Vitronectin in bacterial pathogenesis: a host protein used in complement escape and cellular invasion


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The multifunctional human glycoprotein vitronectin (Vn) plays a significant role in cell migration, tissue repair and regulation of membrane attack complex (MAC) formation. It also promotes neutrophil infiltration and, thus, enhances the inflammatory process during infection. In the host, a balanced homeostasis is maintained by Vn due to neutralization of the self-reactivity of the MAC. On the other hand, Vn bound to the bacterial surface protects from MAC-mediated lysis and enhances adhesion. Gram-negative bacterial pathogens including Moraxella catarrhalis, Haemophilus influenzae and Neisseria gonorrhoeae use Vn recruitment to prevent MAC deposition at their surface. Moreover, Gram-positive bacterial pathogens such as Streptococcus pneumoniae and S. pyogenes utilize Vn for effective adhesion to host cells and subsequent internalization. Vitronectin has an Arg–Gly–Asp (RGD) sequence for binding the host cell integrin receptors and a separate bacterial-binding domain for pathogens, and thus more likely functions to cross-link bacteria and epithelial cells. Once bacteria are attached to the vitronectin–integrin complex, various host cell-signalling events are activated and promote internalization. In this review, we focus on the important roles of vitronectin in bacterial pathogenesis and describe different strategies used by pathogens to evade the host response by the help of this intriguing molecule.


Vitronectin (Vn) was discovered in 1967 and initially called S-protein, but was later renamed by Hayman et al. (1983). It is an important component of the human extracellular matrix (ECM), and is synthesized in the liver and secreted into plasma (Preissner and Seiffert, 1998). The N-terminal part of Vn (43 amino acids) consists of a somatomedin-B (SMB) domain followed by a cell receptor binding site characterized by an Arg–Gly–Asp (RGD) sequence (Lossner et al., 2009) (Fig. 1A). The protein has four haemopexin-like domains that are predicted as putative haem-binding motifs with unknown function. In addition, Vn has three heparin-binding domains (HBD), spanning residues Vn82–137 (HBD-1), Vn175–219 (HBD-2) and Vn348–361 (HBD-3) (Liang et al., 1997). Most of the circulating Vn in blood is a monomer (65 and 75 kDa), whereas the extravascular cell-bound Vn is a multimer (Peterson, 1998). Vn is found at a high concentration in plasma (200–700 µg ml−1) (Boyd et al., 1993; Chauhan and Moore, 2006), and is also present in different human tissues. Particularly high amounts are observed in liver, tonsil, duodenum, heart, skeletal muscle and lung tissues (Fig. 1B–J), as well as in some malignant carcinomas (Berglund et al., 2008).

Figure 1.

Domain arrangement of the Vn molecule and its availability at different tissues.
A. Schematic model of the various domains found in Vn. The N-terminal somatomedin B (SMB) domain (comprising 43 amino acids) binds to uPAR and is inhibited by plasminogen activator inhibitor. An RGD motif is located after the SMB domain that interacts with cellular integrin receptors. Vn has three heparin-binding domains (HBD). The haemopexin-like domains are predicted as putative haem-binding motifs ( The C-terminal region of Vn (aa 312–396) has a PE- and UspA2-binding region including HBD-3. S. pneumoniae binds to HBD-3 whereas, N. meningitidis Opc binds to N-terminal sulphated Y56 and Y59 residues and also to C-terminal HBD-3.
B–J. Vn is present at moderate to high levels in various tissues. Tissue sections probed with anti-vitronectin antibodies using immunohistochemistry. Dark brown area (also indicated by arrows) shows the presence of Vn and blue area shows the cell nuclei. The figures were obtained from

Vitronectin plays a crucial role in many biological processes including cell migration, adhesion and angiogenesis (Preissner and Seiffert, 1998). The interaction of Vn with the urokinase plasminogen activator–urokinase plasminogen activator receptor (uPA–uPAR) complex and integrin receptors is a part of the plasminogen activation system involved in old tissue degradation (pericellular proteolysis), reorganization and wound healing. Hence the uPAR–Vn interaction is a key determinant in homeostatic processes (D'Mello et al., 2009; Smith and Marshall, 2010). Several malignant cells also use Vn for migration and, thus, blocking the Vn interaction in these cases might be therapeutic for cancer treatment (Smith and Marshall, 2010).

Vitronectin is involved in regulation of the terminal pathway of complement activation to limit the self-reactivity of the innate immune response. The role of Vn has been studied in bacterial pathogenesis during the last three decades. Recent findings suggest that many bacterial species interact with Vn, but the functionality of these interactions in pathogenesis has not been fully elucidated. In this review, we discuss the important role of Vn in bacterial serum resistance, adhesion and internalization mediated by host cell signalling.

Bacteria benefit the complement regulatory property of vitronectin

The complement system serves as the first line of the human defence system and consists of 35 different fluid phase and membrane-bound proteins. Exposure of pathogens to the complement system initiates three different pathways: (i) the classical pathway, activated by bacterial LPS, nucleic acids and the antigen–antibody complex, (ii) the lectin mediated pathway, activated by binding of mannose-binding lectin to mannose residues on the bacterial surface, and (iii) the alternative pathway, activated by deposition of C3b after recognizing the bacterial surface and LPS (Blom et al., 2009). These three pathways lead to a common lytic pathway with the formation of C5 convertase that, in turn, assembles the membrane attack complex (MAC) consisting of components C5b6 to C9 (Fig. 2). The complement system thus either tags the pathogens to be killed by phagocytic cells or direct lysis by the MAC. Gram-negative bacteria are killed more effectively by MAC than Gram-positive bacteria because the latter have a thick outer layer of peptidoglycan (Moffitt and Frank, 1994).

Figure 2.

