Role of Streptococcal-Platelet Binding in Endocarditis

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 DESCRIPTION (provided by applicant): The serine-rich repeat (SRR) glycoproteins are a unique family of adhesins that are found in numerous species of Gram-positive bacteria. These cell wall-anchored surface proteins bind a wide range of host ligands, and are associated with increased virulence in a diversity of infections, including endocarditis, meningitis, and pneumonia. The biogenesis of SRR glycoproteins requires both the intracellular glycosylation of the adhesins, and their export to the bacterial cell surface by a specialized transporter, the accessory Sec (aSec) system. This export pathway is dedicated exclusively to the transport of SRR adhesins, and is comprised of SecA2 (the motor protein for transport), SecY2 (the transmembrane channel), and at least three accessory Sec proteins (Asps). The goal of this project is to better define the mechanisms for SRR glycoprotein export by the aSec system and to determine how transport is coordinated with glycosylation. We hypothesize that the biogenesis of SRR glycoproteins requires the precise interplay of glycosylation and export, and that Asps1-3 are bifunctional proteins that serve as a nexus for these two processes. By accurately coordinating glycosylation and transport, the Asps assure that that the binding properties of the SRR adhesins are optimized. To address these hypotheses, we will use GspB (an SRR protein of Streptococcus gordonii) as a model for SRR biogenesis. GspB mediates streptococcal binding to human platelets and enhances virulence in the setting of infective endocarditis. Aim 1 will examine the mechanisms by which Asps1-3 control the interaction of SecA2 with GspB, as measured by changes in SecA2 conformation and ATPase activity. The binding sites on SecA2 for the Asps will also be identified. Aim 2 will explore whether Asp1 modulates Asp3-GspB binding, such that GspB is engaged and then transported by SecA2. Aim 3 will investigate the roles of Asps1-3 in glycosylation, and whether the correct (WT) glycosylation of GspB only occurs during aSec transport. We will generate point mutations in Asps1-3 that have no impact on transport, but result in altered glycoforms of GspB. Analysis of the glycan composition and structure of these variants will reveal the precise roles of each Asp in GspB glycosylation. Aim 4 will assess the importance of accurate glycosylation on virulence, as measured by GspB- mediated binding to human platelets in vitro, and a well-established animal model of infective endocarditis. These studies will provide fundamental insights into the biogenesis of this important family of adhesins, and the roles of the Asps in glycosylation and transport. In turn, this research may lead to innovative antimicrobial therapies that specifically target the glycosylation or transport of SRR adhesins.