Role of the serine-rich surface glycoprotein Srr1 of Streptococcus agalactiae in the pathogenesis of infective endocarditis

Abstract

The binding of bacteria to fibrinogen and platelets are important events in the pathogenesis of infective endocarditis. Srr1 is a serine-rich repeat glycoprotein of Streptococcus agalactiae that binds directly to the Aα chain of human fibrinogen. To assess the impact of Srr1 on the pathogenesis of endocarditis due to S. agalactiae, we first examined the binding of this organism to immobilized human platelets. Strains expressing Srr1 had significantly higher levels of binding to human platelets in vitro, as compared with isogenic Δsrr1 mutants. In addition, platelet binding was inhibited by pretreatment with anti-fibrinogen IgG or purified Srr1 binding region. To assess the contribution of Srr1 to pathogenicity, we compared the relative virulence of S. agalactiae NCTC 10/84 strain and its Δsrr1 mutant in a rat model of endocarditis, where animals were co-infected with the WT and the mutant strains at a 1:1 ratio. At 72 h post-infection, bacterial densities (CFU/g) of the WT strain within vegetations, kidneys, and spleens were significantly higher, as compared with the Δsrr1 mutant. These results indicate that Srr1 contributes to the pathogenesis of endocarditis due to S. agalactiae, at least in part through its role in fibrinogen-mediated platelet binding.

Figure 1. GBS binding to immobilized human platelets is mediated by glycoprotein Srr1.

(A) Platelet binding by GBS strains COH31 and NCTC 10/84, their Δsrr1 isogenic variants, and the mutant strains complemented in trans with srr1 (pSrr1). (B) GBS binding to human platelets was inhibited by pretreating the monolayers with 100 µg/ml of anti-fibrinogen IgG (Anti-Fg). Normal IgG (IgG) served as a control. (C) Inhibition of binding by recombinant Srr1 binding region (Srr1-BR). Levels of binding were calculated as relative to the WT strains (mean ± SD). Values shown represent the means (± S.D.) of triplicate measurements. * = P<0.01.

Figure 2. Recombinant Srr1-BR interacts with human platelets.

(A) Binding of _FLAGSrr1-BR protein to immobilized platelets. (B) Inhibition of _FLAGSrr1-BR binding to platelets by His6 tagged Srr1-BR. Platelets were pretreated with the indicated concentrations of His6 tagged Srr1-BR. (C) Binding of _FLAGSrr1-BR to immobilized platelets pretreated with anti-fibrinogen IgG or preimmune rabbit IgG. Values represent relative binding of _FLAGSrr1-BR binding as compared with untreated platelets. Bars indicate the means (± S.D.). * = P<0.01.

Figure 3. GBS binding to rat fibrinogen.

(A) Alignment of Srr1 binding domain in the human fibrinogen Aα chain, with the homologous region of the rat protein; (B) Rat fibrinogen binding by wild type GBS and their isogenic variants (Δsrr1). (C) Rat fibrinogen binding by _FLAGSrr1-BR protein over a range of concentrations. Casein served as a negative control.

Figure 4. Competitive index (CI) analysis of WT and Δsrr1 mutant obtained in the rat model of endocarditis.

Competition index (CI) was calculated as the ratio of the WT to the Δsrr1 mutant in each tissue, normalized for the ratio of strains within the inoculum. Circles represent data from individual animals. A CI above 10⁰ (dashed line) indicates a competitive disadvantage of Δsrr1 compared with WT. Horizontal black bars indicates means of CIs.