, Can (pdb: 1AMX) [51], but has since been FW213 [62]. Sanguis streptococci are primary colonizers of
observed in other systems [52,53]. Cna contains a number of the oral cavity in humans and specific initial attachment is
intramolecular isopeptide bonds [54], which are now recog- mediated via the salivary components that interact directly
nised to be a functionally important feature of many surface with the tooth surface [63]. Furthermore, these streptococci
proteins in Gram-positive bacteria and have been implicated also behave as ligands for additional oral bacterial species
in stabilizing structures, helping to withstand mechanical and form the substrata within a biofilm on the surface of the
stress and facilitating biofilm formation [55]. teeth: dental plaque [64]. In addition to having a major role
In the crystal structure of ClfA in complex with a 13 in caries and periodontal diseases, these bacteria can also
residue fibrinogen-derived peptide (pdb: 2VR3), the ligand is colonize native and prosthetic heart valves and are a com-
recognized and binds along the interface between the N2 and mon cause of endocarditis [4,65-67].
N3 domains [47]. Comparisons between the two crystal Fap1 is a ~200 kDa surface fibre which is essential for
structures of ClfA are overall very similar, although in the fimbrial biogenesis, adhesion and biofilm formation [62,67-
apo-structure the C-terminal residues fold back into the 70]. Analysis of Fap1 showed it to be composed of an N-
ligand binding site within the N3 domain [50], whilst in fi- terminal signal sequence, followed by a short stretch of
brinogen bound ClfA, this sequence crosses over and forms (E/V/I)S dipeptide repeats, a unique adhesive region, a much
an inter-domain -sheet in N2. The peptide forms a parallel longer dipeptide repeat region and finally at the C-terminus
Fig. (2). Adhesive mechanisms of MSCRAMMs. (A) Schematic of S. aureus ClfA and FnBPA adapted from [46]. The N-terminal signal
sequence (S) is followed by the A-region, B-region and at the C-terminus is the cell wall anchoring region containing the cell wall sorting
region (W) containing the LPXTG motif (star), membrane-spanning hydrophobic domain (M) and the cytoplasmic positively charged C-
terminal tail (C). The fibrinogen binding A-region of both ClfA and FnBPA contain three domains (N1-N3). The B-region of ClfA (R-region)
is composed of mainly serine and aspartate residues whilst in FnBPA this is made up of 11 fibronectin binding domains (FnBDs: numbered 1-
11). (B) Crystal structure of S. aureus ClfA with and without a fibrinogen peptide bound. (C) Model of the FnBR-1 region (residues 508-546)
of FnBPA in complex with fibronectin (2F13F14F15F1). The A-region is shown as a schematic and the FnBR-2-11 region is shown as dashed
line (not to scale).
an LPxTG cell wall anchor sequence [68]. The presence of and 7.1, however, after the ingestion of fermentable carbo-
such extensive regions of alternating serine dipeptide repeats hydrates, microbial acid production can lead to a drop in
(~80% of the overall sequence), which are O-glycosylated plaque pH to below 5.0. Unlike some streptococci, S.
through the serine residues [53,67,70-76], has led to Fap1 parasanguis does not have an acid tolerance response
being termed a ‘serine-rich repeat glycoprotein’ (SRRP). [96,97] and instead it shuts down its metabolic functions [6].
Over the last decade new SRRPs have been discovered and So Fap1 presents a mechanism of reversible adhesion for S.
this ever expanding family of Gram-positive bacterial fim- parasanguis under metabolically active neutral/alkali condi-
briae now also includes Staphylococcus aureus SraP [77], tions, but at low pH whilst metabolically dormant, Fap1 pro-
Staphylococcus agalactiae Srr-1 and Srr-2 [78-80], Strepto- vides a much higher affinity for the salivary pellicle and in
coccus gordonii GspB [52,81], S. gordonii Hsa [82], Staphy- turn the mature biofilm which protects S. parasanguis [53].
