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。 这种相互作用对于血液传播的生物体最初附着在 心内膜，以及随后在心脏瓣膜表面形成肉眼的赘生物。 我们最近发现了一个新的戈登链球菌基因座，它编码一个大细胞GspB 与人血小板结合的壁糖蛋白。该基因座还编码四种蛋白质，它们介导着 GspB的胞内糖基化，以及七种组成专门输出途径的蛋白质( 辅助SEC系统)。这一系统似乎专门用于GspB的出口。它是 未知这一途径的组件如何相互作用来调节GspB的输出。两名成员，SecA2和 SecY2是规范SEC系统的SecA和SecY的同源物，表明它们可能起作用 同样的。其他五个组分(辅助SEC蛋白Asp1-Asp5)与 已知功能的蛋白质，但对GspB出口是必不可少的。这个项目旨在勾勒出 调节GspB输出的辅助SEC系统的相互作用，特别是Asp1的作用， Asp2和AsP3。 我们之前的研究表明，AsP3与Asp1、Asp2及其自身结合，表明这些蛋白 可以形成络合物。目的1探讨Asp1-3结合在GspB输出中的作用。其大小和组成 将分别用层析法和免疫共沉淀法评估天冬氨酸复合体的活性。这个 在体内形成天冬氨酸复合体的重要性也将通过确定AsP3的变体是否 没有形成多聚体的仍然可以支持出口。Aim 2研究Asp1-3是否促进 GspB与出口马达蛋白SecA2的相互作用。Asp1-3(单独或AS)的能力 在体外结合GspB或SecA2的络合物)将通过天然凝胶电泳法进行评估， 表面等离子激元共振和等温滴定量热法。这种绑定对SecA2的影响 运动功能也将被确定。AIM 3研究Asp1-3是否与SecY2/Asp4/Asp5相关， GspB出口的假定渠道(转运子)。Asp1-3与转运子的结合将是 如上所述，进行了检查。此外，通过SecA2将GspB体外转位到蛋白脂体中 将对转位基因以及这一过程是否需要Asp1-3进行评估。 这些实验应该会提供相当大的机械洞察力，以了解组件是如何 附件SEC系统相互作用以形成GspB出口专用路径，尤其是如何 Asp1、Asp2和AsP3参与了这一过程。由于这个系统在众多其他系统中是保守的 对于革兰氏阳性病原体，这些研究应该高度适用于其他生物，并可能识别 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.