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Microbes and Infection 6 (2004) 101–112
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Review
Adhesins and invasins of pathogenic bacteria: a structural view
Hartmut H. Niemann, Wolf-Dieter Schubert, Dirk W. Heinz *
Department of Structural Biology, German Research Center for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany
Abstract
Adhesion and invasion of pathogenic bacteria represent the important initial step of infection. Pathogens utilize surface-located
adhesins/invasins for specific interaction with host cell receptors. The three-dimensional structures of a number of adhesins/invasins show that
many are elongated molecules containing domains commonly found in eukaroytic proteins. Similar folds are employed repeatedly to target
different receptors.
© 2003 Elsevier SAS. All rights reserved.
Keywords: Adhesins; Crystal structure; Invasins; Pathogenic bacteria; Solution structure; Virulence factors
1. Introduction
Adhesion to and invasion of host tissues are crucial steps
in the pathogenesis of many bacteria, parasites and viruses.
Adhesion can help balance clearance by mechanical forces
and is often a prerequisite for successful colonization of
epithelia. Adhesins are specialized surface proteins that mediate bacterial adhesion. They specifically recognize receptors on the surface of target host cells, determining tissue
tropism of the pathogen. Invasion following adhesion allows
bacteria to evade the humoral immune response and to proliferate in a well-protected niche. To invade non-professional
phagocytes, bacteria usually abuse the host cell cytoskeleton
dynamics. They trigger signaling cascades in the host cell,
leading to the assembly of phagocytotic machinery inducing
their uptake. For the facultative intracellular pathogen Listeria monocytogenes, two invasins have been identified and
thoroughly characterized both functionally and structurally.
This review focuses on the three-dimensional structures of
these invasins and their functional implications. We furthermore discuss other bacterial adhesins and invasins of known
Abbreviations: FnI repeat, fibronectin type I repeat; FnIII repeat, fibronectin type III repeat; hEC1, human extracellular E-cadherin domain 1;
HGF, hepatocyte growth factor; Ig, immunoglobulin; InlA′, fragment of
InlA comprising cap, LRR and IR; IR, interrepeat region; LRR, leucine-rich
repeat; LTA, lipoteichoic acid; Omp, outer membrane protein; Tir, translocated intimin receptor.
* Corresponding author. Tel.: +49–531–6181; fax: +49–531–6181–763.
E-mail address: [email protected] (D.W. Heinz).
© 2003 Elsevier SAS. All rights reserved.
doi:10.1016/j.micinf.2003.11.001
three-dimensional structure and compare them with the invasins of L. monocytogenes.
2. Adhesion and invasion by L. monocytogenes
2.1. Internalins of L. monocytogenes
L. monocytogenes is a facultative intracellular Grampositive bacterium that invades phagocytic as well as normally non-phagocytic cells. Usually taken up as a food contaminant, it can cause severe systemic disease in humans
with compromised immune systems. L. monocytogenes is
able to cross three major barriers in the human body: the
intestinal, placental, and blood–brain barriers. Two invasins
have been identified in L. monocytogenes: internalin A (InlA)
[1] and InlB [2]. Both belong to a larger family of surface
proteins in L. monocytogenes, the internalins. Of these, only
InlA and InlB have been identified as invasins. While InlA
and InlB also function as adhesins [1,3], other listerial proteins, like p104 and Ami, also play an important role in
adhesion to host cells [4,5]. InlA is sufficient for uptake into
gut epithelial cells [6] and is required for crossing the intestinal barrier [7]. The receptor for InlA is human E-cadherin
[8]. InlB mediates uptake into a variety of cell types, e.g.
hepatocytes, endothelial cells and some epithelial cell lines
[2,3,9]. Several distinct receptors identified for InlB will be
discussed below.
InlA and InlB have a related domain organization. As
surface or secreted proteins, they share an N-terminal signal
sequence cleaved during maturation. Both, furthermore, have
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two repeat regions: an N-terminal domain consisting of a
variable number (15 repeats in InlA and seven in InlB) of
leucine-rich repeats (LRRs) and a C-terminal repeat region.
The LRR domain is flanked by a cap at its N-terminus, and by
an interrepeat region (IR) at its C-terminus. The LRR domain
consists of tandemly arranged LRRs containing 22 amino
acids. Ten of these 22 residues are conserved, of which seven
are leucine or isoleucine. The IR derives its name from its
position between the N-terminal LRRs and the C-terminal
repeat region. In InlA, three so-called B repeats, each containing about 70 amino acids, make up the C-terminal repeat
region. The B repeats are followed by an LPXTG motif
required for covalent attachment to the bacterial cell wall and
a single-pass transmembrane helix [10,11]. InlB lacks both
the LPXTG motif and the transmembrane helix. Accordingly, it is not covalently bound to the cell wall. Following a
single B-repeat, it contains three GW domains (named for a
conserved Gly–Trp (GW) dipeptide), another type of repeat
unit comprising 70–85 residues, at its C-terminal end.