lococcus saprophyticus UafB [83], Streptococcus pneumo-
niae PsrP [84-87], and Streptococcus sanguinis SrpA [88]. Work in Carlos Orihuela’s group has shown that the
Primary sequence analysis of this family demonstrates the SRRPs PsrP from Streptococcus pneumoniae, SraP from
same overall arrangement within these very large macro- Staphylococcus aureus and GspB from Streptococcus
molecules, although they each retain unique adhesive fea- gordonii strain M99 can all mediate inter-bacterial biofilm
tures (Fig. 3A). interactions in the lungs of infected animals via their unique
non-repeat regions, in addition to the well documented host
SRRPs are glycosylated in the cytoplasm [69,89-91] be- adhesive properties [98]. Recently the crystal structure of the
fore being exported to the cell surface via the SecA2/Y2 ac- GspB adhesive region (GspBBR) from S. gordonii was solved
cessory secretory pathway [72,89,92]. Structures of the (pdb: 3QC6, 3QC5, 3QD1) in Tina Iverson’s group, and this
unique adhesive region of Fap1 have been recently published has exemplified how the basic SRRP architecture can ac-
(pdb: 2X12, 2KUB, 3RGU) and using structural and bio- commodate a plethora of different functions [52]. GspB is an
chemical techniques it has been possible to model the overall important adhesive structure of S. gordonii M99 and loss of
architecture of the SRR (Fig. 3A) [53,93,94]. In mature Fap1 expression leads to a noticeable effect on the pathogenicity
the extensive major SRR region forms a super-coiled struc- of this strain [99]. GspB binds to human platelets through the
ture which projects the N-terminal adhesive domain away membrane glycoprotein GPIb [100] and specifically it rec-
from the cell surface, with the extensive glycosylation pro- ognizes sialyl-T antigen, which is one of the major carbohy-
tecting this highly extended region from proteolysis. We drates of GPIb [101].
have performed biophysical analysis of short synthetic SRR
peptides based on Fap1 that show they are unstructured and The crystal structure of apo-GspBBR is composed of three
this suggests the importance of glycosylation in the correct adjacent domains and has been described as being like beads
folding of SRRPs (unpublished data). on a string [52]. The N-terminal subdomain is reminiscent of
an Ig-like fold and has high tertiary homology with the A-
The ‘non-repeat’ region of Fap1 (Fap1-NR) was charac- region of the S. aureus Cna [51] and S. parasanguinis Fap1-
terized as an adhesive domain after it was shown to interact NR [53]. Whilst the C-terminal subdomain has a unique
with an in vitro tooth model: saliva-coated hydroxylapatite fold, the central subdomain has structural homology with
(SHA) [95]. Furthermore, these adhesive properties can be eukaryotic Siglecs, which are involved in the binding of car-
modulated by pH with a much greater affinity observed un- bohydrates. The crystal structure of GspBBR in complex with
der acidic conditions [53]. Fap1-NR can be further subdi- an analogue of sialyl-T antigen confirmed this function (Fig.
vided into an -helical (Fap1-NR) followed by a -sheet 3C). A deviation of the subdomain orientation within these
(Fap1-NR) region. NMR analysis and SHA assays show two GspBBR structures suggests that these regions display
that whilst both are involved in binding to a yet unknown independent motion. Furthermore, the modular nature of
host ligand in the salivary pellicle, it is the helical subdomain these motifs hints at multiple functions, which may be inde-
that directs the pH-mediated effects [53]. SAXS data from pendent or cooperative as within Fap1-NR [97]. Mutations
Fap1-NR at pH 5.0 and pH 8.0 reveals a ‘boomerang’ shape within the lectin domain of GspBBR cause a significant re-
that ‘opens’ under acidic conditions (Fig. 3B). Comparison duction in bacterial densities within kidneys and spleens and
of the crystal structure of Fap1-NR at pH 5.0 [93,94] and so it is likely that it is this region that facilitates the main S.
the NMR structure at pH 8.0 [53] shows no significant dif- gordonii M99 interactions in endocarditis [96]. However,
ferences, although docking of these structures and the crystal these additional domains may be involved in other virulence
structure of Fap1-NR into these low resolution envelopes strategies such as mediating GspB-GspB interactions during
identifies a mechanism of adhesion modulated by electrostat- later stages of biofilm development.
ics (Fig. 3B) [53,93].
