Pilus Assembly in Gram-Positive Bacteria

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连功能，并在呼吸道、泌尿系统和肠道传染病的建立过程中发挥重要作用。革兰氏阴性菌菌毛组装的机制已被很好地描述，然而，革兰氏阳性病原菌的菌毛组装途径尚未被揭示。这项提议的长期目标是阐明革兰氏阳性微生物中小球组装的机制。我们的具体假设是，革兰氏阳性细菌菌毛通过分类酶与细菌细胞壁共价连接。这一假说基于以下观察结果：i)革兰氏阳性微生物利用细胞壁的肽聚糖作为蛋白质共价附着的表面细胞器，这是一种需要表面蛋白前体的分选信号和分类酶的机制，分类酶在LPXTG基序上切割分选信号，并通过酰胺键将表面蛋白的C-末端连接到肽聚糖交叉桥；ii)白喉棒杆菌中的菌毛组装是通过有序的交联机制进行的，通过菌毛素专一性的分解酶在分选信号中裂解前体蛋白，并涉及菌毛素基序序列的侧链氨基，以产生菌毛素亚单位之间的联系；iii)胞壁酰胺酶处理将菌毛释放到介质中；IV)在许多革兰氏阳性病原菌中发现了具有分选信号和菌毛素基序的索糖酶和菌毛素基因。基于这些观察结果，我们提出了以下具体目标：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，以及组装所需的分类酶和菌毛基序序列。2.棒状杆菌菌毛组装的生化分析。3.革兰氏阳性菌菌毛结构和组装机制的普适性测定我们将通过生化和显微镜方法来确定无乳链球菌是否使用相同的机制来组装菌毛。我们还将基于棒状杆菌菌毛在细菌黏附中的重要作用的观察，利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中的作用。这些研究将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作用。

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.