The molecular basis of fibronectin-mediated bacterial adherence to host cells

Many pathogenic Gram-positive bacteria produce cell wall-anchored proteins that bind to components of the extracellular matrix (ECM) of the host. These bacterial MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) are thought to play a critical role in infection. One group of MSCRAMMs, produced by staphylococci and streptococci, targets fibronectin (Fn, a glycoprotein found in the ECM and body fluids of vertebrates) using repeats in the C-terminal region of the bacterial protein. These bacterial Fn-binding proteins (FnBPs) mediate adhesion to host tissue and bacterial uptake into non-phagocytic host cells. Recent studies on interactions between the host and bacterial proteins at the residue-specific level and on the mechanism of host cell invasion are providing a much clearer picture of these processes.     

Isopeptide bond in collagen- and fibrinogen-binding MSCRAMMs

The internal isopeptide bonds are amide bonds formed autocatalytically between the side chains of Lys and Asn/Asp residues and have been discovered recently. These bonds are well conserved in Gram-positive bacterial pilin proteins and are also observed over a wide range of Gram-positive bacterial surface proteins. The presence of these bonds confers the pilus subunits with remarkable properties in terms of thermal stability and resistance to proteases. Like pili, microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are also surface proteins found only in Gram-positive bacteria. They specifically interact with the extracellular matrix (ECM) molecules like collagen, fibrinogen, fibronectin, laminin, etc. Many biophysical and biochemical studies have been carried out to characterize the isopeptide bonds in pili proteins from Gram-positive bacteria, but no attempts have been made to study the isopeptide bonds in MSCRAMMs. This short review aims to study the significance of the isopeptide bonds in relation to their function, by analyzing the crystal structures of collagen- and fibrinogen-binding MSCRAMMs. In this analysis, interestingly, we observed that the putative isopeptide bonds are restricted to the collagen-binding MSCRAMMs. Based on analogy with bacterial pilus subunits, we hypothesize that the collagen-binding MSCRAMMs possessing putative isopeptide bonds exhibit similar structural properties, which could help the bacteria in colonizing the host and provide resistance against host–defense mechanisms.     

PfbA, a novel plasmin- and fibronectin-binding protein of Streptococcus pneumoniae, contributes to fibronectin-dependent adhesion and antiphagocytosis

Streptococcus pneumoniae is a major causative agent of mortality throughout the world. The initial event in invasive pneumococcal disease is the attachment of pneumococci to epithelial cells in the upper respiratory tract. Several bacterial proteins can bind to host extracellular matrix proteins and function as adhesins and invasins. To identify adhesins or invasins on the pneumococcal cell surface, we searched for several proteins with an LPXTG anchoring motif in the whole-genome sequence of Streptococcus pneumoniae and identified one, which we called PfbA (plasmin- and fibronectin-binding protein A), that bound to human serum proteins. Immunofluorescence microscopy and fluorescence-activated cell sorter analysis revealed that PfbA was expressed on the pneumococcal cell surface. A DeltapfbA mutant strain was only half as competent as the wild-type strain at adhering to and invading lung and laryngeal epithelial cells. In addition, epithelial cells infected with DeltapfbA showed morphological changes, including cell flattening and a loss of microvilli, that did not occur in cells infected with the wild-type strain. The mutant strain also exhibited a weaker antiphagocytotic activity than wild type in human peripheral blood. Moreover, the growth of wild-type bacteria in human whole blood containing anti-PfbA antibodies was reduced by approximately 50% after 3 h compared with its growth without the antibody. These results suggest that PfbA is an important factor in the development of pneumococcal infections.     

Adhesins and invasins of pathogenic bacteria: a structural view

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 eukaryotic proteins. Similar folds are employed repeatedly to target different receptors.     

Co-ordinate action of bacterial adhesins and human carcinoembryonic antigen receptors in enhanced cellular invasion by capsulate serum resistant Neisseria meningitidis

