Role of Streptococcal-Platelet Binding in Endocarditis

描述（由申请人提供）：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。这种相互作用对于血源性生物最初附着于心内膜，以及随后在心脏瓣膜表面形成肉眼可见的植被可能都是重要的。我们最近发现了一个新的gordonii链球菌基因位点，它编码GspB，一种结合人血小板的大的细胞壁糖蛋白。该位点还编码4种介导GspB细胞内糖基化的蛋白，以及7种构成特殊输出途径（附属Sec系统）的蛋白。这个系统似乎是专门用于GspB出口的。目前尚不清楚该途径的成分如何相互作用介导GspB的输出。两个成员SecA2和SecY2是规范Sec系统的SecA和SecY的同源物，这表明它们的功能可能相似。其他5个成分（辅助Sec蛋白Asp1 - Asp5）与已知功能的蛋白没有明显的同源性，但对GspB的输出至关重要。该项目旨在描述介导GspB输出的附属Sec系统的相互作用，特别是Asp1、Asp2和Asp3的作用。我们之前的研究表明，Asp3可以结合Asp1、Asp2和自身，这表明这些蛋白可能形成复合物。目的1探讨Asp1-3结合在GspB输出中的作用。Asp复合物的大小和组成将分别通过色谱法和共免疫沉淀法进行评估。通过确定不形成多聚体的Asp3变体是否仍然支持输出，还将测试Asp复合物在体内形成的重要性。目的2研究Asp1-3是否促进GspB与SecA2（输出的马达蛋白）的相互作用。Asp1-3（单独或作为复合物）在体外结合GspB或SecA2的能力将通过天然凝胶电泳、表面等离子体共振和等温滴定量热法进行评估。这种结合对SecA2运动功能的影响也将被确定。目标3着眼于Asp1-3是否与SecY2/Asp4/Asp5 （GspB输出的假定通道（易位））相关联。如上所述，将检查Asp1-3与转座子的结合。此外，还将评估SecA2和易位子在体外将GspB易位为蛋白脂体，以及该过程是否需要Asp1-3。这些实验应该提供相当多的机制见解，关于附属Sec系统的组件如何相互作用形成GspB输出的专用途径，特别是Asp1， Asp2和Asp3如何促进这一过程。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，并可能为疫苗开发或新型治疗剂确定新的靶点。公共卫生相关性：GspB是链球菌的表面蛋白，可促进这些细菌对心脏瓣膜的感染。该项目探索了一个新系统的内部工作原理，该系统将GspB运输到细菌表面，然后在那里它可以将这些微生物附着在人体组织上。通过研究GspB是如何运输的，这项研究可能为新疫苗或新种类的抗生素确定独特的靶点。

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.