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， 疾病革兰氏阴性菌除了细胞质膜外还有一层外膜， 分泌毒力因子穿过这两个屏障。发生这种情况的机制可能相当复杂， 很复杂，不太了解。我们使用伴侣/引导（CU）途径作为模型系统 探讨革兰氏阴性杆菌蛋白分泌和毒力因子生物合成的机制， 细菌CU途径是一种保守的分泌系统，致力于组装与毒性相关的蛋白质。 称为皮利或菌毛的表面结构。皮利是毛发状聚合物，通常用作粘合剂 细胞器，并在表面定植、生物膜形成、与宿主细胞的相互作用中发挥作用， 发病机制由CU途径组装的原型结构是由表达的P和1型皮利。 尿路致病性大肠杆菌E.大肠杆菌是泌尿道感染的主要病原体， 卫生保健负担，以及P和1型皮利是介导肾脏定植的关键毒力因子 和膀胱。CU途径需要两种组分用于跨OM的分泌： 周质分子伴侣和一个完整的OM蛋白称为引座员。伴侣蛋白指导蛋白质的正确折叠， 亚基蛋白质，并保持蛋白质在组装能力的状态。引座员是个有活力的人 催化亚基-亚基相互作用形成的分子机器， 菌毛纤维的聚合，并为菌毛分泌到细胞表面提供通道。的 本研究的目的是探讨引座员的结构和功能， 控制菌毛生物发生的分子机制，并使用CU 途径作为了解革兰氏阴性菌中毒力因子分泌的模型系统。这 该提案将测试以下假设：引座员编排了一个定义的域运动序列， 蛋白质-蛋白质相互作用以确保功能性皮利的组装，并且引导分子以 在OM中以不对称的方式催化菌毛的生物发生。第一个具体目标将通过以下方式确定机制： 引导者调节并利用蛋白质-蛋白质相互作用来催化有序的菌毛组装。的 第二个具体目标是确定引导分子如何以不对称方式相互作用以催化菌毛 以最大的效率进行生物合成。第三个具体目标将揭示菌毛生物发生的结构基础， 引座员在其本国。这个应用程序将应用多学科的方法来回答问题 与跨生物膜的蛋白质分泌的基本机制相关，毒力因子 生物发生和复杂细胞器的组装。

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 in addition to a cytoplasmic membrane, and must secrete virulence factors across both these barriers. The mechanisms by which this occurs can be quite complex and are not well understood. We are using 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 P and type 1 pili expressed by uropathogenic Escherichia coli. E. coli is the primary causative agent of urinary tract infections, a major healthcare burden, and P and type 1 pili are critical virulence factors that mediate colonization of the kidneys and bladder, 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 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. The goals of this proposal are to probe the structure and function of the usher to gain an understanding of the molecular mechanisms governing pilus biogenesis across the bacterial outer membrane, and to use the CU pathway as a model system for understanding virulence factor secretion in Gram-negative bacteria. This proposal will test the hypothesis that the usher orchestrates a defined sequence of domain movements and protein-protein interactions to ensure the assembly of functional pili, and that usher molecules interact in an asymmetric manner in the OM to catalyze pilus biogenesis. The first specific aim will define mechanisms by which the usher regulates and harnesses protein-protein interactions to catalyze ordered pilus assembly. The second specific aim will determine how usher molecules interact in an asymmetric manner to catalyze pilus biogenesis with maximal efficiency. The third specific aim will reveal the structural basis of pilus biogenesis by the usher in its native state. This application will apply a multidisciplinary approach to answer questions relevant to fundamental mechanisms of protein secretion across biological membranes, virulence factor biogenesis, and the assembly of complex organelles.