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制的核心事件。这种相互作用对于血液的初始附着可能很重要 将生物体带到心内膜，并随后在心脏瓣膜表面形成肉眼可见的赘生物。我们的研究发现了戈登链球菌的一个新基因位点，它编码细胞壁锚定的富含丝氨酸的糖蛋白（GspB），介导链球菌与人类血小板的结合，并增强毒力（通过心内膜感染的动物模型测量）。该基因座还编码四种在细胞内糖基化 GspB 的蛋白质，以及构成 GspB 输出所必需的专门系统（辅助 Sec 系统）的七种蛋白质。两个组件（SecA2 和 SecY2）是规范 Sec 系统的 SecA 和 SecY 的同系物，并且似乎具有类似的功能。该专门系统的其他五个成员 (Asp1-5) 也是 GspB 输出所必需的，但没有已知功能的同源物。 该项目的目标是进一步定义 GspB 运输和输出到细菌表面的机制。我们将专门研究 GspB 和 Asp1、Asp2 和 Asp3 的特征，这些特征有助于辅助 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）对于输出至关重要，但尚不清楚这些区域如何促进这一过程。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识别运输过程中与 N 和 AST 结构域结合的蛋白质。这些研究不仅应该确定辅助 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，这项工作可以确定运输所需的其他新辅助因子。 目标 2 涉及 Asps 1、2 和 3 在 GspB 靶向跨膜通道复合体（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，这些研究将提供有关 Asp1-3 在输出中的作用的进一步信息。 目标 3 探索 Asp2 的双功能特性，即该蛋白质在介导 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有针对性的突变来解决。此外，我们将通过单糖成分分析和质谱法测量 Asp2 的诱变如何改变 GspB 的糖基化。 这些实验将为 GspB 的选择性运输和生物发生提供重要的见解。由于辅助Sec系统在众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微生物和感染，并且还可能产生开发新型治疗剂的靶标。