In Fap1-NR Asp152, Glu154, Asp430 and Asp435 pack 3. PROTEIN MEDIATED INTER-BACTERIAL IN-
against one another within the inter-subdomain face and this TERACTIONS
has the effect of raising the local pKa of these carboxylate
Once prokaryotes have formed a tight adherence to a
groups so that they are protonated at a higher pH (Fig. 3B)
surface they begin to divide and scan their local environment
[93]. These subdomains are connected by a 27 amino acid
for interactions with siblings and genetically distinct part-
linker and NMR relaxation data indicates that there is inde- ners. This marks the initiation of the three-dimensional archi-
pendent motion in both [53]. Therefore the Fap1-NR tecture of a biofilm. At this stage cellular communications
‘open/active’ conformation results from a rearrangement of are mediated by surface exposed protein interactions and
this region and the potential for intra-Fap1-NR salt bridges to while our biochemical and genetic understanding of these
form. The normal resting saliva pH in humans is between 6.5 processes are quite detailed, high resolution structural infor-
Fig. (3). Adhesive mechanisms of SRRPs. (A) Schematic representation of a mature SRRP. The N-terminal unique adhesive region is pro-
jected away from the cell wall via the extensive SRR region. There is also a minor SRR at the N-terminal pole. The C-terminus is attached to
the cell wall peptidoglycan (yellow spheres) through an LPxTG anchor sequence. (B) Conformations of the ‘open’ and ‘closed’ states of S.
parasanguinis Fap1-NR. SAXS electron densities are shown as envelopes, coloured red (pH 5) or Blue (pH 8), and the structures have been
docked into the maps. Acidic residues at the inter-subdomain boundary are highlighted as yellow spheres. (C) Crystal structure of the carbo-
hydrate bound S. gordonii GspBBR.
mation is lacking. Again in this section, structures will only yet as no high-resolution data is available this will not be
be described which allow an atomic understanding of early described. At the time of writing this review, only three
biofilm formation. A recent crystal structure of the B-region bacterial structures had been deposited in the PDB that
of S. aureus SasG (PDB: 3TIP, 3TIQ) from Jennifer Potts describe the atomic details of protein-mediated interactions
group gives a tantalizing suggestion of how these classes of which allow biofilms to begin and take shape. These will be
proteins may form protein-protein or protein-saccharide/ discussed here.
DNA interactions through the sequestration of Zn2+ [102],
3.1. The Haemophilus influenza Hap These inter-Hap mediated interfaces have been rigorously
tested using a number of mutants in a self-association assay
Autotransporters (ATs) belong to the type V secretion
and this presents a valid model for Hap-Hap biofilm activity.
system family and account for the largest number of ex-
Once this initial dimer has formed in vivo it likely acts as a
ported proteins from Gram-negative bacteria [103]. ATs con-
nucleant for the assembly of mega-Dalton complexes of im-
tain a single polypeptide chain consisting of a C-terminal mense stability that overcomes the repulsive force between
outer membrane -barrel pore through which the N-terminal
bacteria [109].
passenger domain is transported. The N-termini contain a
perisplasmic signal sequence that directs them through the In the work by Meng and colleagues [109], they were
Sec apparatus, and whilst for a long time it was believed that able to go one step further and model the functional modula-
insertion of ATs into the outer membrane utilized the Omp85 tion of the Hap biofilm. The serine protease is autoprote-
superfamily [104], new data suggests that they use a dedi- olytic and can release adjacent Hap passenger domains from
cated translocation and assembly module (TAM) [105]. Pas- the bacterial surface to regulate host and inter-bacterial adhe-
senger domains, which usually fold into a -helix like struc- sion [112,113]. Interestingly this proteolytic activity is inhib-
ture, are important virulence factors in Gram-negative ited by the secretory leukocyte protease inhibitor (SLPI)
pathogens and upon export (and often cleavage from the [114], which is present in the upper and lower respiratory
surface) they can function as proteolytic enzymes, adhesins, tract [113]. In the model of Hap-Hap multimerization, the
invasins and toxins [106]. A subset of the AT family are protease domain is still partially solvent exposed and super-
called the self-associating autotransporters (SAATs) and imposition with the elastate/SLPI structure [114] permitted
they share sequence homology and mediate inter-bacterial the positioning of SLPI with respect to Hap (Fig. 4C) [87].
aggregation [107]. These SAATs include AIDA, Ag43, and This suggests that in the absence of SLPI the serine protease
TibA from Escherichia coli and recently Joseph St Geme’s activity will release adjacent Hap from the H. influenza sur-
group has published the structure of Hap from Haemophilus face, whilst in its presence this activity is revoked resulting
influenza (pdb: 3SYJ) and presented a general model for in oligomerization and inter-bacterial aggregation.
SAAT-mediated bacterial auto-aggregation [108,109].