Role of Vn as a regulatory molecule for the complement system. The main complement cascades can be divided into classical, alternative or lectin-mediated pathways. These pathways leads to a common lytic pathway after activation of complement 5 (C5).
A. Complement pathways are controlled by different fluid phase regulators. C4b-binding protein (C4BP) is a fluid-phase regulator that inhibits the formation and accelerates the decay of C3 convertase (C4bC2a). It also serves as a cofactor to factor I in the proteolytic degradation of C4b and C3b. Thus C4BP bound to bacterial surface inhibits the classical and lectin-mediated pathways.
B. Recruitment of factor H (FH) and factor H-like protein (FHL) at the bacterial surface leads to the C3b inactivation and thus inhibits subsequent complement activation.
C. The lytic pathway initiates assembly of the membrane attack complex (MAC). Vn is known to inhibit C5b-7 complex formation and C9 polymerization. Thus, Vn deposited on the bacterial surface inhibits the MAC formation and protects the bacteria from MAC-mediated lysis.

To avoid an excessive response and subsequent self-damage to host tissues, the complement system is tightly regulated by soluble and membrane-bound proteins, such as factor-I, factor H, C4b-binding protein (C4BP), Vn and clusterin (Carroll, 2004; Zipfel and Skerka, 2009). The pathogens Klebsiella pneumoniae, Staphylococcus aureus and Moraxella catarrhalis can directly inhibit or subvert the initiation of the complement cascade by binding to early complement components including C1q and C3 (Albertíet al., 1993; Nordstrom et al., 2005; Hair et al., 2010). Furthermore, several bacteria including Neisseria gonorrhoeae, N. meningitidis, Pseudomonas aeruginosa, Streptococcus pyogenes, S. pneumoniae, Haemophilus influenzae and M. catarrhalis utilize the fluid phase regulator C4BP that regulates the classical complement pathway and inhibits the formation and accelerates the decay of the C3 convertase (C4bC2a). Indirectly, C4BP also acts as a regulator of the alternative pathway by interfering with C3 deposition at the bacterial surface (Blom et al., 2009) (Fig. 2A). Factor H and factor H-like-1 (FHL-1) proteins are also recruited by N. meningitidis, H. influenzae, P. aeruginosa and Borrelia burgdorferi to inhibit initiation of the alternative complement pathway at their surface (Fig. 2B) (Blom et al., 2009; Zipfel and Skerka, 2009).

Vitronectin binds directly to complement proteins and modulates their functions (Fig. 2C). The mechanism of binding and mode of regulation are not fully understood. However, it was postulated that Vn interacts with the C5b-7 complex and, thereby, occupies the meta-stable membrane binding site of the nascent precursor complex C5b-7, so that the newly formed Vn–C5b-7 complex is unable to be inserted into the cell membrane (Milis et al., 1993). The binding affinity and mechanism of Vn to C5b-7 complex are not exactly known but non-heparin-binding domains of Vn are involved. Binding to C9 and inhibition of the terminal complement complex is the best described mechanism of complement regulation controlled by Vn. It has been shown that heparin competes with C9 to bind Vn while HBD(s) of Vn was suggested to interact with C9 (Milis et al., 1993; Sheehan et al., 1995; Attia et al., 2006). Vn therefore contains distinct binding sites for the C5b-7 complex and C9. The effectiveness in MAC inhibition is, however, achieved by C9 inhibition, and Vn recruitment at the bacterial surface effectively inhibits lytic pore formation by C9. Nevertheless, the molecular insight of the Vn interaction with complement components is still unknown due to the lack of ultrastructural data.

Vitronectin-dependent serum resistance has been well studied for many pathogens (Table 1). The respiratory and invasive pathogen encapsulated H. influenzae type b (Hib) binds Vn by the trimeric autotransporter Haemophilus surface fibrils (Hsf) and, hence, acquires serum resistance (Hallström et al., 2006). Hsf is a multifunctional autotransporter/adhesin and adheres to an unknown host cell receptor via two well-defined binding sites. Bacterial autotransporters in general have a ‘lolly-pop’-like structure and comprise an N-terminal signal peptide, an internal passenger domain and finally a C-terminal translocator domain (Cotter et al., 2005a,b). Hsf mutants are significantly serum sensitive and have a decreased Vn binding, which can effectively be inhibited by heparin suggesting that the C-terminal heparin binding regions of Vn are potentially involved (Hallström et al., 2006). Interestingly, Hsf has two distinct Vn binding regions located at amino acid 608–1351 and 1536–2414. We recently also demonstrated that the adhesin protein E (PE), which exists in both non-typable H. influenzae (NTHi) and Hib (Singh et al., 2010a), provides serum resistance for NTHi by binding Vn (Hallström et al., 2009). PE is a high-affinity Vn-binding protein (KD =  4.0 × 10−7 M) and binds Vn equally well both in vitro and in vivo. A peptide mapping approach suggested that PE84–108 binds to Vn, and that the HBDs of Vn are involved in this interaction. Most importantly Vn bound to the surface of NTHi is functionally active and effectively inhibits MAC formation. Further detailed characterization of the PE–Vn interaction has shown that K85 and R86 of PE selectively interact with the C-terminal 312–396 amino acid region of Vn and contribute to serum resistance (B. Singh et al., unpubl. data).

Table 1.  Bacterial pathogens and interactions with vitronectin regarding serum resistance and adhesion.
Name of pathogenProtein/system interaction with vitronectinRole in serum resistanceRole in adhesionReferences
  1. +, positive role in mechanism; −, no direct role reported; (−), not reported/unknown.

