Virulence Genes in Mycobacterium tuberculosis

描述（由申请方提供）：为了鉴定对宿主内存活重要的分枝杆菌基因，将开发肺结核兔模型。通过经胸注射和支气管内滴注分枝杆菌感染家兔。腔体内的细菌增殖将允许分离足够量的RNA，用于使用结核分枝杆菌寡核苷酸微阵列进行分析。比较培养中生长的分枝杆菌和体内生长的分枝杆菌之间的基因表达模式将提供分枝杆菌在宿主内存活的重要基因的信息。第二种方法将同时用于确定毒力基因。一个水手衍生的转座子，已被证明随机插入到M。结核病基因组将用于产生分枝杆菌突变体文库。转座子位点杂交技术将用于比较动物感染前后的突变体库。微阵列分析将用于确定输出池中不存在的缺失突变体。这些突变体可能具有毒力基因的缺失，并且除了通过体内基因表达谱鉴定的那些基因之外，这些基因将被进一步单独表征。

DESCRIPTION (provided by applicant): In order to identify mycobacterial genes important for survival within the host, a rabbit model of pulmonary tuberculosis will be developed. Rabbits will be infected by transthoracic injection and intrabronchial instillation of mycobacteria. Bacillary multiplication within cavities will allow for isolation of sufficient quantities of RNA for analysis using a Mycobacterium tuberculosis oligonucleotide microarray. Comparison of gene expression patterns between mycobacteria growing in culture and those growing in vivo will provide information on genes important in mycobacterial survival within the host. A second approach will be used concurrently to determine virulence genes. A Mariner-derived transposon that has been shown to insert randomly into the M. tuberculosis genome will be used to generate libraries of mycobacterial mutants. The technique of transposon site hybridization will be used to compare pools of mutants before and after animal infection. Microarray analysis will be used to determine deletion mutants which are absent from the output pools. These mutants potentially have deletions in virulence genes, and these genes, in addition to those identified by in vivo gene expression profiling, will be further characterized individually.