Neisseria meningitidis (Nm) is a human specific opportunistic pathogen that occasionally penetrates mucosal barriers via the action of adhesins and invasins and evades host immune mechanisms during further dissemination via capsule expression. From in vitro studies, the primary adhesion of capsulate bacteria is believed to be mediated by polymeric pili, followed by invasion via outer membrane adhesins such as Opa proteins. As the latter requires the surface capsule to be down-modulated, invading bacteria would be serum sensitive and thus avirulent. However, there is recent evidence that capsulate bacteria may interact via Opa proteins when host cells express high levels of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), their target receptors. Such a situation may arise following increased circulation of inflammatory cytokines that upregulate certain adhesion molecules on host cells. In this study, using a tetracycline controlled expression system, we have developed cell lines with inducible CEACAM expression to mimic post-inflammation state of target tissues and analysed the interplay between the three surface components capsule, pili and Opa proteins in cellular interactions. With two distinct cell lines, not only the level but also the rate of adhesion of capsulate Opa-expressing Nm increased concurrently with CEACAM density. Moreover, when threshold levels of receptor were reached, cellular invasion ensued in an Opa-dependent manner. In studies with cell lines intrinsically expressing pilus receptors, notable synergism in cellular interactions between pili and Opa of several meningococcal strains was observed and was independent of capsule type. A number of internalized bacteria were shown to express capsule and when directly isolated from host cells, these bacteria were as serum resistant as the inoculated phenotype. Furthermore, we observed that agents that block Opa-CEACAM binding substantially reduced cellular invasion, while maintaining a low level of cellular adhesion. These studies highlight some of the factors that may determine increased host susceptibility to infection by serum resistant phenotypes; and demonstrate the potential of selective inhibition of key interactions in preventing target tissue penetration while maintaining a level of colonization.     

Identification of Cell-Binding Adhesins of Leptospira interrogans

Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus Leptospira. Infection can lead to illness ranging from mild and non-specific to severe, with jaundice, kidney and liver dysfunction, and widespread endothelial damage. The adhesion of pathogenic Leptospira species (spp.), the causative agent of leptospirosis, to host tissue components is necessary for infection and pathogenesis. While it is well-established that extracellular matrix (ECM) components play a role in the interaction of the pathogen with host molecules, we have shown that pathogenic Leptospira interrogans binds to host cells more efficiently than to ECM components. Using in vitro phage display to select for phage clones that bind to EA.hy926 endothelial cells, we identified the putative lipoproteins LIC10508 and LIC13411, and the conserved hypothetical proteins LIC12341 and LIC11574, as candidate L. interrogans sv. Copenhageni st. Fiocruz L1–130 adhesins. Recombinant LIC11574, but not its L. biflexa homologue LBF1629, exhibited dose-dependent binding to both endothelial and epithelial cells. In addition, LIC11574 and LIC13411 bind to VE-cadherin, an endothelial cell receptor for L. interrogans. Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction, a likely indication that these proteins are outer membrane localized. All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. This work has identified bacterial adhesins that are potentially involved in L. interrogans infection of the mammalian host, and through cadherin binding, may contribute to dissemination and vascular damage. Our findings may be of value in leptospirosis control and prevention, with the bacterial adhesins potentially serving as targets for development of diagnostics, therapeutics, and vaccines.     

The BtaF Trimeric Autotransporter of Brucella suis Is Involved in Attachment to Various Surfaces,Resistance to Serum and Virulence

The adhesion of bacterial pathogens to host cells is an event that determines infection, and ultimately invasion and intracellular multiplication. Several evidences have recently shown that this rule is also truth for the intracellular pathogen Brucella. Brucella suis displays the unipolar BmaC and BtaE adhesins, which belong to the monomeric and trimeric autotransporter (TA) families, respectively. It was previously shown that these adhesins are involved in bacterial adhesion to host cells and components of the extracellular matrix (ECM). In this work we describe the role of a new member of the TA family of B. suis (named BtaF) in the adhesive properties of the bacterial surface. BtaF conferred the bacteria that carried it a promiscuous adhesiveness to various ECM components and the ability to attach to an abiotic surface. Furthermore, BtaF was found to participate in bacterial adhesion to epithelial cells and was required for full virulence in mice. Similar to BmaC and BtaE, the BtaF adhesin was expressed in a small subpopulation of bacteria, and in all cases, it was detected at the new pole generated after cell division. Interestingly, BtaF was also implicated in the resistance of B. suis to porcine serum. Our findings emphasize the impact of TAs in the Brucella lifecycle.     

The use of sortase-mediated ligation for the immobilisation of bacterial adhesins onto fluorescence-labelled microspheres: a novel approach to analyse bacterial adhesion to host cells

Commensal and pathogenic bacteria express adhesive proteins on their cell surface, which are important for colonisation of the host. In Gram-positive bacteria, these adhesins are often covalently anchored to the cell wall by a sortase enzyme. A recent bioinformatic study has revealed a total of 860 predicted cell wall-anchored proteins in 94 completely sequenced genomes. The interaction of adhesins with host cells can be analysed with the use of adhesin-coated microbeads. Here we show that sortase-mediated ligation can be used for the site-specific immobilisation of adhesins to red-fluorescence microspheres. This coupling method allows for the native orientation of the adhesins on the beads. Furthermore, the high substrate specificity of the sortase enzyme allows the use of only partially purified recombinant proteins, which reduces preparation time and costs, and also prevents coupling of any contaminants that might interfere with cell binding.     

