Role of Streptococcal-Platelet Binding in Endocarditis

细菌与血小板的结合是感染性心内膜炎发病机制中的中心事件。 这种相互作用可能对血液传播的生物体最初附着到 内分泌，以及随后在心脏瓣膜表面上形成肉眼可见的赘生物。 我们最近发现了一个新的基因位点的链球菌gordonii编码GspB，一个大的， 结合人血小板的壁糖蛋白。该基因座还编码四种蛋白质， GspB的细胞内糖基化以及构成专门输出途径的七种蛋白质（The 辅助Sec系统）。该系统似乎专门用于出口GspB。是 目前尚不清楚该途径的组分如何相互作用以介导GspB输出。两名成员，SecA 2和 SecY 2是经典Sec系统的SecA和SecY的同源物，表明它们可能起作用 同样的。其他五种组分（辅助Sec蛋白Asp 1-Asp 5）与 已知功能的蛋白质，但对于GspB输出是必需的。该项目旨在描述 介导GspB输出的辅助Sec系统的相互作用，特别是Asp 1的作用， Asp 2和Asp 3。 我们以前的研究表明，Asp 3结合Asp 1，Asp 2和本身，这表明这些蛋白质 可以形成复合物。目的1探索Asp 1 -3结合在GspB输出中的作用。人数和组成 将分别通过色谱法和免疫共沉淀法评估Asp复合物的含量。的 还将通过确定Asp 3的变体是否 不形成多聚体的细胞仍然可以支持输出。目标2检查Asp 1 -3是否有助于 GspB与SecA 2（输出的马达蛋白）的相互作用。Asp 1 -3的能力（单独或作为 复合物）在体外结合GspB或SecA 2的能力，如通过天然凝胶电泳所测量的， 表面等离子体共振和等温滴定量热法。这种约束对SecA 2的影响 还将确定运动功能。目的3观察Asp 1 -3是否与SecY 2/Asp 4/Asp 5相关， GspB输出的假定通道（易位子）。Asp 1 -3与易位子的结合将是 检查，如上所述。此外，SecA 2在体外将GspB转运到蛋白脂质体中， 和易位子，以及该过程是否需要Asp 1 -3，将被评估。 这些实验应该提供相当多的机械见解，如何组成的 辅助Sec系统相互作用，形成GspB输出专用通道，特别是如何 Asp 1、Asp 2和Asp 3都参与了这一过程。由于这个系统是保存在许多其他 革兰氏阳性病原体，这些研究应高度适用于其他生物体，并可能确定 用于疫苗开发的新靶点或新类别的治疗剂。

The binding of bacteria with platelets is a central event in the pathogenesis of infective endocarditis. This interaction may be important both for the initial attachment of blood-borne organisms to the endocardium, and for the subsequent formation of macroscopic vegetations on the cardiac valve surface. We have recently identified a novel genetic locus of Streptococcus gordonii that encodes GspB, a large, cell wall glycoprotein that binds human platelets. The locus also encodes four proteins mediating the intracellular glycosylation of GspB, and seven proteins comprising a specialized export pathway (the accessory Sec system). This system appears to be dedicated to the export of GspB exclusively. It is unknown how the components of this pathway interact to mediate GspB export. Two members, SecA2 and SecY2, are homologs of SecA and SecY of the canonical Sec system, suggesting they may function similarly. The five other components (accessory Sec proteins Asp1 - Asp5) have no significant homology to proteins of known function, but are essential for GspB export. This project seeks to delineate the interactions of the accessory Sec system that mediate GspB export, and in particular, the roles of Asp1, Asp2, and Asp3. Our previous studies indicate that Asp3 binds Asp1, Asp2, and itself, suggesting that these proteins may form complexes. Aim 1 explores the role of Asp1-3 binding in GspB export. The size and composition of Asp complexes will be assessed by chromatography and co-immunoprecipitation, respectively. The importance of Asp complex formation in vivo will also be tested, by determining whether variants of Asp3 that do not form multimers can still support export. Aim 2 examines whether Asp1-3 facilitate the interaction of GspB with SecA2 (the motor protein for export). The ability of Asp1-3 (either individually or as complexes) to bind GspB or SecA2 in vitro will be assessed, as measured by native gel electrophoresis, surface plasmon resonance, and isothermal titration calorimetry. The impact of such binding on SecA2 motor function will also be determined. Aim 3 looks at whether Asp1-3 associate with SecY2/Asp4/Asp5, the putative channel (translocon) for GspB export. The binding of Asp1-3 to the translocon will be examined, as described above. In addition, the in vitro translocation of GspB into proteolipsomes by SecA2 and the translocon, and whether this process requires Asp1-3, will be evaluated. These experiments should provide considerable mechanistic insights as to how the components of the accessory Sec system interact to form a dedicated pathway for GspB export, and in particular, how Asp1, Asp2, and Asp3 contribute to this process. Since this system is conserved among numerous other Gram-positive pathogens, these studies should be highly applicable to other organisms, and may identify novel targets for vaccine development or new classes of therapeutic agents.