Mechanism of the Usher in Assembly and Secretion of Pili

项目总结 病原菌必须聚集和分泌毒力因子，才能与宿主组织相互作用而致病。 革兰氏阴性菌除细胞质膜外还有外膜(OM)，必须分泌 毒力因素跨越了这两个障碍。发生这种情况的机制还没有被很好地理解。 本项目以伴侣-引座者(CU)通路为模型系统，探讨其发病机制 革兰氏阴性杆菌的蛋白质分泌和毒力因子的生物发生。CU途径是一条保守的 专用于组装与毒力相关的表面结构的分泌系统，称为菌毛或菌毛。 菌毛是一种毛发状聚合物，通常起粘性细胞器的作用，并在表面定植中发挥作用， 生物膜的形成，与宿主细胞的相互作用，以及发病机制。CU组装的原型结构 途径是由致尿性大肠埃希菌表达的1型和P菌毛。大肠杆菌是主要的致病因素。 尿路感染的病原体，主要的医疗负担和抗生素耐药性的来源。I型和P型毛发 是关键的毒力因子，分别介导膀胱和肾脏的定植。CU路径 整个OM的分泌需要两个成分：一个周质伴侣和一个完整的OM蛋白 被称为引座员。伴侣引导毛状亚单位蛋白质的正确折叠，并将蛋白质维持在 一个有装配能力的国家。引座者是一个动态的分子机器，催化形成 亚基-亚基相互作用，促进菌毛纤维的有序聚合，并为 菌毛分泌到细胞表面。菌毛是启动和维持感染的关键，铜绿假单胞菌途径 是开发抗毒力疗法的一个有吸引力的靶点，特别是对于感染的 尿路。这项建议的目标是探索引座员的结构和功能，以获得一个完整的 了解细菌OM菌毛生物发生的分子机制，并开发 在开发CU小分子抑制剂的项目过程中获得的知识和专业知识 路径。这项提议将检验引座员区分并组织菌毛亚单位的假设 以确保粘性细胞器的组装，以及引座员和引座员-伴侣-亚单位的接口 是小分子抑制的靶标。这项建议的具体目的是：1)确定引座员如何 被激活并启动菌毛的生物发生；2)确定亚基结合和菌毛的分子细节 以及3)鉴定在菌毛生物发生中引导功能的小分子抑制物。建议数 研究将回答蛋白质分泌和复杂细胞器的调节组装的基本问题 并将为开发具有选择性的新型治疗剂提供基础 破坏毒力因子的生物发生。

PROJECT SUMMARY Pathogenic bacteria must assemble and secrete virulence factors to interact with host tissues and cause disease. Gram-negative bacteria have an outer membrane (OM) in addition to a cytoplasmic membrane and must secrete virulence factors across both these barriers. The mechanisms by which this occurs are not well understood. This project uses the chaperone-usher (CU) pathway as a model system with which to probe mechanisms of protein secretion and virulence factor biogenesis in Gram-negative bacteria. The CU pathway is a conserved secretion system dedicated to the assembly of virulence-associated surface structures termed pili or fimbriae. Pili are hair-like polymers that typically function as adhesive organelles and have roles in colonization of surfaces, biofilm formation, interactions with host cells, and pathogenesis. The prototype structures assembled by the CU pathway are the type 1 and P pili expressed by uropathogenic Escherichia coli. E. coli is the primary causative agent of urinary tract infections, a major healthcare burden and source of antibiotic resistance. Type 1 and P pili are critical virulence factors that mediate colonization of the bladder and kidneys, respectively. The CU pathway requires two components for secretion across the OM: a periplasmic chaperone and an integral OM protein termed the usher. The chaperone directs proper folding of pilus subunit proteins and maintains the proteins in an assembly-competent state. The usher is a dynamic molecular machine that catalyzes the formation of subunit-subunit interactions, promotes ordered polymerization of the pilus fiber, and provides the channel for secretion of the pilus to the cell surface. Pili are critical for initiating and sustaining infection, and the CU pathway represents an attractive target for the development of anti-virulence therapeutics, particularly for infection of the urinary tract. The goals of this proposal are to probe the structure and function of the usher to gain a complete understanding of the molecular mechanisms governing pilus biogenesis at the bacterial OM, and to exploit the knowledge and expertise gained over the course of this project to develop small molecule inhibitors of the CU pathway. This proposal will test the hypothesis that the usher distinguishes among and organizes pilus subunits to guarantee the assembly of adhesive organelles, and that the usher and usher-chaperone-subunit interfaces are targets for small molecule inhibition. The Specific Aims of this proposal are to: 1) Determine how the usher is activated and initiates pilus biogenesis; 2) Define molecular details of the subunit incorporation and pilus extension cycle; and 3) Identify small molecule inhibitors of usher function in pilus biogenesis. The proposed studies will answer fundamental questions of protein secretion and the regulated assembly of complex organelles at the bacterial OM, and will provide a foundation for the development of novel therapeutic agents that selectively disrupt virulence factor biogenesis.