Virulence Factors in Mycobacteria

描述（由申请人提供）：结核病感染了大部分的 它是世界人口的主要来源，每年造成数百万人死亡。的 病原体结核分枝杆菌已被确认为 世纪，但很少有人知道的分子机制所使用的这一点 细菌导致疾病。我建议使用三种方法，我们最近 与结核分枝杆菌基因组序列一起开发，以确定 哪些基因是结核分枝杆菌在体外和体内存活所必需的？ vivo.首先，我们开发了一种新的转座子， 结核分枝杆菌的诱变。第二，我们制作了一个DNA微阵列， 我们可以测量与每个结核分枝杆菌开放阅读框的杂交。第三、 我们开发了转座子连接杂交（TJH），一种定位 在大量的突变体中，使用DNA 微阵列我们建议使用TJH来比较M所需的基因 结核病在体外生长与那些需要在动物中生存。我们将 使用TJH的变体，差异长度杂交，来鉴定 一套完整的基因是必不可少的生长在确定的媒体。我们将 还对来自插入突变体文库的几千个克隆进行测序， 产生一个确定的突变体库这将使我们能够测试个人 TJH鉴定的候选毒力基因中含有突变的菌株。 由于病原体协调调节毒力基因的表达，我们将 关注感染所需的调控基因，并确定哪些下游基因 他们控制的基因这将使我们能够确定两个基因所需的 生存和引起疾病。重要基因的鉴定 感染应导致新的结核病战略的发展 治疗和预防。

DESCRIPTION (provided by the applicant): Tuberculosis infects much of the world's population and is responsible for millions of deaths annually. The causative organism, Mycobacterium tuberculosis, has been recognized for over a century, but little is known about the molecular mechanisms used by this bacterium to cause disease. I propose to use three methods we have recently developed in conjunction with the M tuberculosis genomic sequence to determine which genes are required by M tuberculosis to survive both in vitro and in vivo. First, we have developed a new transposon to perform saturating mutagenesis in M tuberculosis. Second, we have made a DNA microarray with which we can measure hybridization to each M tuberculosis open reading frame. Third, we have developed transposon junction hybridization (TJH), a method for mapping the sites of transposon insertions in large pools of mutants using a DNA microarray. We propose to use TJH to compare the genes required for M tuberculosis in vitro growth with those needed to survive in an animal. We will use a variation of TJH, differential length hybridization, to identify the complete set of genes that are essential for growth in defined media. We will also sequence several thousand clones from an insertion mutant library to produce a bank of defined mutants. This will allow us to test individual strains that contain mutations in candidate virulence genes identified by TJH. Since pathogens coordinately regulate expression of virulence genes, we will focus on regulatory genes required for infection and determine which downstream genes they control. This will enable us to identify both genes required for survival and for causing disease. Identification of genes important in infection should lead to the development of new strategies of tuberculosis treatment and prevention.