Mechanism of the Usher in Assembly and Secretion of Pili

项目总结 病原菌必须组装和分泌毒力因子，才能与宿主组织相互作用并引起 疾病。革兰氏阴性细菌除了细胞质膜外还有外膜，必须 跨越这两个障碍，隐藏毒力因子。发生这种情况的机制可能相当 这些问题很复杂，而且还没有得到很好的理解。我们正在使用伴侣/引导者(CU)途径作为模型系统 探讨革兰氏阴性菌蛋白质分泌和毒力因子的生物发生机制 细菌。铜绿假单胞菌途径是一个保守的分泌系统，专用于组装与毒力相关的 表面结构称为菌毛或菌毛。菌毛是一种毛发状聚合物，通常用作粘合剂 细胞器，在表面的定植、生物膜的形成、与宿主细胞的相互作用以及 发病机制。由CU途径组装的原型结构是由以下表达的P和1型菌毛 泌尿系致病性大肠杆菌。大肠杆菌是尿路感染的主要病原体，是一种主要的 医疗负担，以及P和1型菌毛是调节肾脏定植的关键毒力因素 和膀胱癌。CU途径需要两种成分通过OM进行分泌：A 周质伴侣和一个被称为引座者的完整OM蛋白。监护人指导适当的折叠 亚单位蛋白质，并维持蛋白质在组装能力状态。引座员是一个动态的人 催化亚单位-亚单位相互作用形成的分子机器，促进有序 菌毛纤维聚合，并为菌毛分泌到细胞表面提供通道。这个 这项建议的目的是探讨引座员的结构和功能，以了解 细菌外膜上菌毛生物发生的分子机制，并利用CU 途径作为了解革兰氏阴性菌毒力因子分泌的模式系统。这 提案将测试这样的假设，即引座员协调定义的域移动序列，并 蛋白质-蛋白质相互作用，以确保功能性菌毛的组装，以及引入分子在 在OM中以不对称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定义机制 引座员调节和利用蛋白质-蛋白质相互作用来催化有序的菌毛组装。这个 第二个特定目标将确定引导者分子如何以不对称的方式相互作用以催化菌毛 效率最高的生物发生。第三个具体目标将通过以下方式揭示菌毛生物发生的结构基础 引座者迎来了它的原生状态。此应用程序将应用多学科方法来回答问题 与跨越生物膜的蛋白质分泌的基本机制有关，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.