Functional and Structural Analysis of the Dot/Icm Type IVB Secretion Machine

Dot/Icm型IVB分泌机的功能和结构分析

• 批准号: 10264923
• 负责人: Jun Liu
• 金额: $ 58.63万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-16 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10264923
• 关键词: 3-Dimensional; ATP Hydrolysis; ATP phosphohydrolase; Amaze; Bacteria; Bacterial Proteins; Biochemical; Cell membrane; Cells; Classification; Complex; Coupling; Coxiella burnetii; Cryo-electron tomography; Cytosol; Data; Development; Electron Microscopy; Genetic; Goals; Imaging technology; In Situ; Individual; Infection; Ion Channel; Ions; Legionella pneumophila; Location; Mediating; Membrane; Molecular; Molecular Conformation; Multiprotein Complexes; Pathway interactions; Property; Proteins; Resolution; Site; Structure; System; Time; Tomogram; Travel; Type IV Secretion System Pathway; Vacuole; Virulence; Work; fascinate; human disease; improved; insight; light microscopy; mutant; nanomachine; novel; pathogen; pathogenic bacteria; protein protein interaction; recruit; three dimensional structure

PROJECT SUMMARY/ABSTRACT Legionella pneumophila and Coxiella burnetii are intracellular bacterial pathogens capable of causing human disease. A notable feature of these two pathogens is that they retain a common virulence determinant that is essential for their ability to replicate intracellularly, which is the specialized type IV secretion system (T4SS) called Dot/Icm. The Dot/Icm is an incredibly versatile secretion apparatus that has the capacity to translocate into host cells a repertoire of over 300 different proteins with different biochemical functions and diverse structural properties. The goal of this project is to determine the structure and assembly of the Dot/Icm machine and elucidate how the individual Dot and Icm proteins contribute to machine function at the molecular level. We will combine advanced cryo-electron tomography (cryo-ET) with genetic and biochemical approaches to determine the pathway of Dot/Icm machine assembly (Aim 1) and to determine the mechanism by which cytosolic ATPases recruit effectors and mediate changes in the Dot/Icm structure (Aim 2). Furthermore, we aim to characterize the translocation pore in the host cell membrane that serves as the protein-conducting channel for Dot/Icm effectors (Aim 3).

项目摘要/摘要 肺炎军团菌和Coxiella burnetii是能够引起人的细胞内细菌病原体 疾病。这两种病原体的一个值得注意的特征是它们保留了共同的毒力决定因素 对于它们细胞内复制的能力至关重要，这是专门的IV型分泌系统（T4SS） 称为点/ICM。 DOT/ICM是一种多才多艺的分泌物，具有易位的能力 进入宿主细胞，具有300多种具有不同生化功能和多种结构的不同蛋白质的曲目 特性。该项目的目的是确定点/ICM机器的结构和组装， 阐明单个点和ICM蛋白如何在分子水平上有助于机器功能。我们将 将晚期冷冻电子层析成像（冷冻-ET）与遗传和生化方法相结合以确定 点/ICM机器组件的途径（AIM 1），并确定胞质ATPases的机制 招募效应子并介导DOT/ICM结构的变化（AIM 2）。此外，我们旨在表征 宿主细胞膜中的易位孔，该孔充当点/ICM效应子的蛋白质传导通道 （目标3）。