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

项目摘要 病原菌使用专门的“纳米机器”来识别宿主细胞并与其相互作用。这些机器 是有吸引力的药物靶标，因为它们暴露在表面，分布广泛，对致病性至关重要。而当 其中一台机器，III型分泌系统，已经得到了很好的研究，其他系统仍然相对 人们对此知之甚少。在这里，我们建议使用电子冷冻断层扫描(ECT)来解剖结构和 多种致病纳米机器的功能。ECT是一种强大的技术，可以用来对完整的结构进行成像 细胞内大分子(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它的“弹簧加载”收缩机制。向前迈进一步 此外，我们意识到，我们可以将ECT和高分辨率亚断层图像平均与可用 从其他技术中获得的知识来制作黄色粘球菌型的完整建筑模型 IVA Pilus(T4aP)。这产生了大量新的机械论见解，并激励我们应用同样的观点 致病分泌物系统的探讨。在这个项目中，我们建议使用ECT来揭示结构和 病原IV型菌毛(T4P)、VI型分泌物(T6SS)和IV型分泌物系统(T4SS)的功能 机械制造。对于每个系统，我们将在原地成像完整的结构。在大多数情况下，这将是 第一次对这些结构进行高分辨率成像。然后我们将亚断层图像平均与差值相结合 对单个构件被敲除或标记以产生建筑结构的突变体的分析 结构的模型。在有成分(或同系物)的晶体结构的情况下，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 对于那些非致病亲属(以前或在当前研究中解决的)，我们的目标是识别适应 潜在的毒力功能。我们还将应用最先进的低温聚焦离子束研磨和 低温荧光光学显微镜和ECT相互关联成像分泌结构的作用 感染真核宿主的细菌细胞。这将提供批判性的人类的第一个这样的图像 幽门螺杆菌和嗜肺军团菌病原体，我们希望对它们提供宝贵的见解 它们在体内的致病机制的运作。总之，我们希望这个项目能够产生一个详细的 T4P、T6SS和T4SS纳米机器介导发病机制的机制图，这是第一个重要的 在未来确定治疗靶点的步骤。

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.