The A domain of fibronectin-binding protein B of Staphylococcus aureus contains a novel fibronectin binding site

The fibronectin-binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μM). The binding site for FnBPB in fibrinogen was localized to the C-terminus of the γ-chain. Like clumping factor A, region A of FnBPB bound to the γ-chain of fibrinogen in a Ca(2+)-inhibitable manner. The deletion of 17 residues from the C-terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock-lock-latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with K(D) = 2.5 μM despite lacking any of the known fibronectin-binding tandem repeats. A truncate lacking the C-terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, K(D) of 20 μM, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin-binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression.     

The N-terminal A domain of Staphylococcus aureus fibronectin-binding protein A binds to tropoelastin

Staphylococcus aureus is an important human pathogen. Its virulence factors include a variety of MSCRAMMs (microbial surface component recognizing adhesive matrix molecules), each capable of binding specifically to the host extracellular matrix. The fibronectin-binding protein, FnBPA, has been shown previously to bind immobilized fibronectin, fibrinogen, and alpha-elastin peptides. Here we show that region A of FnBPA (rAFnBPA) binds to recombinant human tropoelastin. Binding occurs to three separate truncates of tropoelastin, encompassing domains 2-18, 17-27, and 27-36, signifying that the interaction occurs at multiple sites. The greatest affinity was for the N-terminal truncate. We observed a pH dependency for the rAFnBPA-tropoelastin interaction with strong, nonsaturable binding at low pH. The interaction ceased at higher pH. These data support a model of surface-surface interactions between the negative charges present on rAFnBPA and the positive lysines of tropoelastin. A protein lacking the negatively charged C-terminal fibronectin-binding motif of the A domain of FnBPA and another construct lacking subdomain N1 were both capable of binding immobilized tropoelastin with a lower affinity. The binding properties of five site-directed mutants of rAFnBPA were compared with wild-type rAFnBPA. There was no decreased affinity for immobilized tropoelastin, in contrast to the defective binding of these mutants to alpha-elastin and fibrinogen. The data indicate novel interactions between tropoelastin and FnBPA that include the use of surface charges. These results demonstrate that FnBPA is capable of directly binding tropoelastin prior to its incorporation into elastin.     

Two different genes encode fibronectin binding proteins in Staphylococcus aureus. The complete nucleotide sequence and characterization of the second gene.

A gene encoding a fibronectin binding protein (FnBP) has recently been isolated and sequenced from Staphylococcus aureus strain 8325-4. In the same bacterial strain, 682 bp downstream to the stop codon of this gene (fnbA), a second gene termed fnbB has not been discovered, encoding another FnBP (FnBPB). The two genes show in large parts striking sequence homologies. The complete amino acid sequence encoded by fnbB has been deduced and compared to that deduced from fnbA. In FnBPB a stretch of 66 amino acids downstream to the signal peptide has 75% identity with the corresponding region in FnBPA. At the C-terminal site another 394 amino acid stretch is almost identical in both gene products. This stretch contains the 38 amino acid long D repeats, the wall spanning Wr repeats and the hydrophobic membrane spanning domain. In FnBPA each of the three D repeats has been identified as a fibronectin binding structure. These structures are highly conserved in FnBPB and most likely represent the major Fn-binding domain of this protein. However, a subclone of gene fnbB lacking the coding region for the D repeats also clearly expresses fibronectin binding activity. This additional binding site is so far unique for FnBPB and interacts like the D domains with the N-terminal 24-31-kDa fragment of fibronectin. The purified recombinant FnBP fragment (not containing the D repeats) completely inhibits the binding of fibronectin to whole cells of S. aureus.     

Fibrinogen and elastin bind to the same region within the A domain of fibronectin binding protein A, an MSCRAMM of Staphylococcus aureus

The fibronectin binding protein, FnBPA, is a multifunctional microbial surface component recognizing adhesive matrix molecule (MSCRAMM) that promotes bacterial adherence to immobilized fibrinogen and elastin via the N-terminal A domain. The binding site for fibrinogen and elastin was localized to subdomains N2N3. A three-dimensional structural model of FnBPA was created based on the known crystal structure of the domains N2N3 of clumping factor A (ClfA). The role of individual residues in the putative ligand binding trench was examined by testing the affinity of mutants for fibrinogen and elastin. Two residues (N304 and F306) were crucial for binding both ligands and are in the equivalent positions to residues known to be important for fibrinogen binding by ClfA. A peptide comprising the C-terminus of the gamma-chain of fibrinogen and a monoclonal anti-rAFnBPA antibody were potent inhibitors of the FnBPA-elastin interaction. This suggests that FnBPA binds to fibrinogen and elastin in a similar manner. Amino acid sequence divergence of 26.5% occurred between the A domains of FnBPA from strains 8325-4 and P1. Most variant residues were predicted to be located on the surface of domains N2N3 while few occurred in the putative ligand binding trench and the latching peptide explaining limited immunocross reactivity while ligand binding activity is conserved.     

