Role of Streptococcal-Platelet Binding in Endocarditis

描述（由申请方提供）：细菌与血小板的结合是感染性心内膜炎发病机制的中心事件。这种相互作用可能是重要的血液传播的生物体的初始附着的内膜，并为随后的心脏瓣膜表面上的宏观赘生物的形成。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，细胞壁糖蛋白，结合人类血小板。该基因座还编码四种介导GspB细胞内糖基化的蛋白质，以及七种包含专门的输出途径（辅助Sec系统）的蛋白质。该系统似乎专门用于出口GspB。尚不清楚该途径的组分如何相互作用以介导GspB输出。SecA 2和SecY 2是经典Sec系统SecA和SecY的同源物，表明它们可能具有相似的功能。其他五种组分（辅助Sec蛋白Asp 1-Asp 5）与已知功能的蛋白质没有显著的同源性，但对GspB输出是必需的。该项目旨在描述介导GspB出口的辅助Sec系统的相互作用，特别是Asp 1，Asp 2和Asp 3的作用。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能形成复合物。目的1探索Asp 1 -3结合在GspB输出中的作用。将分别通过色谱法和免疫共沉淀法评估Asp复合物的大小和组成。还将通过确定不形成多聚体的Asp 3变体是否仍能支持输出来测试体内Asp复合物形成的重要性。目的2检测Asp 1 -3是否促进GspB与SecA 2（用于输出的马达蛋白）的相互作用。通过天然凝胶电泳、表面等离子体共振和等温滴定量热法测定，评估Asp 1 -3（单独或作为复合物）体外结合GspB或SecA 2的能力。还将确定这种结合对SecA 2运动功能的影响。目的3观察Asp 1 -3是否与SecY 2/Asp 4/Asp 5相关，SecY 2/Asp 4/Asp 5是GspB输出的假定通道（易位子）。如上所述，将检查Asp 1 -3与易位子的结合。此外，还将评价GspB通过SecA 2和易位子在体外易位到蛋白脂质体中，以及该过程是否需要Asp 1 -3。这些实验应该提供相当多的机械见解，如何辅助Sec系统的组件相互作用，形成一个专门的途径GspB出口，特别是，如何Asp 1，Asp 2和Asp 3有助于这一进程。由于该系统在许多其他革兰氏阳性病原体中是保守的，因此这些研究应该高度适用于其他生物体，并且可以确定疫苗开发的新靶点或新类别的治疗剂。公共卫生相关性：GspB是链球菌的一种表面蛋白，可促进这些细菌对心脏瓣膜的感染。该项目探索了一种新系统的内部工作原理，该系统将GspB运送到细菌表面，然后它可以将这些微生物附着到人体组织上。通过研究GspB是如何转运的，这项研究可能会为新疫苗或新类别的抗生素确定独特的靶标。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: GspB is a surface protein of streptococci that promotes the infection of heart valves by these bacteria. This project explores the inner workings of a novel system that transports GspB to the bacterial surface, where it can then function to attach these microbes to human tissue. By examining how GspB is transported, this research may identify unique targets for new vaccines or new classes of antibiotics.