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The mechanistic basis for toxin secretion in Clostridioides difficile

艰难梭菌毒素分泌的机制基础

基本信息

批准号：
10445004
负责人：
Shannon L Kordus
金额：
$ 7.23万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10445004
关键词：
3-Dimensional Address Amino Acids Applications Grants Architecture Bacteria Bacterial Physiology Bacterial Toxins Bacteriophages Binding Biochemical Biological Assay Biological Process Carrier Proteins Cell Wall Cell membrane Cells Clostridium difficile Complex Cryo-electron tomography Cytolysis Data Diffusion Disease Electron Microscopy Foundations Genes Gram-Negative Bacteria Gram-Positive Bacteria Image In Situ In Vitro Institution Ions Laboratories Learning Life Light Lighting Lipid A Lipid Bilayers Lipids Measures Membrane Mentorship Microscopy Molecular Molecular Chaperones N-terminal Pathogenicity Peptidoglycan Photobleaching Process Protein Isoforms Proteins Reporting Research Research Personnel Research Proposals Resolution Role Scanning Electron Microscopy Structural Biologist Structure Sugar Acids System Techniques Technology Thick Thinness Time Toxin Training Transmission Electron Microscopy Transport Process VDAC1 gene Viral aqueous bacterial genetics crosslink endolysin experimental study in vivo light microscopy macromolecule medical specialties overexpression particle pathogen prevent programs structural biology three dimensional structure

项目摘要

Project Summary/Abstract Understanding how large macromolecules are transported across a cell wall is a complex and poorly understood biological process. The nosocomial pathogen Clostridioides difficile produces and secretes two large toxins that are responsible for causing disease. Although much is known about the effector functions of the toxins, very little is known about how the toxins are secreted. The toxins are encoded on a pathogenicity locus which also encodes TcdE, a holin-like protein and TcdL, the N-terminal remnant of an endolysin. While bacteriophages use holin/endolysin systems to trigger bacterial cell lysis and escape, multiple reports now suggest that TcdE is used for the secretion of the toxins. This proposal will address the outstanding questions of how TcdE and TcdL interact with C. difficile toxins to create a pore and secrete toxins without causing cell lysis. TcdE and TcdL will be fluorescently tagged, and their localization and oligomerization will be determined in vivo using structured illumination microscopy and stepwise photobleaching. The oligomerization state(s) of TcdE will also be determined in vitro using cryo-transmission electron microscopy (cryo-TEM). TcdL has recently been discovered and its role in toxin secretion has not been fully explored. TcdL will be deleted and overexpressed to determine what, if any, role it has during toxin secretion. Direct binding assays will assess if TcdL can interact with the toxins or TcdE to facilitate toxin secretion. Finally, the cell wall architecture of C. difficile will be assessed during toxin secretion using correlative light and electron microscopy, and focused ion-beam scanning electron microscopy. The 3D structure of TcdE and the cell wall will be determined in situ using cryo-electron tomography. This proposal will shed new light on a basic biological process: the transport of macromolecules across the cell membrane. The experiments proposed here will build upon my strong foundation in bacterial genetics and physiology. I will be training in the laboratory of Dr. D. Borden Lacy, an established leader in the field of structural biology with a specialty in solving high-resolution toxin structures. Her guidance will allow me to learn cutting edge techniques in structural biology and high-resolution light microscopy. The techniques that I will learn and the research program that I will build over the course of this training will allow me to transition into an independent investigator at a R1 research institution.

项目总结/文摘