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Molecular Dissection of Conjugation, Virulence and ESX Secretion in Mycobacteria

分枝杆菌结合、毒力和 ESX 分泌的分子解剖

基本信息

批准号：
8079913
负责人：
KEITH M DERBYSHIRE
金额：
$ 46.12万
依托单位：
WADSWORTH CENTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8079913
关键词：
Address Affect Animal Model Appearance Attenuated Biochemical Biological Assay Biology Cells Cues DNA DNA Sequence Disease Dissection Drug Delivery Systems Drug Design Drug resistance Evolution Genes Genetic Genetic Processes Genetic Screening Genetic Techniques Genome Genus Mycobacterium Goals HIV Horizontal Gene Transfer Immune system Investigation Laboratories Lateral Lead Mediating Modeling Molecular Molecular Genetics Multi-Drug Resistance Mutagenesis Mycobacterium smegmatis Mycobacterium tuberculosis Pathogenesis Pharmaceutical Preparations Physiological Plasmids Play Population Process Protein Secretion Proteins Public Health Publishing Regulation Role Set protein System Tuberculosis Virulence Virulent Work base extracellular genome wide association study genome-wide innovation insight killings macrophage mutant mycobacterial novel pathogen resistant strain response tool vaccine development

项目摘要

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.

这项工作的目的是确定称为 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发挥关键作用：夫妻DNA转移和毒力。该实验室已确定 ESX-1 分泌和 DNA 转移在功能上和基因上相互交织； 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个模型，其中分枝杆菌定制其 ESX-1 分泌谱以响应不同的情况生理和环境线索。这解释了保守型 ESX-1 的功能多样性促进 DNA 转移和巨噬细胞存活的分泌装置。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。接合需要细胞与细胞接触，并涉及 DNA 从供体细胞单向转移到受体细胞，通常会导致大片段染色体 DNA 的转移。 DNA转移耻垢分枝杆菌菌株之间的相互作用通过一种不同于典型的新机制发生，质粒介导的接合系统。这项工作的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 分泌装置对于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、ESX-1 底物及其机制的基因测定分泌的定义不明确。该提案将通过利用独特的优势来解决这些差距我们的观察结果：ESX-1 对于耻垢分枝杆菌受体菌株中的 DNA 转移至关重要。耻垢分枝杆菌 DNA 转移需要完整的 ESX-1 装置，这提供了第一个遗传基因。筛选 ESX-1 突变体的分离。该提案的目标是描述 DNA 的过程转移，利用 DNA 转移作为遗传工具，阐明 ESX-1 分泌机制并确定 ESX-1 分泌的蛋白质在 DNA 转移和 M 调节中的作用。结核病毒力。这些目标将通过分子、遗传和生化测定。具体目标是： 1. 确定供体和受体菌株中接合 DNA 转移的遗传要求。耻垢分枝杆菌，以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。这些目标共同描述了一种剖析 ESX-1 分泌机制的创新方法。此外，分泌蛋白及其细胞外靶标的鉴定将提供重要的信息。关于它们在横向 DNA 转移和结核分枝杆菌毒力中的作用的新见解。