Fig 1.
Structural organization of FnBPA.
Structurally, FnBPA begins with an N-terminal signal sequence (S), which is followed by Region A (37-511), responsible for fibrinogen binding. Region A is composed of three subdomains (N1, N2, and N3). At position 103 inside the N1 subdomain there is a key reactive glutamine, which mediates transglutaminase reactions. As part of our study, this specific residue was replaced with an alanine via site-directed mutagenesis, resulting in the Q103A variant. The C-terminal region, named also Repeated region (511-878) is composed of multiple fibronectin-binding motifs arranged in tandem. Toward the end of the C-terminus there are proline-rich repeats (PRR), followed by wall-associated (W) and membrane-spanning (M) domains. The LPETG motif is recognized by the sortase enzyme for cell wall anchoring.
Fig 2.
Conservation and functional relevance of the Q103 residue in FnBPA.
(A) Multiple sequence alignment of fnbA gene sequences from representative S. aureus strains across major clonal complexes to determine the conservation of the Q103 residue (highlighted), implicated in bacterially-mediated cross-linking with fibrinogen (Fbg). (B) Multiple sequence alignment of fnbB sequences from corresponding clonal complexes with fnbA (subsp.8325). Alignments in Fig 2A and 2B were carried out with ClustalOmega. Gene sequences were retrieved from KEGG GENOME database with the following entries: T00557 (subsp. Newman - CC8, NWMN_2397/ NWMN_2397); T00324 (subsp. 8325 - CC8, SAOUHSC_02803/ SAOUHSC_02802); T00086 (subsp. MW2 – CC1, MW2041/MW2040); T02702 (subsp. CA347 – CC45, CA347_2575/ CA347_2574); T00182 (subsp. MRSA252 – CC30, SAR2580); T00051 (subsp. N315 – CC5, SA2291/SA2290); T02059 (subsp. HO 5096 0412 - CC22, SAEMRSA15_23990); T02071 (subsp. ST398 – CC398, SAPIG2553) (C) Saturation binding curves of recombinant WT and Q103A-mutated FnBPA N1N2N3 to Fbg. Microtiter plates were coated with each protein variant and incubated with increasing concentrations of Fbg. Detection was performed using anti-Fbg antibody and HRP-conjugated secondary antibody. Data are expressed as means ± S.D. of tests performed in triplicate.
Fig 3.
Role of Q103A residue in vWbp-mediated cross-linking of FnBPA to fibrin(ogen).
(A–B) Cross-link formation of recombinant N1N2N3 domain of FnBPA WT (A) or Q103A mutant (B) to fibrin(ogen) by vWbp-activated FXIII was analyzed by Western Blotting. Reaction mixtures were incubated for increasing time points (0–120 min) in the presence of calcium, separated by SDS-PAGE under reducing conditions and then transferred to PVDF membranes, then probed with anti-FnBPA IgG and HRP-conjugated secondary antibody. Lane 8 in both panels lacks fibrin(ogen); lane 1 contains only fibrin(ogen) Arrows show the positions of FnBPA WT (A) or FnBPA Q103A. Molecular mass markers (kDa) are indicated on the left. Data is representative of three independent experiments. (C) ELISA analysis of fibrin(ogen) crosslinking with WT and Q103A N1N2N3 domains in the presence of Ca²⁺ or EDTA. Microtiters wells were coated with fibrinogen and then probed with mixtures containing FnBPA WT or Q103A, vWbp, ProT, FXIII and either calcium chloride or EDTA. Binding was measured using anti-FnBPA IgG and HRP-conjugated secondary antibody. Statistically significant difference is reported (**P < 0.01). Inset: binding of the mutant protein was reduced to approximately 30% of the reactivity observed for the FnBPA WT. Data are expressed as means ± S.D. of triplicate tests. (D–E) Cross-link formation of recombinant N1 subdomain of FnBPA WT (D) or Q103A (E) to fibrin(ogen) by vWbp-activated FXIII, analyzed as described for panels A–B. Arrows show the positions of FnBPA WT or FnBPA Q103A. (F) ELISA analysis of fibrin(ogen) crosslinking with WT and Q103A N1 domains in the presence of Ca²⁺ or EDTA. Statistical analysis is reported (**P < 0.01). Inset: WT signal in presence of Ca² ⁺ ions is > 90% higher than mutant.
