Mechanism of the Usher in Assembly and Secretion of Pili

霹雳虫的组装与分泌机制

• 批准号: 7941574
• 负责人: David G Thanassi
• 金额: $ 7.24万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7941574
• 关键词: Adhesives; Antibiotic Resistance; Bacterial Adhesins; Binding; Biogenesis; Biological Models; Bladder; C-terminal; Cell membrane; Cell surface; Communicable Diseases; Complex; Cryoelectron Microscopy; Crystallography; Development; Disease; Electrons; Environment; Face; Fiber; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Generations; Goals; Gram-Negative Bacteria; Hair; Image; Imaging Techniques; In Vitro; Kidney; Knowledge; Label; Length; Lipid Bilayers; Measures; Membrane; Membrane Proteins; Methods; Molecular; Molecular Chaperones; Molecular Probes; Organelles; Pathway interactions; Peptide Hydrolases; Pilum; Protein Secretion; Proteins; Protocols documentation; Research Personnel; Resolution; Roentgen Rays; Site; Staging; Structure; Surface; Testing; Time; Tissues; Twin Multiple Birth; Urinary tract infection; Uropathogenic E. coli; Virulence; Virulence Factors; Work; X-Ray Crystallography; antimicrobial drug; base; electron crystallography; fimbria; in vivo; mutant; novel; particle; patch clamp; pathogen; pathogenic bacteria; periplasm; prevent; programs; protein protein interaction; prototype; reconstitution; three dimensional structure; tool; two-dimensional

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.

描述（申请人提供）：项目摘要。致病细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。革兰氏阴性细菌除了具有细胞质膜外，还具有外膜，并且必须在这两个障碍物中分泌毒力因子。发生这种情况的机制可能非常复杂，并且尚不清楚。伴侣/usher途径是一种毒力蛋白分泌途径，需要在整个外膜上分泌两个组成部分：周质伴侣蛋白和外膜外膜蛋白称为usher。伴侣指导分泌的蛋白质的正确折叠，并防止围绕道路的相互作用。 Usher在外膜上充当装配平台，并为细胞表面提供了分泌通道。由广泛的病原体组装和分泌毒力相关的表面结构的超家族所必需的伴侣/usher途径。该途径组装的典型细胞器是尿路感染的主要病原体尿道病大肠杆菌表达的P和1 pili。 P和1型Pili是关键的毒力因子，分别允许肾脏和膀胱的结合和定植。该提案的目标是探究usher的结构和功能，以了解对整个OM pilus生物发生的分子机制，并使用伴侣/Usher途径作为了解革兰氏阴性细菌中毒力因子分泌的模型系统。该提案将检验以下假设：usher在OM中形成双孔复合体，该复合物起着PILUS组装和分泌位点的作用。 usher函数的不同域，以控制，坐标和催化伴侣 - 亚基相互作用的交换，从而在usher的周面部面部的亚基 - 亚基相互作用，以驱动pilus纤维通过usher的有序组装和分泌的分泌，并分泌通过usher usher到细胞表面。第一个具体目的是探测usher处菌毛生物发生的分子机制。第二个特定目的是分析体内和体外的Usher-Chaperone-Subunit相互作用。第三个具体目的是使用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进行图像菌毛组装中间体。关联。该提案中描述的工作将阐明病原细菌的细胞器生物发生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的发展创造机会，在这一抗生素耐药性增加的时期急需。