The mechanistic basis for toxin secretion in Clostridioides difficile

项目摘要/摘要 了解大分子如何穿过细胞壁是一个复杂而糟糕的问题 了解生物过程。院内致病菌艰难梭状芽胞杆菌产生和分泌两种 引起疾病的大毒素。尽管对效应器的功能已知很多， 毒素，人们对毒素是如何分泌的知之甚少。毒素被编码在致病性上 该基因还编码TcdE和TCDL，TcdE是一种类似Holin的蛋白，TCDL是内毒素的N端残基。而当 噬菌体使用霍林/内溶素系统来触发细菌细胞裂解和逃逸，现在有多份报告 提示TcdE用于毒素的分泌。这项提案将解决悬而未决的问题 TcdE和TCDL如何在不引起细胞的情况下与艰难梭菌毒素相互作用产生毛孔和分泌毒素 解体。TcdE和TCDL将被荧光标记，并将确定它们的定位和寡聚化 体内使用结构照明显微镜和逐步光漂白。低聚状态(S) TcdE也将在体外使用低温透射电子显微镜(Cryo-TEM)进行测定。TCDL有 最近被发现，但其在毒素分泌中的作用尚未被充分研究。TCDL将被删除并 过度表达，以确定它在毒素分泌过程中的作用(如果有的话)。直接结合分析将评估 TCDL可与毒素或TcdE相互作用，促进毒素分泌。最后，确定了C. 艰难梭菌将在毒素分泌过程中使用相关的光学和电子显微镜进行评估，并聚焦 离子束扫描电子显微镜。TcdE的三维结构和细胞壁将在原位确定 使用冷冻电子断层扫描。这一提议将为一个基本的生物过程提供新的线索：运输 穿过细胞膜的大分子。这里提出的实验将建立在我强大的 细菌遗传学和生理学基础。我将在D.Borden Lacy博士的实验室接受培训 在结构生物学领域确立了领先地位，擅长解决高分辨率的毒素结构。 她的指导将让我学习结构生物学和高分辨率光的尖端技术 显微镜。我将学习的技术和我将在此过程中建立的研究计划 培训将使我过渡到一家R1研究机构的独立调查员。

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.