The accessory Sec system of Gram-positive pathogens

革兰氏阳性病原体的辅助Sec系统

• 批准号: 8597939
• 负责人: PAUL M. SULLAM
• 金额: --
• 依托单位: VETERANS AFFAIRS MED CTR SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-10-01 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8597939
• 关键词: Address; Affect; Amino Acids; Animal Model; Bacteria; Binding; Biogenesis; Blood; Blood Platelets; Carbohydrates; Cell Fractionation; Cell Wall; Complex; Deletion Mutation; Development; Disease; Endocarditis; Endocardium; Event; Fluorescence Microscopy; Genetic; Glycoproteins; Goals; Heart Valves; Homologous Gene; Human; Infection; Infective endocarditis; Lectin; Life; Location; Mass Spectrum Analysis; Measures; Mediating; Membrane Glycoproteins; Membrane Proteins; Microbe; Monosaccharides; Mutagenesis; Mutation; Organism; Pathogenesis; Pathway interactions; Peptide Signal Sequences; Process; Production; Property; Protein Binding; Protein Family; Proteins; Research; Role; Series; Serine; Site; Streptococcus; Streptococcus gordonii; Surface; System; Therapeutic Agents; Virulence; Work; base; crosslink; glycosylation; in vivo; in vivo Model; insight; member; microbial; novel; novel therapeutics; pathogen; protein B; protein transport; public health relevance; receptor; research study; systems research; trafficking

DESCRIPTION (provided by applicant): The binding of bacteria with platelets appears to be 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. Our research has identified a novel genetic locus of Streptococcus gordonii that encodes a cell wall-anchored, serine-rich glycoprotein (GspB) that mediates the binding of streptococci to human platelets, and enhances virulence, as measured by animal models of endocardial infection. The locus also encodes four proteins that glycosylate GspB intracellularly, and seven proteins comprising a specialized system (the accessory Sec system) that are essential for GspB export. Two components (SecA2 and SecY2) are homologs of SecA and SecY of the canonical Sec system, and appear to have analogous functions. The other five members of this specialized system (Asp1-5) are also required for GspB export, but have no homologs of known function. The goal of this project is to further define the mechanisms for GspB trafficking and export to the bacterial surface. We will specifically examine the features of GspB and Asp1, Asp2, and Asp3 that contribute to the selective transport of the substrate by the accessory Sec system. Although both the extended N region of the GspB signal peptide and the novel AST domain (amino acids 91 - 117) of GspB are essential for export, it is unknown how these regions facilitate this process. To address these issues, Aim 1 uses in vivo site-specific photo cross-linking and mass spectroscopy to identify proteins that bind to the N and AST domains during transport. These studies should identify not only which components of the accessory Sec system interact directly with GspB, but will also provide insights into the sequence of these events. In addition, this work could identify additiona novel co-factors needed for transport. Aim 2 addresses the roles Asps 1, 2, and 3 in the targeting of GspB to the transmembrane channel complex (SecA2, SecY2, Asp4/5). The subcellular localization of GspB and the Asps will be examined by cell fractionation and fluorescence microscopy. By examining the location of GspB and the Asps, and how their distribution is affected by selected deletions and mutations, these studies will provide further information as to the roles of Asp1-3 in export. Aim 3 explores the bi-functional properties of Asp2, i.e., the role of this protein in mediating both GspB export and glycosylation. The impact of Asp2 binding on GspB export will be addressed through a series of targeted mutations. In addition, we will assess how mutagenesis of Asp2 alters the glycosylation of GspB, as measured by monosaccharide compositional analysis and mass spectroscopy. These experiments should provide considerable insights into the selective trafficking and biogenesis of GspB. Since the accessory Sec system is conserved among numerous Gram- positive pathogens, these studies should be highly applicable to a broad range of clinically- important organisms and infections, and may also yield targets for the development of novel therapeutic agents.

描述（由申请人提供）： 细菌与血小板的结合似乎是感染性心内膜炎发病机理的中心事件。这种相互作用对于血液的初始依恋可能很重要 传播生物体到心内膜，以及随后在心脏瓣膜表面形成宏观植被。我们的研究已经确定了链球菌Gordonii的一个新的遗传基因座，该基因座编码了细胞锚定的，富含丝氨酸的糖蛋白（GSPB），该糖蛋白（GSPB）介导了链球菌与人血小板的结合，并通过衡量的链球菌与人血小板的结合，并通过衡量的内膜感染动物模型来增强病毒性。该基因座还编码四种糖基酸GSPB细胞内的蛋白质，以及七个包含GSPB导出至关重要的专用系统（附件SEC系统）的蛋白质。两个组件（Seca2和secy2）是Canonical SEC系统的Seca和Secy的同源物，并且似乎具有类似的功能。 GSPB导出也需要该专业系统的其他五个成员（ASP1-5），但没有已知功能的同源物。 该项目的目的是进一步定义GSPB运输的机制并导出到细菌表面。我们将专门检查GSPB和ASP1，ASP2和ASP的功能，这些功能有助于通过附件SEC系统选择性传输基板。尽管GSPB信号肽的扩展N区域和GSPB的新型AST域（氨基酸91-117）都是出口必不可少的，但这些区域如何促进这一过程是未知的。为了解决这些问题，AIM 1在体内特定的照片交叉链接和质谱中使用，以鉴定运输过程中与N和AST结构域结合的蛋白质。这些研究不仅应确定附件SEC系统的哪些组成部分直接与GSPB相互作用，而且还将提供对这些事件序列的见解。此外，这项工作可以识别出运输所需的新颖的新型共同因素。 AIM 2解决了GSPB靶向跨膜通道复合物（SECA2，SECY2，ASP4/5）的ASP 1、2和3的角色。 GSPB和ASP的亚细胞定位将通过细胞分馏和荧光显微镜检查。通过检查GSPB和ASP的位置，以及其分布如何受选定的缺失和突变的影响，这些研究将提供有关ASP1-3在出口中的作用的进一步信息。 AIM 3探讨了ASP2的双功能特性，即该蛋白在介导GSPB输出和糖基化中的作用。 ASP2结合对GSPB导出的影响将通过一系列靶向突变来解决。此外，我们将评估ASP2的诱变如何通过单糖组成分析和质谱法测量，如何改变GSPB的糖基化。 这些实验应提供有关GSPB的选择性运输和生物发生的大量见解。由于辅助SEC系统在众多的阳性病原体中是保守的，因此这些研究应高度适用于广泛的临床重要生物和感染，并且还可能产生靶标，以开发新的治疗剂。