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制中的核心事件。这种相互作用对于血源性生物体最初附着于心内膜以及随后在心脏瓣膜表面形成宏观赘生物可能都很重要。我们最近发现了戈登链球菌的一个新基因位点，它编码 GspB，一种结合人类血小板的大细胞壁糖蛋白。该基因座还编码介导 GspB 细胞内糖基化的四种蛋白质，以及构成专门输出途径（辅助 Sec 系统）的七种蛋白质。该系统似乎专门用于 GspB 的出口。目前尚不清楚该途径的组成部分如何相互作用来介导 GspB 输出。 SecA2 和 SecY2 两个成员是规范 Sec 系统中 SecA 和 SecY 的同源物，表明它们可能具有相似的功能。其他五个成分（辅助 Sec 蛋白 Asp1 - Asp5）与已知功能的蛋白没有显着同源性，但对于 GspB 输出至关重要。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 Asp1、Asp2 和 Asp3 的作用。我们之前的研究表明，Asp3 与 Asp1、Asp2 及其自身结合，表明这些蛋白质可能形成复合物。目标 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免疫共沉淀进行评估。还将通过确定不形成多聚体的 Asp3 变体是否仍支持输出来测试体内 Asp 复合物形成的重要性。目标 2 检查 Asp1-3 是否促进 GspB 与 SecA2（输出马达蛋白）的相互作用。将评估 Asp1-3（单独或作为复合物）在体外结合 GspB 或 SecA2 的能力，通过天然凝胶电泳、表面等离振子共振和等温滴定量热法进行测量。还将确定这种结合对 SecA2 运动功能的影响。目标 3 着眼于 Asp1-3 是否与 SecY2/Asp4/Asp5（GspB 输出的假定通道（易位子））相关联。如上所述，将检查 Asp1-3 与易位子的结合。此外，还将评估 SecA2 和易位子在体外将 GspB 易位至脂蛋白体，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，了解辅助 Sec 系统的组件如何相互作用以形成 GspB 输出的专用途径，特别是 Asp1、Asp2 和 Asp3 如何参与此过程。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，并且可以确定疫苗开发或新型治疗剂的新靶标。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型系统的内部运作机制，该系统将 GspB 运输到细菌表面，然后它可以将这些微生物附着到人体组织上。通过检查 GspB 的运输方式，这项研究可能会确定新疫苗或新类别抗生素的独特靶点。