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Microbes and Infection 6 (2004) 101–112 www.elsevier.com/locate/micinf 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 102 H.H. Niemann et al. / Microbes and Infection 6 (2004) 101–112 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. 103 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). 104 H.H. Niemann et al. / Microbes and Infection 6 (2004) 101–112 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 105 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 106 H.H. Niemann et al. / Microbes and Infection 6 (2004) 101–112 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 107 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). 108 H.H. Niemann et al. / Microbes and Infection 6 (2004) 101–112 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. 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