BackgroundFibronectin 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.ResultsSeven different fnbB allelic variants were identified. Some strains that cluster by phylogenetic analysis contain different fnbB variants, whereas more divergent strains contain the same fnbB variant. The phylogeny of fnbB alleles does not match the phylogeny of fnbA alleles. Some FnBPA and FnBPB isotypes that are specified by human S. aureus strains are also found in bovine strains. The seven fnbB allelic variants encode seven distinct isotypes of the FnBPB A domain that are 61 to 85% identical in amino acid sequence. Variant amino acid residues were mapped on a three-dimensional model of the FnBPB A domain and were predicted to be surface-exposed. They are responsible for the antigenic diversity detected with polyclonal antibody and a monoclonal antibody raised against isotype I. Ligand binding by recombinant FnBPB N23 isotypes was compared by ELISA-based solid phase assays and surface plasmon resonance. Each bound to immobilized fibrinogen, elastin and fibronectin dose-dependently and saturably with similar affinities. Binding to fibronectin was surprising because the A domains do not contain any known motifs that mediate binding to fibronectin. This raises the possibility that the A domain of FnBPB contains a novel fibronectin binding motif that binds fibronectin by a novel mechanism.ConclusionsSeven different isoforms of FnBPB A domain retain ligand-binding functions but are antigenically distinct. The variation in FnBPA and FnBPB occurs in human and bovine S. aureus strains and may act as an immune evasion mechanism. All seven isotypes of FnBPB are capable of binding fibronectin though none contain any known fibronectin-binding motifs. These results have implications for the development of vaccines or immunotherapeutics that target FnBPB

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 Ca2+‐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 KD = 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, KD 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. Structured digital abstract  http://www.uniprot.org/uniprot/P02751 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 to http://www.uniprot.org/uniprot/Q53682 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0049 http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8173367  http://www.uniprot.org/uniprot/P02751 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 to http://www.uniprot.org/uniprot/Q53682 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0107 (View Interaction http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8173300, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8173347)

Fibronectin-binding proteins A and B are multifunctional LPXTG staphylococcal adhesins, comprising an N-terminal region that binds fibrinogen and elastin, and a C-terminal domain that interacts with fibronectin. The C-terminal domain of fibronectin-binding protein A is organized into 11 tandem repeats, six of which bind the ligand with high affinity; other sites bind more weakly. Fibronectin-binding protein B has been postulated to harbor 10 rather than 11 repeats, but it contains the same number of highaffinity fibronectin-binding sites as fibronectin-binding protein A. In this study, we confirm this prediction and show that six of 10 sites bind with dissociation constants in the nanomolar range. We also found that the fulllength repetitive region of fibronectin-binding protein B stimulated the production of a mAb (15E11) that binds with high affinity to an epitope shared by repeats 9 and 10 from both adhesins. With the use of truncated fragments of repeat 9 of fibronectin-binding protein A, we mapped the antibody epitope to the N-terminal segment and the fibronectin-binding site to the C-terminal segment of the repeat. The distinct localization of the 15E11 epitope and the fibronectin-binding site suggests that the interfering effect of the antibody might result from steric hindrance or a conformational change in the structure that reduces the accessibility of fibronectin to its binding determinant. The epitope is well exposed on the surface of staphylococcal cells, as determined by genetic analyses, fluorescence microscopy, and flow cytometry. When incubated with cells of Staphylococcs aureus strains, 15E11 inhibits attachment of bacteria to surface-coated fibronectin by almost 70%.