Catalytic mechanism of the usher in pilus biogenesis by uropathogenic E. coli

泌尿道致病性大肠杆菌引导菌毛生物发生的催化机制

• 批准号: 9061399
• 负责人: Glenn Thomas Werneburg
• 金额: $ 4.25万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-15 至 2018-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9061399
• 关键词: Activation Analysis; Address; Adhesions; Adhesives; Affinity; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Biogenesis; Biological Assay; Biological Models; Bladder; C-terminal; Catalysis; Cell membrane; Cell surface; Complex; Cryoelectron Microscopy; Cystitis; DAG/PE-Binding Domain; Development; Disease; Electron Microscopy; Escherichia coli; Fiber; Fluorescence; Goals; Gram-Negative Bacteria; Hair; Health Care Costs; Healthcare; Hemagglutination; Kidney; Lead; Mediating; Medical; Membrane; Membrane Proteins; Molecular; Molecular Chaperones; N Domain; N-terminal; Organelles; Pathway interactions; Periplasmic Proteins; Pilum; Positioning Attribute; Protein Secretion; Proteins; Pyelonephritis; Recruitment Activity; Research; Role; Structure; Surface; System; Techniques; Testing; Time; Tissues; Urinary tract; Urinary tract infection; Uropathogenic E. coli; Usher Proteins; Vaccination; Virulence; Virulence Factors; Work; X-Ray Crystallography; attenuation; base; beta barrel; cost; fimbria; in vivo; insight; microscopic imaging; mutant; nanomachine; new therapeutic target; novel; pathogenic bacteria; periplasm; premature; prevent; public health relevance; research study; vaccine development

 DESCRIPTION (provided by applicant): Urinary tract infections (UTIs) are among the most common bacterial infections in the world and result in more than $1.5 billion USD in associated medical costs. The most common cause of UTIs is uropathogenic Escherichia coli (UPEC). UPEC use the chaperone/usher (CU) pathway to assemble and secrete pili onto their cell surfaces. These pili are virulence-associated structures that project away from the cell surface in a hairlike fashion and mediate adhesion to the urinary tract. The chaperone and usher proteins of the CU pathway work in unison to construct these pili. The chaperone, a periplasmic protein, facilitates subunit folding, prevents premature subunit-subunit interactions, and targets subunits to the outer membrane usher protein. The usher is a multifunctional pilus assembly and secretion platform, catalyzing the exchange of chaperone-subunit for subunit-subunit interactions to assemble the pilus fiber and providing the channel for secretion of the fiber to th cell surface. The usher is a large protein consisting of a periplasmic N-terminal domain (N), a transmembrane beta-barrel domain that is gated by an internal Plug domain, and two periplasmic C-terminal domains (C1 and C2). These domains act together to facilitate the ordered assembly and secretion of the pilus. The overall goal of this proposal is to structurally and mechanistically characterize the usher's role in pilus biogenesis. This proposal will test the hypothesis that the usher is both a gated secretion channel and catalytic nanomachine, and that its catalytic activity is due to a carefully coordinated sequence of usher-chaperone-subunit interactions and the accurate placement of chaperone-subunit complexes relative to one another. I propose to use fluorescence techniques to understand how the usher transfers pilus subunits from its N domain to its C domains, a necessary step to clear the N domain for incorporation of the next pilus subunit. I will use similar techniques to understand the Plug's interaction with the C domains of the inactivated usher and how and when the Plug domain is removed from the usher channel upon usher activation. Finally, I propose to probe the structural basis for usher-catalyzed pilus assembly and secretion. I will use molecular techniques to generate pilus assembly intermediates suitable for analysis by cryo-electron microscopy and X-ray crystallography. The information gained by the experiments described in this proposal will lead to new advances in the field of protein secretion. The results will aid in the identification f physical and mechanistic targets for novel therapeutics against UPEC, a particularly important endeavor in the current era of rampant antibiotic resistance.

 描述(申请人提供)：尿路感染(UTI)是世界上最常见的细菌感染之一，导致超过15亿美元的相关医疗费用。尿路感染最常见的原因是尿路致病性大肠杆菌(UPEC)。UPEC利用伴侣/Usher(CU)途径将菌毛组装并分泌到其细胞表面。这些菌毛是与毒力相关的结构，以毛发的方式从细胞表面伸出，并介导与尿路的粘连。CU途径的伴侣蛋白和引座者蛋白协同工作来构建这些菌毛。伴侣蛋白是一种周质蛋白，它促进亚基折叠，防止亚基与亚基的过早相互作用，并将亚基靶向外膜引导蛋白。引座器是一个多功能的菌毛组装和分泌平台，催化分子伴侣-亚单位之间的相互作用来组装菌毛纤维，并为纤维分泌到TH细胞表面提供通道。USHER是一个大的蛋白质，由一个周质N末端结构域(N)、一个由内部插入域控制的跨膜β桶结构域和两个周质C末端结构域(C1和C2)组成。这些结构域共同作用，促进菌毛的有序组装和分泌。这项提议的总体目标是从结构和机械上描述引座员在菌毛生物发生中的作用。这一提议将检验这样的假设，即引座者既是门控分泌通道又是催化纳米机器，其催化活性是由于引导者-伴侣-亚单位相互作用的仔细协调序列以及伴侣-亚单位复合体相对于彼此的准确放置。我建议使用荧光技术来了解引座者如何将毛状亚单位从其N结构域转移到其C结构域，这是清除N结构域以并入下一个毛状亚单位的必要步骤。我将使用类似的技术来了解插头与未激活的引座器的C域的交互，以及在引座器激活时如何以及何时从引座器通道中移除插头域。最后，我建议探索引座者催化的菌毛组装和分泌的结构基础。我将使用分子技术产生适合低温电子显微镜和X射线结晶学分析的菌毛组装中间体。这项建议中描述的实验所获得的信息将导致蛋白质分泌领域的新进展。这一结果将有助于确定针对UPEC的新疗法的物理和机械靶点，这在当前抗生素耐药性猖獗的时代是一项特别重要的努力。