Structural information for the internalin LRR and its
flanking regions (cap and IR) is available for several constructs of InlB [12–14], InlA [15], InlH [13] and InlE [16].
InlE and InlH are two members of the internalin family not
further discussed here. For InlB, structural information is
also available for the GW domains [14]. Cap, LRR and IR of
InlA (in the following, called InlA′) and InlB (InlB′) are in
principle very similar. The LRRs create an extended yet
curved superhelix, giving rise to a curved structure (Fig. 1).
The different number of repeat units leads to different overall
shapes: while the LRR domain of InlB is only slightly
curved, the corresponding domain of InlA shows a pronounced sickle-shaped structure. An extended, parallel
b-sheet forms the concave side, where the individual
b-strands are oriented perpendicularly to the molecular axis.
Each b-strand is followed by a short 310 helix on the convex
side. A single LRR thus consists of a b-strand, a loop, a 310
helix and another loop that links it to the next LRR. The seven
conserved leucines and isoleucines face inward, forming the
elongated hydrophobic core between the b-strands and the
310 helices (inset in Fig. 1). At the N- and C-terminal ends of
the LRR domain, the hydrophobic core needs to be shielded
from the hydrophilic surrounding. This is achieved by the cap
and the IR domains, respectively. Cap, LRR and IR form a
single structural unit and have a common hydrophobic core
[13]. The cap adopts the fold of a severely truncated
calmodulin-like EF-hand domain with three short a-helices.
The IR domain is a b-sandwich consisting of a four- and a
three-stranded b-sheet [13]. The four-stranded b-sheet of the
IR directly extends the last b-sheet on the concave side of the
LRR. The IR domain is structurally related to immunoglobulin (Ig) domains but does not strictly adopt the Ig fold. It is,
therefore, referred to as Ig-like.
2.2. The InlA/E-cadherin complex
Human E-cadherin was initially identified as receptor for
InlA by affinity chromatography [8]. The extracellular part of
E-cadherin is rod-shaped and consists of five Ig-like domains
(EC1–EC5; recently reviewed in [17]). The largely unstructured cytoplasmic tail links E-cadherin to the actin cytoskeleton through adaptor proteins, like catenins [18]. An InlA
fragment comprising cap, LRR and IR is sufficient and necessary to induce internalization in an uptake assay using latex
beads coated with purified protein [6]. The N-terminal domain of human E-cadherin (hEC1) is involved in binding
InlA [19] and has recently been crystallized in complex with
InlA′ [15]. The crystal structure of the complex shows that
hEC1, forming a 1:1 complex with InlA′, almost fills the
Fig. 1. Ribbon diagram of the fused cap, LRR and interrepeat (IR) domain of InlA (left) and InlB (right). The cap is shown in turquoise, the Ig-like IR is shown
in green. The LRR domain consists of 15 and 7 repeats in InlA and InlB, respectively. Each repeat contributes a b-strand (dark blue) and a helix (red) to the LRR
domain. The inset shows a top view on a single repeat (LRR10) of InlA. Side chains of leucines and isoleucines forming the hydrophobic core are yellow. All
figures generated with PyMOL [94].
H.H. Niemann et al. / Microbes and Infection 6 (2004) 101–112
central void of the curved LRR domain (Fig. 2). Although a
large surface area is buried upon complex formation, the
affinity in the micromolar range is rather weak. Besides
numerous solvent molecules, a calcium ion is present at the
interface between InlA′ and hEC1, explaining the observed
increase in stability of the complex by an order of magnitude
in the presence of >20 mM Ca2+ [15]. Only the LRR domain
and not the cap or the Ig-like domain of InlA′ are engaged in
interactions with hEC1, which is in excellent agreement with
the observation that LRR domains generally function as
modular structural scaffolds designed for protein–protein
interactions [20]. InlA′ mostly utilizes residues located in the
LRR b-strands to bind hEC1. This includes several surfaceexposed aromatic residues.
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The sixth LRR of InlA is unusual in that it consists of only
21 rather than 22 residues. The single amino acid deletion
shortens b-strand six by one residue, forming a shallow
hydrophobic pocket at the surface of InlA′, which accommodates Pro16 of hEC1 in the complex (inset in Fig. 2). This
interaction is crucial in determining the species specificity of
a Listeria infection. In contrast to human E-cadherin, murine
E-cadherin contains a glutamate in position 16 and is not
recognized by InlA [15,19]. Binding to InlA can be achieved
using an E16P mutant of murine E-cadherin [19]. The pivotal
importance of Pro16 is underscored by in vivo experiments.
