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: 7174307
• 负责人: PAUL M. SULLAM
• 金额: $ 39.11万
• 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 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7174307
• 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制中假定的核心机制。细菌-血小板结合对于血源性生物体最初附着到瓣膜表面以及随后形成肉眼可见的植被都很重要。 我们之前的研究表明，轻链球菌 SF100 菌株与血小板的结合部分是由温带噬菌体 (SM1) 编码的两种细胞壁相关蛋白（PblA 和 PblB）介导的。 SF100 最大限度地结合血小板需要两种蛋白在细菌表面的表达。 该提案的总体目标是进一步描述这些蛋白质促进血小板结合的机制，并确定 PblA 和 PblB 介导的结合在心内膜炎发病机制中的作用。我们将首先纯化PblA和PblB，并在体外检查每种蛋白与人血小板的结合特性，以确定其中一种或两种蛋白是否可以在体外直接结合人血小板。将进行正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 还将用于通过蛋白质印迹法和免疫沉淀或亲和层析来鉴定其各自的血小板结合位点。然后根据需要通过多种方法（例如，N 端测序或质谱）来鉴定与 PblA 或 PblB 结合的血小板膜蛋白。我们还将评估 PblA 和 PblB 输出到细菌表面的机制，以及这些蛋白质是否与其他噬菌体结构蛋白形成血小板结合复合物。为了阐明这些粘附素在心内膜炎发病机制中的作用，我们将在兔心内膜感染模型中比较 SF100 和选定突变体的毒力。通过表征血小板的链球菌粘附素，这项研究将进一步明确血小板结合在心内膜炎发病机制中的作用。此外，它还可以确定新的预防或治疗策略的新靶标。