Molecular Dissection of Conjugation, Virulence and ESX Secretion in Mycobacteria

这项工作的目的是确定一种名为ESX-1的分枝杆菌分泌装置如何发挥作用 在重要但明显不同的分子过程中的关键作用：配偶DNA转移和 致命性。本实验室已证实ESX-1的分泌和DNA转移在功能上和 遗传上相互交织；ESX-1突变体在DNA转移方面存在缺陷。我们的研究使我们能够 提出了一种模型，在该模型中，分枝杆菌根据不同的情况定制其ESX-1分泌谱 生理和环境方面的线索。这就解释了保守的ESX-1的功能多功能性 促进巨噬细胞DNA转移和存活的分泌器。拟议的研究将 阐明这两个过程对分枝杆菌生物学和发病机制的影响。 接合需要细胞间的接触，涉及DNA从供体细胞单向转移到 一种受体细胞，通常导致大段染色体DNA的转移。DNA转移 污垢分枝杆菌菌株之间通过一种不同于原型的新机制发生， 质粒介导的接合系统。这项工作的一个长期目标是了解 与分枝杆菌进化的结合，包括耐药性和毒力决定因素的传播。 ESX-1分泌器对结核分枝杆菌的毒力至关重要。没有 结核分枝杆菌ESX-1活性的遗传检测、ESX-1底物及其作用机制 分泌物的定义不明确。这项提案将通过利用独特的 我们观察到：ESX-1对于耻垢分枝杆菌受体菌株的DNA转移是必不可少的。 耻垢分枝杆菌DNA转移对完整ESX-1装置的要求提供了第一个基因 筛选分离ESX-1突变体。这项提议的目标是描述DNA的过程 转移，利用DNA转移作为遗传工具，阐明ESX-1的分泌机制，并 确定ESX-1分泌的蛋白质在DNA转移和M。 肺结核的致死性。这些目标将通过结合分子、基因和 生化分析。具体目标是： 1.确定供体和受体品系配偶DNA转移的遗传要求。 为了描述这种新的基因交换机制。 2.利用DNA转移进行全基因组遗传筛选，以确定ESX-1所需的基因 活性，并确定它们在DNA转移和毒力中的作用。 总之，这些目标描述了一种剖析ESX-1分泌机制的创新方法。 此外，对分泌蛋白及其胞外靶点的鉴定将提供重要的 对它们在横向DNA转移和结核分枝杆菌毒力中的作用的新见解。

The objective of this work is to determine how a Mycobacterial secretion apparatus called ESX-1 plays critical roles in important, but apparently disparate, molecular processes: conjugal DNA transfer and virulence. This laboratory has established that ESX-1 secretion and DNA transfer are functionally and genetically intertwined; ESX-1 mutants are defective in DNA transfer. Our studies have allowed us to propose a model in which Mycobacteria customize their ESX-1 secretion profile in response to different physiological and environmental cues. This explains the functional versatility of the conserved ESX-1 secretory apparatus in promoting DNA transfer and survival in a macrophage. The proposed studies will elucidate the impact of these two processes on the biology and pathogenesis of Mycobacteria. Conjugation requires cell-cell contact and involves the unidirectional transfer of DNA from a donor cell to a recipient cell, often resulting in the transfer of large segments of chromosomal DNA. DNA transfer between strains of Mycobacterium smegmatis occurs by a novel mechanism distinct from the prototypical, plasmid-mediated conjugation systems. A long-term goal of the work is to understand the contributions of conjugation to mycobacterial evolution, including the spread of drug resistance and virulence determinants. The ESX-1 secretory apparatus is essential for virulence of Mycobacterium tuberculosis. There is no genetic assay for M. tuberculosis ESX-1 activity, and both ESX-1 substrates and the mechanism of secretion are poorly defined. This proposal will address these gaps by taking advantage of a unique observation that we have made: ESX-1 is essential for DNA transfer in the M. smegmatis recipient strain. The requirement for an intact ESX-1 apparatus for DNA transfer in M. smegmatis provides the first genetic screen for the isolation of ESX-1 mutants. The goals of this proposal are to characterize the process of DNA transfer, to exploit DNA transfer as a genetic tool, to elucidate the mechanism of ESX-1 secretion and to determine the roles of the proteins secreted by ESX-1 in both regulation of DNA transfer and M. tuberculosis virulence. These goals will be achieved using a combination of molecular, genetic and biochemical assays. The specific aims are to: 1. Determine the genetic requirements for conjugal DNA transfer in donor and recipient strains of M. smegmatis in order to characterize this novel mechanism of genetic exchange. 2. Utilize DNA transfer to perform a genome-wide genetic screen to identify genes required for ESX-1 activity, and determine their role in both DNA transfer and virulence. Together, these aims describe an innovative approach to dissect the mechanism of ESX-1 secretion. Moreover, the identification of the secreted proteins, and their extracellular targets will provide significant new insights into their roles in both lateral DNA transfer and M. tuberculosis virulence.