Molecular Dissection of Conjugation, Virulence and ESX Secretion in Mycobacteria

这项工作的目的是确定如何分枝杆菌分泌装置称为ESX-1发挥 在重要但明显不同的分子过程中起关键作用：配偶体DNA转移和 毒性该实验室已经确定ESX-1分泌和DNA转移在功能上是相互关联的， 基因交织在一起; ESX-1突变体在DNA转移中有缺陷。我们的研究使我们能够 提出了一个模型，其中分枝杆菌定制其ESX-1分泌谱，以响应不同的 生理和环境线索。这解释了保存的ESX-1的功能多样性 在促进巨噬细胞中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的生物学和发病机制的影响。 缀合需要细胞-细胞接触，并且涉及DNA从供体细胞到供体细胞的单向转移。 受体细胞，通常导致染色体DNA的大片段转移。DNA转移 在耻垢分枝杆菌菌株之间通过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 接合分枝杆菌的进化，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 遗传测定M.结核病ESX-1活性，以及两种ESX-1底物和 分泌物定义不明确。该提案将利用独特的 我们已经观察到：ESX-1对于M.丝霉受体菌株。 在M.包皮环切术提供了第一个基因 筛选ESX-1突变体的分离。这项提案的目标是描述DNA的过程， 转移，利用DNA转移作为遗传工具，阐明ESX-1分泌的机制， 确定ESX-1分泌的蛋白质在调节DNA转移和M. 结核病毒力这些目标将通过分子、遗传和 生化分析具体目标是： 1.确定供体和受体M菌株接合DNA转移的遗传要求。 为了表征这种新的遗传交换机制， 2.利用DNA转移进行全基因组遗传筛查，以确定ESX-1所需的基因 活性，并确定其在DNA转移和毒力中的作用。 总之，这些目标描述了一种创新的方法来剖析ESX-1分泌的机制。 此外，分泌蛋白及其胞外靶标的鉴定将提供重要的 新的见解，他们的作用，在横向DNA转移和M。结核病毒力

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.