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：
GRANT J JENSEN
金额：
$ 36.95万
依托单位：
BRIGHAM YOUNG UNIVERSITY
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

来源：
https://reporter.nih.gov/project-details/10604243
关键词：
3-Dimensional Adhesions Antibiotic Resistance Archaea Architecture Award Bacteria Bdellovibrio Bioinformatics Caulobacter crescentus Cells Cholera Toxin Competence Complex Cryoelectron Microscopy Cytoplasmic Structures DNA Development Disparate Docking Drug Design Drug Targeting Electron Microscopy Electrons Escherichia coli Evolution Family First Independent Research Support and Transition Awards Fishes Flavobacteria Floods Future Genetic Conjugation Gram-Negative Bacteria Grant Helicobacter pylori Hemagglutinin Homologous Gene Horizontal Gene Transfer Human Image Imaging Techniques Imaging technology In Situ Individual Infection Ions Knock-out Legionella pneumophila Location Mannose Maps Mediating Membrane Modeling Molecular Motor Myxococcus xanthus Neptunium Pathogenesis Pathogenicity Peptide Hydrolases Periodontal Diseases Pilum Porphyromonas gingivalis Process Proteins Pseudomonas aeruginosa Public Health Recording of previous events Resolution Role Secretin Serotyping Shewanella Signal Pathway Structure Surface System Techniques Technology Testing Thermococcus Time Toxin Tube Type II Secretion System Pathway Type IV Secretion System Pathway Vaccines Vibrio cholerae Virulence Work X-Ray Crystallography cell envelope cell motility combat comparative genomics cryogenics density genetic information high resolution imaging human pathogen imaging system in vivo insight light microscopy member mutant nanomachine nanosystems novel novel therapeutics operation particle pathogen pathogenic bacteria periplasm reconstruction therapeutic target

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标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，并建立其结构模型属于 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，使我们能够成像起作用的致病分泌系统：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）

In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System.

幽门螺杆菌 cag IV 型分泌系统的体内结构。

DOI：
发表时间：
2018-04-17
期刊：
影响因子：
8.8
作者：
Chang, Yi;Shaffer, Carrie L;Rettberg, Lee A;Ghosal, Debnath;Jensen, Grant J
通讯作者：
Jensen, Grant J

The Atlas of Bacterial & Archaeal Cell Structure: an Interactive Open-Access Microbiology Textbook.

细菌图谱

DOI：
发表时间：
2021
期刊：
影响因子：
0
作者：
Oikonomou, Catherine M;Jensen, Grant J
通讯作者：
Jensen, Grant J

Structure and Assembly of the Proteus mirabilis Flagellar Motor by Cryo-Electron Tomography.

通过冷冻电子断层扫描技术研究奇异变形杆菌鞭毛马达的结构和组装。

DOI：
发表时间：
2023-05-05
期刊：
影响因子：
5.6
作者：
Kaplan, Mohammed;Yao, Qing;Jensen, Grant J
通讯作者：
Jensen, Grant J

In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography.

通过电子冷冻断层扫描技术观察军团菌 Dot/Icm IV 型分泌系统的原位结构。

DOI：
发表时间：
2017
期刊：
影响因子：
7.7
作者：
Ghosal, Debnath;Chang, Yi;Jeong, Kwangcheol C;Vogel, Joseph P;Jensen, Grant J
通讯作者：
Jensen, Grant J

Rubisco forms a lattice inside alpha-carboxysomes.

Rubisco 在 α-羧基体内形成晶格。

DOI：
发表时间：
2022-08-18
期刊：
影响因子：
16.6
作者：
Metskas, Lauren Ann;Ortega, Davi;Oltrogge, Luke M;Blikstad, Cecilia;Lovejoy, Derik R;Laughlin, Thomas G;Savage, David F;Jensen, Grant J
通讯作者：
Jensen, Grant J