Gram-negative bacteria
Haemophilus influenzae type b (Hib)Haemophilus surface fibrils (Hsf)++Cotter et al. (2005a); Hallström et al. (2006)
Non-typable H. influenzae (NTHi)Protein E (PE)++Ronander et al. (2009); Hallström et al. (2009)
H. ducreyiDucreyi serum resistance protein A (DsrA)+(−)Leduc et al. (2009)
Moraxella catarrhalisUbiquitous surface protein A2 (UspA2)++Attia et al. (2006); Singh et al. (2010b)
Pseudomonas aeruginosaComplement regulator-acquiring surface proteins (CRASP)-2++Schiller and Joiner (1986); Chhatwal et al. (1987); Leroy-Dudal et al. (2004); Hallström et al. (2010)
Escherichia coliUnknown+Chhatwal et al. (1987); Shen et al. (1995)
Yersinia pseudotuberculosisUnknown(−)+Gustavsson et al. (2002)
Neisseria meningitidisOpacity protein (Opc)++Virji et al. (1994); Carbonnelle et al. (2009); Griffiths et al. (2009); Sa E Chunha et al. (2010)
N. gonorrhoeaeOpacity-associated outer membrane protein (Opa)++Arko et al. (1991); Duensing and Putten (1998); Dehio et al. (1998)
Chlamydia trachomatisUnknown(−)+Kihlstrom et al. (1992)
Clostridium difficileFibronectin-binding protein (Fbp), surface-layer protein (SLP) A(−)+Calabi et al. (2002); Hennequin et al. (2003)
Helicobacter pyloriUnknown+Ringnér et al. (1992; 1994); Chmiela et al. (1997); Dubreuil et al. (2002)
Porphyromonas gingivalisFimbriae
Gingipain enzymes
+Nakamura et al. (1999); Nakagawa et al. (2005); McAlister et al. (2009)
Gram-positive bacteria
Streptococcus pyogenesUnknown(−)+Kostrzynska et al. (1992); Liang et al. (1997)
S. pneumoniaeUnknown(−)+Eberhard and Ullberg (2002); Bergmann et al. (2009)
S. bovisUnknown(−)+Styriak et al. (1999a)
S. suisUnknown(−)+Styriak et al. (1999b); Esgleas et al. (2005)
S. dysgalactiaeunknown(−)+Filippsen (1999)
Staphylococcus epidermidisAutolysin (Aae)+Li et al. (2001); Heilmann et al. (2003)
Staph. aureusUnknown(−)+Chhatwal et al. (1987); Liang et al. (1993); Styriak et al. (1999b)
Enterococcus faecalisUnknown(−)+Zareba et al. (1997); Tyriak and Ljungh. (2003)

The urogenital pathogen, Haemophilus ducreyi is resistant to serum and ducreyi serum resistance protein A (DsrA) plays an important role in this context as proven by dsrA mutants (Elkins et al., 2000). DsrA is a trimeric autotransporter and abundantly expressed on the bacterial cell surface. In a recent study, truncation of DsrA suggested that the C-terminal region of the passenger domain binds to fibronectin (Fn) and Vn, and hence contributes to serum resistance (Leduc et al., 2009). DsrA also inhibits deposition of serum bactericidal IgM on the surface of H. ducreyi to prevent serum-mediated killing of bacteria. Inhibition of IgM deposition by DsrA was found to be more effective than Vn-meditated serum resistance. Importantly, H. ducreyiΔdsrA mutants are also unable to infect human volunteers, suggesting that the Vn-mediated serum resistance contributed to bacterial virulence (Bong et al., 2001).

Vitronectin-mediated serum resistance is very effectively utilized by M. catarrhalis through ubiquitous surface protein (Usp) A2 that directly interacts with Vn. The UspA2-dependent Vn interaction leads to inhibition of C9 polymerization and MAC deposition. In contrast, M. catarrhalisΔuspA2 mutants are serum sensitive and high amounts of C9 are deposited at the surface (Attia et al., 2006). In relation to these findings, we recently reported that UspA2 is a high-affinity Vn-binding protein (KD = 2.3 × 10−8 M), and that UspA2 amino acids 30–177 are involved in this interaction (Singh et al., 2010b). UspA2 exists as a surface fibril-like structure in which amino acids 30–177 of the head region bind Vn. Moreover, the Vn amino acids 312–396 region forms the UspA2-binding domain. Pre-treatment of normal human serum (NHS) with UspA2 quenches Vn and, thus, M. catarrhalis becomes significantly more serum sensitive. These findings confirmed that Vn is a crucial factor in serum resistance of M. catarrhalis (Singh et al., 2010b). In addition, UspAs also interact with other ECM proteins such as Fn and laminin. However, these interactions only contribute to M. catarrhalis adherence to host tissues (Tan et al., 2005; 2006).

Pseudomonas aeruginosa serum resistance has been directly inferred by the deposition of C9 at the bacterial surface, i.e. significantly less C9 was deposited on serum resistant strains as compared with serum sensitive strains. Both types of strains activate complement cascades equally; the remarkable property of resistant strains is the reduced insertion of C5b-9 in the membrane and hence results in increased survival (Schiller and Joiner, 1986). In a more recent report, P. aeruginosa complement regulator-acquiring surface proteins (CRASP-2) showed direct binding to Vn heparin-binding domains and inhibited the complement mediated lysis (Hallström et al., 2010).

Neisseria meningitidis expresses the Opacity protein (Opc) and Opacity-associated outer membrane protein (Opa) that function as major adhesins, interact with Vn and contribute to serum resistance (Griffiths et al., 2009; Sa E Cunha et al., 2010). Vn directly binds to N. gonorrhoeae and agglutinates the bacteria. In an interesting study, the N. gonorrhoeae strains causing only disseminated gonococcal infection (DGI) were able to bind Vn, whereas CSF, blood and septic joint isolates were unable to interact with Vn. The majority of the DGI isolates (= 35; 92%) were highly serum resistant together with their Vn-binding capacity in comparison with septic joint and CSF isolates. Intriguingly, Vn binding was suggested as the major factor in survival and serum resistance of DGI isolates of N. gonorrhoeae (Arko et al., 1991).