Mice are not susceptible to oral infection with L. monocytogenes, as the bacteria are unable to cross the intestinal barrier.
Expression of a human E-cadherin transgene in enterocytes
Fig. 2. Ribbon diagram of the structure of InlA′ in complex with the N-terminal domain of its receptor, human E-cadherin (hEC1). Color coding of InlA is as
in Fig. 1. hEC1 (orange) occupies the central cavity of the InlA LRR domain. Aromatic residues of InlA involved in receptor-binding are shown as ball-and-stick
model in pink. The yellow sphere indicates a Ca2+ in the InlA/hEC1 interface. Pro16 of hEC1, which is crucial for binding specificity, fits into a hydrophobic
pocket of the InlA LRR domain (inset).
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Fig. 3. Ribbon diagram of the structure and receptor-binding sites of full-length InlB. Aromatic residues at the concave face of the InlB LRR domain are crucial
for binding of c-Met (inset). The maroon oval represents the B-repeat, which is not resolved in the crystal structure. The B-repeat may bind an as yet unidentified
receptor. The three C-terminal GW modules (yellow) are responsible for non-covalent binding of InlB to the bacterial cell wall through interaction with LTA.
The receptors gC1q-R and heparin compete with each other and with LTA for binding to the GW modules.
renders mice susceptible to InlA-dependent oral infection
with L. monocytogenes [7]. These data show that InlA has
been evolutionarily optimized as a human pathogen by a
single deviation from the canonical 22-residue LRRs present
in all internalins to provide an optimal fit for Pro16 of human
E-cadherin.
In the InlA′/hEC1 complex, the C-termini of both molecules point in opposite directions. This is biologically significant, as InlA and E-cadherin are attached to the cell
surfaces by their C-termini. The observed orientation puts the
bacterium and the host cell on opposite sides of the recognition complex (Fig. 4A). In vivo, the moderate affinity of the
InlA/E-cadherin interaction may be compensated for by a
larger number of molecules being involved in binding, as
both InlA and E-cadherin are present in high local copy
numbers. The Ca2+ dependence of the InlA/E-cadherin interaction also provides a rationale of how the bacterium can be
released into the cytosol after uptake. Once inside the cell,
the strength of the InlA/E-cadherin complex is diminished
due to lower intracellular Ca2+ levels, liberating the bacterium from attachment with the host membrane.
2.3. InlB and its receptors
InlB presently is the only internalin crystallized in its
entirety [14]. It is an elongated molecule with the cap–
LRR–IR domain at one end and the GW domains at the other
(Fig. 3). The B-repeat, which is poorly ordered in the crystal
and could, therefore, not be modeled, spans a distance of
50 Å, linking the remaining domains. Recombinant protein
comprising only the cap and the LRR is sufficient to promote
uptake of latex beads into cells [21,22]. Uptake is accompanied by stimulation of PI3-kinase and rearrangements of the
actin cytoskeleton, also known as membrane ruffling [21].
Purified full-length InlB additionally leads to the tyrosine
phosphorylation of host cell proteins [23]. These signaling
events are also observed following binding of growth factors
to their receptors [24]. Subsequently, the tyrosine kinase
c-Met, the receptor for hepatocyte growth factor (HGF), was
identified as InlB receptor on mammalian cells [25]. c-Met is
expressed on both epithelial and endothelial cells. Upon
HGF-binding, c-Met is activated by autophosphorylation of
its cytoplasmic tyrosine kinase domain.
c-Met is a disulfide-linked heterodimer with an extracellular a-chain and a transmembrane b-chain. While largely
extracellular, the b-chain bears the cytoplasmic tyrosine kinase domain at its C-terminus [26]. Ligand-binding is
thought to cause dimerization of the c-Met extracellular
domain, leading to autophosphorylation of the cytoplasmic
domain. Structural information is currently not available for
c-Met. Likewise, the binding sites on c-Met for InlB and for
its natural ligand, HGF, are unknown, but they appear to be
non-overlapping, as the ligands do not compete with each
other in a bioassay [25].
The c-Met-binding determinants of InlB are well characterized. A fragment of cap and LRR is sufficient for c-Met
activation [25], and the c-Met-binding site on InlB has been
mapped to the concave face of the LRR, where surfaceexposed aromatic residues play a crucial role in receptorbinding (inset in Fig. 3) [14,22]. The InlB′/c-Met interaction
is strong, with a dissociation constant in the nanomolar range
measured in vitro using purified components [22]. In cultured cells, soluble, full-length InlB activates c-Met in a
concentration range similar to that of HGF (0.15 nM or
higher). While the cap-LRR region alone is both necessary
and sufficient for activation of c-Met, the presence of the GW
domains is required to achieve full activation at concentrations below 1.5 nM [25].
