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:
    10604243
  • 负责人:
  • 金额:
    $ 36.95万
  • 依托单位:
  • 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 2027-03-31
  • 项目状态:
    未结题

项目摘要

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.
项目概要 致病细菌利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。这些机器是有吸引力的药物靶点 因为它们是表面暴露的、广泛保守的并且具有特异性的致病性。然而不幸的是, 许多这些关键系统的结构仍然知之甚少。在这里我们描述我们将如何 继续使用电子冷冻断层扫描(cryoET)来剖析致病菌的结构和功能 纳米机器。 CryoET 是一种革命性的成像技术,能够揭示内部的天然结构 具有大分子(2-5 nm)分辨率的 3D 完整细胞。相同结构的亚断层图平均 在最有利的情况下,一张或多张冷冻断层图可以将该分辨率提高到 1 nm 以上,从而使 将组件放置在整个机器中的上下文中。我的团队率先开发 这种革命性的成像技术,在我们第一个奖项的不到四年的时间里,我们已经使用了 冷冻电子断层扫描(cryoET)可产生数十种新的致病性分泌系统结构,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)家族,产生了大量新的机制见解。通过利用新的cryoET技术 我们这几年才刚刚发展起来,在此我们建议在下一个奖励期内继续我们的工作 这些复合物的不同功能状态、关键相关系统和新目标:致病性 IX 型 分泌系统(T9SS)。此外,我们还将把整个工作推向更高的分辨率。对于每个目标, 我们将在现场对整个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,我们将把次断层图平均与突变体的差异分析结合起来,其中 各个组件被敲除或用额外的密度标记,以产生建筑 复合体的模型。如果组件(或同系物)的原子模型可用,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 与非致病亲属的那些(之前或在拟议的工作中解决),我们的目标是确定 毒力功能背后的适应性。我们还将应用最先进的低温相关光和 电子显微镜(cryo-CLEM)引导低温聚焦离子束(FIB)铣削,使我们能够成像 致病性分泌系统的作用:在感染真核宿主的细菌细胞中。这将提供第一个 此类关键人类病原体的图像,我们希望这些图像能够为我们的运作提供宝贵的见解 它们在体内的毒力机制。我们共同期望这个项目能够产生详细的机制图 介导发病机制的 T4SS、T4P 和 T9SS 纳米机器,这是识别的重要第一步 未来的治疗目标。

项目成果

期刊论文数量(28)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Programmed Flagellar Ejection in Caulobacter crescentus Leaves PL-subcomplexes.
  • DOI:
    10.1016/j.jmb.2021.167004
  • 发表时间:
    2021-06-25
  • 期刊:
  • 影响因子:
    5.6
  • 作者:
    Kaplan, Mohammed;Wang, Yuhang;Chreifi, Georges;Zhang, Lujia;Chang, Yi-Wei;Jensen, Grant J.
  • 通讯作者:
    Jensen, Grant J.
Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET.
  • DOI:
    10.1016/j.str.2023.03.011
  • 发表时间:
    2023-05-04
  • 期刊:
  • 影响因子:
    5.7
  • 作者:
    Dutka, Przemysaw;Metskas, Lauren Ann;Hurt, Robert C.;Salahshoor, Hossein;Wang, Ting-Yu;Malounda, Dina;Lu, George J.;Chou, Tsui-Fen;Shapiro, Mikhail G.;Jensen, Grant J.
  • 通讯作者:
    Jensen, Grant J.
The Atlas of Bacterial & Archaeal Cell Structure: an Interactive Open-Access Microbiology Textbook.
Subtomogram averaging for biophysical analysis and supramolecular context.
  • DOI:
    10.1016/j.yjsbx.2022.100076
  • 发表时间:
    2022
  • 期刊:
  • 影响因子:
    2.9
  • 作者:
    Metskas, Lauren Ann;Wilfong, Rosalie;Jensen, Grant J.
  • 通讯作者:
    Jensen, Grant J.
Rubisco forms a lattice inside alpha-carboxysomes.
  • DOI:
    10.1038/s41467-022-32584-7
  • 发表时间:
    2022-08-18
  • 期刊:
  • 影响因子:
    16.6
  • 作者:
  • 通讯作者:
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GRANT J JENSEN其他文献

GRANT J JENSEN的其他文献

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{{ truncateString('GRANT J JENSEN', 18)}}的其他基金

Expansion of the ‘Getting Started in Cryo-EM’ course into a comprehensive theory and practice curriculum
将“Cryo-EM 入门”课程扩展为综合理论和实践课程
  • 批准号:
    10223807
  • 财政年份:
    2021
  • 资助金额:
    $ 36.95万
  • 项目类别:
Expansion of the 'Getting Started in Cryo-EM' course into a comprehensive theory and practice curriculum
将“冷冻电镜入门”课程扩展为综合理论和实践课程
  • 批准号:
    10834296
  • 财政年份:
    2021
  • 资助金额:
    $ 36.95万
  • 项目类别:
Expansion of the 'Getting Started in Cryo-EM' course into a comprehensive theory and practice curriculum
将“冷冻电镜入门”课程扩展为综合理论和实践课程
  • 批准号:
    10798674
  • 财政年份:
    2021
  • 资助金额:
    $ 36.95万
  • 项目类别:
Expansion of the ‘Getting Started in Cryo-EM’ course into a comprehensive theory and practice curriculum
将“Cryo-EM 入门”课程扩展为综合理论和实践课程
  • 批准号:
    10437759
  • 财政年份:
    2021
  • 资助金额:
    $ 36.95万
  • 项目类别:
Imaging large macromolecular complexes inside cells with electron cryotomography
使用电子冷冻断层扫描对细胞内的大分子复合物进行成像
  • 批准号:
    10013429
  • 财政年份:
    2017
  • 资助金额:
    $ 36.95万
  • 项目类别:
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
  • 财政年份:
    2016
  • 资助金额:
    $ 36.95万
  • 项目类别:
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
  • 财政年份:
    2016
  • 资助金额:
    $ 36.95万
  • 项目类别:
Structural Basis of Chemoreception
化学感受的结构基础
  • 批准号:
    8643266
  • 财政年份:
    2013
  • 资助金额:
    $ 36.95万
  • 项目类别:
Structural Basis of Chemoreception
化学感受的结构基础
  • 批准号:
    9022486
  • 财政年份:
    2013
  • 资助金额:
    $ 36.95万
  • 项目类别:
Structural Basis of Chemoreception
化学感受的结构基础
  • 批准号:
    8507076
  • 财政年份:
    2013
  • 资助金额:
    $ 36.95万
  • 项目类别:

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