Recently Vn has been described to be involved in the induction of host inflammatory responses by binding to bacterial lipopeptides (BLP) and involvement of Toll-like receptor 2 (TLR2) (Gerold et al., 2008). Vn acts as a direct receptor for BLP and functions as a bridging molecule between bacteria (BLP-mediated)–integrin β3 complex, and the lipid moiety of BLP is involved in Vn binding. In Glanzmann's thrombasthenia, which is related to the lack of integrins, BLP-mediated activation of monocytes could not be observed. This observation strongly suggested the involvement of β3 integrin in BLP-mediated activation of monocytes. Finally, it was proposed that TLR2 binds to integrin β3 and makes a TLR2–β3 complex that dissociates after stimulation with BLP (Gerold et al., 2008). The inflammatory response initiated by the BLP–Vn–integrin complex has been proven to be beneficial to bacterial colonization and subsequent infection.

Vitronectin mediates bacterial adherence

Adhesion of pathogens to host cells is a first crucial step in colonization that subsequently leads to infection. Attachment is mainly mediated by pili and several other surface-exposed membrane proteins, designated as adhesins (Boyle and Finlay, 2003), which recognize host cell surface receptors. ECM proteins are often used by pathogens for adherence, but under normal physiological conditions the ECM is not freely exposed (Pizarro-Cerdá and Cossart, 2006). However, after tissue damage due to bacterial/viral infection, proteolytic activities of toxins or mechanical injury, the pathogen may gain access to the ECM (Fig. 3A). On the other hand, ECM proteins are also components of plasma, circulate in various forms and play an important role in physiology, and invasive blood stream pathogens thus have better access to the ECM proteins for their survival and successful colonization. Vn significantly facilitates adhesion of both Gram-positive and Gram-negative bacteria to host cells (Table 1). Importantly, Vn has distinct binding sites for pathogens and epithelial cells, and most likely functions as a bridge between bacteria and epithelial cells that, for some species, promotes internalization (Fig. 3B). It has been reported that Vn bound to Eschericiha coli, Staph. aureus and S. pneumoniae provides a more efficient bacterial adhesion to epithelial cells (Chhatwal et al., 1987). Staph. aureus exhibits high-affinity binding to Vn, which helps in adhesion during colonization (Kostrzynska et al., 1992; Liang et al., 1993). Moreover, other Streptococcous spp. are also known to bind Vn (Esgleas et al., 2005). Vn potentiates the adherence of pathogens not only to host cells but also to synthetic materials. For example, it increases adherence of Staphylococcus epidermidis to polyvinylchloride (PVC) catheters (Lundberg et al., 1997). The major Vn-binding protein of Staph. epidermidis was identified as an autolysin (Aae) (Li et al., 2001). In parallel, experiments with immobilized Vn on a glass surface, a several-fold decreased attachment of PE-deficient NTHi mutants was observed in comparison with wild-type NTHi (Hallström et al., 2009).

Figure 3.

Localization of ECM proteins in tissues and contribution to bacterial internalization (an example model).
A. Epithelial cells secrete ECM components to synthesize basal lamina that function as a support and anchorage platform for cells. Cells are connected to each other by the tight junctions, adherent junctions, desmosomes and other protein–protein interactions that join together and seal the cell boundaries. Integrins are clustered at the basolateral and basal surface of polarized epithelial cells where they make hemidesmosomes like adhering structures with focal adhesions in order to make contact with the basal lamina. Underneath the basal lamina is the interstitial space, which consists of ECM proteins and blood capillaries, forming the connective tissues. During the infection, bacteria can breach the tight junctions and degrade focal adhesion complexes between epithelial cells by using toxins and several secretory systems, followed by the degradation of basal lamina and exposure of ECM. Meanwhile, host inflammatory and tissue repair systems will also stimulate the degradation of ECM that will be available for bacteria. Vn promotes bacterial adherence to epithelial cells and confers serum resistance.
B. Some pathogenic bacteria such as Yersiniapestis binds directly to integrins via surface adhesins, e.g. YadA, while others including S. pneumoniae and P. aeruginosa interact with integrin by using Vn as bridging molecule. However, Vn-mediated bacterial interaction to integrins is inhibited by glycosylaminoglycans (heparin), as the heparin masks the bacterial-binding domain of Vn molecule. In contrast, N. meningitidis has heparin-binding protein (Opc) that bind simultaneously to both heparin and Vn. The Opc–heparin–Vn–integrin complex thus enhance N. meningitidis adherence to host cell. Vn binding to integrin receptors induces an intracellular signal that leads to actin polymerization, resulting in membrane protrusions around the bacteria. Bacteria trapped inside the protrusions pocket are internalized as a phagosome surrounded by a plasma membrane and actin coat. Finally, as an escape strategy from phagosome, some bacteria release virulence factors that induce actin depolymerization by host proteins such as vinculin or cofilin. The detailed description of integrin-mediated signalling is shown in Fig. 4.

Escherichia coli causing colonic infection produces high concentrations of surface proteins that bind to host ECM proteins including Vn and thus promotes bacterial adherence to host cells (Shen et al., 1995). Clostridium difficile, which is associated with antibiotic-associated diarrhoea, adheres to the gastrointestinal tract mucosa via several adhesins during colonization. High-molecular-weight surface-layer proteins (SLPs) of C. difficile have been shown to interact with Vn and other ECM proteins under in vivo and in vitro conditions (Calabi et al., 2002; Cerquetti et al., 2002). In addition, another adhesin of C. difficile, fibronectin-binding protein (Fbp) 68, also has been identified as a Vn-binding protein (Hennequin et al., 2003). The gastrointestinal pathogen Helicobacter pylori attracts Vn with high affinity via sialic acid haemagglutinins and promotes bacterial adherence to the host (Ringnér et al., 1992; 1994). Another study proposed that binding of Vn at the surface of H. pylori inhibits the complement-mediated phagocytosis by macrophages (Chmiela et al., 1997; Dubreuil et al., 2002). In parallel, the obligate intracellular human pathogen, Chlamydia trachomatis interacts with several ECM proteins including Vn, and these interactions have important roles in colonization (Kihlstrom et al., 1992).

