Prophage-Encoded Binding of S. mitis to Human Platelets

原噬菌体编码的轻链球菌与人血小板的结合

• 批准号: 7342140
• 负责人: PAUL M. SULLAM
• 金额: $ 38.37万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7342140
• 关键词: Address; Affinity Chromatography; Animal Model; Bacteria; Bacterial Adhesins; Bacteriophages; Binding; Binding Sites; Blood; Blood Platelets; Cell Wall; Cell surface; Complex; Development; Embolism; Endocarditis; Endocardium; Far-Western Blotting; Genetic; Goals; Human; Immunoprecipitation; In Vitro; Infection; Infective endocarditis; Lesion; Ligands; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Methods; Modeling; N-terminal; Organism; Oryctolagus cuniculus; Pathogenesis; Property; Prophages; Protein Binding; Proteins; Research; Role; Streptococcus; Streptococcus Viridans Group; Streptococcus mitis; Structural Protein; Surface; Therapeutic; Virulence; Work; human tissue; mutant; novel; receptor; septic

DESCRIPTION (provided by applicant): The binding of streptococci to human platelets is a postulated central mechanism in the pathogenesis of infective endocarditis. Bacterium-platelet binding may be important both for the initial attachment of blood-borne organisms to the valve surface, and for the subsequent formation of macroscopic vegetations. Our previous research has shown that platelet binding by Streptococcus mitis strain SF100 is mediated in part by two cell wall-associated proteins (PblA and PblB) that are encoded by a temperate bacteriophage (SM1). Expression of both proteins on the bacterial surface is required for maximum levels of platelet binding by SF100. The overall goal of this proposal is to delineate further the mechanisms by which these proteins contribute to platelet binding, and to determine the role of PblA and PblB mediated binding in the pathogenesis of endocarditis. We will first purify PblA and PblB, and examine the binding properties of each protein with human platelets in vitro, to determine whether either or both proteins can bind human platelets directly in vitro. Formal binding analysis will be done to determine whether binding resembles a receptor-ligand interaction. Purified PblA and PblB will also be used to identify their respective platelet binding sites, by far western blotting and by immunoprecipitation or affinity chromatography. Platelet membrane proteins bound by either PblA or PblB will then be identified by several methods as needed (e.g., N-terminal sequencing, or mass spectroscopy). We will also assess the mechanisms for the export of PblA and PblB to the bacterial surface, and whether these proteins form platelet-binding complexes with other phage structural proteins. To address the role of these adhesins in the pathogenesis of endocarditis, we will compare the virulence of SF100 and selected mutants in a rabbit model of endocardial infection. By characterizing streptococcal adhesins for platelets, this research will further define the role of platelet binding in the pathogenesis of endocarditis. In addition, it may identify novel targets for new preventative or therapeutic strategies.

描述（由申请人提供）：链球菌与人血小板的结合是感染性心内膜炎发病机理的一种假定的中心机制。细菌 - 血小板结合对于血液传播生物在瓣膜表面的初始附着以及随后的宏观植被的形成可能很重要。 我们先前的研究表明，链球菌菌株SF100的血小板结合部分是由两种由温带噬菌体（SM1）编码的细胞壁相关蛋白（PBLA和PBLB）介导的。 SF100的最大血小板结合水平需要两种蛋白在细菌表面上的表达。 该提案的总体目标是进一步描述这些蛋白质有助于血小板结合的机制，并确定PBLA和PBLB介导的结合在心内膜炎发病机理中的作用。我们将首先纯化PBLA和PBLB，并在体外检查每种蛋白质与人血小板的结合特性，以确定任何一种蛋白是否可以直接在体外结合人血小板。将进行形式的结合分析，以确定结合是否类似于受体配体相互作用。纯化的PBLA和PBLB也将用于鉴定其各自的血小板结合位点，远处的蛋白质印迹以及免疫沉淀或亲和色谱法。然后，由PBLA或PBLB结合的血小板膜蛋白将根据需要（例如N末端测序或质谱）鉴定出几种方法。我们还将评估将PBLA和PBLB导出到细菌表面的机制，以及这些蛋白是否与其他噬菌体结构蛋白形成血小板结合复合物。为了解决这些粘附素在心内膜炎发病机理中的作用，我们将比较SF100的毒力和心内膜感染模型中选定的突变体的毒力。通过表征血小板的链球菌粘附素，这项研究将进一步定义血小板结合在心内膜炎发病机理中的作用。此外，它可以确定新的预防或治疗策略的新目标。