Structurally, the three C-terminal GW domains are related
to SH3 domains, but the binding site for proline-rich ligands
of true SH3 domains is obstructed [14]. The GW domains
mediate non-covalent attachment of InlB to the cell wall [27].
H.H. Niemann et al. / Microbes and Infection 6 (2004) 101–112
InlB reversibly associates with the bacterial surface by binding to lipoteichoic acid (LTA; [28]). The interaction between
LTA and the GW domain is at least partly a result of charge
complementarity between the positively charged GW domains (isoelectric point of ~10) and LTA.
Two further host cell receptors have been described for
InlB in addition to c-Met: gC1q-R [29] and the heparan
sulfate proteoglycans or heparin [30]. Both gC1q-R and
HPSGs interact with the GW domains. gC1q-R, a receptor
for the complement component C1q, is a multifunctional and
multicompartmental protein [31] forming a doughnutshaped trimer, one side of which is negatively charged [32].
The exact role of gC1q-R in InlB-mediated uptake of bacteria
is currently unclear. gC1q-R competes with LTA for InlBbinding, and the addition of gC1q-R to bacteria leads to the
release of InlB from the cell surface into the medium [14].
The same effect is seen with heparin [30]. Consequently,
heparin competes for InlB-binding with LTA and qC1q-R
[14]. Like LTA and gC1q-R, heparin is negatively charged
under physiological conditions, explaining in part the interaction with the GW domains. The in vivo relevance of glycosaminoglycans is demonstrated by the fact that L. monocytogenes invades cells deficient in glycosaminoglycan
synthesis by an order of magnitude less efficiently than
wild-type cells [30]. The heparin and c-Met receptors may
thus act synergistically, closely resembling HGF, which also
interacts with both receptors.
Recently, the existence of a fourth, as yet unidentified,
receptor has been postulated. A construct containing the
cap–LRR–IR domain and the single B repeat unit elicits
activation of c-Met similarly to HGF but leads to a stronger
activation of the downstream MAP kinase pathway [33]. This
superactivation of the MAP kinase pathway is not observed
with a shorter construct lacking the B repeat unit. There may
thus be an alternative pathway for MAP activation, using an
unknown receptor that might be recognized by the B repeat
of InlB. While much more is known about downstream signaling for InlB than InlA, the structural basis for the interaction of InlB with its multiple receptors remains elusive.
3. Structures of other bacterial adhesins and invasins
3.1. Yersinia pseudotuberculosis: invasin
Y. pseudotuberculosis is a Gram-negative enteropathogenic bacterium causing gastroenteritis in humans. Y. pseudotuberculosis crosses the intestinal epithelium by translocating across M cells to enter Peyer’s patches. Uptake into M
cells requires invasin, a chromosomally encoded outer membrane protein (Omp) [34]. Invasin binds to members of the b1
integrin family [35] with higher affinity than their natural
ligands and induces formation of pseudopods that envelop
the bacteria. The N-terminal part (about 500 out of the
986 amino acid residues of invasin) anchors the protein in the
outer membrane and is believed to form a b-barrel. The
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crystal structure of a fragment comprising the C-terminal
497 amino acids shows an elongated, rod-like structure consisting of five tandem domains (Fig. 4B) [36]. Structurally,
the four N-terminal domains (termed D1–D4) belong to the
Ig superfamily. The C-terminal D5 domain adopts the fold of
C-type lectins that typically bind carbohydrate or polypeptide ligands [37]. Invasin lacks the binding site for a Ca2+ that
is involved in carbohydrate recognition in many C-type lectins, and may therefore, favor a polypeptide over a sugar
ligand. The 192 C-terminal amino acids of invasin are sufficient for integrin-binding [38] and to induce bacterial uptake
by mammalian cells [39]. Domains D4 and D5 comprise this
C-terminal integrin-binding fragment. Physiologically, integrins bind two domains of fibronectin, the fibronectin type III
(FnIII) repeats 9 and 10. The primary integrin-binding determinant of fibronectin, the RGD motif [40], is located in
domain 10, while domain 9 contains the ‘synergy region’,
which supports integrin-binding [41,42]. Structurally, domains D4 and D5 of invasin appear to mimic FnIII repeats
9 and 10. Asp911 located in domain D5 (Fig. 4B) is crucial
for bacterial uptake [43] and may mimic the conserved Asp
from the RGD motif in repeat 10 of FnIII. Residues centered
around another aspartate in domain D4 of invasin are also
crucial for integrin-binding [44]. They may correspond to
residues from the synergy region in FnIII repeat 9. The
surface area buried between domains D4 and D5 of invasin is
large, rigidly linking them through extensive interactions.
