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

• 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
• 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

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.

项目摘要 致病细菌使用专门的“纳米机器”来识别和与宿主细胞相互作用。这些机器 是有吸引力的药物靶标，因为它们表面暴露，宽度且对致病性至关重要。尽管 这些机器之一是III型分泌系统，已经进行了很好的研究，其他系统仍然相对 理解不佳。在这里，我们建议使用电子冷冻理学（ECT）剖析结构和 多种致病纳米机器的功能。 ECT是一种强大的技术，可用于图像完整结构 细胞内部大分子（2-5 nm）分辨率。以前，我们将ECT应用于VI型分泌系统 （T6SS），我们的图像立即揭示了其“弹簧加载”的收缩机制。迈出一步 此外，我们意识到我们可以将ECT和高分辨率子图平均图与可用 来自其他技术的知识，以产生粘膜粘膜类型的完整建筑模型 Iva Pilus（T4AP）。这产生了大量的新机械见解，并激发了我们应用相同的 致病性分泌系统的方法。在这个项目中，我们建议使用ECT揭示结构和 致病性IV型菌毛（T4P），VI型分泌（T6SS）和IV型分泌系统（T4SS）的功能 机械。对于每个系统，我们将原位映像整个完整的结构。在大多数情况下，这将是 这些结构的首次高分辨率成像。然后，我们将结合亚图平均值与差异 分析突变体的分析，其中单个组件被淘汰或标记以产生建筑 结构的模型。如果可以使用组件（或同源物）的晶体结构，我们将 将它们扩展到我们的地图中，以生成每台机器的伪原子模型。通过比较这些结构 与非致病亲戚的那些（以前或在当前研究中解决），我们旨在确定适应性 潜在的病毒功能。我们还将采用最新以低温的离子光束铣削和 相关的低温荧光光学显微镜和ECT与作用形象分泌结构 - 在 细菌细胞感染真核宿主。这将提供关键人类的第一个这样的图像 病原体幽门螺杆菌和肺虫军团菌，我们期望为此提供宝贵的见解 他们在体内的致病机械操作。我们在一起，我们希望这个项目能产生详细的 T4P，T6SS和T4SS纳米机械的机械图，介导发病机理的纳米机器，这是一个重要的第一 一步识别将来的治疗靶标。