Recently it was reported that Vn increases the binding of S. pneumoniae by several-fold to epithelial cells. Multimeric forms of Vn bind to S. pneumoniae significantly higher in comparison with monomeric forms, indicating that cell-bound multimeric Vn is a preferable target for S. pneumoniae during adhesion (Bergmann et al., 2009). Thus, S. pneumoniae-dependent binding to multimeric Vn (found on the epithelial cell surface) might be selectively responsible for the bacterial attachment to host cells. Both isoforms of Vn have almost a similar heparin-binding capacity, but some of the cryptic heparin and bacterial binding sites are better exposed in multimeric (refolded or partially denatured) Vn (Zhuang et al., 1997; Sa E Cunha et al., 2010). In a recent study, the multimeric (oligomeric) form of Vn was shown to efficiently support the adherence and migration of human endometrial stromal fibroblasts, and the C-terminal HBD was required for Vn oligomerization (Chillakuri et al., 2010). This study also confirmed that the cell-bound form of Vn is multimeric. Moreover, heparin inhibits Vn-mediated adherence and internalization of S. pneumoniae to lung epithelial cells, implying that HBD of Vn is involved in the host–pathogen interaction. Further analysis also suggested that an RGD-containing peptide and antibodies directed against αvβ3 integrin inhibit the bacterial adhesion and internalization (Bergmann et al., 2009). Thus, Vn is a bridging molecule between an unknown adhesin of S. pneumoniae and the host cell receptor αvβ3 integrin. In contrast, we showed that multimeric and monomeric Vn binds to a similar extent to M. catarrhalis UspA2 (Singh et al., 2010b). Our further observations with H. influenzae also revealed the similar binding to both isoforms of Vn (our unpublished data). Nevertheless, while the role of Vn in adherence to host cells, however, is still unproven for H. influenzae and M. catarrhalis, the role in serum resistance has been well established.

Pseudomonas aeruginosa also potentially binds to Vn, and this interaction increases bacterial adherence and internalization into A549 alveolar epithelial cells. Here αvβ5 integrin is the major receptor for the Vn since blocking with antibodies directed against Vn and αvβ5 significantly reduced the bacterial binding and internalization (Chhatwal et al., 1987; Leroy-Dudal et al., 2004). Vn signalling involves the Src-like tyrosine kinase and MEK/ERK pathways in P. aeruginosa uptake, and therefore inhibitors against MEK and ERK significantly reduce bacterial entry into epithelial cells (Evans et al., 2002; Leroy-Dudal et al., 2004). Furthermore, a virulence factor of P. aeruginosa, LasB (secreted metalloproteinase), cleaves uPAR (consists of three domains; D1–D3) at two locations and releases the N-terminal (D1) and C-terminal (D3) domains from a GPI anchor. Cleaved uPAR therefore falls apart from the host cell surface. This proteolytic cleavage of uPAR reduces the capacity of cells to bind to urokinase and results in impaired cell repair, healing and cell migration. It also diminishes the interaction between uPAR and the matrix adhesive protein Vn, which is also involved in tissue repair during inflammation (Leduc et al., 2007). It has earlier been shown that in a mouse pulmonary clearance model, uPAR-deficient mice had impaired recruitment and activation of leucocytes at P. aeruginosa infection site, resulting in defective bacterial clearance and increased host mortality (Gyetko et al., 2004). Thus P. aeruginosa benefits the defective cellular repair and healing conditions triggered by the LasB protease during the course of colonization.

Vitronectin is also utilized by N. meningitidis for survival in serum. Interestingly, the concentration of circulating Vn is diminished in plasma of patients suffering from N. meningitidis sepsis due to the extensive consumption by bacteria. However, the plasma Vn is restored to a normal physiological level after recovery from the infection (Høgåsen et al., 1994). Pre-incubation of serum with N. meningitidis causes Vn depletion, demonstrating the high Vn attraction capacity of meningococci. The adhesin Opc of N. meningitidis was shown to be involved in the bacterial binding to Vn. The Opc–Vn interaction mediates bacterial adherence to host cells by using Vn as a bridging molecule. Specific antibodies directed against the Opc, integrins αvβ3 and αvβ5, or RGD peptide, significantly decreased the attachment and internalization of meningococci (Virji et al., 1994). In a more recent study, the nature of Vn binding to Opc was analysed in detail revealing a distinguished Opc–Vn interaction. The N. meningitidis (both capsulated and non-capsulated strains) bound serum Vn efficiently while their respective opc- mutants were unable to attract Vn. Vn bound to the meningococcal surface caused significantly increased adhesion and internalization of human brain endothelial cells (HBMEC). The role of Vn in adhesion is highly important for this pathogen as the adhesive capacity of pili-deficient N. meningitidis mutants can be restored when Vn is supplemented (Sa E Cunha et al., 2010). However, this study showed that the interaction is only restricted to the activated form of Vn, whereas the native Vn-binding capacity is relatively low. In contrast to meningococci, M. catarrhalis and NTHi bind to both native and activated Vn (Hallström et al., 2009; Singh et al., 2010b). In fact, activated Vn is partially in denatured conformation for the exposure of the cryptic binding sites, such as heparin binding regions and C-terminal domains (Preissner and Seiffert, 1998). On the basis of Vn–Opc binding properties, Sa E Cunha and co-workers proposed dual binding mechanisms: Opc binds at the residues Y56S/Y59S of Vn and the C-terminal HBD-3 (Sa E Cunha et al., 2010).

