Virulence Factors in Mycobacteria

描述(由申请人提供)：结核病感染了大部分 它是世界人口的主要来源，每年造成数百万人死亡。这个 致病微生物，结核分枝杆菌，已经被认识了一年多 世纪，但人们对这种病毒所使用的分子机制知之甚少 细菌导致疾病。我建议使用我们最近使用的三种方法 与结核分枝杆菌基因组序列一起开发以确定 结核分枝杆菌需要哪些基因才能在体外和体内存活 活着。首先，我们开发了一种新的转座子来执行饱和 结核分枝杆菌的诱变。第二，我们已经制作了DNA微阵列， 我们可以对每个结核分枝杆菌开放阅读框进行杂交测量。第三, 我们开发了转座子连接杂交(TJH)，这是一种定位的方法 利用DNA在大量突变体库中插入转座子的位置 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，与那些需要在动物身上生存的人一起。我们会 使用TJH的一种变体，差异长度杂交，来鉴定 在特定的介质中生长所必需的一套完整的基因。我们会 还对插入突变体文库中的数千个克隆进行了测序 产生一批明确的突变体。这将允许我们测试个人 含有TJH鉴定的候选毒力基因突变的菌株。 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 它们控制的基因。这将使我们能够确定所需的两个基因 生存和致病。重要基因的鉴定 感染应该导致结核病新策略的发展 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.