The long-term goal of this research is to improve our understanding of the pathogenesis of tuberculosis. This research will address this long-term goal by examining the role of sigma factors in the regulation of mycobacterial gene expression. A clearer picture of the molecular biology of mycobacterial gene expression may provide insight into mechanisms that these organisms use to survive the hostile intracellular environment of the host cell. Sigma factors whose activity is essential under conditions relevant to in vivo replication and survival may also provide a means to identify regulated genes that are essential for mycobacterial virulence. A better understanding of mycobacterial gene expression and disease pathogenesis at the molecular level will provide opportunities to develop new approaches to treatment and prevention of mycobacterial infection and disease. The long-term goals of this research will be approached through three specific aims. First, sigma factor genes of M. tuberculosis will be cloned and sequenced. Dominant negative mutations will then be created in these cloned sigma factor genes by site-directed or PCR mutagenesis, focusing on the -10 recognition region of these sigma factors. Homologues in M. fortuitum of these M. tuberculosis sigma factor genes will be cloned, and M. fortuitum strains with null mutations in these genes will be created by allelic exchange. These M. tuberculosis and M. fortuitum mutants will be used to examine the role of specific sigma factors on patterns of mycobacterial gene expression by two-dimensional gel electrophoresis. Second, these mutant sigma factor genes in M. tuberculosis and M. fortuitum will be used to examine the role of sigma factor function on replication and survival a) in culture under various conditions, b) after uptake by macrophages in vitro, and c) in mice in vivo. Third, initial characterization of the promoters of genes whose expression is regulated by specific sigma factors will be undertaken by identifying a limited number of these regulated genes. Consensus promoter elements in these genes will be determined through searching of the M. tuberculosis sequence database and through cloning and sequencing these genes.
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