This is a revised application in which the PI proposes to analyze an alternate sigma factor of Mycobacterium tuberculosis which may be involved in the intracellular survival of the tubercle bacilli. The PI points out that the ability of M. tuberculosis to adapt to a variety of environmental conditions is an important feature of its pathogenicity. Many bacteria use alternate sigma factors for RNA polymerase to upregulate response genes specific for a needed adaptation. Using degenerate PCR, the PI has identified an alternate sigma factor from M. tuberculosis which he has called SigF. M. tuberculosis SigF is homologous to the SigF and SigB proteins of Bacillus subtilis, which are involved in regulation of sporulation and general stress response gene expression, respectively. Not only does M. tuberculosis SigF have homology with B. subtilis SigF and SigB, but it is preceded by an antisigma and a possible anti-antisigma homologue, just as are the B. subtilis sigma factors. In vitro (i.e., broth-grown cultures), the expression of the M. tuberculosis SigF is upregulated by stress conditions and entry into stationary phase. In vivo, its expression increases 10-fold upon entry into murine macrophages. Hence, the PI hypothesizes that the M. tuberculosis SigF may govern an intracellular survival regulon. In this application, the PI proposes to determine (i) the in vitro and in vivo phenotypes of an M. tuberculosis sigF mutant, (ii) how SigF is regulated (i.e., is an antisigma partner switching mechanism involved?), and (iii) the identities of SigF-dependent genes. The PI anticipates that understanding how M. tuberculosis adapts for intracellular survival may lead to novel drug and vaccine targets for tuberculosis.
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