Mechanism of the Usher in Assembly and Secretion of Pili

霹雳虫的组装与分泌机制

• 批准号: 9335873
• 负责人: David G Thanassi
• 金额: $ 45.1万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9335873
• 关键词: Address; Adhesives; Antibiotic Resistance; Bacteria; Binding; Biogenesis; Biological; Biological Models; Bladder; Carrier Proteins; Cell membrane; Cell surface; Cells; Complex; Cryoelectron Microscopy; Development; Disease; Energy-Generating Resources; Ensure; Escherichia coli; Fiber; Fluorescence; Funding; Goals; Gram-Negative Bacteria; Hair; Healthcare; Kidney; Knowledge; Lipids; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Microbial Biofilms; Molecular; Molecular Chaperones; Molecular Machines; Movement; Organelles; Pathogenesis; Pathway interactions; Physiological; Pilum; Polymers; Process; Protein Secretion; Protein Subunits; Protein translocation; Proteins; Regulation; Resolution; Role; Site; Structure; Surface; System; Techniques; Testing; Therapeutic Agents; Time; Tissues; Urinary tract infection; Uropathogenic E. coli; Virulence; Virulence Factors; Work; X-Ray Crystallography; antimicrobial drug; base; cell envelope; insight; interdisciplinary approach; membrane assembly; nanodisk; novel; novel therapeutics; pathogenic bacteria; periplasm; polymerization; premature; prevent; protein protein interaction; prototype

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.

项目概要 致病菌必须组装并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，并且必须 跨越这两个障碍分泌毒力因子。发生这种情况的机制可能相当 复杂且不太容易理解。我们使用陪伴/引座员 (CU) 途径作为模型系统 用于探究革兰氏阴性菌蛋白质分泌和毒力因子生物发生的机制 细菌。 CU途径是一个保守的分泌系统，致力于毒力相关的组装 称为菌毛或菌毛的表面结构。菌毛是毛发状聚合物，通常用作粘合剂 细胞器并在表面定植、生物膜形成、与宿主细胞相互作用等方面发挥作用 发病。 CU途径组装的原型结构是P和1型菌毛，表达为 尿路致病性大肠杆菌。大肠杆菌是尿路感染的主要病原体，是尿路感染的主要病原体。 医疗负担，P 菌毛和 1 型菌毛是介导肾脏定植的关键毒力因子 和膀胱，分别。 CU 途径需要两个成分才能穿过 OM 分泌： 周质伴侣和称为引座员的完整 OM 蛋白。监护人指导正确折叠 亚基蛋白质并维持蛋白质处于可组装状态。引座员是动态的 催化亚基-亚基相互作用形成的分子机器，促进有序 菌毛纤维的聚合，并提供菌毛分泌到细胞表面的通道。这 该提案的目标是探讨引座员的结构和功能，以了解 控制菌毛跨细菌外膜生物发生的分子机制，并使用 CU 途径作为了解革兰氏阴性细菌毒力因子分泌的模型系统。这 提案将测试引座员精心安排域移动的定义序列的假设，以及 蛋白质-蛋白质相互作用，以确保功能性菌毛的组装，并引导分子在 OM 中的不对称方式催化菌毛生物发生。第一个具体目标将通过以下方式定义机制： 引座员调节并利用蛋白质-蛋白质相互作用来催化有序的菌毛组装。这 第二个具体目标将确定引导分子如何以不对称方式相互作用以催化菌毛 以最大效率进行生物发生。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： 迎来其原生状态。该应用程序将采用多学科方法来回答问题 与蛋白质跨生物膜分泌的基本机制、毒力因子相关 生物发生和复杂细胞器的组装。