Structure and function of pathogenesis-associated bacterial structures by electron cryotomography

通过电子冷冻断层扫描研究发病机制相关细菌结构的结构和功能

基本信息

批准号：
9357518
负责人：
GRANT J JENSEN
金额：
$ 41.08万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-23 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9357518
关键词：
Adhesions Antibiotic Resistance Archaea Architecture Bacteria Bacteriophages Biological Models Cells Complex Contracts Crystallization DNA Docking Drug Design Drug Targeting Electrons Epithelial Cells Eukaryotic Cell Fiber Filament Floods Fluorescence Microscopy Future Gram-Negative Bacteria Helicobacter pylori Hemagglutinin Human Hybrids Image Imaging Techniques In Situ Individual Infection Injection of therapeutic agent Ions Knock-out Knowledge Legionella Legionella pneumophila Location Mannose Maps Mediating Modeling Molecular Molecular Conformation Molecular Structure Myxococcus xanthus Pathogenesis Pathogenicity Pilum Prokaryotic Cells Proteins Public Health Regulation Resolution Signal Transduction Stomach Structure Surface System Tail Techniques Time Toxin Type II Secretion System Pathway Type III Secretion System Pathway Type IV Secretion System Pathway Vesicle Vibrio cholerae Virulence Virulence Factors Work X-Ray Crystallography appendage cell envelope cell motility cryogenics extracellular high resolution imaging image processing imaging system in vivo insight member mutant nanomachine new therapeutic target novel operation pathogen pathogenic bacteria prevent resistant strain therapeutic target

项目摘要

Project Summary Pathogenic bacteria use specialized “nanomachines” to identify and interact with host cells. These machines are attractive drug targets because they are surface-exposed, widespread, and vital for pathogenicity. While one of these machines, the Type III Secretion System, has been well studied, other systems remain relatively poorly understood. Here, we propose to use electron cryotomography (ECT) to dissect the structures and functions of multiple pathogenic nanomachines. ECT is a powerful technique to image intact structures with macromolecular (2-5 nm) resolution inside cells. Previously, we applied ECT to the Type VI Secretion System (T6SS), where our images immediately revealed its "spring-loaded" contractile mechanism. Going a step further, we realized that we could combine ECT and high-resolution subtomogram averaging with available knowledge from other techniques to produce a complete architectural model of the Myxococcus xanthus Type IVa Pilus (T4aP). This produced a flood of new mechanistic insights and inspired us to apply the same approach to pathogenic secretion systems. In this project, we propose to use ECT to reveal the structure and function of pathogenic Type IV Pilus (T4P), Type VI Secretion (T6SS), and Type IV Secretion System (T4SS) machineries. For each system, we will image the entire, intact structure in situ. In most cases, this will be the first high-resolution imaging of these structures. We will then combine subtomogram averaging with difference analysis of mutants in which individual components are knocked out or tagged in order to produce architectural models of the structures. In cases where crystal structures of components (or homologs) are available, we will dock them into our maps to produce pseudo-atomic models of each machine. By comparing these structures with those of non-pathogenic relatives (solved previously or in the current study), we aim to identify adaptations underlying virulence functions. We will also apply state-of-the-art cryogenic-focused ion beam milling and correlated cryogenic fluorescence light microscopy and ECT to image secretion structures in action – in bacterial cells infecting eukaryotic hosts. This will provide the first such images of the critical human pathogens Helicobacter pylori and Legionella pneumophila, which we expect to provide invaluable insights into the operation of their pathogenic machinery in vivo. Together, we expect this project to produce a detailed mechanistic picture of the T4P, T6SS, and T4SS nanomachines that mediate pathogenesis, an important first step in identifying therapeutic targets in the future.

项目概要致病细菌使用专门的“纳米机器”来识别宿主细胞并与这些机器相互作用。是有吸引力的药物靶点，因为它们是表面暴露的、广泛的并且对于致病性至关重要。其中一台机器，III型分泌系统，已得到充分研究，其他系统仍然相对在这里，我们建议使用电子冷冻断层扫描（ECT）来解剖结构和 ECT 是一种对完整结构进行成像的强大技术。细胞内的大分子（2-5 nm）分辨率之前，我们将 ECT 应用于 VI 型分泌系统。（T6SS），我们的图像立即揭示了它的“弹簧加载”收缩机制。此外，我们意识到我们可以将 ECT 和高分辨率次断层图平均与可用的来自其他技术的知识，用于生成黄色粘球菌类型的完整建筑模型 IVa Pilus (T4aP)。这产生了大量新的机制见解，并激励我们应用同样的见解。在这个项目中，我们建议使用 ECT 来揭示致病性分泌系统的结构和致病性 IV 型菌毛 (T4P)、VI 型分泌物 (T6SS) 和 IV 型分泌系统 (T4SS) 的功能对于每个系统，我们将在原位对整个完整的结构进行成像。在大多数情况下，这将是完整的结构。然后，我们将首先对这些结构进行高分辨率成像，然后将次断层图平均与差值结合起来。分析突变体，其中单个组件被敲除或标记以产生结构如果组件（或同系物）的晶体结构可用，我们将提供结构模型。通过比较这些结构，将它们对接到我们的地图中以生成每台机器的伪原子模型。与非致病性亲属的那些（之前或当前研究中已解决），我们的目标是确定适应性我们还将应用最先进的低温聚焦离子束铣削和将低温荧光光学显微镜和 ECT 与活动中的分泌结构图像相关联感染真核宿主的细菌细胞这将提供关键人类的第一个此类图像。幽门螺杆菌和嗜肺军团菌病原体，我们希望能够提供宝贵的见解我们希望这个项目能够共同研究它们体内致病机制的运作。介导发病机制的 T4P、T6SS 和 T4SS 纳米机器的机制图，这是重要的第一确定未来治疗靶点的一步。