Pilus Assembly in Gram-Positive Bacteria

革兰氏阳性细菌中的菌毛组装

• 批准号: 7048723
• 负责人: Hung Ton-That
• 金额: $ 34.36万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-15 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7048723
• 关键词: Corynebacterium diphtheriae; Streptococcus agalactiae; Streptococcus infection; aminoacyltransferase; bacteria infection mechanism; bacterial genetics; bacterial proteins; cell wall; crosslink; electron microscopy; enzyme activity; gram positive bacteria; host organism interaction; laboratory mouse; lysozyme; molecular assembly /self assembly; peptidoglycan; pilus; respiratory infections; septicemia

DESCRIPTION (provided by applicant): Pili or fimbriae provide adhesive functions for the pathogenesis of bacterial infections and play important roles during the establishment of respiratory, urinary, and intestinal infectious diseases. The mechanism of pilus assembly in gram-negative bacteria has been well characterized; however, an assembly pathway for pili of gram-positive pathogens has not yet been revealed. The long-term goal of this proposal is to elucidate the mechanisms of pillus assembly in gram-positive microbes. Our specific hypothesis is that gram-positive bacterial pili are covalently linked by sortase to the bacterial cell wall. This hypothesis is based on the following observations: i) Gram-positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, a mechanism that requires sorting signals of surface protein precursors and sortase, which cleaves sorting signals at the LPXTG motif and links the C-terminus of surface proteins via an amide bond to the peptidoglycan cross-bridge; ii) Pilus assembly in Corynebacterium diphtheriae occurs by a mechanism of ordered cross-linking, whereby pilin-specific sortases cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits; iii) Muramidase treatment releases pili into the medium; iv) Sortase and pilin genes with sorting signal and pilin motif are found in many gram-positive pathogens. Based on these observations, we propose the following specific aims: 1. Determination of the molecular architecture of corynebacterial pili. The proposed experiments will characterize the biochemical linkages between subunits of corynebacterial pili and the requirements of sortases and pilin motif sequences for assembly. 2. Biochemical analysis of pilus assembly in corynebacteria. 3. Determination of the universality of pilus architecture and assembly mechanisms in gram-positive bacteria. We will determine, by biochemical and microscopic methods, whether Streptococcus agalactiae employes the same mechanism for pilus assembly. We will also evaluate the role of pilus-specific genes in S. agalactiae pathogenesis using rodent models of infection based on the observation that corynebacterial pili plays an important role in bacterial adherence. Together these studies will reveal the assembly mechanism of pili in gram-positive bacteria and the contribution of these organelles during the pathogenesis of infection.

描述（申请人提供）：菌毛或菌毛为细菌感染的发病机制提供粘附功能，并在呼吸道、泌尿道和肠道传染病的发生过程中发挥重要作用。革兰氏阴性细菌中菌毛组装的机制已得到很好的表征；然而，革兰氏阳性病原体菌毛的组装途径尚未被揭示。该提案的长期目标是阐明革兰氏阳性微生物菌毛组装的机制。我们的具体假设是革兰氏阳性细菌菌毛通过分选酶与细菌细胞壁共价连接。该假设基于以下观察： i) 革兰氏阳性微生物利用细胞壁肽聚糖作为蛋白质共价附着的表面细胞器，这种机制需要表面蛋白质前体和分选酶的分选信号，分选酶在LPXTG 基序并通过酰胺键将表面蛋白的 C 末端连接至肽聚糖跨桥； ii) 白喉棒状杆菌中的菌毛组装通过有序交联机制发生，其中菌毛蛋白特异性分选酶在分选信号处切割前体蛋白并涉及菌毛蛋白基序序列的侧链氨基以在菌毛蛋白亚基之间产生连接； iii) 胞壁质酶处理将菌毛释放到培养基中； iv) 在许多革兰氏阳性病原体中发现了具有分选信号和菌毛蛋白基序的分选酶和菌毛蛋白基因。基于这些观察，我们提出以下具体目标： 1. 确定棒状杆菌菌毛的分子结构。拟议的实验将表征棒状杆菌菌毛亚基之间的生化联系以及分选酶和菌毛蛋白基序序列组装的要求。 2.棒状杆菌中菌毛组装的生化分析。 3.确定革兰氏阳性菌菌毛结构和组装机制的普遍性。我们将通过生化和显微镜方法确定无乳链球菌是否采用相同的菌毛组装机制。我们还将根据棒状杆菌菌毛在细菌粘附中发挥重要作用的观察结果，使用啮齿动物感染模型来评估菌毛特异性基因在无乳链球菌发病机制中的作用。这些研究将共同​​揭示革兰氏阳性菌中菌毛的组装机制以及这些细胞器在感染发病机制中的贡献。