Competitive index (CI) analysis of WT and Δ<i>srr1</i> mutant obtained in the rat model of endocarditis.

<p>Competition index (CI) was calculated as the ratio of the WT to the Δ<i>srr1</i> 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 Δ<i>srr1</i> compared with WT. Horizontal black bars indicates means of CIs.</p

GBS binding to rat fibrinogen.

<p>(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 (Δ<i>srr1</i>). (C) Rat fibrinogen binding by _FLAGSrr1-BR protein over a range of concentrations. Casein served as a negative control.</p

Recombinant Srr1-BR interacts with human platelets.

<p>(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.</p

Impact of Srr1 on virulence in an animal model of endocarditis.

<p>Infective endocarditis was induced in rats, using an inoculum of 5×10⁵ CFU containing GBS NCTC10/84 and its isogenic Δ<i>srr1</i> mutant, at a 1∶1 ratio. Animals were sacrified 72 h post-infection, and log₁₀ CFU/g of tissue for each strain was determined by plating onto selective media.</p

Strains and plasmids.

a<p>Cm^R, chloramphenicol resistance; ErmR, erythromycin resistance; Amp^R, ampicillin resistance; Kan^R, kanamycin resistance.</p

Effects of transport upon sWGA reactivity of GspB736flag.

<p>A) Upper panel shows Western blot analysis of GspB736flag exported from a <i>nss</i> and <i>gly</i> deletion (Δ<i>gn</i>) variant derivative strain PS3309 (lane 1) and intracellular GspB736flag expressed in the aSec deficient <i>nss</i> and <i>gly</i> deletion variant strains PS3310 (lane 2) and PS3584 (lane 3). Anti-FLAG antibodies were used to measure GspB736flag protein levels, while GlcNAc reactivity was assessed by lectin blot analysis using biotinylated sWGA as a GlcNAc probe. Lower panel shows densitometry analysis of GspB736flag levels and GlcNAc reactivity was determined by band intensity analysis via LI-COR imaging. B) Western blot analysis of GspB736flag exported from strains PS3309 (Δ<i>gn</i>)(lane 1), PS3310 (Δ<i>gn</i>Δ<i>secA2</i>)(lane 2) and PS3605 (Δ<i>gn</i>Δ<i>secA2</i>::<i>asp2</i>^S362A)(lane 3). (M) media fraction, (P) protoplast fraction. GspB736flag was detected with anti-FLAG antibodies. C) GlcNAc reactivity of GspB736flag before and after saponfication of GspB736flag obtained from protoplasts in the defined aSec background. Non-exported GspB736flag was expressed in the following accessory Sec/Δ<i>gn</i> deletion strains, PS3310 (lane 1), PS3605 (lane 2). Saponified intracellular GspB736flag was achieved through incubation with 100 mM NaOH to release ester-linked acetate. Western and lectin blot analysis of secreted GspB variants, together with corresponding densitometry analysis, are representative of at least three different genetic transformants analyzed for each strain.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Effects of mutating the Asp2 catalytic triad upon the export of GspB736flag.

<p>Western blot analysis of Asp2-dependent export of GspB736flag by <i>S</i>. <i>gordonii</i> Δ<i>asp2</i> strain PS1244 (lane 1), parental strain PS1225 (lane 2) and derivative strains harboring the designated alanine substitution within the catalytic triad of Asp2, PS3539 (lane 3), PS3551 (lanes 4), PS3552 (lane 5), PS3553 (lane 6), PS3554 (lane 7). Culture media (M) and protoplasts (P) were collected from exponentially growing strains. Proteins were separated by SDS-PAGE and analyzed by Western blotting, using anti-FLAG antibody to detect GspB736flag. The data shown is representative of at least three different genetic transformants for each strain.</p

O-acetylation of the serine-rich repeat glycoprotein GspB is coordinated with accessory Sec transport

<div><p>The serine-rich repeat (SRR) glycoproteins are a family of adhesins found in many Gram-positive bacteria. Expression of the SRR adhesins has been linked to virulence for a variety of infections, including streptococcal endocarditis. The SRR preproteins undergo intracellular glycosylation, followed by export via the accessory Sec (aSec) system. This specialized transporter is comprised of SecA2, SecY2 and three to five accessory Sec proteins (Asps) that are required for export. Although the post-translational modification and transport of the SRR adhesins have been viewed as distinct processes, we found that Asp2 of <i>Streptococcus gordonii</i> also has an important role in modifying the SRR adhesin GspB. Biochemical analysis and mass spectrometry indicate that Asp2 is an acetyltransferase that modifies <i>N</i>-acetylglucosamine (GlcNAc) moieties on the SRR domains of GspB. Targeted mutations of the predicted Asp2 catalytic domain had no effect on transport, but abolished acetylation. Acetylated forms of GspB were only detected when the protein was exported via the aSec system, but not when transport was abolished by <i>secA2</i> deletion. In addition, GspB variants rerouted to export via the canonical Sec pathway also lacked <i>O</i>-acetylation, demonstrating that this modification is specific to export via the aSec system. Streptococci expressing GspB lacking O-acetylated GlcNAc were significantly reduced in their ability bind to human platelets <i>in vitro</i>, an interaction that has been strongly linked to virulence in the setting of endocarditis. These results demonstrate that Asp2 is a bifunctional protein involved in both the post-translational modification and transport of SRR glycoproteins. In addition, these findings indicate that these processes are coordinated during the biogenesis of SRR glycoproteins, such that the adhesin is optimally modified for binding. This requirement for the coupling of modification and export may explain the co-evolution of the SRR glycoproteins with their specialized glycan modifying and export systems.</p></div

List of GspB736flag glycopeptides identified by Q-TOF LC/MS.

<p>List of GspB736flag glycopeptides identified by Q-TOF LC/MS.</p