Structure and function of prokaryotic appendages by cryo-electron tomography

冷冻电子断层扫描原核附属物的结构和功能

• 批准号: 8421785
• 负责人: ELIZABETH R WRIGHT
• 金额: $ 28.79万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8421785
• 关键词: ATP phosphohydrolase; Address; Adherence; Algorithms; Animals; Architecture; Area; Automation; Bacteria; Bacterial Pili; Bacteriophages; Biodiversity; Biological; Biological Assay; Biological Models; Campylobacter jejuni; Caulobacter crescentus; Cell membrane; Cells; Cessation of life; Complex; Computer software; Cryoelectron Microscopy; Data; Data Collection; Development; Diarrhea; Electron Microscope; Engineering; Exhibits; Filament; Film; Fimbriae Proteins; Flagella; Flagellin; Freezing; Gastroenteritis; Goals; Homologous Gene; Human; Imaging technology; In Situ; Individual; Macromolecular Complexes; Maintenance; Membrane; Membrane Proteins; Microbial Biofilms; Modeling; Molecular Genetics; Molecular Weight; Motor; Organism; Peptidoglycan; Phase; Pilum; Process; Prokaryotic Cells; Proteins; Resistance; Resolution; Role; Salmonella typhimurium; Secretin; Structure; Structure-Activity Relationship; Sum; Surface; System; Technology; Tomogram; Type III Secretion System Pathway; Variant; Vibrio; Vibrio cholerae; Vibrio vulnificus; Virulence; Virulence Factors; Work; appendage; cell motility; comparative; electron tomography; environmental adaptation; extracellular; improved; kinetosome; mutant; pathogen; pathogenic bacteria; pressure; public health relevance; reconstruction; research study; response; stoichiometry; success; three dimensional structure; trafficking

DESCRIPTION (provided by applicant): Many prokaryotes use extracellular appendages, notably the flagella and pili, for motility and colonization of biotic and abiotic substrates. Both flagella and pili are well-known virulence factors for numerous pathogenic bacterial species. Here we aim to determine two things: 1) how redundancy of the flagellins in multi-component flagellar filaments alters overall flagellum structure and function, and 2) the structural variatio that exists between several classes of type IV pili at the macromolecular level. To answer these questions, we are using several bacterial species, Vibrio cholerae and Vibrio vulnificus, human pathogens that cause severe gastroenteritis, diarrhea, and death; and Caulobacter crescentus, a well-established non-pathogenic model system. We are using these organisms to build upon our previous structural studies of the polar appendages of C. crescentus and to develop Vibrio species as model systems for structural studies of pathogens. Together, these studies will provide new information regarding the structural and functional diversity of prokaryotic flagella and pili. The three areas to be investigated in this project are: 1. Determine the structural and functional implications of multi-flagellin flagella in pathogenic and non-pathogenic bacteria. Experiments will determine the structure of flagella from wild type and mutant C. crescentus, V. cholerae, and V. vulnificus in situ through cryo-ET of frozen hydrated cells. Cryo-EM and helical reconstruction approaches will define the high-resolution structure of flagella isolated from C. crescentus, V. cholerae, and V. vulnificus. 2. Determine the structures of several classes of type IV pili in intact bacterial cells. Experiments in this aim will determine the in situ structure of he T4bP of V. cholerae and V. vulnificus and Flp-type pili of C. crescentus through cryo-ET and sub-tomogram averaging. We will characterize structural changes resulting from mutants to the pilus filament or pilus complex and their impact on pilus structure, pilin secretion and possible retraction, and complex integration into the cell membrane. 3. Continue the development of cryo-electron tomography technology to generate improved structures of prokaryotes and their appendages. We aim to optimize our newly installed Zernike phase contrast electron microscope for cryo-ET applications. Our goal is to develop ZPC cryo-ET by optimizing the thin films used for Zernike phase plates and developing full automation of biological data collection. In addition, this state of the art technology calls for the development of new software and improvement of already existing algorithms for a more efficient and biologically relevant filtering and 'denoising' of cryo-ET data.

描述（由申请人提供）：许多原核生物使用细胞外附属物，尤其是鞭毛和菌毛，用于生物和非生物底物的运动和定殖。两个都 鞭毛和菌毛是许多致病细菌物种的众所周知的毒力因子。在这里，我们的目的是确定两件事：1）多组分鞭毛细丝中鞭毛蛋白的冗余如何改变整体鞭毛结构和功能，以及2）在大分子分子水平的几类IV类Pili之间存在的结构变量。为了回答这些问题，我们使用了几种细菌，弧菌霍乱和弧菌，引起严重胃肠炎，腹泻和死亡的人类病原体；和Caulobacter Crescentus，这是一个完善的非致病模型系统。我们正在使用这些生物来建立我们先前对C. c. c. c. c. c. c. c.c。的极性附属物的结构研究，并开发颤音物种作为病原体结构研究的模型系统。这些研究将共同​​提供有关原核生物鞭毛和pili的结构和功能多样性的新信息。在该项目中要研究的三个区域是：1。确定多氟蛋白鞭毛在致病性和非致病细菌中的结构和功能意义。实验将通过冷冻水合细胞的冷冻ET来确定野生型和突变体C. C. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c. c.c。冷冻EM和螺旋重建方法将定义从C. crescentus，V。Cholerae和V. vulnificus中分离出的鞭毛结构。 2。确定完整细菌细胞中几种IV型Pili的结构。此目标中的实验将确定霍乱弧菌和V. vulnificus和V. vulnificus和flp型的原位结构，并通过冷冻和子图平均图。我们将表征从突变体到菌毛细丝或菌毛复合物引起的结构变化及其对菌毛结构，PILIN分泌以及可能的缩回以及复杂整合到细胞膜中的影响。 3。继续开发冷冻电子层析成像技术，以产生改进的原核生物结构及其附属物。我们旨在优化针对低温ET应用的新安装的Zernike相对比电子显微镜。我们的目标是通过优化用于Zernike相板的薄膜并发展生物数据收集的完全自动化来开发ZPC Cryo-ET。此外，这种最先进的技术需要开发新软件，并改善现有算法，以进行更高效且与生物学相关的过滤 和冷冻数据的“ denoising”。