The proposed project will examine the adaptive responses m. tuberculosis undergoes after phagocytosis by a macrophage. Genes associated with infection are likely to be regulated and are likely to be under the control of specific promoters responsive to environmental cues. The main objective of the project will be to assess activation and inactivation of gene transcription by analysis of promoter activity; a reporter vector system will be developed for this purpose. Genomic M. tuberculosis DNA will be fragmented and ligated into the reporter vector, which will then be used to transform M. tuberculosis. Transformants will be subjected to phagocytosis by a macrophage cell line; promoters with altered activity will be identified by differential expression of the reporter genes. Sequences that flank these promoters will be assessed for protein coding sequences and regulatory regions. Genes identified from these sequences will be evaluated for their relevance to virulence, which will be tested ultimately by site-specific mutagenesis of wild-type M. tuberculosis. The proposed work will provide fundamental information regarding the structure and regulation of mycobacterial promoters, genes, and operans. In addition, understanding M. tuberculosis virulence factors could identify new targets for vaccine or drug development.
