Molecular characterization of a novel fibronectin-binding protein of Streptococcus pyogenes strains isolated from toxic shock-like syndrome patients

Group A Streptococcus pyogenes has surface-located fibronectin (Fn)-binding proteins known to be a major virulence factor, which adheres to and invades host cells. We present a novel Fn-binding protein of group A streptococcus serotype M3 and M18 strains isolated from patients with toxic shock-like syndrome (TSLS). By searching the whole genome sequence of an M3 strain from a TSLS patient, an open reading frame was found among the putative surface proteins. It possessed an LPXTG motif and Fn-binding repeat domains in the C-terminal region and was designated as FbaB (Fn-binding protein of group A streptococci type B). The fbaB gene was found in all M3 and M18 strains examined, although not in other M serotypes. Furthermore, FbaB protein was expressed on the cell surface of TSLS strains but not on non-TSLS ones. Enzyme-linked immunosorbent assay and ligand blotting revealed that recombinant FbaB exhibits a strong Fn-binding ability. An FbaB-deficient mutant strain showed 6-fold lower adhesion and invasion efficiencies to HEp-2 cells than the wild type. Moreover, mortality was decreased in mice infected with the mutant strain in comparison to the wild type. These data suggest that FbaB is etiologically involved in the development of invasive streptococcal diseases.     

Protein F2, a novel fibronectin-binding protein from Streptococcus pyogenes, possesses two binding domains

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)-binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn-binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene ( prtF2 ) from GAS strain 100076 encoding a novel Fn-binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high-affinity Fn binding. prtF2 -related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn-binding protein in GAS. Protein F2 is highly homologous to Fn-binding proteins from Streptococcus dysgalactiae and Strep-tococcus equisimilis , particularly in its carboxy-terminal portion. Two domains are responsible for Fn binding by protein F2. One domain (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non-repeated stretch of approximately 100 amino acids and is located 100 amino acids amino-terminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.     

Evidence for Steric Regulation of Fibrinogen Binding to Staphylococcus aureus Fibronectin-binding Protein A (FnBPA)

The adjacent fibrinogen (Fg)- and fibronectin (Fn)-binding sites on Fn-binding protein A (FnBPA), a cell surface protein from Staphylococcus aureus, are implicated in the initiation and persistence of infection. FnBPA contains a single Fg-binding site (that also binds elastin) and multiple Fn-binding sites. Here, we solved the structure of the N2N3 domains containing the Fg-binding site of FnBPA in the apo form and in complex with a Fg peptide. The Fg binding mechanism is similar to that of homologous bacterial proteins but without the requirement for “latch” strand residues. We show that the Fg-binding sites and the most N-terminal Fn-binding sites are nonoverlapping but in close proximity. Although Fg and a subdomain of Fn can form a ternary complex on an FnBPA protein construct containing a Fg-binding site and single Fn-binding site, binding of intact Fn appears to inhibit Fg binding, suggesting steric regulation. Given the concentrations of Fn and Fg in the plasma, this mechanism might result in targeting of S. aureus to fibrin-rich thrombi or elastin-rich tissues.     

Identification and characterization of a new ligand-binding site in FnbB,a fibronectin-binding adhesin from Streptococcus dysgalactiae

Streptococcus dysgalactiae S2, a bovine mastitis isolate, expresses the fibronectin (Fn)-binding adhesin FnbB. Here, we describe a new fibronectin-binding domain called UFnBD, located 100 amino acid N-terminal to the primary repetitive Fn-binding domain (FnBRD-B) of FnbB. UFnBD interacted with N-terminal region of Fn (N29) and this binding was mostly mediated by type I module pair 2-3 of N29 fragment, whereas FnBRD-B mainly bound to type I module pair 4-5. Furthermore, UFnBD inhibited adherence of S. dysgalactiae to Fn but at lower level as compared to FnBRD-B. UFnBD exclusively shared antigenic properties with the Fn-binding unit Du of FnbpA from Staphylococcus aureus but not with ligand-binding domains or motifs of other adhesins, while Fn-induced determinants of FnBRD-B and other adhesins appeared to be conformationally related. Consistent with this, a monoclonal antibody 7E11 generated from a mouse immunized with FnbB, and that recognized UFnBD did not cross-react with FnBRD-B. The epitope for 7E11 was mapped to 40 amino acid long segment within UFnBD and interaction between the antibody and the epitope was specifically induced by Fn or N29. A similar antibody epitope was observed in Streptococcus pyogenes strains suggesting the presence of an adhesin bearing epitope related to FnbB.     

