Mechanism of the Usher in Assembly and Secretion of Pili

描述(申请人提供)：项目摘要。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。革兰氏阴性细菌除了细胞质膜外还有一层外膜，必须跨越这两个屏障分泌毒力因子。发生这种情况的机制可能相当复杂，而且还没有被很好地理解。伴侣/引座者途径是一种毒力蛋白分泌途径，需要两种成分通过外膜分泌：周质伴侣和称为引座者的外膜蛋白。伴侣引导分泌蛋白质的正确折叠，并防止非途径相互作用。引座器在外膜处用作组装平台，并向细胞表面提供分泌通道。伴侣/引导者途径是广泛的病原菌组装和分泌与毒力相关的表面结构超家族所必需的。由该途径组装的原型细胞器是由尿路感染的主要病原体致病大肠杆菌表达的P和1型菌毛。P和1型菌毛是关键的毒力因子，分别允许肾脏和膀胱的结合和定植。该方案的目的是探索引座器的结构和功能，以了解整个OM中控制菌毛生物发生的分子机制，并利用伴侣/引座器途径作为了解革兰氏阴性菌毒力因子分泌的模型系统。这一提议将检验以下假设：引座者在OM中形成一个双孔复合体，其功能是毛孔组装和分泌部位。引导者功能的不同区域控制、协调和催化在引导者周质面上亚单位-亚基相互作用的伴侣交换，从而驱动毛状纤维的有序组装和分泌通过引导者到达细胞表面。第一个具体目的是探索引座处菌毛生物发生的分子机制。第二个具体目标是分析引座者-伴侣-亚单位在体内和体外的相互作用。第三个具体目标是使用X射线和电子结晶学确定引座器的结构，并使用冷冻电子显微镜对菌毛组装中间体进行成像。关联性。这项建议中描述的工作将阐明致病细菌细胞器生物发生和毒力因子分泌的机制。从这项提议中获得的知识将为开发新的抗菌剂创造机会，这是在抗生素耐药性日益增加的时期迫切需要的。

DESCRIPTION (provided by applicant): 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. The chaperone/usher pathway is a virulence protein secretion pathway that requires two components for secretion across the outer membrane: a periplasmic chaperone and an outer membrane protein termed an usher. The chaperone directs proper folding of the secreted proteins and prevents off-pathway interactions. The usher serves as an assembly platform at the outer membrane and provides a secretion channel to the cell surface. The chaperone/usher pathway is required for assembly and secretion of a superfamily of virulence-associated surface structures by a broad range of pathogens. The prototypical organelles assembled by this pathway are the P and type 1 pili expressed by uropathogenic Escherichia coli, the primary causative agent of urinary tract infections. P and type 1 pili are critical virulence factors, allowing binding and colonization of the kidney and bladder, respectively. 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 OM and to use the chaperone/usher pathway as a model system for understanding virulence factor secretion in Gram-negative bacteria. This proposal will test the following hypotheses: The usher forms a twin-pore complex in the OM that functions as a pilus assembly and secretion site. Distinct domains of the usher function to control, coordinate, and catalyze the exchange of chaperone-subunit interactions for subunit-subunit interactions at the periplasmic face of the usher to drive the ordered assembly and secretion of the pilus fiber through the usher to the cell surface. The first specific aim is to probe the molecular mechanisms of pilus biogenesis at the usher. The second specific aim is to analyze usher-chaperone-subunit interactions both in vivo and in vitro. The third specific aim is to determine the structure of the usher using X-ray and electron crystallography, and image pilus assembly intermediates using cryo-electron microscopy. Relevance. The work described in this proposal will elucidate mechanisms of organelle biogenesis and virulence factor secretion by pathogenic bacteria. Knowledge gained from this proposal will create opportunities for the development of novel antimicrobial agents, urgently needed during this time of increasing antibiotic resistance.