Localization and assembly of the M. tuberculosis ESX-1 secretory apparatus

描述(申请人提供)：最近描述的III型分泌系统，以结核分枝杆菌的ESX-1原型装置为例，是许多革兰氏阳性细菌的关键毒力决定因素。ESX-1是一个高度保守的器官，它分泌一组特定的蛋白质，通过调节免疫反应和帮助细菌通过宿主传播来促进毒力。结核分枝杆菌ESX-1突变体减毒。尽管这种装置很重要，但人们对结构蛋白、它们如何或在哪里组装以及分泌机制知之甚少。这项提案将解决这些赤字问题。本实验室已经确定了ESX-1分泌在调节耻垢分枝杆菌模式中的配偶DNA转移中的作用。我们所有的遗传和生化研究表明，污垢分枝杆菌和结核分枝杆菌之间的ESX-1装置及其分泌底物在功能上是可以互换的；污垢分枝杆菌将分泌结核分枝杆菌ESX底物，而结核分枝杆菌esx-1基因将补充耻垢分枝杆菌ESX-1突变体。此外，污垢分枝杆菌的快速生长速度、遗传易感性和非致病性使其成为解剖ESX-1装置的理想系统。我们将建立在初步数据的基础上，这些数据表明ESX-1装置定位于细胞极点。利用荧光显微镜和分子遗传学的结合，我们将确定那些组装成核心机制的蛋白质，它们的组装机制，并寻找分泌的小分子抑制剂。其具体目的是：1.确定耻垢分枝杆菌ESX-1相关蛋白的细胞定位。2.确定ESX-1的组装顺序以及参与这一过程的蛋白质。3.建立一种简单的基于荧光的方法来监测ESX-1的分泌。革兰氏阳性细菌之间VII型分泌系统的相似性将确保这项工作开发的模型将广泛适用于其他细菌病原体，包括炭疽杆菌和白喉杆菌。最重要的是，最近出现了极具抗药性的结核分枝杆菌菌株，这增加了对这种每年导致160多万人死亡的疾病的新疗法的需求：ESX-1设备代表着合理药物设计的潜在新目标。 公共卫生相关性：结核分枝杆菌每年造成160多万人死亡。为了确定合理的新药靶点来对抗这种微生物，有必要了解它的生物学和它如何在宿主细胞中生存。结核分枝杆菌分泌的蛋白质可以调节宿主的反应。这项工作的目标是确定这一分泌过程所需的蛋白质，以及它们如何组装形成分泌机器。由于分泌物对毒力至关重要，我们预计该装置将提供一个新的药物靶点。

DESCRIPTION (provided by applicant): The recently described type VII secretion system, exemplified by the archetypal ESX-1 apparatus in Mycobacterium tuberculosis, is a key virulence determinant in many Gram-positive bacteria. ESX-1 is a highly conserved apparatus, which secretes a specific set of proteins that promote virulence by modulating the immune response and helping to disseminate the bacteria through the host. M. tuberculosis ESX-1 mutants are attenuated. Despite the importance of this apparatus, little is known about the structural proteins, how, or where they are assembled and the mechanism of secretion. This proposal will address these deficits. This laboratory has defined a role of ESX-1 secretion in modulating conjugal DNA transfer in the model mycobacterium M. smegmatis. All of our genetic and biochemical studies indicate that the ESX- 1 apparatus and its secreted substrates are functionally interchangeable between M. smegmatis and M. tuberculosis; M. smegmatis will secrete M. tuberculosis ESX substrates, and M. tuberculosis esx-1 genes will complement M. smegmatis ESX-1 mutants. Moreover, the fast-growth rate, its genetic tractability and the non-pathogenic nature of M. smegmatis make it an ideal system to dissect the ESX- 1 apparatus. We will build upon preliminary data that suggest the ESX-1 apparatus localizes to the cell poles. Using a combination of fluorescence microscopy and molecular genetics we will define those proteins assembled into the core machinery, their mechanism of assembly, and look for small molecule inhibitors of secretion. The specific aims are to: 1. Identify the cellular location of ESX-1 associated proteins in M. smegmatis. 2. Determine the order of assembly of ESX-1 and the proteins mediating this process. 3. Develop a simple fluorescence-based assay to monitor ESX-1 secretion. The similarities of type VII secretion systems among Gram-positive bacteria will ensure that models developed from this work will be broadly applicable to other bacterial pathogens including B. anthracis and C. diphtheriae. Most importantly, the recent emergence of extremely-drug resistant strains of M. tuberculosis has increased the demand for new treatments for a disease that kills over 1.6 million people a year: the ESX-1 apparatus represents a potential new target for rational drug design. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis accounts for over 1.6 million deaths per year. In order to define rational new drug targets to combat this organism, there is a need to understand its biology and how it survives within host cells. M. tuberculosis secretes proteins that are known to modulate the host response. The goal of this work is to identify proteins required for this secretion process and how they assemble to form a secretion machine. As secretion is critical for virulence we anticipate that the apparatus will provide a new drug target.