Surface-associated proteins of Staphylococcus aureus: Their possible roles in virulence

Abstract A class of proteins that are associated with the cell surface of Gram-positive bacteria has been recognised. Common structural features which are implicated in the proper secretion and attachment of these proteins to the cell surface occur in the C-termini. N-terminal domains interact with the host by binding to soluble host proteins, to matrix proteins or to host cells. They probably have important roles in pathogenicity by allowing bacteria to avoid host defences and by acting as adhesins. Four such proteins of Staphylococcus aureus have been characterised: protein A (immunoglobulin binding protein), fibronectin binding proteins, collagen binding protein and the fibrinogen binding protein (clumping factor). Site-specific mutants are being used to define their roles in pathogenesis in in vitro and in vivo models of adherence and infection.     

Evaluation of Cell Binding Activities of Leptospira ECM Adhesins

Pathogenic spirochetes of the genus Leptospira are the causative agents of leptospirosis, a zoonotic infection that occurs globally. The bacteria colonize the renal proximal tubules of many animals and are shed in the urine. Contact with the urine, or with water contaminated with the urine of infected animals can cause infection of new host animals, including humans. Mechanisms of colonization of the proximal tubule and other tissues are not known, but specific interactions between bacterial adhesins and host substrates are likely to be critical in this process. Several extracellular matrix (ECM) adhesins have been previously identified, but more recently, it has been shown that Leptospira bind more efficiently to cells than ECM. In this work, recombinant forms of five putative Leptospira ECM adhesins, namely LipL32, Loa22, OmpL1, p31/LipL45, and LenA were evaluated for binding to cells as well as an expanded variety of ECM components. Reproducible and significant adhesin activity was demonstrated only for OmpL1, which bound to both mammalian cell lines tested and to glycosaminoglycans (GAGs). While determination of biologically significant bacterial adhesion activity will require generation of site-directed mutant strains, our results suggest that OmpL1 is a strong candidate for future evaluation regarding the roles of the adhesin activity of the protein during L. interrogans infection.     

Identification and characterization of a cell surface protein of Prevotella intermedia 17 with broad-spectrum binding activity for extracellular matrix proteins

Prevotella intermedia binds and invades a variety of host cells. This binding is most probably mediated through cell surface proteins termed adhesins. To identify proteins binding to the host extracellular matrix (ECM) component, fibronectin, and study the molecular mechanism underlying bacterial colonization, we applied proteomic approaches to perform a global investigation of P. intermedia strain 17 outer membrane proteins. 2-DE followed by Far Western Blot analysis using fibronectin as a probe revealed a 29-kDa fibronectin-binding protein, designated here AdpB. The molecular identity of the protein was determined using PMF followed by a search of the P. intermedia 17 protein database. Database searches revealed the similarity of AdpB to multiple bacterial outer membrane proteins including the fibronectin-binding protein from Campylobacter jejuni. A recombinant AdpB protein bound fibronectin as well as other host ECM components, including fibrinogen and laminin, in a saturable, dose-dependent manner. Binding of AdpB to immobilized fibronectin was also inhibited by soluble fibronectin, laminin, and fibrinogen, indicating the binding was specific. Finally, immunoelectron microscopy with anti-AdpB demonstrated the cell surface location of the protein. This is the first cell surface protein with a broad-spectrum ECM-binding abilities identified and characterized in P. intermedia 17.     

Pneumococcal Adhesins PavB and PspC Are Important for the Interplay with Human Thrombospondin-1

The human matricellular glycoprotein thrombospondin-1 (hTSP-1) is released by activated platelets and mediates adhesion of Gram-positive bacteria to various host cells. In staphylococci, the adhesins extracellular adherence protein (Eap) and autolysin (Atl), both surface-exposed proteins containing repeating structures, were shown to be involved in the acquisition of hTSP-1 to the bacterial surface. The interaction partner(s) on the pneumococcal surface was hitherto unknown. Here, we demonstrate for the first time that pneumococcal adherence and virulence factor B (PavB) and pneumococcal surface protein C (PspC) are key players for the interaction of Streptococcus pneumoniae with matricellular hTSP-1. PavB and PspC are pneumococcal surface-exposed adhesins and virulence factors exhibiting repetitive sequences in their core structure. Heterologously expressed fragments of PavB and PspC containing repetitive structures exhibit hTSP-1 binding activity as shown by ELISA and surface plasmon resonance studies. Binding of hTSP-1 is charge-dependent and inhibited by heparin. Importantly, the deficiency in PavB and PspC reduces the recruitment of soluble hTSP-1 by pneumococci and decreases hTSP-1-mediated pneumococcal adherence to human epithelial cells. Platelet activation assays suggested that PavB and PspC are not involved in the activation of purified human platelets by pneumococci. In conclusion, this study indicates a pivotal role of PavB and PspC for pneumococcal recruitment of soluble hTSP-1 to the bacterial surface and binding of pneumococci to host cell-bound hTSP-1 during adhesion.     

