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的一种假定的中心机制。细菌-血小板结合对于血液传播的生物体最初附着在瓣膜表面以及随后的宏观植物的形成都可能是重要的。我们先前的研究表明，米氏链球菌SF100株的血小板结合部分是由温和噬菌体(SM1)编码的两个细胞壁相关蛋白(PblA和PblB)介导的。这两种蛋白在细菌表面的表达是SF100最大限度地结合血小板所必需的。 这项建议的总体目标是进一步阐明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。我们将首先提纯PblA和PblB，并在体外检测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都能在体外直接与人血小板结合。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也将被用来鉴定它们各自的血小板结合部位，目前为止，通过免疫印迹和免疫沉淀或亲和层析。然后，将根据需要通过几种方法(例如，N-末端测序或质谱学)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，以及这些蛋白是否与其他噬菌体结构蛋白形成血小板结合复合体。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和选定的突变体的毒力。通过表征链球菌黏附素对血小板的作用，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，它还可能为新的预防或治疗策略确定新的靶点。