Role of Streptococcal-Platelet Binding in Endocarditis

 描述（由申请人提供）：富含丝氨酸重复（SRR）糖蛋白是一个独特的粘附素家族，存在于许多革兰氏阳性细菌物种中。这些细胞壁锚定的表面蛋白结合广泛的宿主配体，并与多种感染中的毒力增加相关，包括心内膜炎、脑膜炎和肺炎。SRR糖蛋白的生物合成需要粘附素的细胞内糖基化，以及它们通过专门的转运蛋白，辅助Sec（aSec）系统输出到细菌细胞表面。该输出途径专门用于SRR粘附素的转运，并且由SecA 2（用于转运的马达蛋白）、SecY 2（跨膜通道）和至少三种辅助Sec蛋白（Asps）组成。 该项目的目标是更好地定义aSec系统的SRR糖蛋白输出机制，并确定转运如何与糖基化协调。我们假设SRR糖蛋白的生物合成需要糖基化和出口的精确相互作用，并且Asps 1 -3是作为这两个过程的联系的双功能蛋白。通过精确协调糖基化和转运，Asps确保SRR粘附素的结合特性得到优化。为了解决这些假设，我们将使用GspB（戈登链球菌的SRR蛋白）作为SRR生物发生的模型。GspB介导链球菌与人血小板的结合并增强感染性心内膜炎的毒力目的1将研究Asps 1 -3控制SecA 2与GspB相互作用的机制，如SecA 2构象和ATP酶活性的变化所测量的。还将鉴定Asps在SecA 2上的结合位点。目的2将探索Asp 1是否调节Asp 3-GspB结合，使得GspB被SecA 2接合并随后被SecA 2转运。目的3将研究Asps 1 -3在糖基化中的作用，以及GspB的正确（WT）糖基化是否仅发生在aSec转运过程中。我们将在Asps 1 -3中产生点突变，这些点突变对转运没有影响，但会导致GspB的糖型改变。这些变体的聚糖组成和结构的分析将揭示每个Asp在GspB糖基化中的精确作用。目的4将评估准确的糖基化对毒力的重要性，如通过GspB介导的体外与人血小板的结合以及良好建立的感染性心内膜炎动物模型所测量的。这些研究将为这个重要的粘附素家族的生物起源以及Asps在糖基化和转运中的作用提供基本的见解。反过来，这项研究可能会导致创新的抗菌治疗，专门针对糖基化或运输的SRR粘附素。

 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.