Fibronectin-binding protein B variation in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Background  

Fibronectin binding proteins A and B (FnBPA and FnBPB) mediate adhesion of S. aureus to fibrinogen, elastin and fibronectin. We previously identified seven different isotypes of FnBPA based on divergence in the fibrinogen- and elastin-binding A domains. The variation created differences in antigenicity while ligand binding functions were retained. Here, FnBPB variation was examined in both human and bovine isolates and compared to that of FnBPA.     

The fibronectin binding proteins of Staphylococcus aureus are required for adhesion to and invasion of bovine mammary gland cells

We recently described adhesion to and invasion of bovine mammary gland cells by Staphylococcus aureus in vitro. Here, we show that the levels of adhesion and invasion are dependent on the bacterial growth phase and are controlled by the agr locus. Incubation of exponential growth phase cells of S. aureus with mammary gland cells resulted in bacterial cell clumping. Strains of S. aureus deficient in expression of the fibronectin binding proteins (FnBPA and FnBPB) failed to clump and their ability to adhere to and to invade the bovine mammary gland cells is strongly reduced. This indicates that the fibronectin binding proteins are essential for S. aureus adhesion to and invasion of bovine mammary gland cells.     

The N-terminal domain of elastin-binding protein of Staphylococcus aureus changes its secondary structure in a membrane-mimetic environment

Elastin-binding protein of Staphylococcus aureus (EbpS) has been identified as an adhesin that can bind to soluble elastin or tropoelastin. However, the structure and exact function of EbpS remain to be elucidated. To gain insight into the molecular characteristics of EbpS, we investigated the physical properties of its N-terminal extracellular domain in various environments. CD spectroscopy showed that this protein was soluble and unstructured under aqueous conditions. Non-native secondary structures, however, were induced by several alcohols that provided membrane-mimetic environments. These changes may have some correlation with the function of this protein.     

Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor

Staphylococcus aureus is a leading cause of infective endocarditis (IE). Platelet activation promoted by S. aureus resulting in aggregation and thrombus formation is an important step in the pathogenesis of IE. Here, we report that the fibrinogen/fibronectin-binding proteins FnBPA and FnBPB are major platelet-activating factors on the surface of S. aureus from the exponential phase of growth. Truncated derivatives of FnBPA, presenting either the fibrinogen-binding A domain or the fibronectin-binding BCD region, each promoted platelet activation when expressed on the surface of S. aureus or Lactococcus lactis, indicating two distinct mechanisms of activation. FnBPA-promoted platelet activation is mediated by fibrinogen and fibronectin bridges between the A domain and the BCD domains, respectively, to the low affinity form of the integrin GPIIb/IIIa on resting platelets. Antibodies recognizing the FnBPA A domain or the complex between the FnBPA BCD domains and fibronectin were essential for activation promoted by bacteria expressing the A domain or the BCD domain respectively. Activation was inhibited by a monoclonal antibody (IV-3) specific for the FcgammaRIIa IgG receptor on platelets. We propose that the activation of quiescent platelets by bacteria expressing FnBPs involves the formation of a bridge between the bacterial cell and the platelet surface by (i) fibronectin and fibrinogen interacting with the low affinity form of GPIIb/IIIa and (ii) by antibodies specific to FnBPs that engage the platelet Fc receptor FcgammaRIIa. Platelet activation by S. aureus clinical IE isolates from both the exponential and stationary phases of growth was completely inhibited by monoclonal antibody IV-3 suggesting that the IgG-FcgammaRIIa interaction is of fundamental importance for platelet activation mediated by this organism. This suggests new avenues for development of therapeutics against vascular infections.     