Effect of adsorbed fibronectin on the differential adhesion of osteoblast-like cells and Staphylococcus aureus with and without fibronectin-binding proteins

The influence of fibronectin (Fn) coated surfaces patterned with poly(ethylene glycol) microgels having inter-gel spacings between 0.5 and 3.0 μm on the adhesion of Staphylococcus aureus strains with and without Fn-binding proteins and cellular adhesion/spreading was investigated. Quantitative force measurements between a S. aureus cell and a patterned surface showed that the adhesion force between the bacterium and the patterned surface increased substantially after Fn adsorption, regardless of the strain used, but decreased with decreasing inter-gel spacing. In flow-chamber experiments, the Fn-binding strain adhered at a higher rate after Fn adsorption than the strain lacking Fn-binding proteins. In both cases, the adhesion rates decreased with decreasing inter-gel spacing. Osteoblast-like cells could bind to patterned surfaces despite the microgels, and adsorbed Fn substantially amplified this effect. Even under highly non-adhesive conditions associated with closely spaced microgels, adsorbed Fn preserves a window of inter-gel spacing around 1 μm where the adhesion of staphylococcal cells is hindered while cells can still adhere and spread.     

Novel mycobacteria antigen 85 complex binding motif on fibronectin

The members of the antigen 85 protein family (Ag85), consisting of members Ag85A, Ag85B, and Ag85C, are the predominantly secreted proteins of mycobacteria and possess the ability to specifically interact with fibronectin (Fn). Because Fn-binding proteins are likely to be important virulence factors of Mycobacterium spp., Ag85 may contribute to the adherence, invasion, and dissemination of organisms in host tissue. In this study, we reported the Fn binding affinity of Ag85A, Ag85B, and Ag85C from Mycobacterium avium subsp. paratuberculosis (MAP) (K(D) values were determined from 33.6 to 68.4 nm) and mapped the Ag85-binding motifs of Fn. Fn14, a type III module located on the heparin-binding domain II (Hep-2) of Fn, was discovered to interact with Ag85 from MAP. The peptide inhibition assay subsequently demonstrated that a peptide consisting of residues 17-26 from Fn14 ((17)SLLVSWQPPR(26), termed P17-26) could interfere with Ag85B binding to Fn (73.3% reduction). In addition, single alanine substitutions along the sequence of P17-26 revealed that the key residues involved in Ag85-Fn binding likely contribute through hydrophobic and charge interactions. Moreover, binding of Ag85 on Fn siRNA-transfected Caco2 cells was dramatically reduced (44.6%), implying the physiological significance of the Ag85-Fn interaction between mycobacteria and host cells during infection. Our results indicate that Ag85 binds to Fn at a novel motif and plays a critical role in mycobacteria adherence to host cells by initiating infection. Ag85 might serve as an important colonization factor potentially contributing to mycobacterial virulence.     

Identification of a fibronectin-binding domain within the Campylobacter jejuni CadF protein

The binding of Campylobacter jejuni to fibronectin (Fn), a component of the extracellular matrix, is mediated by a 37 kDa outer membrane protein termed CadF for Campylobacter adhesion to Fn. Previous studies have indicated that C. jejuni binds to Fn on the basolateral surface of T84 human colonic cells. To further characterize the interaction of the CadF protein with Fn, enzyme-linked immunosorbent assays were performed to identify the Fn-binding domain (Fn-BD). Using overlapping 30-mer and 16-mer peptides derived from translated cadF nucleotide sequence, maximal Fn-binding activity was localized to four amino acids (AA 134-137) consisting of the residues phenylalanine-arginine-leucine-serine (FRLS). A mouse alpha-CadF peptide polyclonal antibody (M alpha-CadF peptide pAb) was generated using FRLS containing peptides and found to react with viable C. jejuni as judged by indirect fluorescent microscopy, suggesting that the FRLS residues are surface-exposed. Binding of CadF to purified Fn and INT 407 human epithelial cells was significantly inhibited with peptides containing the Fn-BD. Moreover, a CadF recombinant variant protein, in which the Phe-Arg-Leu residues (CadF AA 134-136) were altered to Ala-Ala-Gly, exhibited a 91% decrease in Fn-binding activity as compared with the wild-type CadF protein. Collectively, these data indicate that the FRLS residues (CadF AA 134-137) of the C. jejuni CadF protein possess Fn-binding activity.     

