Analysis of Group B Streptococcal Fibrinogen Cleavage

B 族链球菌纤维蛋白原裂解分析

• 批准号: 6874477
• 负责人: Daniel W Shelver
• 金额: $ 10.8万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-15 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6874477
• 关键词: Bacillus subtilis; SDS polyacrylamide gel electrophoresis; Streptococcus agalactiae; affinity chromatography; chemical cleavage; endopeptidases; fibrinogen; mass spectrometry; neutrophil; opsonin; phagocytosis; polymerase chain reaction; protein purification; protein structure function; thrombin; two dimensional gel electrophoresis; virulence; western blottings

DESCRIPTION (provided by applicant): Group B steptococcus is an important human pathogen, and is the leading cause of invasive neonatal disease including pneumonia, sepsis, and meningitis. GBS has also been recently recognized as a cause of invasive disease in the immunocompromised and eldedy. While some key virulence factors of GBS have been characterized, such as the polysaccharide capsule and the hemolysin, relatively few other virulence factors have been thoroughly characterized. In this proposal, we characterize a novel streptococcal virulence factor. We have discovered a streptococcal protease (CspA) that is necessary for full virulence in a neonatal rat sepsis model, is necessary for full resistance to opsonophagocytosis by human neutrophils, and mediates cleavage of human fibrinogen. In this proposal, we seek to prove that CspA is the protease responsible for the cleavage of human fibrinogen by purification of the CspA protease and characterization of how this important virulence factor cleaves fibrinogen. The first aim seeks to define the biochemical reaction mediated by CspA by determining the site of CspA-mediated cleavage of fibrinogen. It is our hypothesis that CspA cleaves fibrinogen in a manner similar to thrombin to generate a substance that coats the bacterium and protects it from opsonophagocytosis. We will investigate the characteristics of the fibrinogen cleavage reaction by two-dimensional electrophoresis, N-terminal sequencing of the fibrinogen reactants and products, and characterization by mass spectrometry of the fibrinogen peptide. The second aim seeks to prove that CspA is the protease responsible for the cleavage of fibrinogen by over expressing the protein in Bacillus subtilis, purifying the protease to homogeneity, and demonstrating that purified CspA can promote the fibrinogen cleavage reaction characterized in the first aim. The availability of the purified protein will facilitate future biochemical structure-function studies on this important virulence factor of GBS.

描述（由申请人提供）：B族链球菌是一种重要的人类病原体，是肺炎、败血症和脑膜炎等侵袭性新生儿疾病的主要原因。最近，GBS 也被认为是免疫功能低下和老年患者侵袭性疾病的原因。虽然 GBS 的一些关键毒力因子（例如多糖荚膜和溶血素）已得到表征，但其他毒力因子相对较少得到彻底表征。在本提案中，我们描述了一种新型链球菌毒力因子。我们发现了一种链球菌蛋白酶（CspA），它对于新生大鼠脓毒症模型中的完全毒力是必需的，对于人中性粒细胞完全抵抗调理吞噬作用是必需的，并且介导人纤维蛋白原的裂解。在本提案中，我们试图通过纯化 CspA 蛋白酶并表征这一重要毒力因子如何裂解纤维蛋白原，来证明 CspA 是负责裂解人纤维蛋白原的蛋白酶。第一个目标是通过确定 CspA 介导的纤维蛋白原裂解位点来定义 CspA 介导的生化反应。我们假设 CspA 以类似于凝血酶的方式裂解纤维蛋白原，产生一种包裹细菌并保护其免受调理吞噬作用的物质。我们将通过二维电泳、纤维蛋白原反应物和产物的 N 末端测序以及纤维蛋白原肽的质谱表征来研究纤维蛋白原裂解反应的特征。第二个目标旨在通过在枯草芽孢杆菌中过表达该蛋白、将蛋白酶纯化至同质性来证明CspA是负责纤维蛋白原裂解的蛋白酶，并证明纯化的CspA可以促进第一个目标中表征的纤维蛋白原裂解反应。纯化蛋白的获得将有助于未来对 GBS 这一重要毒力因子的生化结构功能研究。