Structure-Function Relationships in the Spirochetal Flagellar Motor

描述（由申请人提供）：细菌运动及其驱动力鞭毛运动是伯氏疏螺旋体（莱姆病的病原体）和许多其他细菌的重要毒力因素。鞭毛马达是一种非凡的纳米机器，由细胞质膜上的质子（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。鞭毛电机由定子和转子两大部分组成。尽管先前的结构研究已经揭示了鞭毛转子的惊人复杂性，但在分子水平上对鞭毛马达的能量耦合机制仍然知之甚少，这主要是因为缺乏关于鞭毛旋转中膜结合定子和转子-定子相互作用的结构信息。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旋转和反转机制模型所需的重要结构信息。本应用程序的目的是通过结合新的高通量冷冻电子断层扫描（crao - et）方法和遗传分析来研究模型系统B. burgdorferi，以确定原位完整鞭毛马达的结构/功能关系。通过与博士合作。史蒂文·诺里斯，奈尔斯·卡龙。MD Motaleb， Chunhao Li和Hanspeter Winkler，我们提出了两个具体目标：具体目标1 -通过分析完整的鞭毛电机和纯化的鞭毛转子在2nm分辨率下的三维结构来确定扭矩发电单元的详细结构。特定目标2 -通过对比分析野生型生物和鞭毛基因突变体，确定单个鞭毛蛋白的结构和功能作用。我们相信，对原地转子/定子组件的详细分析可能为理解鞭毛旋转和细菌运动的机制提供最清晰的途径，这将反过来适用于所有螺旋体和其他运动细菌的发病机制。此外，作为该项目的一部分，高通量Cryo-ET的进一步发展将很容易应用于了解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，并在生物体内提供广泛的分子分辨率的重要生物医学信息。

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.