Mechanism of the Usher in Assembly and Secretion of Pili

项目摘要 病原菌必须组装并分泌毒力因子与宿主组织相互作用并引起疾病。 革兰氏阴性菌除了细胞质膜外还有一层外膜（OM）， 毒力因子跨越这两个屏障。发生这种情况的机制还不清楚。 本项目使用分子伴侣-引导（CU）通路作为模型系统，以探讨 革兰氏阴性菌中的蛋白分泌和毒力因子生物合成。CU途径是一种保守的 分泌系统，专门用于组装称为皮利或菌毛的毒性相关表面结构。 皮利是毛发状聚合物，其通常用作粘附细胞器并在表面定殖中起作用， 生物膜形成、与宿主细胞的相互作用和发病机制。CU组装的原型结构 途径是由尿路致病性大肠杆菌表达的1型和P皮利。E.大肠杆菌是主要致病菌 尿路感染是一种主要的医疗负担，也是抗生素耐药性的来源。1型和P皮利 是分别介导膀胱和肾脏定植的关键毒力因子。CU途径 需要两种成分来分泌穿过OM：周质伴侣蛋白和完整的OM蛋白 叫引座员。伴侣蛋白指导菌毛亚基蛋白的正确折叠，并维持蛋白质在 一个有议会资格的国家。引座员是一种动态分子机器， 亚基-亚基相互作用，促进菌毛纤维的有序聚合，并提供通道， 菌毛分泌到细胞表面。皮利是启动和维持感染的关键，CU途径 代表了用于开发抗毒性治疗剂的有吸引力的靶标，特别是用于感染 泌尿系统本研究的目的是探讨引座员的结构和功能，以获得一个完整的 了解细菌OM中菌毛生物发生的分子机制，并利用 知识和专业知识获得了在这个项目的过程中，以开发小分子抑制剂的CU 通路这项提议将检验引座员区分和组织菌毛亚单位的假设 以保证粘附细胞器的组装，并且引导器和引导器-伴侣蛋白-亚基界面 是小分子抑制的靶点。本建议的具体目标是：1）确定如何引座员 被激活并启动菌毛生物发生; 2）定义亚单位掺入和菌毛的分子细节 延伸周期;和3）鉴定菌毛生物发生中引导功能的小分子抑制剂。拟议 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 在细菌OM，并将提供一个新的治疗药物，选择性地开发基础 破坏毒力因子的生物合成。

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.