Role of Streptococcal-Platelet Binding in Endocarditis

链球菌-血小板结合在心内膜炎中的作用

• 批准号: 7742599
• 负责人: PAUL M. SULLAM
• 金额: $ 38.36万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7742599
• 关键词: ATP phosphohydrolase; Antibiotics; Bacteria; Bacterial Adhesins; Binding; Blood; Blood Platelets; Calorimetry; Cell Wall; Chromatography; Co-Immunoprecipitations; Complex; Development; Endocarditis; Endocardium; Event; Genetic; Glycoproteins; Goals; Heart Valves; Homologous Gene; Human; In Vitro; Infection; Infective endocarditis; Ion Channel; Measures; Mediating; Membrane; Membrane Proteins; Microbe; Motor; Organism; Pathogenesis; Pathway interactions; Process; Protein Binding; Protein Export Pathway; Proteins; Research; Role; Streptococcus; Streptococcus gordonii; Surface; Surface Plasmon Resonance; System; Testing; Therapeutic Agents; Titrations; Variant; Work; gel electrophoresis; glycosylation; human tissue; in vivo; insight; member; novel; novel vaccines; pathogen; protein protein interaction; public health relevance; research study; vaccine development

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.

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理的中心事件。这种相互作用对于最初的血传播生物与心内膜的结合以及随后在心脏瓣膜表面形成宏观植被。我们最近确定了链球菌Gordonii的一个新型遗传基因座，该基因座编码GSPB，该GSPB是一种结合人血小板的大细胞壁糖蛋白。该基因座还编码介导GSPB的细胞内糖基化的四种蛋白质，以及七个蛋白质的蛋白质，其中包括专门的导出途径（附件SEC系统）。该系统似乎专门用于GSPB的导出。尚不清楚该途径的组件如何相互作用以介导GSPB导出。两个成员SECA2和SECY2是SECA和SECY的同源物，表明它们的功能可能相似。其他五个组件（辅助SEC蛋白ASP1 -ASP5）与已知功能的蛋白质没有显着同源性，但对于GSPB导出至关重要。该项目旨在描述介导GSPB导出的附件SEC系统的交互，尤其是ASP1，ASP2和ASP3的角色。我们先前的研究表明，ASP3结合ASP1，ASP2和本身，表明这些蛋白质可能形成复合物。 AIM 1探讨了ASP1-3结合在GSPB导出中的作用。 ASP复合物的大小和组成将分别通过色谱和共免疫沉淀评估。通过确定未形成多聚体的ASP3变体是否仍然可以支持导出，体内也将测试ASP复合体形成的重要性。 AIM 2检查ASP1-3是否促进GSPB与Seca2（导出的运动蛋白）的相互作用。通过天然凝胶电泳，表面等离子体的共振和等温滴定热量法测量，将评估ASP1-3（单独或作为复合物）在体外结合GSPB或SECA2结合的能力。这种结合对SECA2运动功能的影响也将得到确定。 AIM 3查看ASP1-3是否与Secy2/Asp4/Asp5（用于GSPB导出的推定通道（ClressOcon））相关。如上所述，将检查ASP1-3与转运的结合。此外，将评估GSPB的体外易位，将GSPB通过SECA2和ClressOcon易位，以及该过程是否需要ASP1-3。这些实验应提供有关辅助SEC系统的组件如何相互作用以形成GSPB导出的专用途径，特别是ASP1，ASP2和ASP3如何促进此过程的专用途径的相当机械见解。由于该系统是在许多其他革兰氏阳性病原体中保守的，因此这些研究应高度适用于其他生物体，并可能确定疫苗开发或新的治疗剂类型的新靶标。公共卫生相关性：GSPB是链球菌的表面蛋白，可促进这些细菌的心脏瓣膜感染。该项目探讨了一种新型系统的内部运作，该系统将GSPB传输到细菌表面，然后可以在其中运作以将这些微生物连接到人体组织。通过检查GSPB的运输方式，这项研究可能会确定针对新疫苗或新的抗生素类别的独特靶标。