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

结核分枝杆菌 ESX-1 分泌装置的定位和组装

• 批准号: 7871120
• 负责人: KEITH M DERBYSHIRE
• 金额: $ 18.63万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-15 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7871120
• 关键词: Accounting; Address; Attenuated; Bacillus (bacterium); Bacillus anthracis; Bacteria; Biochemical; Biological Assay; Biological Models; Biology; Cells; Cessation of life; Collection; Color; Complement; Corynebacterium; DNA; Data; Diphtheria; Disease; Drug Delivery Systems; Drug Design; Drug resistance; Ensure; Fluorescence; Fluorescence Microscopy; Genes; Genetic; Genus Mycobacterium; Genus staphylococcus; Goals; Gram-Positive Bacteria; Growth; Immune response; Knowledge; Laboratories; Listeria; Location; Mediating; Methods; Modeling; Molecular Genetics; Monitor; Mycobacterium tuberculosis; Nature; Organism; Pathogenesis; Pathway interactions; Process; Proteins; Role; Set protein; Signal Transduction; Staphylococcus aureus; Streptococcus; Structural Protein; System; Virulence; Work; base; combat; high throughput screening; inhibitor/antagonist; innovation; insight; killings; mutant; pathogen; public health relevance; resistant strain; secretion process; small molecule

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.

描述（由申请人提供）：最近描述的VII型分泌系统，以结核分枝杆菌中的原型ESX-1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 是一种高度保守的装置，它分泌一组特定的蛋白质，这些蛋白质通过调节免疫反应并帮助在宿主中传播细菌来增强毒力。结核分枝杆菌 ESX-1 突变体被减毒。尽管这个装置很重要，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。该提案将解决这些缺陷。 该实验室已经确定了 ESX-1 分泌在耻垢分枝杆菌模型中调节配偶 DNA 转移中的作用。我们所有的遗传和生化研究表明，ESX-1 装置及其分泌底物在耻垢分枝杆菌和结核分枝杆菌之间可以功能互换；耻垢分枝杆菌将分泌结核分枝杆菌 ESX 底物，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，耻垢分枝杆菌的快速生长速度、遗传易​​处理性和非致病性使其成为解剖 ESX-1 装置的理想系统。 我们将基于表明 ESX-1 设备定位于电池极的初步数据。结合荧光显微镜和分子遗传学，我们将定义那些组装成核心机器的蛋白质、它们的组装机制，并寻找分泌的小分子抑制剂。 具体目标是： 1. 确定 ESX-1 相关蛋白在耻垢分枝杆菌中的细胞位置。 2. 确定 ESX-1 的组装顺序以及介导该过程的蛋白质。 3. 开发一种简单的基于荧光的检测方法来监测 ESX-1 的分泌。 革兰氏阳性菌之间 VII 型分泌系统的相似性将确保从这项工作中开发的模型将广泛适用于其他细菌病原体，包括炭疽芽孢杆菌和白喉杆菌。最重要的是，最近出现的极度耐药的结核分枝杆菌菌株增加了对这种每年导致超过 160 万人死亡的疾病的新疗法的需求：ESX-1 装置代表了合理药物设计的潜在新目标。 公共卫生相关性：结核分枝杆菌每年导致超过 160 万人死亡。为了确定合理的新药物靶点来对抗这种生物体，需要了解其生物学特性以及它如何在宿主细胞内生存。结核分枝杆菌分泌已知可调节宿主反应的蛋白质。这项工作的目标是确定该分泌过程所需的蛋白质以及它们如何组装形成分泌机器。由于分泌对于毒力至关重要，我们预计该装置将提供新的药物靶点。