Borrelia burgdorferi binds fibronectin through a tandem beta-zipper, a common mechanism of fibronectin binding in staphylococci, streptococci, and spirochetes

BBK32 is a fibronectin-binding protein from the Lyme disease-causing spirochete Borrelia burgdorferi. In this study, we show that BBK32 shares sequence similarity with fibronectin module-binding motifs previously identified in proteins from Streptococcus pyogenes and Staphylococcus aureus. Nuclear magnetic resonance spectroscopy and isothermal titration calorimetry are used to confirm the binding sites of BBK32 peptides within the N-terminal domain of fibronectin and to measure the affinities of the interactions. Comparison of chemical shift perturbations in fibronectin F1 modules on binding of peptides from BBK32, FnBPA from S. aureus, and SfbI from S. pyogenes provides further evidence for a shared mechanism of binding. Despite the different locations of the bacterial attachment sites in BBK32 compared with SfbI from S. pyogenes and FnBPA from S. aureus, an antiparallel orientation is observed for binding of the N-terminal domain of fibronectin to each of the pathogens. Thus, these phylogenetically and morphologically distinct bacterial pathogens have similar mechanisms for binding to human fibronectin.     

Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes

Staphylococcus aureus 8325-4 has the potential to express two distinct cell wall-associated fibronectin-binding proteins called FnBPA and FnBPB. In order to test if both proteins are expressed in S. aureus and if both are required for promoting bacterial adhesion to fibronectin-coated surfaces, insertion mutations were isolated in each gene. A DNA fragment encoding tetracycline resistance was inserted into fnbA and a fragment encoding erythromycin resistance was inserted into fnbB . A double fnbA fnbB mutant was also constructed. The fnbA and fnbB single mutants showed no significant reduction in their adhesion to polymethylmethacrylate coverslips that had been coated in vitro with fibronectin. However, the double mutant was completely defective in adhesion. Monospecific antibodies directed against the non-conserved N-terminal regions of both proteins confirmed the lack of expression of FnBPs in the mutant strains. Wild-type fnbA and fnbB genes cloned seperately on a multicopy plasmid were each able to restore fully the adhesion-defective phenotype of the 8325-4 fnbA fnbB mutant. This demonstrates that both fnb genes are expressed in S. aureus and that both contribute to the ability of strain 8325-4 to adhere to fibronectin-coated surfaces. The double mutant was also defective in adhesion to coverslips that had been removed from tissue cages implanted subcutaneously in guinea-pigs, which suggests that fibronectin is important in promoting attachment of S. aureus to biomaterial in vivo .     

Group B streptococci adhere to a variant of fibronectin attached to a solid phase

Group B streptococci (GBS) are the leading cause of neonatal pneumonia and meningitis. Adherence of GBS to host tissues may play an important role in the pathogenesis of infection. The host molecules which mediate GBS adherence to host tissues are unknown. Many bacterial pathogens adhere to fibronectin, an important component of the extracellular matrix (ECM). Some pathogens adhere to both immobilized and soluble fibronectin, while others adhere to immobilized fibronectin, but not to soluble fibronectin. Previous data indicated that GBS do not adhere to soluble fibronectin. We studied the ability of GBS to adhere to immobilized fibronectin. Forty-five per cent of the input inoculum of COH1, a virulent GBS isolate, adhered to fibronectin immobilized on polystyrene. COH1 did not adhere to the other ECM proteins tested (laminin, type I collagen, vitronectin, and tenascin). Nine out of nine GBS strains from human sources tested adhered specifically to fibronectin at levels varying from 4–60%. We considered the possibility that GBS were adherent to a contaminant in the fibronectin preparation. Properties of fibronectin, including the presence of an immunologic epitope of fibronectin and binding to collagen, were verified to be properties of the molecule to which GBS adhere. COH1 adhered to fibronectin captured by a monoclonal antibody to fibronectin (FN-15), confirming that the molecule to which GBS adhere bears immunologic determinants of fibronectin. Adherence of COH1 to fibronectin was inhibited by collagen, confirming that the molecule to which GBS adhere binds to collagen. These data strongly suggest that GBS adhere to fibronectin, and not to a contaminant. Protein blot analysis revealed that GBS were adherent to a high-molecular-weight variant of non-reduced fibronectin monomers and dimers. GBS did not adhere to reduced fibronectin monomers. We conclude that GBS adhere to a variant of plasma fibronectin when attached to a solid phase.     