Domains D4 and D5 hence form an integrin-binding superdomain. This contrasts with the situation found for FnIII
repeats 9 and 10. Their interface has little buried surface,
suggesting considerable interdomain flexibility [45]. The
rigid arrangement of invasin domains D4 and D5 allows for
the presentation of residues from both domains competent
for integrin-binding, possibly explaining the higher affinity
for integrins when compared with the more flexible fibronectin.
3.2. EPEC: the intimin/Tir complex
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are human pathogens responsible
for high morbidity and mortality of infants in developing
countries. The extracellular pathogens intimately adhere to
the host intestinal epithelium, inducing the formation of
actin-rich pedestals, on which they reside [46]. Pedestal
formation requires intimin, a chromosomally encoded adhesin of EPEC and EHEC [47,48], and its receptor, translocated intimin receptor (Tir; [49]). Tir is an effector molecule
of the bacterial type III secretion system, which upon translocation into the host cytosol and phosphorylation, is inserted
into the host cell membrane. Tir has two transmembrane
helices, with the intervening sequence forming the extracellular intimin-binding domain. The Tir N- and C-termini are
located in the cytoplasm of the host cell and are involved in
signaling to the actin cytoskeleton. Intimin is related to invasin (see above), both in terms of sequence and structure. The
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H.H. Niemann et al. / Microbes and Infection 6 (2004) 101–112
N-terminal membrane anchor domain of intimin shares more
than 30% sequence identity with the corresponding region of
invasin. Structures were determined of a 280-amino-acid
fragment of the extracellular domain of intimin alone and in
complex with the intimin-binding domain of Tir [50,51].
Similarly to invasin, the structure shows two Ig-like domains
and a C-terminal C-type lectin domain (Fig. 4B). Again, the
most distal Ig domain and the C-type lectin domain (D3 and
D4 in intimin) form a rigid superdomain binding Tir [52]. A
third Ig-like domain (D1) is presumably positioned between
the membrane anchor and the described 280-amino-acid
fragment. Tir interacts directly with the C-type lectin domain
of intimin. A short a-helix involved in Tir-binding is not
present in the related C-type lectin domain of invasin. The
extracellular domain of Tir forms two antiparallel a-helices
connected by a b-hairpin. Intimin-binding by Tir involves
mainly residues from this b-hairpin. In the crystal structure,
the intimin-binding domain of Tir is a dimer, with its long,
roughly parallel a-helices forming a four-helix bundle [51].
The large surface area buried in the interface suggests this
dimerization to be physiologically relevant. The intimin rod
would, as a result, lie alongside the bacterial membrane
rather than pointing away from it. This is plausible, as the
intimin/Tir complex (lacking one intimin Ig-like domain) has
a combined length of more than 140 Å, while the gap between intimately attached EPEC and the host cell is only
100 Å.
3.3. Bordetella pertussis: pertactin
B. pertussis, the causative agent of whooping cough, uses
several proteins to adhere to ciliated epithelial cells in the
human respiratory tract [53]. The surface protein pertactin
(also called P.69 because of its apparent molecular weight) is
one of the factors involved in adhesion [54]. Pertactin belongs to the family of autotransporter proteins, many of
which are virulence factors [55]. Autotransporter proteins of
Gram-negative bacteria have a signal peptide for translocation across the inner membrane, a specialized C-terminal
domain, which forms a pore in the outer membrane, and a
passenger domain that is transported across the membrane,
forming the extracellular part of the mature protein. Translated as a 93-kDa precursor protein, pertactin is cleaved to
yield the P.69 extracellular domain that remains associated
with the P.30 C-terminal fragment embedded in the outer
membrane [56].
Emsley et al. [57] have determined the crystal structure of
P.69 pertactin. Pertactin is an elongated molecule forming a
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16-stranded parallel b-helix (Fig. 4B). Much like the LRR
domain of internalins, this fold is made of tandem units
containing b-strands that stack to form the parallel b-sheet.
While the LRR domain has a single parallel b-sheet, there are
three parallel sheets in pertactin, leading to a triangular
molecular cross-section. Typically, a repeat unit (13 residues)
contributes a single strand to each sheet. The repetitive fold is
not reflected by a correspondingly repetitive sequence. Nevertheless, ladders of aliphatic residues are formed inside the
b-helix formed by side chains from residues in equivalently
positioned residues. An N-terminal cap helix is missing in
pertactin, although it is present in many proteins forming
b-helices [58].
