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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.

项目摘要病原菌利用专门的分泌系统来识别宿主细胞并与宿主细胞相互作用，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，并且对致病性具有特异性。然而不幸的是，对其中许多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）来解剖致病性组织的结构和功能，纳米机器CryoET是一种革命性的成像技术，具有揭示内部天然结构的能力完整的细胞在3D与大分子（2-5 nm）的分辨率。相同结构的子断层图像平均值，在最有利的情况下，一个或多个冷冻断层照片可以将该分辨率推到优于1 nm，这些组件将被放置在整个机器中的上下文中。我的团队率先开发了这项革命性的成像技术，在我们第一个奖项期的不到四年时间里，我们已经使用了 cryoET产生了数十种致病分泌系统的新结构，并建立了属于IV型菌毛（T4 P）、VI型分泌系统（T6 SS）和IV型分泌的关键系统系统（T4 SS）家族，产生了大量新的机械见解。通过开发新的冷冻ET技术我们在过去的几年里刚刚发展起来，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，关键相关系统和一个新的目标：致病性IX型分泌系统（T9 SS）。此外，我们将推动整个工作的更高分辨率。对于每个目标，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，这将是第一个高分辨率成像的这些结构。然后，我们将联合收割机子断层图像平均与突变体的差异分析相结合，单个组件被敲除或标记有额外的密度，以产生建筑复合体的模型。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。通过比较这些结构与那些非致病性的亲属（以前解决或在拟议的工作），我们的目标是确定适应潜在的毒力功能。我们还将应用最先进的低温相关光，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分泌系统的作用：在细菌细胞感染真核宿主。这将提供第一个这些关键的人类病原体的图像，我们希望提供宝贵的见解的运作，它们在体内的毒力机制。总之，我们希望这个项目能产生一个详细的机械图，介导发病机制的T4 SS、T4 P和T9 SS纳米机器，这是识别未来的治疗目标。

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：
JOURNAL OF MOLECULAR BIOLOGY
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：
STRUCTURE
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.

DOI：
10.1128/jmbe.00128-21
发表时间：
2021
期刊：
Journal of microbiology & biology education
影响因子：
1.9
作者：
Oikonomou CM;Jensen GJ
通讯作者：
Jensen GJ

Subtomogram averaging for biophysical analysis and supramolecular context.

DOI：
10.1016/j.yjsbx.2022.100076
发表时间：
2022
期刊：
JOURNAL OF STRUCTURAL BIOLOGY-X
影响因子：
2.9
作者：
Metskas, Lauren Ann;Wilfong, Rosalie;Jensen, Grant J.
通讯作者：
Jensen, Grant J.

Loss of the Bacterial Flagellar Motor Switch Complex upon Cell Lysis.

DOI：
10.1128/mbio.00298-21
发表时间：
2021-06-29
期刊：
mBio
影响因子：
6.4
作者：
Kaplan M;Tocheva EI;Briegel A;Dobro MJ;Chang YW;Subramanian P;McDowall AW;Beeby M;Jensen GJ
通讯作者：
Jensen GJ