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: 10463690
• 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/10463690
• 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).

项目概要/摘要 嗜肺军团菌和伯内氏柯克斯体是细胞内细菌病原体，能够引起人类 疾病。这两种病原体的一个显着特征是它们保留了共同的毒力决定因素，即 对于它们的细胞内复制能力至关重要，这是专门的 IV 型分泌系统 (T4SS) 称为点/Icm。 Dot/Icm 是一种极其多功能的分泌装置，具有易位的能力 将 300 多种具有不同生化功能和不同结构的不同蛋白质带入宿主细胞 特性。该项目的目标是确定 Dot/Icm 机器的结构和组装以及 阐明单个 Dot 和 Icm 蛋白如何在分子水平上对机器功能做出贡献。我们将 将先进的冷冻电子断层扫描 (cryo-ET) 与遗传和生化方法相结合，以确定 Dot/Icm 机器组装的途径（目标 1）并确定胞质 ATP 酶的机制 招募效应器并介导 Dot/Icm 结构的变化（目标 2）。此外，我们的目标是描述 宿主细胞膜上的易位孔，充当 Dot/Icm 效应器的蛋白质传导通道 （目标 3）。