Inhibition of fibronectin binding of some bacterial cells by subtle pH increase within the physiological range

The fibronectin (Fn)-binding ability of microorganisms is considered to be involved in their pathogenicities. Granulicatella adiacens, a member of the oral flora and a causative agent of culture-negative infective endocarditis, showed nearly maximum binding to immobilized Fn at pH 7.2 but greatly reduced binding at a slightly higher pH 7.4 and almost no binding at pH 7.6 in the presence of physiological concentration of NaCl (0.15 M). A similar pH-sensitive Fn-binding property was noted with Escherichia coli and Abiotrophia defectiva, but not with Streptococcus pyogenes nor Staphylococcus aureus. In contrast, bindings to laminin and fibrinogen observed for some of these strains were unaffected by the same pH changes. This fastidious pH-dependency of Fn-binding abilities of some bacteria warns that the pH condition must be seriously considered in the in vitro assay of bacterial adherence to fibronectin.     

Effects of depleting the essential central metabolic enzyme fructose-1,6-bisphosphate aldolase on the growth and viability of Candida albicans: implications for antifungal drug target discovery

The central metabolic enzyme fructose-1,6-bisphosphate aldolase (Fba1p) catalyzes a reversible reaction required for both glycolysis and gluconeogenesis. Fba1p is a potential antifungal target because it is essential in yeast and because fungal and human aldolases differ significantly. To test the validity of Fba1p as an antifungal target, we have examined the effects of depleting this enzyme in the major fungal pathogen Candida albicans. Using a methionine/cysteine-conditional mutant (MET3-FBA1/fba1), we have shown that Fba1p is required for the growth of C. albicans. However, Fba1p must be depleted to below 5% of wild-type levels before growth is blocked. Furthermore, Fba1p depletion exerts static rather than cidal effects upon C. albicans. Fba1p is a relatively abundant and stable protein in C. albicans, and hence, Fba1p levels decay relatively slowly following MET3-FBA1 shutoff. Taken together, our observations can account for our observation that the virulence of MET3-FBA1/fba1 cells is only partially attenuated in the mouse model of systemic candidiasis. We conclude that an antifungal drug directed against Fba1p would have to be potent to be effective.     

The R28 protein of Streptococcus pyogenes is related to several group B streptococcal surface proteins, confers protective immunity and promotes binding to human epithelial cells

The R28 protein is a surface molecule expressed by some strains of Streptococcus pyogenes (group A streptococcus). Here, we present evidence that R28 may play an important role in virulence. Sequence analysis demonstrated that R28 has an extremely repetitive sequence and can be viewed as a chimera derived from the three surface proteins Rib, alpha and beta of the group B streptococcus (GBS). Thus, the gene encoding R28 may have originated in GBS. The R28 protein promotes adhesion to human epithelial cells, as shown by experiments with an R28-negative mutant and by the demonstration that antibodies to highly purified R28 inhibited adhesion. In a mouse model of lethal intraperitoneal S. pyogenes infection, antibodies to R28 conferred protective immunity. However, the virulence of an R28-negative mutant was similar to that of the parental strain in the intraperitoneal infection model. Together, these data indicate that R28 represents a novel type of adhesin expressed by S. pyogenes and that R28 may also act as a target for protective antibodies at later stages of an infection. We consider the hypothesis that R28 played a pathogenetic role in the well-known epidemics of childbed fever (puerperal fever), which were caused by S. pyogenes. A role for R28 in these epidemics is suggested by epidemiological data.     

