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: 8434422
• 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/8434422
• 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制的中心事件。这种相互作用对于血液的初始附着 生物体附着于内膜，随后在心脏瓣膜表面形成肉眼可见的赘生物。我们的研究已经确定了一个新的遗传基因座的戈登链球菌编码细胞壁锚定，富含丝氨酸的糖蛋白（GspB），介导的结合链球菌对人类血小板，并增强毒力，作为测量的动物模型内分泌感染。该基因座还编码四种蛋白质，糖基化GspB细胞内，和七种蛋白质，包括一个专门的系统（辅助Sec系统）是必不可少的GspB出口。两个组分（SecA2和SecY2）是经典Sec系统的SecA和SecY的同系物，并且似乎具有类似的功能。这个专门系统的其他五个成员（Asp1 - 5）也需要GspB输出，但没有已知功能的同源物。 该项目的目标是进一步确定GspB运输和出口到细菌表面的机制。我们将具体研究GspB和Asp1，Asp2和Asp3的功能，有助于辅助Sec系统选择性转运底物。尽管GspB信号肽的延伸N区和GspB的新AST结构域（氨基酸91 - 117）对于输出是必需的，但尚不清楚这些区域如何促进该过程。为了解决这些问题，Aim 1使用体内位点特异性光交联和质谱来鉴定在运输过程中与N和AST结构域结合的蛋白质。这些研究不仅应确定辅助Sec系统的哪些组件直接与GspB相互作用，而且还将提供对这些事件顺序的见解。此外，这项工作可以确定额外的运输所需的新的辅助因子。 目的2阐述Asps 1、2和3在GspB靶向跨膜通道复合物（SecA2、SecY2、Asp4/5）中的作用。GspB和Asps的亚细胞定位将通过细胞分级分离和荧光显微镜检查。通过检查GspB和Asps的位置，以及它们的分布如何受到选定的缺失和突变的影响，这些研究将提供关于Asp1 - 3在出口中的作用的进一步信息。 目的3探索Asp2的双功能性质，即，该蛋白在介导GspB输出和糖基化中的作用。Asp2结合对GspB输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的诱变如何改变GspB的糖基化，通过单糖组成分析和质谱测定。 这些实验应该提供相当多的见解GspB的选择性贩运和生物起源。由于辅助Sec系统在许多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广泛的临床重要生物体和感染，并且还可以产生用于开发新治疗剂的靶点。