3.2. The Escherichia Coli Common Pilus
H. influenza is an agent of bacteremia, pneumonia and
acute bacterial meningitis. Hap is involved in the adhesion of In 2001 Timo Korhonen’s group published data on a
H. influenza to epithelial cells and extracellular matrix pro- novel fimbria isolated at low temperatures from Escherichia
teins, invasion of epithelial cells and inter-bacterial aggrega- coli associated with newborn meningitis and septicaemia
tion during early biofilm development [110]. The N-terminus (NMEC) [115]. This was called the meningitis associated
contains the usual signal peptide, followed by a passenger and temperature regulated (Mat) fimbria, although over the
domain (Haps) consisting of a serine protease, an ECM- past decade Jorge Girón and José Puente’s groups have
binding/SAAT domain and finally the C-terminus comprises shown it to be ubiquitous across most E. coli strains and it is
of the outer membrane -barrel motif (Fig. 4A). The ECM- now usually referred to as the E. coli common pilus (ECP)
binding domain is responsible for recognizing fibronectin, [116-119]. E. coli are primarily commensal colonizers of the
laminin and collagen IV [111] and the SAAT region is in- human and other animal bowels and they contribute to a
volved in interactions with epithelial cells [112] and also healthy immune system of the host. There are also a number
Hap-Hap mediated biofilm processes [112,113]. The crystal of virulent strains that can cause diarrheal diseases such as
structure of the full Hap passenger domain is described as a hemorrhagic colitis [120]. Furthermore, if they enter ex-
‘Dane Axe’-like assembly (Fig. 4A). The main spine of the traintestinal sites these strains can also lead to neonatal men-
structure is formed from a -helix and has at the C-terminus ingitis, urinary tract infections, sepsis, and pneumonia [121].
the SAAT domain, overlapped with the ECM-binding do- ECP fibres are assembled via a variant of the CU path-
main. The N-terminal region of the -helix acts as a scaffold way and as with all members of this superfamily, filaments
for the positioning of the serine protease at the extreme N- are formed from polymerisation of several different/identical
terminus of the passenger domain. pilin domains (Fig. 5A) [18,122]. The tip of ECP is uniquely
The SAAT domain of Hap is entirely -helical and dis- composed from a polymerized array of a novel 60 kDa adhe-
plays a hydrophilic edge of stacked Asn/Asp residues [109]. sive domain EcpD, which recognizes an unknown ligand on
Furthermore, this region is unusually straight and forms a the host cell surface [122]. The majority of ECP is composed
striking triangular prism composed of approximately nine of an 18 kDa domain called EcpA [115,116], which func-
strands per face. Remarkably, the crystal lattice packing re- tions in binding hydrophobic surfaces and mediating inter-
lates two molecules in trans configuration by a crystallo- bacterial aggregation in early biofilm formation [122,123].
graphic 2-fold screw axis (Fig. 4B). Inter-dimer interactions
The crystal structure of EcpA (pdb: 3QS2, 3QS3) from
are mediated in the main by a ladder of Asn/Asp hydrogen
uropathogenic E. coli (UPEC) has been recently solved by
bonds from one monomer to the face of another, with also
our group [122]. Like other CU major pilin domains, EcpA
some burial of hydrophobicity. A secondary dimer interface
is formed from an incomplete Ig-like fold, where an adjacent
is formed by a region flanking either end of the SAAT do-
molecule in the fibre donates its N-terminal strand (N-
main. As this dimer forms with a single monomer’s Asn/Asp
terminal extension; NTE) to fill a hydrophobic groove run-
ladder, the other remains exposed for higher ordered oli-
ning along the full length of EcpA, completing the very sta-
gomerization. Further symmetry relations build an array of
ble Ig-like motif (Fig. 5B). EcpA is fashioned from ap-
multimerized Hap molecules with the N-terminal -helix
proximately 50% hydrophobic residues and the surface is
facilitating significant inter-molecular interactions (Fig. 4C).
scattered with hydrophobic patches including a number of
Fig. (4). Hap-Hap mediated biofilm formation. (A) Crystal structure of the H. influenza Hap passenger domain (Haps) with the C-terminal
pore shown as a schematic. (B) Crystallographic relationship between Haps molecules in the crystal. The interface of a dimer of Haps in trans
(coloured orange and green) shows that a run of Asp/Asn residues (the Asp/Asn ladder) from one subunit packs against a complimentary but
alternative surface of the other molecule (shown as electrostatic surfaces : dark regions). (C) The remaining Asp/Asn ladder from the latter
molecule of the dimer is still accessible and with the burying of hydrophobicity, translations of these dimers can lead to great multimers form-
ing. The modelled SLPI bound to each serine protease domain is coloured blue.