Pertactin has an RGD motif in a loop region protruding
from the b-helix, but no receptor has been identified for
pertactin so far. The role of pertactin in pathogenesis, adhesion and invasion is still controversial [54,59–61]. The molecular mechanism of pertactin-mediated adhesion and the
importance of the RGD motif are not clear either [54,59].
3.4. Adhesins of type 1 and P pili bound to their receptors
Uropathogenic E. coli (UPEC), the main cause of urinary
tract infections, can adhere to and invade the uroepithelial
cells of bladder and kidney [62]. The adhesive structures of
UPEC include, among others, type 1 and P pili. Pili are long
(up to over 1 µm), hair-like structures sticking out from the
bacterial surface. Type 1 and P pili share a similar architecture. They are formed by a helical assembly of pilin subunits
forming a thick, 60–70 Å wide, proximal rod. At the distal
end, there is a thinner fibrillum (20–30 Å) with the adhesin
sitting at its tip [63]. Formation of pili is structurally well
characterized and has been reviewed elsewhere [64].
FimH, the adhesin at the tip of type 1 pili, binds to
mono-mannose and mannose oligosaccharides and is required for colonization of the bladder [65,66]. FimH consists
of two domains connected by a short linker (Fig. 4B) [67].
The C-terminal Ig-like pilin domain, details of which will not
be discussed here, is responsible for anchoring the adhesin to
the pilus. The N-terminal sugar-binding receptor domain is
an elongated 11-stranded b-barrel without any significant
structural homologues. Mono-mannose binds at the tip of the
receptor-binding domain into a pocket that is shaped by
residues invariant among more than 200 UPEC isolates but
not conserved in EHEC, explaining tissue tropism [68].
P pili mediate attachment to the uroepithelium in the
kidney. PapG is the adhesin at the tip of P pili, and it binds the
Fig. 4. Three-dimensional structures of bacterial adhesins and invasins that have been determined so far. All ribbon diagrams are drawn to scale. Domains not
resolved in the structure are shown schematically as ovals or cylinders. Color coding is as follows: host cell receptors in orange, and Ig-like domains not involved
in receptor-binding and the b-barrels of Omps in green. Within receptor-binding domains, b-strands are dark blue and helices red. The cap domains of InlA and
InlB are in turquoise, the extracellular parts of the Omps possibly involved in receptor-binding are shown in violet. The individual structures are described in the
text. (A) Invasins of Gram-positive bacteria. For InlA/hEC1, the orange arrow indicates the position of the host cell. For the large fibronectin-binding protein and
fibronectin, only the parts resolved in the NMR structure are shown, other domains are omitted. (B) Invasins and adhesins of Gram-negative bacteria. Side chains
of the following important residues are shown in ball-and-stick representation: Asp911 and residues from D4 of invasin; the RGD motif of pertactin; basic
residues of OpcA potentially involved in heparin-binding (blue); aromatic girdles of Omps (pink).
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glycolipid receptor globoside (GbO4), consisting of a tetrasaccharide linked to ceramide [69]. A two-domain structure similar to that of FimH with an N-terminal receptorbinding domain and a C-terminal pilin domain was
anticipated for PapG. The structure of the N-terminal
receptor-binding domain of PapG alone and bound to GbO4
(Fig. 4B) [70,71] shows that the receptor-binding domain of
PapG is an elongated eight-stranded b-sandwich that can be
divided into an upper and a lower layer. There is no significant homology to other structures, but the topology of the
lower layer is similar to that of the corresponding part in
FimH. In PapG, the receptor-binding domain is considerably
larger than in FimH, and the substrate-binding site, formed
by the upper layer, is positioned along the long axis of the
molecule, not at the tip as observed for FimH.
3.5. Fibronectin-binding protein bound to fibronectin
The Gram-positive bacteria Staphylococcus aureus and
Streptococcus pyogenes are important human pathogens,
utilizing, among other adhesins, cell-wall-anchored
fibronectin-binding proteins for adhesion and invasion
[72–75]. These target the N-terminal fibronectin domain containing five tandem fibronectin type I (FnI) repeats. The
bacterial fibronectin-binding proteins themselves contain
multiple unstructured fibronectin-binding repeats. The solution structure of a peptide from a fibronectin-binding protein
of Streptococcus dysgalactiae in complex with two FnI repeats reveals a striking mode of interaction [76]: each FnI
repeat forms a b-sandwich structure consisting of two- and
three-stranded b-sheets. The bacterial peptide (36 amino
acids) laterally extends both three-stranded b-sheets by contributing a fourth antiparallel strand forming a tandem
b-zipper interaction (Fig. 4A). Sequence analysis showed
that the fibronectin-binding proteins of S. aureus and S.
pyogenes contain several C-terminal repeats, each with putative binding motifs for four to five consecutive FnI repeats.