Structural biology of bacterial pathogenesis

Recent years have seen a rapid increase in structural information on proteins implicated in bacterial pathogenesis. The different modes by which bacteria establish contact with their host tissues are exemplified by the structures of bacterial adhesins in complex with their cognate host receptor. A more detailed structural understanding of the various Gram-negative secretion systems has emerged with the determination of the structures of type I and type IV secretion system components, and with the elucidation of the mechanism of fibre formation in the chaperone-usher pathway of pilus biogenesis. Finally, the structures of complexes of secreted virulence factors bound to their host targets have unravelled the mechanisms by which bacterial pathogens exploit cellular processes to their advantage.     

Bacterial adhesion and entry into host cells

Successful establishment of infection by bacterial pathogens requires adhesion to host cells, colonization of tissues, and in certain cases, cellular invasion-followed by intracellular multiplication, dissemination to other tissues, or persistence. Bacteria use monomeric adhesins/invasins or highly sophisticated macromolecular machines such as type III secretion systems and retractile type IV pili to establish a complex host/pathogen molecular crosstalk that leads to subversion of cellular functions and establishment of disease.     

Steered molecular dynamics study reveals insights into the function of the repetitive B region of collagen- and fibrinogen-binding MSCRAMMs

MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) are modular proteins covalently anchored in the bacterial cell wall of many Gram-positive bacteria. The N-terminal region of most MSCRAMMs carries the ligand-binding domains (A region) which specifically target the host extracellular matrix (ECM) proteins such as collagen, fibrinogen and fibronectin. In Staphylococcus aureus Cna, the prototype collagen-binding MSCRAMM, the A region is followed by a repetitive B region which is found to be conserved among many Gram-positive bacteria. This conservation signifies an important functional role for the B region which is made of repetitive domains. It was suggested that this region could act as a ‘stalk’ as well as a ‘spring’ to present the ligand-binding A region, away from the bacterial surface. But there is no clear functional implication of this region available till date. Each repetitive domain in the B region possesses a variant of the Ig fold called the CnaB fold. Additionally, the B repeats are also paired and the pairs are clustered together. To investigate if the B domains have a function similar to the Ig domains in the I-band region of the giant muscle protein, titin, steered molecular dynamics simulations of one, two and four B repeats of Cna were carried out. The results of the simulations suggest that the B region could provide mechanical stability, extensibility and elasticity to Cna due to the CnaB fold as well as the clustered arrangement of their domains. This study thus provided further insights into the biological underpinnings of adhesin–host interaction.     

Structural biology of Gram-positive bacterial adhesins

The structural biology of Gram-positive cell surface adhesins is an emerging field of research, whereas Gram-negative pilus assembly and anchoring have been extensively investigated and are well understood. Gram-positive surface proteins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) and individual proteins that assemble into long, hair-like organelles known as pili have similar features at the primary sequence level as well as at the tertiary structural level. Some of these conserved features are essential for their transportation from the cytoplasm and for cell wall anchoring. More importantly, the MSCRAMMs and the individual pilins are assembled with building blocks that are variants of structural modules used for human immunoglobulins. MSCRAMMs target the host's extracellular matrix proteins, such as collagen, fibrinogen, and fibronectin, and they have received considerable attention from structural biologists in the last decade, who have primarily been interested in understanding their interactions with host tissue. The recent focus is on the newly discovered pili of Gram-positive bacteria, and in this review, we highlight the advances in understanding of the individual pilus constituents and their associations and stress the similarities between the individual pilins and surface proteins.     

