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Identification of the Staphylococcus aureus MSCRAMM clumping factor B (ClfB) binding site in the alphaC-domain of human fibrinogen.

ABSTRACT: Clumping factor B (ClfB) of Staphylococcus aureus binds to cytokeratin 10 and to fibrinogen. In this study the binding site in human fibrinogen was localized to a short region within the C terminus of the Aalpha-chain. ClfB only bound to the Aalpha-chain of fibrinogen in a ligand-affinity blot and in solid-phase assays with purified recombinant fibrinogen chains. A variant of fibrinogen with wild-type Bbeta- and gamma-chains but with a deletion that lacked the C-terminal residues from 252-610 of the Aalpha-chain did not support adherence of S. aureus Newman expressing ClfB. A series of truncated mutants of the recombinant Aalpha-chain were tested for their ability to support adherence of S. aureus Newman ClfB(+), which allowed the binding site to be localized to a short segment of the unfolded flexible repeated sequence within the C terminus of the Aalpha-chain. This was confirmed by two amino acid substititions within repeat 5 of the recombinant Aalpha-chain which did not support adherence of Newman ClfB(+). Lactococcus lactis expressing ClfB mutants with amino acid substitutions (N256 and Q235) located in the putative ligand-binding trench between domains N2 and N3 of the A-domain were defective in adherence to immobilized fibrinogen and cytokeratin 10, suggesting that both ligands bind to the same or overlapping regions.

SUBMITTER: Walsh EJ

PROVIDER: S-EPMC2885624 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Identification of the Staphylococcus aureus MSCRAMM clumping factor B (ClfB) binding site in the alphaC-domain of human fibrinogen.

Walsh Evelyn J EJ Miajlovic Helen H Gorkun Oleg V OV Foster Timothy J TJ

Microbiology (Reading, England) 20080201 Pt 2

Clumping factor B (ClfB) of Staphylococcus aureus binds to cytokeratin 10 and to fibrinogen. In this study the binding site in human fibrinogen was localized to a short region within the C terminus of the Aalpha-chain. ClfB only bound to the Aalpha-chain of fibrinogen in a ligand-affinity blot and in solid-phase assays with purified recombinant fibrinogen chains. A variant of fibrinogen with wild-type Bbeta- and gamma-chains but with a deletion that lacked the C-terminal residues from 252-610 of ...[more]

PMID: 18227259

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Project description:Essentials Staphylococcus aureus (S. aureus) binds to endothelium via von Willebrand factor (VWF). Secreted VWF-binding protein (vWbp) mediates S. aureus adhesion to VWF under shear stress. vWbp interacts with VWF and the Sortase A-dependent surface protein Clumping factor A (ClfA). VWF-vWbp-ClfA anchor S. aureus to vascular endothelium under shear stress. SUMMARY:Objective When establishing endovascular infections, Staphylococcus aureus (S. aureus) overcomes shear forces of flowing blood by binding to von Willebrand factor (VWF). Staphylococcal VWF-binding protein (vWbp) interacts with VWF, but it is unknown how this secreted protein binds to the bacterial cell wall. We hypothesized that vWbp interacts with a staphylococcal surface protein, mediating the adhesion of S. aureus to VWF and vascular endothelium under shear stress. Methods We studied the binding of S. aureus to vWbp, VWF and endothelial cells in a micro-parallel flow chamber using various mutants deficient in Sortase A (SrtA) and SrtA-dependent surface proteins, and Lactococcus lactis expressing single staphylococcal surface proteins. In vivo adhesion of bacteria was evaluated in the murine mesenteric circulation using real-time intravital vascular microscopy. Results vWbp bridges the bacterial cell wall and VWF, allowing shear-resistant binding of S. aureus to inflamed or damaged endothelium. Absence of SrtA and Clumping factor A (ClfA) reduced adhesion of S. aureus to vWbp, VWF and activated endothelial cells. ADAMTS-13 and an anti-VWF A1 domain antibody, when combined, reduced S. aureus adhesion to activated endothelial cells by 90%. Selective overexpression of ClfA in the membrane of Lactococcus lactis enabled these bacteria to bind to VWF and activated endothelial cells but only in the presence of vWbp. Absence of ClfA abolished bacterial adhesion to the activated murine vessel wall. Conclusions vWbp interacts with VWF and with the SrtA-dependent staphylococcal surface protein ClfA. The complex formed by VWF, secreted vWbp and bacterial ClfA anchors S. aureus to vascular endothelium under shear stress.

Project description:UnlabelledTreatment of Staphylococcus aureus infections has become increasingly difficult because of the emergence of multidrug-resistant isolates. Development of a vaccine to prevent staphylococcal infections remains a priority. To determine whether clumping factor A (ClfA) is a good target protein for inclusion in a multivalent vaccine, we evaluated its efficacy in a variety of relevant staphylococcal infection models, challenging with different S. aureus strains. ClfA adsorbed to Alhydrogel and mixed with Sigma Adjuvant System was more immunogenic and stimulated a more robust Th17 response than ClfA administered with alum alone. ClfA immunization induced the production of functional antibodies in rabbits and mice that blocked S. aureus binding to fibrinogen and were opsonic for S. aureus strains that produced little or no capsular polysaccharide. Mice immunized with ClfA showed a modest reduction in the bacterial burden recovered from subcutaneous abscesses provoked by S. aureus USA300 strain LAC. In addition, the ClfA vaccine reduced lethality in a sepsis model following challenge with strain Newman, but not ST80. Vaccination with ClfA did not protect against surgical wound infection, renal abscess formation, or bacteremia. Passive immunization with antibodies to ClfA did not protect against staphylococcal bacteremia in mice or catheter-induced endocarditis in rats. Some enhancement of bacteremia was observed by ClfA immunization or passive administration of ClfA antibodies when mice were challenged by the intraperitoneal route. Although rodent models of staphylococcal infection have their limitations, our data do not support the inclusion of ClfA in an S. aureus multivalent vaccine.ImportanceAntibiotics are often ineffective in eradicating Staphylococcus aureus infections, and thus, a preventative vaccine is sorely needed. Two single-component vaccines and two immunoglobulin preparations failed to meet their designated endpoints in phase III clinical trials. Importantly, recipients of an S. aureus surface protein (iron surface determinant B) vaccine who developed a staphylococcal infection experienced a higher rate of multiorgan failure and mortality than placebo controls, raising safety concerns. Multicomponent S. aureus vaccines have now been generated, and several include surface protein clumping factor A (ClfA). We immunized mice with ClfA and generated a robust T cell response and serum antibodies that were functional in vitro. Nonetheless, ClfA was not protective in a number of relevant animal models of S. aureus infection, and high levels of ClfA antibodies enhanced bacteremia when mice were challenged with community-acquired methicillin-resistant S. aureus strains. Evidence supporting ClfA as a vaccine component is lacking.

Dataset Information

Identification of the Staphylococcus aureus MSCRAMM clumping factor B (ClfB) binding site in the alphaC-domain of human fibrinogen.

Publications

Identification of the Staphylococcus aureus MSCRAMM clumping factor B (ClfB) binding site in the alphaC-domain of human fibrinogen.

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