The mechanistic basis for toxin secretion in Clostridioides difficile

艰难梭菌毒素分泌的机制基础

• 批准号: 10231080
• 负责人: Shannon L Kordus
• 金额: $ 6.64万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 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/10231080
• 关键词: 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; Cleaved cell; 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

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.

项目摘要/摘要 了解大型大分子在细胞壁上的运输是一个复杂且较差的 了解生物过程。医院病原体梭状芽胞杆菌艰难梭菌产生并分泌两个 负责引起疾病的大型毒素。虽然对效应子功能的了解众所周知 毒素，对毒素的分泌知之甚少。毒素是在致病性上编码的 还编码TCDE，一种霍林样蛋白和TCDL，即内olysin的N末端残留物。尽管 噬菌体使用霍林/内溶素系统触发细菌细胞裂解和逃脱，现在有多个报告 建议将TCDE用于分泌毒素。该提案将解决未悬而未决的问题 TCDE和TCDL如何与艰难梭菌毒素相互作用以产生孔内并分泌毒素而不会引起细胞 裂解。 TCDE和TCDL将被荧光标记，并将确定它们的定位和寡聚化 在体内使用结构化照明显微镜和逐步光漂白。的寡聚状态 TCDE还将在体外使用冷冻传输电子显微镜（Cryo-TEM）确定。 TCDL有 最近被发现及其在毒素分泌中的作用尚未得到充分探索。 TCDL将被删除，并且 过表达以确定毒素分泌过程中它具有什么（如果有）。直接约束分析将评估是否 TCDL可以与毒素或TCDE相互作用，以促进毒素分泌。最后，C的细胞壁结构。 使用相关光和电子显微镜在毒素分泌过程中评估艰难梭菌，并聚焦 离子梁扫描电子显微镜。 TCDE和细胞壁的3D结构将原位确定 使用冷冻电子层析成像。该建议将为基本生物学过程提供新的启示：运输 跨细胞膜的大分子的。这里提出的实验将建立在我的坚强的基础上 细菌遗传学和生理学的基础。我将在D. Borden Lacy博士的实验室接受培训， 在结构生物学领域建立的领导者，专门解决高分辨率毒素结构。 她的指导将使我能够学习结构生物学和高分辨率光的最先进技术 显微镜。我将要学习的技术和我将在此过程中建立的研究计划 培训将使我能够过渡到R1研究机构的独立研究者。