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

项目总结/摘要 嗜肺军团菌和贝氏柯克斯体是能够引起人类肺损伤的细胞内细菌病原体。 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子， 对于它们在细胞内复制的能力至关重要，这是专门的IV型分泌系统（T4 SS） 称为Dot/ICM。Dot/Icm是一种非常通用的分泌器官， 将300多种具有不同生化功能和不同结构的不同蛋白质的库导入宿主细胞中， 特性.本项目的目标是确定Dot/ICM机器的结构和装配， 阐明个别点和Icm蛋白质如何有助于在分子水平上的机器功能。我们将 联合收割机先进的冷冻电子断层扫描（cryo-ET）与遗传和生物化学方法相结合， Dot/Icm机器组装的途径（目的1），并确定细胞溶质ATP酶 募集效应子并介导Dot/Icm结构的变化（目标2）。此外，我们的目标是表征 宿主细胞膜上的转运孔，作为Dot/Icm效应物的蛋白传导通道 (Aim 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).