Sequence diversity in the A domain of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>fibronectin-binding protein A

Background  

Fibronectin-binding protein A (FnBPA) mediates adhesion of Staphylococcus aureus to fibronectin, fibrinogen and elastin. We previously reported that S. aureus strain P1 encodes an FnBPA protein where the fibrinogen/elastin-binding domain (A domain) is substantially divergent in amino acid sequence from the archetypal FnBPA of S. aureus NCTC8325, and that these variations created differences in antigenicity. In this study strains from multilocus sequence types (MLST) that spanned the genetic diversity of S.aureus were examined to determine the extent of FnBPA A domain variation within the S. aureus population and its effect on ligand binding and immuno-crossreactivity.     

The tandem beta-zipper model defines high affinity fibronectin-binding repeats within Staphylococcus aureus FnBPA

Binding of the fibronectin-binding protein FnBPA from Staphylococcus aureus to the human protein fibronectin has previously been implicated in the development of infective endocarditis, specifically in the processes of platelet activation and invasion of the endothelium. We recently proposed a model for binding of fibronectin to FnBPA in which the bacterial protein contains 11 potential binding sites (FnBPA-1 to FnBPA-11), each composed of motifs that bind to consecutive fibronectin type 1 modules in the N-terminal domain of fibronectin. Here we show that six of the 11 sites bind with dissociation constants in the nanomolar range; other sites bind more weakly. The high affinity binding sites include FnBPA-1, the sequence of which had previously been thought to be encompassed by the fibrinogen-binding A domain of FnBPA. Both the number and sequence conservation of the type-1 module binding motifs appears to be important for high affinity binding. The in vivo relevance of the in vitro binding studies is confirmed by the presence of antibodies in patients with S. aureus infections that specifically recognize complexes of these six high affinity repeats with fibronectin.     

Subdomains N2N3 of Fibronectin Binding Protein A Mediate Staphylococcus aureus Biofilm Formation and Adherence to Fibrinogen Using Distinct Mechanisms

Health care-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) forms biofilm in vitro that is dependent on the surface-located fibronectin binding proteins A and B (FnBPA, FnBPB). Here we provide new insights into the requirements for FnBP-dependent biofilm formation by MRSA. We show that expression of FnBPs is sustained at high levels throughout the growth cycle in the HA-MRSA strain BH1CC in contrast to laboratory strain SH1000, where expression could be detected only in exponential phase. We found that FnBP-mediated biofilm accumulation required Zn²⁺, while the removal of Zn²⁺ had no effect on the ability of FnBPA to mediate bacterial adherence to fibrinogen. We also investigated the role of FnBPA expressed on the surface of S. aureus in promoting biofilm formation and bacterial adhesion to fibrinogen. The minimum part of FnBPA required for ligand binding has so far been defined only with recombinant proteins. Here we found that the N1 subdomain was not required for biofilm formation or for FnBPA to promote bacterial adherence to fibrinogen. Residues at the C terminus of subdomain N3 required for FnBPA to bind to ligands using the “dock, lock, and latch” mechanism were necessary for FnBPA to promote bacterial adherence to fibrinogen. However, these residues were not necessary to form biofilm, allowing us to localize the region of FnBPA required for biofilm accumulation to residues 166 to 498. Thus, FnBPA mediates biofilm formation and bacterial adhesion to fibrinogen using two distinct mechanisms. Finally, we identified a hitherto-unrecognized thrombin cleavage site close to the boundary between subdomains N1 and N2 of FnBPA.     

Variation and association of fibronectin‐binding protein genes fnbA and fnbB in Staphylococcus aureus Japanese isolates

Fibronectin‐binding proteins A and B (FnBPA and FnBPB) mediate adhesion of Staphylococcus aureus to fibrinogen, elastin and fibronectin. FnBPA and FnBPB are encoded by two closely linked genes, fnbA and fnbB, respectively. With the exception of the N‐terminal regions, the amino acid sequences of FnBPA and FnBPB are highly conserved. To investigate the genetics and evolution of fnbA and fnbB, the most variable regions, which code for the 67th amino acids of the A through B regions (A67–B) of fnbA and fnbB, were focused upon. Eighty isolates of S. aureus in Japan were sequenced and 19 and 18 types in fnbA and fnbB, respectively, identified. Although the phylogeny of fnbA and fnbB were found to be quite different, each fnbA type connected with a specific fnbB type, indicating that fnbA and fnbB mutate independently, whereas the combination of both genes after recombination is stable. Hence those fnbA–fnbB combinations were defined as FnBP sequence types (FnSTs). Representative isolates of each FnST were assigned distinct STs by multilocus sequence typing, suggesting correspondence of FnST with genome lineage. Linkage disequilibrium (LD) analysis of the A67–B region revealed that subdomains N2, N3 and FnBR1 form a LD block in fnbA, whereas N2 and N3 form two independent LD blocks in fnbB. N2–N3 three‐dimensional structural models indicated that not only the variable amino acid residues, but also well‐conserved amino acid residues between FnBPA and FnBPB, are located on the surface of the protein. These results highlight a molecular process of the FnBP that has evolved by mingled mutation and recombination with retention of functions.     

Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by enzyme-linked immunosorbent assay

Abstract Antibody titres against fibronectin-binding protein (FnBP) of Staphylococcus aureus were determined in sera from rabbits immunized with staphylococcal whole cells or purified native fibronectin receptor. An ELISA technique for detection of antibody titres blocking the binding of soluble fibronectin to immobilized FnBP was developed. A recombinant staphylococcal FnBP fused to E. coli β-galactosidase (gal-FnBp) was used as the immobilized antigen in this test. Serum samples from two different rabbits immunized with native fibronectin receptor gave significant blocking titres, whereas the blocking titres of antisera against staphylococcal whole cells were about 4- to 5-fold lower. Using the gal-FnBP fusion protein, the sensitivity for detection of fibronectin by ELISA was also determined. The detection limit is around 5 ng. The findings are discussed with a view to developing an anti-staphylococcal adherence vaccine and quantitating fibronectin in solution.     

Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by enzyme-linked immunosorbent assay

Antibody titres against fibronectin-binding protein (FnBP) of Staphylococcus aureus were determined in sera from rabbits immunized with staphylococcal whole cells or purified native fibronectin receptor. An ELISA technique for detection of antibody titres blocking the binding of soluble fibronectin to immobilized FnBP was developed. A recombinant staphylococcal FnBP fused to E. coli beta-galactosidase (gal-FnBp) was used as the immobilized antigen in this test. Serum samples from two different rabbits immunized with native fibronectin receptor gave significant blocking titres, whereas the blocking titres of antisera against staphylococcal whole cells were about 4- to 5-fold lower. Using the gal-FnBP fusion protein, the sensitivity for detection of fibronectin by ELISA was also determined. The detection limit is around 5 ng. The findings are discussed with a view to developing an anti-staphylococcal adherence vaccine and quantitating fibronectin in solution.     

The elastin-binding protein of Staphylococcus aureus (EbpS) is expressed at the cell surface as an integral membrane protein and not as a cell wall-associated protein.

The elastin-binding proteins EbpS of Staphylococcus aureus strains Cowan and 8325-4 were predicted from sequence analysis to comprise 486 residues. Specific antibodies were raised against an N-terminal domain (residues 1-267) and a C-terminal domain (residues 343-486) expressed as recombinant proteins in Escherichia coli. Western blotting of lysates of wild-type 8325-4 and Newman and the corresponding ebpS mutants showed that EbpS migrated with an apparent molecular mass of 83 kDa. The protein was found exclusively in cytoplasmic membrane fractions purified from protoplasts or lysed cells, in contrast to the clumping factor ClfA, which was cell-wall-associated. EbpS was predicted to have three hydrophobic domains H1-(205-224), H2-(265-280), and H3-(315-342). A series of hybrid proteins was formed between EbpS at the N terminus and either alkaline phosphatase or beta-galactosidase at the C terminus (EbpS-PhoA, EbpS-LacZ). PhoA and LacZ were fused to EbpS between hydrophobic domains H1-H2 and H2-H3, and distal to H3. Expression of enzymatic activity in E. coli showed that EbpS is an integral membrane protein with two membrane-spanning domains H1 and H3. N-terminal residues 1-205 and C-terminal residues 343-486 were predicted to be exposed on the outer face of the cytoplasmic membrane. The ligand-binding domain of EbpS is known from previous studies to be present in the N terminus between residues 14-34 and probing whole cells with anti-EbpS1-267 antibodies indicated that this region is exposed on the surface of intact cells. This was also confirmed by the observation that wild-type S. aureus Newman cells bound labeled tropoelastin whereas the ebpS mutant bound 72% less. In contrast, the C terminus, which carries a putative LysM peptidoglycan-binding domain, is not exposed on the surface of intact cells and presumably remains buried within the peptidoglycan. Finally, expression of EbpS was correlated with the ability of cells to grow to a higher density in liquid culture, suggesting that EbpS may have a role in regulating cell growth.