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.

项目摘要致病细菌采用专门的分泌系统来识别并与宿主细胞相互作用，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目标因为它们是表面暴露，广泛保守的，并且针对致病性。但是不幸的是，许多关键系统的结构仍然很少理解。在这里，我们描述了我们将如何继续使用电子冷冻理学（冷冻）来剖析致病性的结构和功能纳米机器。冷冻物是一种革命性的成像技术，具有揭示内部本地结构的力量具有大分子（2-5 nm）分辨率的3D中的完整细胞。相同结构的亚图平均在最有利的情况下，一个或多个冷冻图可以将此分辨率提高到比1 nm更好要将其放置在完整的机器中的组件。我的小组开创了发展在这项革命性的成像技术中，在我们第一个奖励期的不到的四年中，我们已经使用了冷冻材料生产许多致病性分泌系统的新结构，并建立属于IV型Pilus（T4P），VI类型分泌系统（T6SS）和IV类型分泌的关键系统系统（T4SS）家庭，产生了大量新的机械见解。通过利用新的冷冻技术在过去的几年中，我们刚刚发展，我们建议在下一个奖励期内扩展我们的工作对于这些复合物，关键相关系统和新目标的不同功能状态：病原IX 分泌系统（T9SS）。此外，我们将把整个工作都推向更高的分辨率。对于每个目标，我们将原位图像整个完整的结构。在大多数情况下，这将是第一个高分辨率成像这些结构。然后，我们将将亚图平均图与突变体的差分分析相结合，其中单个组件被淘汰或用额外的密度标记以产生建筑复合物的模型。如果可以使用组件（或同源物）的原子模型，我们将将它们扩展到我们的地图中，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戚的那些（以前或在拟议的工作中解决），我们旨在确定适应性毒力功能的基础。我们还将应用最新的低温相关光和电子显微镜（冷冻-CLEM）引导低温聚焦离子束（FIB）铣削以使我们能够成像病原体分泌系统的作用：在感染真核宿主的细菌细胞中。这将提供第一个这些关键人类病原体的图像，我们希望这些图像可为运作提供宝贵的见解他们在体内的毒力机械。我们在一起，我们希望这个项目能够产生详细的机械图片 T4SS，T4P和T9SS纳米机器介导发病机理，这是识别的重要第一步将来的治疗靶标。

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）

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.

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.

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