A novel bacterial resistance mechanism against human group IIA-secreted phospholipase A2: role of Streptococcus pyogenes sortase A

Human group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) is a bactericidal molecule important for the innate immune defense against Gram-positive bacteria. In this study, we analyzed its role in the host defense against Streptococcus pyogenes, a major human pathogen, and demonstrated that this bacterium has evolved a previously unidentified mechanism to resist killing by sPLA(2)-IIA. Analysis of a set of clinical isolates demonstrated that an ~500-fold higher concentration of sPLA(2)-IIA was required to kill S. pyogenes compared with strains of the group B Streptococcus, which previously were shown to be sensitive to sPLA(2)-IIA, indicating that S. pyogenes exhibits a high degree of resistance to sPLA(2)-IIA. We found that an S. pyogenes mutant lacking sortase A, a transpeptidase responsible for anchoring LPXTG proteins to the cell wall in Gram-positive bacteria, was significantly more sensitive (~30-fold) to sPLA(2)-IIA compared with the parental strain, indicating that one or more LPXTG surface proteins protect S. pyogenes against sPLA(2)-IIA. Importantly, using transgenic mice expressing human sPLA(2)-IIA, we showed that the sortase A-mediated sPLA(2)-IIA resistance mechanism in S. pyogenes also occurs in vivo. Moreover, in this mouse model, we also showed that human sPLA(2)-IIA is important for the defense against lethal S. pyogenes infection. Thus, we demonstrated a novel mechanism by which a pathogenic bacterium can evade the bactericidal action of sPLA(2)-IIA and we showed that sPLA(2)-IIA contributes to the host defense against S. pyogenes infection.     

Elongation factor Tu and E1 beta subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae

The interactions between pathogenic bacteria and extracellular matrix (ECM) components markedly influence the initiation and establishment of infection. We have identified two surface proteins of virulent Mycoplasma pneumoniae with molecular masses of 45 and 30 kDa that bind to the ECM constituent, fibronectin (Fn). These Fn-binding proteins (FnBPs) were purified to near homogeneity using Fn-coupled Sepharose 4B-affinity column chromatography, and amino acid sequence analysis of the 45 and the 30 kDa proteins identified them as elongation factor Tu (EF-Tu) and pyruvate dehydrogenase E1 beta subunit (PDH-B) respectively. The genes for EF-Tu and PDH-B were cloned, and the entire EF-Tu gene and NH2-terminus of PDH-B (NPDH (pyruvate dehydrogenase E1 beta subunit from amino acid 1-244)-B) gene were overexpressed in Escherichia coli. The recombinant proteins, rEF-Tu and rNPDH-B, were purified to homogeneity by His-tag affinity column chromatography and used to immunize rabbits. Purified rEF-Tu and rNPDH-B bound to Fn using a ligand immunoblot assay and ELISA. Immunogold electron microscopy with polyclonal antibodies reactive against rEF-Tu (antirEF-Tu) and rNPDH-B (antirNPDH-B) and whole cell radioimmunoprecipitation (WCRIP) revealed the surface location of these proteins. Adherence of viable M. pneumoniae to immobilized Fn was inhibited by antirEF-Tu and antirNPDH-B antisera in a dose-dependent and cumulative manner. These results demonstrate that M. pneumoniae EF-Tu and PDH-B, in addition to their major cytoplasmic biosynthetic and metabolic roles, can be surface translocated, which confers additional important biological functions.     

Structural and functional analysis of the tandem β-zipper interaction of a Streptococcal protein with human fibronectin

Bacterial fibronectin-binding proteins (FnBPs) contain a large intrinsically disordered region (IDR) that mediates adhesion of bacteria to host tissues, and invasion of host cells, through binding to fibronectin (Fn). These FnBP IDRs consist of Fn-binding repeats (FnBRs) that form a highly extended tandem β-zipper interaction on binding to the N-terminal domain of Fn. Several FnBR residues are highly conserved across bacterial species, and here we investigate their contribution to the interaction. Mutation of these residues to alanine in SfbI-5 (a disordered FnBR from the human pathogen Streptococcus pyogenes) reduced binding, but for each residue the change in free energy of binding was <2 kcal/mol. The structure of an SfbI-5 peptide in complex with the second and third F1 modules from Fn confirms that the conserved FnBR residues play equivalent functional roles across bacterial species. Thus, in SfbI-5, the binding energy for the tandem β-zipper interaction with Fn is distributed across the interface rather than concentrated in a small number of "hot spot" residues that are frequently observed in the interactions of folded proteins. We propose that this might be a common feature of the interactions of IDRs and is likely to pose a challenge for the development of small molecule inhibitors of FnBP-mediated adhesion to and invasion of host cells.     

Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of fibronectin to facilitate cell binding by Yersinia pestis

The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.     

The fibronectin-binding MSCRAMM FnbpA of Staphylococcus aureus is a bifunctional protein that also binds to fibrinogen

Staphylococcus aureus is an important pathogen capable of causing a wide spectrum of diseases in humans and animals. This bacterium expresses a variety of virulence factors that participate in the process of infection. These include MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that mediate the adherence of the bacteria to host extracellular matrix components, such as collagen, fibronectin (Fn), and fibrinogen (Fg). Two Fn-binding MSCRAMMs, FnbpA and FnbpB, have been previously identified. The Fn binding activity has been localized to the approximately 40-amino acid residue D repeats in the C-terminal part of these proteins. However, no biological activity has yet been attributed to the N-terminal A regions of these proteins. These regions exhibit substantial amino acid sequence identity to the A regions of other staphylococcal MSCRAMMs, including ClfA, ClfB, and SdrG (Fbe), all of which bind Fg. This raises the question of whether the Fn-binding MSCRAMMs can also bind specifically to Fg. In this report, we show that a recombinant form of the A region of FnbpA does specifically recognize Fg. We localize the binding site in Fg for recombinant FnbpA to the gamma-chain, in particular to the C-terminal residues of this polypeptide, the site also recognized by ClfA. In addition, we demonstrate that recombinant FnbpA can compete with ClfA for binding to both immobilized and soluble Fg. By the use of surface plasmon resonance spectroscopy and fluorescence polarization, we determine the dissociation equilibrium constant for the interaction of recombinant FnbpA with intact immobilized Fg and with a synthetic C-terminal gamma-chain peptide, respectively. Finally, by overexpressing FnbpA in a mutant strain of S. aureus that lacks the expression of both ClfA and ClfB, we show that native FnbpA can mediate the interaction of S. aureus with soluble Fg.     

Interaction of calcium-bound C-reactive protein with fibronectin is controlled by pH: in vivo implications

C-reactive protein (CRP) binds with high affinity to fibronectin (Fn), a major component of the extracellular matrix (ECM), but at physiological pH the binding is inhibited by calcium ions (Ca2+). Because CRP circulates in the blood in Ca2+ -bound form, the occurrence of CRP-Fn interactions in vivo has been doubtful. To define the basis of inhibition of CRP-Fn interaction by Ca2+ at pH 7.0, we hypothesized that Fn-binding site on CRP consisted of amino acids co-ordinating Ca2+. Site-directed mutagenesis of amino acids co-ordinating Ca2+ drastically decreased the binding of CRP to Fn, indicating that the Ca2+ -binding site indeed formed the Fn-binding site. To determine the requirements for possible interaction between Ca2+ -bound CRP and Fn, we investigated inhibition of CRP-Fn interaction by Ca2+ as a function of pH. Ca2+ did not inhibit binding of CRP to Fn at pH 6.5 and lower. The contrasting Fn binding properties of CRP at physiological and mildly acidic pH indicated that the interaction of Ca2+ -bound CRP with Fn was controlled by pH. We conclude that the inhibition of binding of CRP to Fn by Ca2+ at pH 7.0 is a mechanism to prevent CRP-Fn interactions under normal conditions. CRP, in its Ca2+ -bound state, is capable of binding Fn but only at the inflammatory sites and tumors with low pH. CRP, Fn, and the ECM all have been implicated in cancer. Taken together our data raise the possibility that CRP-Fn interactions may change the architecture of ECM to modify the development of tumors.     

