Structure and Function of Pathogenesis-Associated Bacterial Structures by Electron Cryotomography

项目摘要 病原菌使用专门的分泌系统来识别宿主细胞并与其相互作用，并 通过水平基因转移交换遗传信息。这些机器是诱人的毒品目标 因为它们暴露在表面，被广泛保守，并且具有致病性。然而，不幸的是， 其中许多关键系统的结构仍然知之甚少。在这里，我们将描述我们将如何 继续使用电子冷冻断层摄影术(CryoET)解剖致病细胞的结构和功能 纳米机器。Cryoet是一种革命性的成像技术，具有揭示内部自然结构的能力 完整的三维细胞，大分子(2-5 nm)分辨率。相同结构的亚断层图像平均化 在最有利的情况下，一个或多个冷冻断层摄影术可以将分辨率提高到1纳米以下，从而实现 要放置在整机的上下文中的组件。我的团队是发展的先驱 这项革命性的成像技术，在我们第一次获奖的不到四年时间里，我们使用了 将生产数十种新的致病分泌物系统结构，并建立 属于IV型菌毛(T4P)、VI型分泌系统(T6SS)和IV型分泌的关键系统 系统(T4SS)家庭，产生了大量新的机械见解。通过开发新的低温电子管技术 我们在过去的几年里刚刚发展起来，在这里我们建议在下一个获奖期延长我们的工作 这些复合体的不同功能状态、关键相关系统和一个新的靶点：致病类型IX 分泌系统(T9SS)。此外，我们将推动整个工作向更高的分辨率发展。对于每个目标， 我们将在原地对完整的结构进行成像。在大多数情况下，这将是第一次高分辨率成像 这些结构。然后，我们将结合亚断层图像平均和突变的差异分析，其中 单个组件被挖出或贴上额外的密度标签，以产生建筑 建筑群的模型。在组件(或同系物)的原子模型可用的情况下，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 对于非致病亲属的问题(以前解决的或在拟议的工作中解决的)，我们的目标是识别 毒力功能基础上的适应。我们还将应用最先进的低温相关光和 引导低温聚焦离子束(FIB)研磨的电子显微镜(Cryo-Clem)使我们能够成像 致病分泌系统的作用：在感染真核宿主的细菌细胞中。这将提供第一个 这些关键的人类病原体的图像，我们希望能为人类的运作提供宝贵的洞察力 它们在体内的毒力机制。总而言之，我们希望这个项目能够产生一个详细的机械图景 介导发病机制的T4SS、T4P和T9SS纳米机器，是识别 未来的治疗靶点。

Project Summary Pathogenic bacteria employ specialized secretion systems to identify and interact with host cells and to exchange genetic information through horizontal gene transfer. These machines are attractive drug targets because they are surface-exposed, widely conserved, and specific for pathogenicity. Unfortunately, however, the structures of many of these critical systems remain poorly understood. Here we describe how we will continue to use electron cryotomography (cryoET) to dissect the structures and functions of pathogenic nanomachines. CryoET is a revolutionary imaging technique with the power to reveal native structures inside intact cells in 3D with macromolecular (2-5 nm) resolution. Subtomogram averaging of identical structures from one or more cryotomograms can push this resolution to better than 1 nm in the most favorable cases, enabling components to be placed in their context in the complete machine. My group has pioneered the development of this revolutionary imaging technology, and in just under four years of our first award period, we have used cryoET to produce tens of new structures of pathogenic secretion systems and build architectural models of key systems belonging to the type IV pilus (T4P), type VI secretion system (T6SS) and type IV secretion system (T4SS) families, producing a flood of new mechanistic insights. By exploiting new cryoET technologies we have just developed in the past couple years, here we propose to extend our work in the next award period to different functional states of these complexes, key related systems, and a new target: the pathogenic type IX secretion system (T9SS). In addition, we will push the whole body of work to higher resolution. For each target, 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 with additional density in order to produce architectural models of the complexes. In cases where atomic models 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 proposed work), we aim to identify adaptations underlying virulence functions. We will also apply state-of-the-art cryogenic correlated light and electron microscopy (cryo-CLEM) to guide cryogenic focused ion beam (FIB) milling to enable us to image pathogenic secretion systems in action: in bacterial cells infecting eukaryotic hosts. This will provide the first such images of critical human pathogens, which we expect to provide invaluable insights into the operation of their virulence machinery in vivo. Together, we expect this project to produce a detailed mechanistic picture of the T4SS, T4P, and T9SS nanomachines that mediate pathogenesis, an important first step in identifying therapeutic targets in the future.