Mechanism of the Usher in Assembly and Secretion of Pili

描述（由申请人提供）：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具有外膜，并且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，而且还没有得到很好的理解。伴侣蛋白/引导蛋白途径是毒力蛋白分泌途径，其需要两种组分用于跨外膜分泌：周质伴侣蛋白和称为引导蛋白的外膜蛋白。伴侣蛋白指导分泌蛋白的正确折叠并防止非途径相互作用。引导器用作外膜处的组装平台，并提供通向细胞表面的分泌通道。分子伴侣/引导途径是广泛的病原体组装和分泌毒力相关表面结构超家族所必需的。通过该途径组装的原型细胞器是由尿路感染的主要病原体致尿病性大肠杆菌表达的P和1型皮利。P和1型皮利是关键的毒力因子，分别允许肾脏和膀胱的结合和定殖。该提案的目标是探测引导器的结构和功能，以了解整个OM的菌毛生物发生的分子机制，并使用分子伴侣/引导器途径作为了解革兰氏阴性菌中毒力因子分泌的模型系统。该提案将测试以下假设：引座形成一个双孔复合体的OM，作为菌毛组装和分泌网站的功能。引导器的不同结构域的功能是控制、协调和催化在引导器的周质面处的伴侣-亚基相互作用与亚基-亚基相互作用的交换，以驱动菌毛纤维的有序组装和分泌通过引导器到达细胞表面。第一个具体的目的是探讨在引座员菌毛生物发生的分子机制。第二个具体目标是分析usher分子伴侣亚基在体内和体外的相互作用。第三个具体的目标是确定使用X射线和电子晶体学的引座员的结构，并使用冷冻电子显微镜的图像菌毛组装中间体。本案无关本研究将阐明病原菌细胞器生物合成和毒力因子分泌的机制。从该提案中获得的知识将为开发新型抗菌剂创造机会，这在抗生素耐药性增加的时期迫切需要。

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.