The expected interaction was verified by isothermal titration
calorimetry using bacterial peptides containing the predicted
fibronectin-binding motifs and constructs consisting of two
or five FnI repeats [76]. This implies that fibronectin-binding
proteins contain tandemly arranged high-affinity fibronectinbinding sites that change from an unstructured to a b-strand
conformation upon binding to FnI modules.
3.6. Outer membrane proteins from E. coli and Neisseria
meningitidis
Crossing the blood–brain barrier by E. coli is the leading
cause of meningitis in neonates [77]. The E. coli outer
membrane protein OmpA contributes to invasion of brain
microvascular endothelial cells, which constitute the blood–
brain barrier [78] by causing actin condensation [79]. OmpA
binds Ecgp, a 96-kDa glycoprotein [80] present on the cell
surface [81]. For OmpX, another Omp from E. coli, no
adhesive phenotype was observed upon deletion so far [82].
However, several homologues of OmpX in other Gramnegative bacteria are adhesins. Overexpression of Enterobacter cloacae OmpX increases invasion of rabbit ileal tissue
[83]. A role in invasion was also shown for the OmpX
homologue Ail from Yersinia enterocolitica [84].
The crystal structures of OmpA [85] and OmpX [86] show
that both are integral membrane proteins comprising eightstranded antiparallel b-barrels (Fig. 4B). On the outside,
mainly aliphatic hydrophobic residues interacting with the
membrane form a central ribbon flanked by girdles of aromatic residues at the bottom and top of the barrel, which
position the barrel in the membrane. Inside the barrels, hydrophilic residues form an intricate network of hydrogen
bonds, and there are large water-filled cavities, but the proteins do not form distinct pores. The N- and C-termini lie on
the periplasmic side, and OmpA has an additional C-terminal
periplasmic domain of unknown structure. The b-strands are
linked by short turns at the periplasmic side, while long
flexible loops connect the individual b-strands on the extracellular side. In OmpX, two of the loops form an unusual
single-layer b-sheet made of four strands. This b-sheet is the
predicted site of receptor-binding by extending an existing
receptor b-sheet. An indication of this is seen in the crystal of
OmpX, where two OmpX molecules align with each other,
leading to an extended eight-stranded antiparallel sheet [86].
N. meningitidis can cause meningitis and septicemia. Adherence to and invasion of human epithelial and endothelial
cells by partially capsule-deficient N. meningitidis strains
lacking pili depend on the Omp OpcA [87]. OpcA binds the
serum protein vitronectin, which in turn binds integrins [88].
Heparan sulfate proteoglycans were identified as a second
OpcA receptor on epithelial cells [89]. OpcA forms an elongated b-barrel consisting of 10 strands with an ellipsoidal
cross-section (Fig. 4B) and bears features very similar to
those of OmpA and OmpX [90]. There are long protruding
loops on the extracellular side with contributions from
b-strands that extend above the predicted membrane surface.
The protruding loops form a continuous surface with a
groove containing basic residues that may be involved in
binding of heparan sulfate proteoglycans (Fig. 4B).
4. Conclusions
Although the structural database of bacterial adhesins and
invasins is still very limited in extent, a number of common
themes are nevertheless emerging. Most of the proteins involved in adhesion and invasion are anchored to the surface
of the bacteria. They are attached to the cell wall in Grampositive bacteria and positioned in the outer membrane by a
b-barrel domain in Gram-negative bacteria. InlB may be the
notable exception. It is not covalently attached to the bacterial cell wall and can act as a soluble molecule. Interactions
of receptors with the GW modules of InlB lead to release into
the medium. Consequently, the role of InlB as adhesin has
been put into question, and it was instead suggested that it
H.H. Niemann et al. / Microbes and Infection 6 (2004) 101–112
mimics a mammalian growth factor [30]. The receptorbinding domain of the adhesin or invasin is frequently presented at the tip of an elongated structure. This is especially
apparent in the case of pili, which are up to a few micrometers in length. The integrin-binding domain of invasin and the
Tir-binding domain of intimin reside at the distal end of a
rod-like structure, and the B-repeats of InlA can be envisaged
to elevate the E-cadherin-binding LRR domain above the
bacterial surface (Fig. 4A). InlB, again, seems to be an
exception. While also having an extended structure, the
C-terminal GW repeats are receptor-binding rather than
spacer domains, and even the central B-repeat seems to
activate signaling cascades in the host cell. Pertactin also
forms an elongated molecule, but the RGD motif suggested
to play a role in receptor-binding is positioned roughly in the
middle (Fig. 4B). However, no receptor is currently known
for pertactin, and the location of receptor-binding residues in
pertactin is likewise unclear. The receptor-binding sites of
the Omps are long loops extending above the outer bacterial
membrane. They partly exhibit considerable flexibility, causing them to be disordered in some crystal structures. The
Omps, however, do not have spacer domains, and therefore,
need to bind their receptors very close to the bacterial outer
membrane.