aromatic residues. This likely promotes a less-specific con- inter-domain salt bridges form within these aggregates, but
tact with a wide range of hydrophobic substrates and poly- some of these dimers are formed solely by the burial of hy-
mers. drophobic surfaces, whereas others also involve patches of
negative charge (Fig. 6B). This is consistent with the obser-
Pili assembled via the CU pathway vary greatly in size
and function. Afa-III fimbrils are very flexible and are com- vations that Fap1 tip interactions can form at pH 8.0 but be-
come much more prevalent at pH 5.0 [53]. Furthermore,
posed of a head-to-tail polymerisation of subunits ~2 nm in
within this aggregate a number of the Fap1-NR host-binding
diameter [124], whilst type I pili are rigid structures formed
sites are not fully occluded and may allow the dual role of
by the major pilin molecules packing about a central axis
salivary pellicle recognition and inter-cellular negotiation.
giving rise to a hollow fibre of ~7 nm in diameter [125]. ECP
are quite flexible with a width ~6 nm, which consistently 4. EPS-MEDIATED INTERACTIONS
varies along the fibre length [122]. Crystal structures of do-
nor strand complemented EcpA reveals a fibre-like arrange- In the middle stages of biofilm formation, cells develop
ment of domains with single filament dimensions matching into a three-dimensional array and start producing the sticky
those observed under EM (Fig. 5C) [122]. conglomerate EPS, of which the composition can be very
diverse between different species. On average 90% of a
EM images of E. coli producing ECP show these fibres biofilms dry mass will be accounted for by the EPS, and this
form a mesh that encapsulates the whole microcolony. ECP is the immediate environment which these prokaryotes sam-
interacts with itself through pili crossing over one another, ple [14]. Biofilms can take on a plethora of architectures and
parallel fibre entwining and antiparallel entwining [116,122]. this is also crucially dependent on the localized production
The crystal lattice of EcpA also revealed an intertwining of and quantity of EPS. The biofilm matrix is mainly composed
antiparallel fibres giving rise to a super helical diameter of of exopolysaccharides, DNA, protein and lipids. Although
~12 nm (Fig. 5D). Inter-ECP interactions are conducted in no experimental high resolution structures are available for
the main via burial of the loop residues Ala147, Val148 and these components, due to the nature of polysaccharides,
Thr149 across the central axis and mutations of these resi- DNA and some of the protein based EPS (i.e. amyloids) it is
dues to bulky amino acids results in dramatic reductions in possible to use modelling techniques (i.e. X-ray fibre diffrac-
cell mass in biofilm assays. EcpA is highly conserved tion [126,127] and NMR [128,129]). However, whilst this
amongst a range of other enteric bacterial species including information is invaluable, from an atomic perspective we
Serratia proteamaculans, Serratia odorifera, Klebsiella sp., know very little about how the matrix interacts within itself.
Klebsiella pneumoniae, and Enterobacter cancerogenus,
which suggests a role for ECP in establishing contacts be- The major contributors of the bacterial EPS matrix are
tween multiple species [122]. polysaccharides [130] and many mutants that cannot synthe-
size exopolysaccharides are unable to form mature biofilms
3.3. Streptococcus parasanguis Fap1 [131,132]. These are mainly heteropolysaccharides, attached
to the cell surface forming long linear and branched struc-
Fap1 is a model system to study the biogenesis, export tures, which establish a complex mesh. A number of well-
and general architecture of SRRPs; however, in its own right known exopolysaccharides include alginate, Pel and Psl from
it displays some very unique properties. As has been detailed Pseudomonas aeruginosa [133]. Depending on the composi-
above, the binding of Fap1 to SHA is affected greatly by the tion of saccharides these polymers can display more or less
pH of the environment and this can be attributed to a survival hydrophobicity or ionic nature. This gives rise to a very large
mechanism of S. parasanguis when experiencing long peri- and durable structure with variable functionality. These
ods of acidity. Moreover, there is much evidence that Fap1 polysaccharides can interact with biotic/abiotic surfaces,
also functions in mid and later stages of biofilm develop- further increasing the affinity of the bio-mass whilst also
ment. The heavy glycosylation has been implicated in some cross linking the sessile cells through sugar-sugar and sugar-
of these processes [67] and the extreme N-terminal SRR lectin contacts. Very recently the first direct evidence for
region may also contributes here. This role likely plays out interactions between the T4P PilA and exopolysaccharides of