In parallel to meningococci, N. gonorrhoeae OpaA binds Vn and functions as an adhesin as well as plays a role in bacterial internalization. OpaA also binds to heparin that functions as a bridging molecule in the OpaA/heparin/Vn complex. For this reason Vn binding to N. gonorrhoeae is poor in the absence of heparin. Interestingly, certain mammalian cells [e.g. Chinese hamster ovarian (CHO) cells] lacking the expression of glycosylaminoglycans such as heparin, are not infected with N. gonorrhoeae (Duensing and Putten, 1998). These findings have also been confirmed by a similar study suggesting that Vn and αvβ integrins together with protein kinase C (PKC) are involved in N. gonorrhoeae uptake (Dehio et al., 1998). The heparin-binding property is an exclusive feature for Neisseria spp. since heparin usually acts as an antagonist in most of bacterial protein/Vn complexes by masking the HBDs (Hallström et al., 2009; Singh et al., 2010b). In contrast, Neisseria spp. requires glycosylaminoglycans, that is, heparin for successful adhesion and internalization via an OpaA/heparin/Vn/integrin complex.

Porphyromonas gingivalis causes human periodontal disease and the pathogen adheres to gum and mucosal epithelial cells by using different adhesins, fimbriae and protease/adhesin complex (designated gingipains) (Baba et al., 2001; Olczak et al., 2001). Fimbriae of P. gingivalis interacts with ECM proteins including laminin, elastin, fibrinogen, Fn, collagen I, thrombospondin and Vn. Surface plasmon resonance showed a high affinity of fimbriae interacting with Vn (association constant, Ka = 3.79 × 106) (Nakamura et al., 1999). In another study, the effect of fimbriae was analysed in Vn–αvβ3 and Fn–α5β1 complex formation that is also a part of wound healing and a tissue repair mechanism. Vn and Fn bound to the bacterial surface provide an increased adherence of P. gingivalis to integrin-expressing CHO cell lines. However, the fimbriae compete with Vn/Fn to interact with the integrins in a dose-dependent manner. This study suggested that fimbriae of P. gingivalis act as inhibitors of the integrin-ECM-associated cellular functions, inducing a prominent damage to periodontal tissues (Nakagawa et al., 2005). Recently, the role of gingipains in ECM-mediated adhesion of P. gingivalis was evaluated. The gingipains and P. gingivalis bind to immobilized Vn, Fn and fibrinogen preferentially in comparison with the soluble/fluid forms (McAlister et al., 2009). In fact, gingipains are known to facilitate bacterial attachment to gum matrix when it is associated on the bacterial surface while the cell-free gingipains are involved in the degradation of ECM proteins, causing tissue damage and inflammation. Moreover, Vn and Fn are abundantly present in periodontal tissues and thus may have a role in bacterial adhesion to epithelial cells facilitating colonization (Rautemaa and Meri, 1996; Nakamura et al., 1999; Baba et al., 2001).

Vitronectin mediated host cell signalling and bacterial opportunities

Vitronectin engages with members of the integrin family (α3β1, αvβ1, αvβ3, αvβ5 and αIIbβ3) (Preissner and Jenne, 1991; Wei et al., 2001; Smith and Marshall, 2010). In adherent cells, integrins are usually clustered at cell attachment sites called focal adhesions (Fig. 3A). The integrins integrate the attachment outside of the cell with reorganization of the intracellular actin cytoskeleton via talin and vinculin. The integrin-rich focal adhesion sites consist of thick actin bundles within the cells that control cell shape and tension, and signalling molecule of protein tyrosine kinases (PTKs), adaptor molecules and small Rho GTPases that transmit signals to the cell interior as well as receive signals from within the cells (Brunton et al., 2004). Integrin engagement stimulates the activity of signalling molecules, including numerous PTKs such as Src and focal adhesion kinase (FAK), tyrosine phosphatases, cAMP-dependent protein kinase, PKC and stimulates production of phosphatidylinositol 4,5-biphosphate (PIP2) (Meyer et al., 2000; Parsons et al., 2000; Ling et al., 2002). The signalling events occur at the basal region of polarized epithelial cells. Integrin-mediated signalling also involves Fn containing RGD sequence as a co-receptor.

Several pathogenic bacteria exploit the integrin-signalling cascades for their entry into non-phagocytic host cells and for intracellular or intercellular spreading, by binding to the integrins directly or by using Vn as a cross-link molecule between bacteria and host cells (Isberg et al., 2000; Wang et al., 2006) (Fig. 3B). The interaction between bacterial adhesins and integrins triggers a cascade of signalling that resembles those stimulated by host physiological effectors. These include tyrosine protein phosphorylation, recruitment of adaptor proteins, lipid metabolism or activation of small G-protein and cytoskeletal components that culminate in phagocytic cup closure and bacterial internalization through a so-called ‘zipper’ mechanism (Swanson and Baer, 1995).

The signalling mechanisms involved in bacterial interaction with integrins have been thoroughly studied for Yersinia spp. Invasin (InvA) of Yersinia interacts directly with αβ1–integrins at higher affinity than its natural substrate Fn (Tran Van Nhieu and Isberg, 1993; Hamzaoui et al., 2004) (Fig. 4). This interaction leads to multimerization of β1 integrins, which induces Pyk2 phosphorylation of cell adhesion proteins (e.g. p130cas) and autophosphorylation of FAK followed by activation of Src kinase, Rac GTPase, Crk-associated substrate and Crk, and lastly the ARP2/3 complex; results in remodelling of the mammalian cell surface and uptake of the bacteria through the reorganization of the actin cytoskeleton (Fig. 4A) (Alrutz et al., 2001; Bruce-Staskal et al., 2002). Another Yersinia spp. adhesin, YadA, uses Fn instead of Vn as a bridging molecule to target β1 integrins in order to promote tight adhesion and invasion into mammalian cells (El Tahir and Skurnik, 2001). Cell invasion and interleukin-8 (IL8) production triggered by YadA require common signalling molecules, i.e. phosphorylation of FAK and c-Src as well as induction of small GTPase Ras which leads to the stimulation of mitogen-activated kinases (MAPKs)-dependent IL-8 production or phosphatidylinositol 3-kinase (PI3K)-dependent invasion (Eitel et al., 2005).

Figure 4.