Distinct mechanisms of integrin binding by Yersinia pseudotuberculosis adhesins determine the phagocytic response of host macrophages

The enteropathogenic yersiniae express two outer membrane adhesins, invasin and YadA, that contribute to pathogenesis. While invasin binds directly to beta1 integrin receptors with high affinity, YadA binds indirectly through extracellular matrix (ECM) components. In this study, Yersinia pseudotuberculosis inv and yadA mutants were used to investigate how these distinct binding mechanisms compare and potentially compete in activating signalling pathways and promoting bacterial uptake by host macrophages. The efficiency of adhesin-mediated phagocytic responses was found to be dependent on the relative expression of invasin and YadA on the bacterial surface as well as the expression of ECM proteins in the extracellular milieu. Under conditions of low concentrations of ECM, invasin was found to be the dominant adhesin, promoting high levels of phagocytosis coincident with robust and sustained activation of the protein tyrosine kinases Fak and Pyk2, phosphorylation of the adaptor molecule Cas and activation of the small GTPase Rac1. In the presence of higher concentrations of ECM, YadA became the dominant functional adhesin through its ability to engage integrin receptors via an ECM bridge. We propose a model whereby invasin promotes robust and prolonged activation of phagocytic signalling cascades by inducing a 'high-affinity' integrin conformation as well as integrin clustering. We postulate that YadA-ECM promotes phagocytosis through a more transient activation of signalling cascades that arises from integrin clustering in the context of a cross-linked fibrillar ECM network.     

Identification and biochemical characterization of two novel collagen binding MSCRAMMs of Bacillus anthracis

Cell wall-anchored proteins play critical roles in the pathogenesis of infections caused by Gram-positive bacteria. Through the analysis of the genome of Bacillus anthracis Ames strain, we identified two novel putative cell wall-anchored proteins, BA0871 and BA5258, which have sequence homology to CNA, a cell wall-anchored collagen adhesin of Staphylococcus aureus. The two proteins have similar domain organization to that of CNA, with typical signal peptide sequences, a non-repetitive A region followed by repeats, and a characteristic cell wall-anchoring region. They are expressed on the surface of B. anthracis. The A regions of the two proteins were predicted to adopt similar structural folds as CNA. Circular dichroism analysis of the recombinant A regions of the two proteins (rBA0871A and rBA5258A) indicate that their secondary structure compositions are similar to those of the A regions of CNA and other cell wall-anchored adhesins. We demonstrate through solid phase binding assays and surface plasmon resonance analyses that rBA0871A and rBA5258A specifically bound type I collagen in a dose-dependent and saturable manner. Their dissociation constants (KD) for collagen are 1.6-3.2 microm for rBA0871A and 0.6-0.9 microm for rBA5258A, respectively. We further demonstrate that BA0871 and BA5258 can mediate cell attachment to collagen when expressed on the surface of a heterologous host bacterium. To our knowledge these are the first two adhesins of B. anthracis described, which may have important implications for our understanding of the pathogenic mechanisms explored by this organism.     

Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells.

Entry into host cells is an essential feature in the pathogenicity of Salmonella spp. The inv locus of Salmonella typhimurium encodes several proteins which are components of a type III protein secretion system required for these organisms to gain access to host cells. We report here the identification of several proteins whose secretion into the culture supernatant of S. typhimurium is dependent on the function of the inv-encoded translocation apparatus. Nucleotide sequence analysis of the genes encoding two of these secreted proteins, SipB and SipC, indicated that they are homologous to the Shigella sp. invasins IpaB and IpaC, respectively. An additional gene was identified, sicA, which encodes a protein homologous to IpgC, a Shigella protein that serves as a molecular chaperone for the invasins IpaB and IpaC. Nonpolar mutations in sicA, sipB, and sipC rendered S. typhimurium unable to enter cultured epithelial cells, indicating that these genes are required for bacterial internalization.     

A novel T. cruzi heparin-binding protein promotes fibroblast adhesion and penetration of engineered bacteria and trypanosomes into mammalian cells.

T. cruzi invades mammalian cells in various organs after migrating through the ECM. These activities appear to be mediated by a unique 60 kd protein exposed on the T. cruzi surface, which promotes selective adhesion of trypomastigotes to three ECM components: heparin, heparan sulfate, and collagen. The affinity-purified protein binds to host fibroblasts in a saturable and glycosaminoglycan- and collagen-inhibitable manner. When adsorbed to plastic, it promotes adhesion and spreading of fibroblasts, as does the recombinant protein expressed in E. coli. The endogenous protein, and reactive ECM proteins, are very effective in preventing T. cruzi invasion of culture cells. The recombinant protein localizes on the E. coli surface and induces the bacteria that express it to adhere to and penetrate nonphagocytic Vero cells in a proteoglycan- and collagen-inhibitable manner. Therefore, the protein, named penetrin, could play a critical role in T. cruzi binding to the ECM and to cells, and in host cell invasion.