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

项目摘要/摘要 嗜肺军团菌和伯氏柯克斯体是能引起人类 疾病。这两种病原体的一个显著特征是它们保留了一个共同的毒力决定因素，即 对于它们在细胞内复制的能力来说是必不可少的，这是专门的IV型分泌系统(T4SS) 称为Dot/ICM。Dot/ICM是一种令人难以置信的多功能分泌装置，具有移位能力 进入宿主细胞的300多种不同的蛋白质，具有不同的生化功能和不同的结构 属性。该项目的目标是确定Dot/ICM机器的结构和组装，并 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。我们会 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)和确定胞浆ATPase的机制 招募效应者并调节Dot/ICM结构的变化(目标2)。此外，我们的目标是描述 宿主细胞膜上的转位孔，作为Dot/ICM效应分子的蛋白质传导通道 (目标3)。

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).