Schematic outline of Vn mediated signalling in epithelial cells and possible opportunities taken by pathogens to promote adhesion and internalization.
A. Bacterial entry based upon integrin- or Vn/integrin-mediated signalling. Vn is proposed to mediate the interaction between uPA/uPAR and integrin and to generate outside-in signalling. Glycosyl phosphatidylinositol (GPI)-anchored urokinase receptor (uPAR) resided within focal adhesion site binds Vn at the SMB domain. Binding of uPA at the central cleft of uPAR enhances the binding of Vn to uPAR. Formation of the uPAR–Vn–integrin (i.e. αvβ3 and α3β1) complex activates the Src kinases that subsequently phosphorylate p130cas (an FAK-associated adaptor protein). Binding of SH2 domain of CRK adaptor protein to the phosphorylated p130cas forms pI3K–CRK complex, brings the dedicator of cytokinesis protein 180 (DOCK180) to the focal adhesion site of integrin and activates Rac. Activated Rac signals to the WAVE complex to promote F-actin assembly at the leading edge of migrating cells through the ARP2/3 actin nucleation factor. This signalling cascade stimulates actin polymerization and membrane protrusion, leading to cell motility and invasion (Savagner, 2001; Wei et al., 2001; 2008; Stradal and Scita, 2006; Rocca et al., 2008; Smith and Marshall, 2010). This complex signalling also leads to actin remodelling by ERK and AKT, followed by the appearance of micro-spike-like structures at the cell surface. During infection, bacterial cells bound to integrin or Vn–integrin might be trapped in the protrusion pocket and subsequently been internalized. Bacteria might also breach the cell–cell barrier when the cell–cell junctions are loose.
B. Bacterial invasion via other non-integrin cell adhesion molecules (CAMs). In addition to the integrins and Vn, many bacterial pathogens have also evolved other invasion strategies by interacting with other host cell adhesion molecules (CAM), including cadherin (e.g. E-cadherin), immunoglobulin receptors (e.g. carcinoembryonic antigen-related cell adhesion molecule 3; CEACAM3), protein phosphatase receptor (e.g. c-Met receptor), selectins and hyaluronate receptors. (i) Internalin A (InlA) of Listeria monocytogenes binds the extracellular N-terminal domain 1 of E-cadherin (Mengaud et al., 1996; Cossart et al., 2003). This interaction is regulated by extracellular calcium levels and signals from inside cells that modulate the stability of cadherin-associated complexes (including β-catenin, α-catenin, γ-catenin, p120ctn, ZO-1, vinculin and α-actinin). The complexes link the cytoplasmic domain of cadherin to actin cytoskeleton and activate zippering internalization of bacteria (Mengaud et al., 1996). (ii) The second major invasin of Listeria, internalin B (InlB), binds c-Met receptor and simulates recruitment and phosphorylation of several signalling molecules including PI3K, growth factor receptor-bound protein-2 (GRB2), GRB2-associated binding protein-1 (GAB1) and Rho GTPase. Activation of PI3K affects cytoskeletal dynamic (Shen et al., 2000; Pentecost et al., 2010). (iii) Model of bacterial entry via IgCAM is based upon interaction of Opa and Opc of Neisseria spp. with CEACAM3, a member of IgCAM protein family expressed by granulocytes. Activated Src-family kinases (Hck and Fgr) are recruited to the tyrosine-phosphorylated ITAM motif of cytoplasmic domain of CEACAM3. Syk then transmits the signal that activates Rac and Cdc42, which lead to actin polymerization (Billker et al., 2002; Booth et al., 2003). (iv) H. pylori produces adhesin BabA and SabA that bind to host cells selectin, Lewis B and sialyl-dimeric-Lewis x (Lex) respectively. The Lex expression of gastric mucosa was simulated by H. pylori during infection, which in turn promoted H. pylori adherence and internalization (Ilver et al., 1998; Mahdavi et al., 2002).

Vn- and Fn-engaged integrin signalling are generally sharing common signal transduction mechanisms that mainly involve activation of FAK, PI3K and integrin-linked kinase (ILK) (Eitel et al., 2005; Scibelli et al., 2007; Wang et al., 2007) (Fig. 4A). These pathways eventually lead to the net effect, actin remodelling and bacterial internalization through a ‘zipper’ or ‘trigger’ mechanism, and the production of pro-inflammatory cytokines triggering pathophysiology trauma in the host during infection (Scibelli et al., 2007).

In addition to interaction with membrane protein heparan sulphate proteoglycans (HSPGs), Opa of N. gonorrhoea and N. meningitidis also utilize Vn as a bridging ligand to interact with αvβ3 and αvβ5 (Figs 3B and 4A); and Fn to engage with α5β1 (Hauck and Meyer, 2003). These interactions are prominent in providing additional host–pathogen interactions and inducing integrin-mediated uptake into host cells expressing low level of HSPG. The combined signalling of HSPG and integrins directs PKC activation that leads to cytoskeletal rearrangements and endocytosis of Vn–bacteria complexes (Dehio et al., 1998). However, the exact component or signalling pathway involved downstream of PKC activation is currently unknown. Another functional homologue of Opa, but an unrelated outer membrane protein, meningoccocal Opc, interacts with Vn and Fn, mediating the attachment to human umbilical vein or brain microvascular endothelial cells (HBMEC) respectively (De Vries et al., 1998; Unkmeir et al., 2002). Bacterial binding to the HBMEC triggers phosphorylation and activation of c-Jun N-terminal kinases 1 and 2 (JNK1 and JNK2) and p38 MAPK (Sokolova et al., 2004). Upon bacterial adherence, the p38 MAPK pathway plays a crucial role in controlling the release of IL-6 and IL-8 by HBMEC. While the specific virulence factor remains unknown, internalization of P. aeruginosa by mammalian cells involves Src, MAPK and ERK following Vn-mediated bacterial engagement with the integrin αvβ5 (Esen et al., 2001; Evans et al., 2002; Leroy-Dudal et al., 2004).

