Pilus Assembly in Gram-Positive Bacteria

性状（由申请人提供）：皮利或菌毛为细菌感染的发病机制提供粘附功能，并在呼吸道、泌尿道和肠道感染性疾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得到很好的表征，然而，革兰氏阳性病原体的皮利组装途径尚未被揭示。这项提案的长期目标是阐明革兰氏阳性菌中绒毛组装的机制。我们的特定假设是革兰氏阳性细菌的皮利通过分选酶共价连接到细菌细胞壁。i）革兰氏阳性微生物利用细胞壁肽聚糖作为表面细胞器用于蛋白质的共价附着，这是一种需要表面蛋白质前体和分选酶的分选信号的机制，分选酶在LPXTG基序处切割分选信号并通过酰胺键将表面蛋白的C末端连接到肽聚糖横桥; ii）白喉棒状杆菌中的菌毛装配通过有序交联机制发生，由此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产生菌毛蛋白亚基之间的连接; 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，我们提出了以下具体目标：1。棒状杆菌皮利分子结构的测定。拟议的实验将表征棒状杆菌皮利亚基之间的生化联系和组装的分选酶和菌毛基序序列的要求。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普遍性的测定。我们将确定，通过生物化学和显微镜的方法，是否无乳链球菌employes相同的机制为菌毛组装。我们还将评估菌毛特异性基因在S。基于棒状杆菌皮利在细菌粘附中起重要作用的观察，使用啮齿动物感染模型的无乳症发病机制。这些研究将揭示革兰氏阳性菌中皮利的组装机制以及这些细胞器在感染发病过程中的作用。

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.