Congugal DNA transfer into M. tuberculosis

描述（由申请人提供）：本提案的长期目标是在结核分枝杆菌中建立接合DNA转移，以操纵这种主要人类病原体的基因组。虽然转化和转导在分枝杆菌遗传学的发展中起着重要的作用，但在分枝杆菌中还没有建立接合DNA转移。结核缀合为在分枝杆菌之间转移DNA提供了一种重要的替代方法，特别是作为一种基因递送系统，用于在菌株之间移动标记物并产生靶向突变。本实验室在模式分枝杆菌中建立了一个有效的接合DNA转移系统。在这里，我们建议运用我们的专业知识和技术，以促进遗传操作的M。结核病基因组转移将建立从一个超接合菌株的M。megaenopsis进入M.结核分枝杆菌和M.结核具体目标是：1.建立染色体DNA转移到M.结核分枝杆菌M.利用遗传选择和反选择的组合的smeglobulin。2.优化质粒转化M.结核病，以开发一种新的和有效的方法，在体内基因工程。3.创建一个M。结核病捐赠者，将允许转移菌株之间的M。结核建立了M.结核病将是在基因分析这一有机体的能力方面向前迈出的重要一步。此外，遗传交换的证明将暗示接合在M的进化中起着重要作用。结核病，并将提高邪恶的可能性，耐药基因之间交换M。肺结核细胞，从而产生多药耐药菌株。结核分枝杆菌每年造成200多万人死亡。此外，由于结核病与艾滋病毒的致命联系以及耐多药菌株的出现，增加了对新疗法的需求，结核病给全球造成的负担更加沉重。新药和疫苗的设计需要了解分枝杆菌的生物学，并开发遗传工具来操纵其基因组，以确定发病机制和耐药性的分子基础。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to establish conjugal DNA transfer in Mycobacterium tuberculosis in order to manipulate the genome of this major human pathogen. Although both transformation and transduction have had important roles in the development of mycobacterial genetics, conjugal DNA transfer has yet to be established in M. tuberculosis. Conjugation offers an important alternative for transferring DNA between mycobacteria and, in particular, as a gene delivery system for moving markers between strains and generating targeted mutations. This laboratory has established an efficient conjugal DNA transfer system in the model mycobacterial organism Mycobacterium smegmatis. Here, we propose to apply our expertise and technology to facilitate genetic manipulation of the M. tuberculosis genome. Transfer will be established from from a hyper- conjugative strain of M. smegmatis into M. tuberculosis and between strains of M. tuberculosis. The specific aims are: 1. To establish chromosomal DNA transfer into M. tuberculosis from M. smegmatis utilizing a combination of genetic selections and counter-selections. 2. To optimize plasmid transfer into M. tuberculosis in order to develop a novel and efficient method for in vivo genetic engineering. 3. To create an M. tuberculosis donor that will allow transfer between strains of M. tuberculosis. The establishment of DNA transfer into M. tuberculosis would represent a significant step forward in the ability to genetically analyse this organism. Moreover, demonstration of genetic exchange would implicate conjugation as a significant player in the evolution of M. tuberculosis, and would raise the sinister possibility of drug resistance genes being exchanged between M. tuberculosis cells in the lung, thereby creating multi-drug resistant strains. Mycobacterium tuberculosis accounts for over 2 million deaths per year. Furthermore, the global burden of tuberculosis has been compounded by its deadly association with the AIDS virus and by the emergence of multi-drug resistant strains, which have increased the demand for new treatments. The design of new drugs and vaccines requires an understanding of the biology of mycobacteria, and the development of genetic tools to manipulate their genomes in order to determine the molecular basis of pathogenesis and drug resistance.