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.