ILK is another major protein involved in actin remodelling (Fig. 4A) as ILK knock-down in epithelial cells showed impaired F-actin cytoskeleton formation (Grashoff et al., 2004). ILK knock-down showed significantly reduced internalization of S. pneumoniae due to lack of microspike formations (Bergmann et al., 2009). Furthermore, in signalling events, protein kinase B (Akt) is phosphorylated at Ser473 by ILK, and this phosphorylation is also indispensible for bacterial internalization (Bergmann et al., 2009; Weber et al., 2009). ILK is the link between integrin and further signalling to FAK, PI3K, Akt, Src or Ras/Raf pathways (Wang et al., 2006; Bergmann et al., 2009; Smith and Marshall, 2010). Thus structural and functional activity of ILK is crucial for integrin-mediated bacterial entry into the host cells.

Many studies have characterized the role of Vn/integrin-mediated signalling in the uptake of intact bacteria. However, the contribution of the signalling pathway in the entry of bacterial outer membrane vesicles (OMV) or other virulence factors into epithelial cells remains unknown. The secreted P. gingivalis OMV contain major virulence factors, including major fimbriae (type II FimA) that binds both Vn and integrin α5β1 with high affinity (Nakagawa et al., 2002). However, the FimA acts as an antagonist between the interaction of Vn and integrin α5β1; possibly they are sharing the same binding domain (Amano, 2003). Instead of using Vn as a bridging molecule, the FimA could just hook the OMV on the epithelial cells by direct interaction with integrins. However, the role of FimA in OMV uptake has been excluded as it was recently reported that the OMV swiftly enter epithelial via an endocytosis pathway mediated by a novel pathway, a Rac1-regulated pinocytic pathway that is independent of caveolin, dynamin and clathrin (Furuta et al., 2009a). Interestingly the internalized OMV degraded cellular integrin-related signalling molecules such as paxillin and FAK, resulting in inhibition of cellular migration and immortalization human gingival epithelial cells (Furuta et al., 2009b).

Taken together, present findings suggest that Vn signalling induces cytoskeleton remodelling, which is a common biological process involved in cell migration or tissue repair. The Vn–integrin signalling is similar in the presence or absence of pathogens. The most crucial aspect is microbial adaptation and their opportunistic ability to benefit from the host signalling processes in order to colonize successfully.

Concluding remarks and future perspectives

Bacterial pathogens have developed several different survival strategies in order to escape the innate defence system and proliferate inside the host. Bacteria coated with Vn evade the complement attack and, hence, survive better. Furthermore, Vn cross-links pathogens and host cells, and the bridge functions as a potential adhesin. Once the N-terminal domain of Vn binds to integrin receptors of host cells, and C-terminus with the bacteria, signalling for actin remodelling in host cells is stimulated and this causes the bacterial uptake. Thus, although Vn is produced by the host, it is eventually beneficial to the pathogen.

What is the availability of Vn at a site of infection? Answering this question would help to evaluate the in vivo importance of Vn during infection. It is known that lung and kidney parenchyma have large amounts of Vn (Fig. 1B–J), and infection-induced damage to epithelial cells also causes secretion of plasma exudates that contain Vn. On the other hand, invasive pathogens in the blood stream encounter an excess of Vn, whereas the availability of Vn could be limited in the gastrointestinal tract, skin and other infection prone tissues.

Many recent studies have suggested potential roles of Vn in microbial colonization and serum resistance. However, the precise mechanism involved in bacterial protein(s)–Vn interactions has not been studied in detail. The lack of structural data on Vn also limits the better understanding about Vn–bacterial interactions. The HBD-3 of Vn recognizes S. pneumoniae (Bergmann et al., 2009), whereas an overlapping C-terminal region (Vn312–396) interacts with M. catarrhalis UspA2 (Singh et al., 2010b). Further investigation of H. influenzae also suggested that the same region as the Moraxella–Vn binding site is involved in the interaction with PE (B. Singh et al., unpubl. data). Recent data thus suggest a common bacterial binding site on the Vn molecule.

The exact binding mechanism of Vn to the C5b-7 complex and C9 is still not known, hence a crucial question arose that whether Vn remains active after binding to the bacterial outer membrane. Our recently published studies of Vn binding to the respiratory pathogens H. influenzae and M. catarrhalis suggest that Vn is highly active after binding to the bacterial surface and inhibits MAC deposition (Hallström et al., 2009; Singh et al., 2010b). This suggests that the bacteria–Vn interaction should not mask the possible C5b-7 and C9 binding sites of Vn, since the MAC inhibitory activity is preserved.

The selective peptides derived from ECM have an in vitro antimicrobial activity and might be used to control Gram-negative and Gram-positive bacteria. It has been observed that the HBD of Vn as well as laminin and Fn have antimicrobial activity against E. coli, P. aeruginosa and Enterococcus faecalis (Andersson et al., 2004; Malmsten et al., 2006). Thus, Vn peptides and their derivatives are potential novel antimicrobial agents.

It has been known for many years that interactions between bacterial pathogens and epithelial cells cause induction of pro-inflammatory cytokines, and lead to changes in expression of epithelial surface markers. Exploration of the role of Vn in the epithelial cell host response after bacterial exposure just recently has begun but more work needs to be done. Most of the recent studies have been performed in vitro and have evaluated the role of Vn in serum resistance, bacterial adhesion and internalization. These interesting findings must be extended under in vivo conditions by using suitable models in order to find novel antimicrobial therapies.


This work was supported by grants from the Alfred Österlund, the Anna and Edwin Berger, the Marianne and Marcus Wallenberg, and the Greta and Johan Kock, the Janne Elgqvist, and the Krapperup Foundations, the Swedish Medical Research Council, the Cancer Foundation at the University Hospital in Malmö, and Skåne County Council's research and development foundation.