via hydrophobic stacking of saccharides and/or recognition Myxococcus xanthus were observed under EM [134]. Some
by lectins within the mature biofilm matrix. Interestingly biofilms also have a very hydrophobic nature due to the lipi-
though, visualization of Fap1 displayed on the surface of S. dation of carbohydrates and this property is essential for the
parasanguis using EM also shows a pH-dependence to the adherence of Thiobacillus ferrooxidans to pyrite surfaces
auto-aggregation of these fibres, specifically at their N- [135]
terminal pole [53]. In addition, biofilm assays performed Nucleic acids in the form of DNA can be in abundance in
with S. parasanguis over a range of pH values show a clear biofilms, although whilst in S. aureus it is a major compo-
increase in cellular mass correlated to acidity, mirroring the nent of the matrix structure, it is less so in S. epidermis
binding of Fap1-NR to SHA [53,93]. [136]. The source of extracellular DNA (eDNA) comes from
The crystal structure of Fap1-NR has been very insight- the lysis of bacteria within the biofilm and it has been shown
ful in terms of our understanding of how Fap1 may form that Enterococcus faecalis are able to specifically lyse a frac-
inter-filamentous aggregation via the N-terminal tip (Fig. tion of the bacterial population using a protein GelE [132]. In
6A) [93]. The arrangement of Fap1-NR in the crystal lattice P. aeruginosa eDNA is used to connect cells and treatment
clearly demonstrates an order that can accommodate full with DNase inhibits biofilm formation [137,138], whilst in
Fap1-NR domains and allow the major SRR region to pro- Bacillus cereus eDNA has also been shown to function as an
ject back to a bacterial surface from multiple orientations adhesin [139]. Furthermore, DNA is a versatile molecule for
[93], in a similar fashion to that observed under EM [53]. No specific recognition by proteins such as the T4P of P. aeru-
Fig. (5). ECP-ECP mediated biofilm formation. (A) Model of the E. coli common pilus displayed on the cell surface with the usher pore
(EcpC), the major pilus (EcpA) and the polymerized tip adhesin (EcpD) annotated. (B) Schematic representation of donor strand exchange
between EcpA domains. One EcpA subunit (green) donates its N-terminal extension (NTE) to the adjacent EcpA subunit (purple) where it
lines the hydrophobic groove. (C) Atomic model of ECP fibres. A single fibre from crystals of EcpA is shown as a surface (left) and as a car-
toon with adjacent subunits coloured green and purple. The direction of polymerization in the fibre is shown with black arrows. Four subunits
have been expanded to highlight the zig-zagging and helicity of EcpA along the fibre length. (D) Representation of ECP-ECP mediated anti-
parallel interactions. Cells 1-3 are shown with two ECP fibres intertwined by a half helical turn. One of these regions has been boxed and
expanded, showing the atomic model which describes this event. Two antiparallel entwined ECP are shown from the side and top.
ginosa [140], resulting in a heterogeneous protein-DNA [86]. These are intriguing and novel molecules that have
network. This localization of eDNA also represents a poten- been identified as key contributors to many aspects of the S.
tial reservoir for the horizontal transfer of genes and the in- aureus biofilm maturation process [152] including the for-
creased virulence/persistence of strains within bacterial com- mation of channels, biofilm detachment, biofilm expansion
munities. and dissemination.
Another important type of macromolecule involved in the 6. THERAPEUTIC TARGETS OF BIOFILM DIS-
maturation and shaping of biofilms are microbial amyloid SEMINATION
fibres [141]. Amyloids were historically thought to be a con-
sequence of protein misfolding in neurodegenerative dis- Biofilms represent the dominant microbial life style in
eases such as Alzheimer’s and Parkinson’s [142], but it is aquatic environments, providing nutrients, purification of
now understood that they fulfil an important role in a number water, sequestration of carbon and biogeochemical fluxes;
of organisms. Amyloid are -strands that are orientated per- i.e. roles that are essential for these and other habitats to exist
pendicular to the fibre axis [143] and the Enterobacteriaceae [165]. Compounds such as Triclosan have been reported to
curli are a model system to study this family [144,145]. disseminate biofilms in a global fashion by targeting EPS
Small curli subunits (CsgA) are secreted to the extracellular secretion [166]. These reagents are very effective in one
space where they polymerize into the amyloid and contribute sense, but obviously they are highly detrimental to ecosys-
the major proteinaceous component of the E. coli and Sal- tems and better strategies are needed that target essential
monella enterica serovars Typhimurium biofilm matrix. processes within specific species of biofilm.
