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: 9765150
• 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/9765150
• 关键词: Adhesions; Antibiotic Resistance; Archaea; Architecture; Bacteria; Bacteriophages; Biological Models; Cells; Complex; Contracts; Cryoelectron Microscopy; 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; Injections; 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; Structural Models; 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; human pathogen; 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”来识别宿主细胞并与之相互作用。这些机器 是有吸引力的药物靶标，因为它们是表面暴露的，广泛的，并且对于致病性至关重要。而 这些机器之一，III型分泌系统，已经得到了很好的研究，其他系统仍然相对 不太了解。在这里，我们建议使用电子冷冻断层扫描（ECT）来解剖结构， 多种病原性纳米机器的功能。ECT是一种强大的技术，可以对完整的结构进行成像， 细胞内的大分子（2-5 nm）分辨率。以前，我们将ECT应用于VI型分泌系统 （T6 SS），我们的图像立即揭示了其“弹簧加载”的收缩机制。迈进了一步 此外，我们意识到，我们可以将联合收割机ECT和高分辨率子断层图像平均与可用的 从其他技术的知识，以产生一个完整的结构模型的粘球菌xanthus型 IVa菌毛（T4 aP）。这产生了大量新的机械论见解，并激励我们应用同样的方法。 病原体分泌系统的方法。在这个项目中，我们建议使用ECT来揭示结构， 致病性IV型菌毛（T4 P）、VI型分泌（T6 SS）和IV型分泌系统（T4 SS）的功能 机械。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 首次对这些结构进行高分辨率成像。然后，我们将联合收割机子断层图像平均与差异相结合 分析突变体，其中单个组分被敲除或标记，以产生结构 结构的模型。如果有组分（或同系物）的晶体结构，我们将 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。通过比较这些结构 与那些非致病性亲属（以前或在本研究中解决），我们的目标是确定适应 潜在的毒性功能。我们还将采用最先进的低温聚焦离子束铣削技术， 相关的低温荧光显微镜和ECT图像分泌结构在行动中， 感染真核宿主的细菌细胞。这将提供第一个这样的图像的关键人类 病原体幽门螺杆菌和嗜肺军团菌，我们希望提供宝贵的见解， 它们体内致病机制的运作。我们希望这个项目能产生一个详细的 T4 P，T6 SS和T4 SS纳米机器介导发病机制的机制图，这是一个重要的第一个 在未来确定治疗目标的一步。