Mechanism of the Usher in Assembly and Secretion of Pili

描述（由申请人提供）：项目概述。致病菌必须聚集并分泌毒力因子才能与宿主组织相互作用而致病。革兰氏阴性菌除了细胞质膜外还有外膜，必须跨越这两层屏障分泌毒力因子。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。伴蛋白/usher途径是一种毒力蛋白分泌途径，需要两种成分才能跨外膜分泌：质周伴蛋白和称为usher的外膜蛋白。伴侣指导分泌蛋白的适当折叠，并防止通路外相互作用。usher作为外膜的组装平台，并为细胞表面提供分泌通道。伴侣/usher通路是多种病原体组装和分泌毒力相关表面结构超家族所必需的。通过该途径组装的典型细胞器是尿路感染的主要病原体尿路致病性大肠杆菌表达的P型和1型菌毛。P型菌毛和1型菌毛是关键的毒力因子，分别允许结合和定植肾脏和膀胱。本研究的目的是通过探索引子的结构和功能来了解毛菌生物发生的分子机制，并将伴侣/引子通路作为理解革兰氏阴性菌毒力因子分泌的模型系统。该提案将测试以下假设：usher在OM中形成双孔复合物，作为菌毛组装和分泌位点。不同的usher结构域的功能是控制、协调和催化伴侣-亚基相互作用在usher的质周表面进行亚基-亚基相互作用的交换，以驱动毛纤维通过usher到细胞表面的有序组装和分泌。第一个具体目的是探讨毛菌在诱导期生物发生的分子机制。第二个具体目标是分析体内和体外的引子-伴侣-亚基相互作用。第三个具体目标是使用x射线和电子晶体学确定usher的结构，并使用冷冻电子显微镜成像毛毛组装中间体。的相关性。本研究将阐明病原菌细胞器生物发生和毒力因子分泌的机制。从这一建议中获得的知识将为开发新的抗微生物药物创造机会，这在抗生素耐药性日益增加的时期是迫切需要的。

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.