Curli are crucial in these biofilms and mediate initial surface One point of entry to combat biofilms is to stop them
attachment and provide a scaffold for the community before they can take hold through inhibition of the initial
[144,146,147]. Whilst highly stable models of bacterial amy- attachment. A number of strategies have been investigated
loids have been proposed [143], the molecular details that that target both the adhesive mechanisms and also the bio-
underlie these processes are poorly understood. genesis pathways from which these adhesins are produced.
An example of the latter are Gram-negative bacteria that use
5. STRUCTURING AND DISPERSAL OF BIOFILMS CU assembled pili as adhesive filaments: compounds termed
The controlled restructuring of biofilm architecture re- ‘pilicides’ have been shown to disrupt the biogenesis ma-
sults in a heterogeneous arrangement of matter and the pro- chinery of the type I pilus and inhibit biofilm formation in
duction of cavities and channels. This process can be under- UPEC [167,168]. Antimicrobial peptides coated on surfaces
taken by both targeted cell lysis and cell dispersion. Another have also exhibited activity against S. aureus and P. aerugi-
process that is closely linked to these restructuring events is nosa [169]. It is an essential requirement of modern medical
the partial breakdown of the matrix to allow the release of devices to possess antimicrobial properties and it is now evi-
cells, which are free to migrate and inhabit new environ- dent that both the component material (i.e. copper, gold, zinc
ments, i.e. when nutrients become limiting and when waste and single walled nanotubes) [170,171] and the sub-
products accumulate [148]. These effectors or dispersins can micropatterning of the surface [172] both dictate bacterial
often have enzymatic activity towards polysaccharides [149], growth.
proteins [150] or DNA [151]; or function as surfactant-like Once a biofilm has become established, modern antibiot-
molecules [152,153]. Furthermore, bacteriophages also play ics have little effect on their displacement [173] and can
an important role and can induce cell death and provide en- make them more resilient [174]. The EPS acts as a barrier to
zymes that help dissolve the biofilm matrix [154]. drug delivery [175] and quorum sensing within these dense
Aggregatibacter actinomycetemcomitans is a Gram- communities can up-regulate gene expression linked to an-
negative non-motile commensal of the oral cavity, associated timicrobial resistance [176]. Therefore two possibilities for
with gum disease. Dispersin B is an extracellular enzyme therapeutic advances on mature biofilms are available; direct
(PDB: 1YHT) [155] secreted by A. actinomycetemcomitans dispersal mechanisms and communication pathways.
and can degrade matrix polysaccharides [156]. This is a clas- Enzymes perform functions in biofilm restructuring and
sic example of enzymatic disruption of the biofilm matrix. whilst they are highly specific, they are expensive to produce
Whilst dispersin B has been identified as an essential factor and can be unstable. Furthermore, in a medical setting they
for the dispersal of A. actinomycetemcomitans biofilms may also cause an unwanted immune response, therefore the
[157], it has also been demonstrated to induce dispersal of a direct use of enzymes in therapeutics does not seem viable.
range of other bacteria that contain an poly-N- Phages, however, have been reported to improve the normal
acetylglucosamine substrate [158,159]. antimicrobial activity in biofilm related infections [177] and
moreover, the Bacillus licheniformis lipopeptide biosurfac-
An alternative strategy of restructuring/dispersal is ex-
tant has been shown to have a marked effect on increasing
emplified by the P. aeruginosa rhamnolipid [153,160] and
antibiotic activity against E. coli biofilms [178]. Although
staphylococcal phenol-soluble modulins (PSMs) [152,161].
these strategies are feasible, much development is needed
The immense stability of bacterial biofilms can be attributed
before they can be used in any real applications. Richard
to the extensive burial of hydrophobic material within the
matrix. Therefore, surfactant-like reagents such as rham- Losick’s group have shown that Bacillus subtillis release a
factor that inhibits the formation of, and initiates the break-
nolipid and PSMs have the ability to disrupt these interac-
down of existing biofilms [179]. This was identified as a
tions in a non-specific manner. PSMs are -helical, amphi-
mixture of the amino acids D-tyrosine, D-leucine, D-
pathic peptides ranging from ~20-50 amino acids [162-164]
tryptophan and D-methionine. Furthermore, a number of D-
and their expression is controlled through quorum sensing
Fig. (6). Fap1-Fap1 mediated biofilm formation. (A) Representation of Fap1-NR mediated Fap1-Fap1 interactions. Cells 1-5 are displayed
with Fap1 filaments on their surfaces. A number of these fibres are interacting via their tips, and this has been expanded to show the surfaces
of Fap1-NR orientated based on the Fap1-NR crystal structure. The Fap1-NR region has been further expanded to show the packing of five
molecules from these crystals. The numbers represent the unique molecules of the asymmetric unit. (B) Fap1-NR crystals are formed from 3
types of dimer (A-C). Each dimer interface is drawn as an electrostatic surface and the specific contact areas are circled.
