Structure-Function Relationships in the Spirochetal Flagellar Motor

螺旋体鞭毛运动的结构与功能关系

• 批准号: 8025970
• 负责人: Jun Liu
• 金额: $ 35.86万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8025970
• 关键词: 3-Dimensional; Architecture; Bacteria; Binding; Biological Models; Borrelia burgdorferi; Cell membrane; Charon; Complement Factor B; Coupling; Data; Development; Environment; Event; Flagella; Freezing; Fusion Protein Expression; Generations; Genes; Genetic; Goals; Human; Image Analysis; In Situ; Individual; Infection; Interdisciplinary Study; Knock-in Mouse; Knock-out; Life; Lyme Disease; Mechanics; Membrane; Methodology; Methods; Modeling; Molecular; Molecular Machines; Molecular Structure; Motor; Movement; Mutation; Order Spirochaetales; Organelles; Organism; Outcome; Pathogenesis; Proteins; Proteomics; Protons; Research; Research Personnel; Resolution; Resources; Role; Rotation; Sodium; Structure; Structure-Activity Relationship; Study models; Techniques; Torque; United States; Virulence; Virulence Factors; Work; base; cell envelope; cell motility; comparative; driving force; electron tomography; experience; fascinate; flexibility; genetic analysis; imaging modality; innovation; insight; kinetosome; macromolecular assembly; mutant; nanomachine; novel; pathogen; pathogenic bacteria; public health relevance; structural biology; tomography; vector

DESCRIPTION (provided by applicant): Bacterial motility and its driving force, the flagellar motor, are important virulence factors of B. burgdorferi (the causative agent of Lyme disease) and many other bacteria. The flagellar motor is a remarkable nano-machine, powered by the proton (or sodium) gradient across the cytoplasmic membrane. The coupling of an electrochemical gradient to mechanical rotation is one of the most fascinating features of this molecular machine. The flagellar motor is composed of two major components: the stator and the rotor. Although prior structural studies have revealed the stunning complexity of the flagellar rotor, the mechanism of energy coupling in the flagellar motor remains poorly understood at the molecular level, mainly because of the lack of structural information about the membrane-bound stator and the rotor- stator interactions involved in flagellar rotation. The central hypothesis is that a high- resolution structure of the rotor-stator-C ring interface will provide vital structural information needed to propose models on the mechanisms of rotation and reversal. The objective of this application is to determine the structure/function relationship of the intact flagellar motor in situ by combining novel high throughput Cryo-Electron Tomography (Cryo-ET) approaches with genetic analysis to study the model system, B. burgdorferi. By collaborating with Drs. Steven Norris, Nyles Charon. MD Motaleb, Chunhao Li and Hanspeter Winkler, we propose to focus on two specific aims: Specific Aim 1 - Determine the detailed structure of the torque-generating unit by analyzing the 3-D structures of the complete flagellar motor and the purified flagellar rotor at 2 nm resolution. Specific Aim 2 - Determine the structural and functional roles of individual flagellar proteins by comparative analysis of wild-type organisms and flagellar gene mutants. We believe that the detailed analysis of the rotor/stator assembly in situ may provide the clearest avenue yet available to understanding of the mechanism of flagellar rotation and bacterial motility, which will in turn be applicable to the pathogenesis of all spirochetes and other motile bacteria. In addition, the further development of high-throughput Cryo-ET as part of this project will be readily applied to gain new insights into the structural and functional relationship of macromolecular machines related to pathogenesis of a variety of human pathogens, and offer a wide spectrum of important biomedical information at molecular resolution in living organisms. PUBLIC HEALTH RELEVANCE: Project Narrative/Relevance B. burgdorferi is a highly motile and invasive pathogen causing Lyme disease, the most common vector-borne infection in the United States. The bacterial motility and its driving force, the flagellar motor, are important virulence factors of B. burgdorferi and many other bacteria. By elucidating the molecular architecture of flagellar motor and the structural basis of mechanochemical coupling in flagellar rotation, we could gain insight into how the flagellar motor works and contributes to bacterial pathogenesis. The information resulting from these studies can be readily applied to understanding the motility of many pathogenic bacteria that depend on flagella-based movement to colonize, establish infection, and disseminate in humans and other hosts.

性状（由申请方提供）：细菌运动性及其驱动力（鞭毛马达）是B的重要毒力因子。莱姆病的病原体）和许多其他细菌。鞭毛马达是一台非凡的纳米机器，由跨细胞质膜的质子（或钠）梯度提供动力。电化学梯度与机械旋转的耦合是这种分子机器最迷人的特征之一。鞭毛马达由两个主要部件组成：定子和转子。虽然先前的结构研究已经揭示了鞭毛转子的惊人的复杂性，在鞭毛电机的能量耦合的机制仍然知之甚少，在分子水平上，主要是因为缺乏结构信息的膜结合定子和转子定子的相互作用参与鞭毛旋转。中心假设是转子-定子-C环界面的高分辨率结构将提供提出关于旋转和反转机制的模型所需的重要结构信息。本申请的目的是通过将新的高通量冷冻电子断层扫描（Cryo-ET）方法与遗传分析相结合来研究模型系统B，从而原位确定完整鞭毛马达的结构/功能关系。burgdorferi。通过与史蒂文·诺里斯博士，奈尔斯·卡隆博士合作。MD Motaleb，Chunhao Li和Hanspeter Winkler，我们建议专注于两个具体目标：具体目标1-通过分析完整鞭毛马达和纯化鞭毛转子的3-D结构，以2 nm分辨率确定扭矩产生单元的详细结构。 具体目标2-通过野生型生物和鞭毛基因突变体的比较分析，确定单个鞭毛蛋白的结构和功能作用。 我们相信，详细的分析转子/定子组件在原位可能提供最清晰的途径，但可用于了解鞭毛旋转和细菌运动的机制，这将反过来适用于所有螺旋体和其他能动细菌的发病机制。此外，作为该项目的一部分，高通量Cryo-ET的进一步开发将很容易应用于获得与各种人类病原体发病机制相关的大分子机器的结构和功能关系的新见解，并在活生物体的分子分辨率上提供广泛的重要生物医学信息。 公共卫生相关性：项目叙述/相关性B。伯氏螺旋体是一种引起莱姆病的高度运动性和侵入性病原体，莱姆病是美国最常见的媒介传播感染。细菌的运动性及其驱动力鞭毛马达是B的重要毒力因子。伯氏菌和许多其他细菌。通过阐明鞭毛马达的分子结构和鞭毛旋转中机械化学耦合的结构基础，我们可以深入了解鞭毛马达是如何工作的，并有助于细菌的致病机制。从这些研究中得到的信息可以很容易地应用于了解许多致病菌的运动，这些致病菌依赖于基于鞭毛的运动来定殖，建立感染，并在人类和其他宿主中传播。