Fig 4.
Role of Q103A residue in TG2- mediated cross-linking of FnBPA to fibrinogen.
(A–B) Cross-link formation of recombinant N1N2N3 domain of FnBPA WT (A) or Q103A mutant (B) to fibrinogen by TG2 was analyzed by Western Blotting. Arrows show the position of the N1 WT (A) or Q103A (B) subdomain of FnBPA. Reaction mixtures were incubated for increasing time intervals (0–120 min) in the presence of calcium, separated by SDS-PAGE and transferred to PVDF membranes, then probed with anti-FnBPA IgG and HRP-conjugated secondary antibody. Lane 8 in both panels lacks fibrinogen; lane 1 contains only fibrinogen. Molecular mass markers (kDa) are indicated on the left. Data is representative of three independent experiments. (C) ELISA analysis of fibrin(ogen) crosslinking with WT and Q103A N1N2N3 domains in the presence of Ca²⁺ or EDTA by TG2. Microtiters wells were coated with fibrinogen and then incubated with mixtures containing FnBPA WT or Q103A, vWbp, ProT, FXIII and either calcium chloride or EDTA. Binding was measured using anti-FnBPA IgG and HRP-conjugated secondary antibody. Statistically significant difference is reported (**P < 0.01). Inset: binding of the mutant protein was reduced to about 30% of the interaction of WT FnBPA. Data are expressed as means ± S.D. of triplicate tests.(D–E) Cross-link formation of recombinant N1 subdomain of FnBPA WT (D) or Q103A (E) to fibrinogen by TG2, analyzed as described for panels A–B.(F) ELISA analysis of fibrin(ogen) crosslinking with WT and Q103A N1 domains by the action of TG2 in the presence of Ca²⁺ or EDTA. Statistically significant differences are reported (***P < 0.001). Inset: binding of WT protein was about 90% greater of the FnBPA Q103A.
Fig 5.
Impact of the Q103A substitution in FnBPA on S. aureus SH10004X binding to fibrinogen and cross-linking to fibrin.
(A) Binding of fibrinogen (Fbg) to S. aureus SH10004X strains expressing either WT or Q103A-mutated FnBPA. Microtiter wells were coated with diluted bacterial suspensions (OD = 1) and incubated with serial dilutions of Fbg. Binding was detected using an anti-Fbg antibody and an HRP-conjugated secondary antibody. Data are expressed as means ± S.D. of tests performed in triplicate. (B) FXIII-mediated incorporation of S. aureus SH10004X (pFnBA4_WT) or (pFnBA4_Q103A) fibrin in presence of calcium or EDTA. Microtiter wells were coated with Fbg and incubated with mixtures containing S. aureus SH10004X strains expressing either WT or Q103A-mutated FnBPA, vWbp, FXIII, ProT and either calcium ions or EDTA. Binding of bacteria to fibrin was then detected using an anti-S. aureus antibody and an HRP-conjugated secondary antibody. Statistically significant differences are reported (*P < 0.05). Data are expressed as means ± S.D. of triplicate tests.
Fig 6.
In vivo impact of Q103A mutation in a murine dermonecrosis model.
(A) Mean lesions size (mm²) were measured in mice infected with S. aureus SH10004X strains expressing either WT or Q103A-mutated FnBPA at day 7 post-infection. (B) Bacterial load per biopsy was quantified by CFU counts at day 7 post-infection. Each dot represents an individual mouse. Bars indicate the means. Statistically significant differences were calculated (**p < 0.01). (C) Photographs of lesions at day 7 post-infection in mice infected with S. aureus SH10004X strains expressing either WT or Q103A-mutated FnBPA.
Table 1.
Bacterial strains used in this study.