amino acids also have similar effects on S. aureus and P. ring [185]. AIP has long been known to control virulence
aeruginosa biofilms. These amino acids function through factor expression but it also mediates biofilm detachment
incorporation into the cell wall peptidoglycan where they through activation of an EPS proteases [150]. Furthermore,
stimulate release of the amyloid TasA from the matrix P. aeruginosa produce an organic compound, cis-2-decenoic
[180,181]. In addition, another secreted Bacillus subtillis acid, which can disseminate established biofilms and also
compound, norspermidine, seems to act in a complimentary inhibit biofilm development [186]. This may or may not op-
manner by targeting exopolysaccharides [182]. erate through manipulating quorum-sensing pathways, but
none-the-less in addition to P. aeruginosa this highly potent
Bacteria have developed highly robust cell-cell signalling
or quorum-sensing mechanisms. In S. aureus quorum- fatty acid can also disrupt many other Gram-negative
biofilms. A number of these natural dispersal mechanisms
sensing inhibits biofilm development [183,184] via the agr
have been coordinated with current antibiotic therapies and
(accessory gene regulator) locus, which produces and senses
have shown great promise in dissemination of medically
AIP (autoinducing peptide). AIP is an eight residue peptide
relevant biofilms [187].
where the C-terminal five residues form a cyclic thiolactone
CONCLUDING REMARKS [18] Waksman, G.; Hultgren, S.J. Structural biology of the chaperone-
usher pathway of pilus biogenesis. Nat. Rev. Microbiol., 2009, 7,
Biofilms represent the most frequent mode of growth for 765-774.
many microbes. While headway is being made in under- [19] Geibel, S.; Waksman, G. Crystallography and electron microscopy
of chaperone/usher pilus systems. Adv. Exp. Med. Biol., 2011, 715,
standing their formation and development, we are still far 159-174.
from being able to describe all of these processes from a [20] Piatek, R.; Zalewska, B.; Bury, K.; Kur, J. The chaperone-usher
molecular perspective. As further insights into this compli- pathway of bacterial adhesin biogenesis -- from molecular
cated life style are made available, both at the atomic and mechanism to strategies of anti-bacterial prevention and modern
cellular level, new targets to be exploited will arise, giving vaccine design. Acta. Biochim. Pol., 2005, 52, 639-646.
[21] Remaut, H.; Tang, C.; Henderson, N.S.; Pinkner, J.S.; Wang, T.;
us a much wider scope to address problematic biofilms. Hultgren, S.J.; Thanassi, D.G.; Waksman, G.; Li, H. Fiber
formation across the bacterial outer membrane by the
CONFLICT OF INTEREST chaperone/usher pathway. Cell, 2008, 133, 640-652.
[22] Phan, G.; Remaut, H.; Wang, T.; Allen, W.J.; Pirker, K.F.;
The author(s) confirm that this article content has no con- Lebedev, A.; Henderson, N.S.; Geibel, S.; Volkan, E.; Yan, J.;
flicts of interest. Kunze, M.B.; Pinkner, J.S.; Ford, B.; Kay, C.W.; Li, H.; Hultgren,
S.J.; Thanassi, D.G.; Waksman, G. Crystal structure of the fimd
ACKNOWLEDGEMENTS usher bound to its cognate fimc-fimh substrate. Nature, 2011, 474,
49-53.
We thank the Wellcome Trust for financial support. [23] Wertheim, H.F.L.; Vos, M.C.; Ott, A.; van Belkum, A.; Voss, A.;
Kluytmans, J.A.J.W.; van Keulen, P.H.J.; Vandenbroucke-Grauls,
C.M.J.E.; Meester, M.H.M.; Verbrugh, H.A. Risk and outcome of
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Received: June 18, 2012 Revised: July 16, 2012 Accepted: August 03, 2012

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Interactions in Bacterial Biofilm Development: A Structural Perspective

James A. Garnett and Steve Matthews*

1. INTRODUCTION plex sessile form, planktonic and biofilm phenotypes have

1875-5550/12 $58.00+.00 © 2012 Bentham Science Publishers

gies and materials. In this review we will be concentrating on 2.1. MSCRAMMs

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