When looking at the protein folds utilized by the bacterial
adhesins and invasions, it becomes obvious that bacteria
reuse (or reinvent) domain structures well known from other
contexts. The Ig superfamily is one of the major families in
eukaryotes [91], and its members are also present in some
bacteria. In bacterial adhesins and invasins, Ig-like domains
are mostly used as spacer units. Proteins forming pili have
Ig-like folds and so has the domain responsible for anchoring
the receptor-binding domain of FimH or PapG to the pilus.
The Ig-like domains of InlA and InlB are similarly not
involved in receptor-binding but rather stabilize the preceding LRR domains through a common hydrophobic core.
Likewise, domains D1–D3 of invasin and D1–D2 of intimin
do not interact with the receptors. Only Ig-like domain D4 of
invasin and D3 of intimin participate in receptor-binding.
Both form a superdomain with the terminal C-type lectin
domain that provides a scaffold to bind oligosaccharides or
polypeptide moieties in a variety of proteins [37].
LRRs are found mainly in eukaryotic proteins with diverse cellular functions, and most if not all are engaged in
protein–protein interactions [20]. In bacteria, LRRcontaining proteins are mainly (if not exclusively) found in
pathogenic bacteria and often constitute virulence factors.
The binding mode of the InlA/hEC1 complex is reminiscent
of that between the eukaryotic proteins ribonuclease inhibitor, another horseshoe-shaped LRR protein, and its binding
partner, ribonuclease A [16,92]. The b-helix of pertactin is
structurally related to LRR proteins because it also has a
repetitive fold, with each repeat contributing a b-strand to a
parallel b-sheet. b-Helices are found in various other proteins, many of which are enzymes of bacterial origin [58].
The b-barrel, finally, is neither restricted to adhesins or inva-
109
sins but is a fold likely to be present in all integral membrane
proteins of the bacterial outer membrane [93]. Within the
scope of this review, proteins with a b-barrel include not only
OmpA, OmpX and OpcA but also invasin, intimin and pertactin.
Similar folds found in evolutionarily related adhesins and
invasins are adapted to bind different receptors. InlA binds
E-cadherin via its LRR domain, while the LRRs of the
related InlB bind to c-Met, however, with very different
affinities. It is notable that the affinity to its receptor is about
three orders of magnitude lower for the larger InlA than for
the smaller InlB. Invasin and intimin are also evolutionarily
and structurally related but again bind different receptors.
Invasin targets the host cell receptor integrin, while intimin
recognizes a bacterial protein (Tir) translocated “back” to the
host cell surface. Subversion of the host cell machinery starts
in the extracellular space for invasin-mediated signaling but
in the cytoplasm for intimin-triggered signals. The b-barrel
again represents a common structural scaffold used to bind a
variety of receptors in OmpA, OmpX and OpcA.
On the other hand, bacteria employ multiple ways to target
the same receptor. Both InlB and OpcA bind to heparan
sulfate proteoglycans. Integrins are the target of several invasins, bound either directly, e.g. in the case of invasin, or
indirectly, as in the cases of OpcA (binding to vitronectin) or
the fibronectin-binding proteins. While there is a clear case
of molecular mimicry for invasin resembling the integrinbinding site of fibronectin, the situation is different for the
internalins. InlB is unlikely to structurally mimic HGF. The
two c-Met ligands do not compete for binding and will most
likely have different binding sites. Likewise, InlA does not
structurally mimic the homophilic interactions between
E-cadherins forming adherence junctions.
The rich diversity of adhesive and invasive strategies developed by bacteria pose a multitude of seemingly unrelated
problems to cell biologists and medical professionals. However, understanding each mechanism at a structural level will
provide a possible key in combating a particular infection at a
very early stage. On the other hand, the precise knowledge of
the specific interactions between bacteria and host may allow
bacterial infection strategies to be adapted, providing tools
specifically targeting individual cells or tissues. Strategies
developed by the deadliest of bacteria may, therefore, become vehicles of choice in combating or preventing these
infections.
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