Actions of the functional upstream domain of protein F1 of Streptococcus pyogenes on the conformation of fibronectin

Fibronectin (Fn), discovered by Harvard's Plasma Protein Program as plasma "cold-insoluble globulin" in the 1940s, has attracted much interest over the past three decades. One of the most interesting features of Fn is its ability to change shape in response to various environmental conditions and interactions with other substances found in the extra-cellular space. Here we examine the potential of the functional upstream domain (FUD) of Streptococcus pyogenes protein F1 to bring about changes in structure of Fn. In particular, we investigate the accessibility of Fn's 10th type III module that contains the integrin binding RGD motif. By use of monoclonal antibodies in a competitive ELISA assay, we found that FUD interacts with the amino-terminal type I modules of Fn to unveil the cell-binding region of Fn. This conformational change was achieved at sub-equimolar ratios of FUD/Fn monomer. We discuss the functional relevance of the interaction for both Fn and S. pyogenes and correlate the results with a conformational model of Fn that arose out of a collaboration between our laboratory and that of John Ferry.     

Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein

The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment. The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no. M31161). The spiralin gene was identified by the size and amino acid composition of its translational product. Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs). The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk. The product of the fifth ORF remains to be identified and was named protein X (X gene). The order of the above genes was tsf--X--spiralin gene--pfk--pyk. These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction.     

Functional and structural investigations of fibronectin-binding protein Apa from Mycobacterium tuberculosis

BackgroundAlanine and proline-rich protein (Apa) is a secreted antigen of Mycobacterium spp. which involves in stimulating immune responses and adhering to host cells by binding to fibronectin (Fn). Here, we report the crystal structure of Apa from Mycobacterium tuberculosis (Mtb) and its Fn-binding characteristics.MethodsThe crystal structure of Mtb Apa was determined at resolutions of 1.54 Å. The dissociation constants (K_D) of Apa and individual modules of Fn were determined by surface plasmon resonance and enzyme-linked immunosorbent assay. Site-directed mutagenesis was performed to investigate the putative Fn-binding motif of Apa.ResultsMtb Apa folds into a large seven-stranded anti-parallel β-sheet which is flanked by three α-helices. The binding affinity of Mtb Apa to individual Fn modules was assessed and the results indicated that the Mtb Apa binds to FnIII-4 and FnIII-5 of Fn CBD segment. Notably, structure analysis suggested that the previously proposed Fn-binding motif ²⁵⁸RWFV²⁶¹ is buried within the protein and may not be accessible to the binding counterpart.ConclusionsThe structural and Fn-binding characteristics we reported here provide molecular insights into the multifunctional protein Mtb Apa. FnIII-4 and FnIII-5 of CBD are the only two modules contributing to Apa-Fn interaction.General significanceThis is the first study to report the structure and Fn-binding characteristics of mycobacterial Apa. Since Apa plays a central role in stimulating immune responses and host cells adhesion, these results are of great importance in understanding the pathogenesis of mycobacterium. This information shall provide a guidance for the development of anti-mycobacteria regimen.     

Characterization of a novel fibronectin-binding surface protein in group A streptococci

Streptococcus pyogenes interacts with host fibronectin via distinct surface components. One of these components is the Sfbl protein (streptococcal fibronectin-binding protein, now specified as class I), an adhesin that represents a protein family with characteristic features. Here we present the complete structure of a novel fibronectin-binding protein of S. pyogenes , designated SfbII, which is distinct from the previously described Sfbl proteins. The sfbII gene originated from a λ EMBL3 library of chromosomal DNA from group A streptococcal strain A75 and coded for a 113kDa protein exhibiting features of membrane-anchored surface proteins of Gram-positive cocci. The expression of biologically active fusion proteins allowed the determination of the location of the fibronectin-binding domain within the C-terminal part of the protein. It consisted of two and a half repeats which share common motifs with fibronectin-binding repeats of other streptococcal and staphylococcal proteins. Purified recombinant fusion protein containing this domain competitively inhibited the binding of fibronectin to the parental S. pyogenes strain. Furthermore, polyclonal antibodies against the binding domain specifically blocked the Sfbll receptor site on the streptococcal surface. No cross-reactivity could be detected between anti-Sfbll antibodies and the sfbl gene product, and vice versa, indicating that the two proteins do not share common immunogenic epitopes. Southern hybridization experiments performed with specific sfbll gene probes revealed the presence of the sfbll gene in more than 55% of 93 streptococcal isolates tested. The majority of the strains also harboured the sfbl gene, and 86% carried at least one of the two sfb genes.