- Objectives To research the genome structure and differences in response to environmental stimuli of selected strains of M tuberculosis which differ in their clinical characteristics (standard laboratory strain H37Rv; a strain which has recently caused a major outbreak of tuberculosis in the UK and a strain from South India which has been shown to have a large number of genomic deletions). These strains will be compared in different terms: - Genomic comparisons: DNA will be extracted and compared firstly by DNA micro array analysis. This will reveal the identity and extent of any deletions in the two clinical isolates, compared to the reference strain, H37Rv. In order to investigate whether there are genes present in the clinical strains which are absent in the reference strain, we will use subtractive DNA hybridization and PCR. Subtracted DNAs will be directly inserted into cloning vectors, sequenced, and sequences novel to the clinical strains identified.
- Infectivity and interactions with host cells: The strains will be compared for their ability to produce progressive infections in mice using a standard intravenous infection model and for their ability to infect and grow within both murine and human macrophages.
- Comparison of gene expression: The transcriptional response of these strains following exposure to a range of environmental stresses will be investigated using the DNA M tuberculosis micro array. The gene expression analysis is performed by doing a competitive hybridization between cDNA prepared from the bacteria exposed To the stress and that prepared from the bacteria grown under conventional conditions.