The identification of genes controlling mycobacterial persistence and the signals that induce them is of paramount importance in developing new therapeutic agents and improved diagnostics for latent tuberculosis. The genetic mechanisms by which M tuberculosis survives for many years in a stationary state are poorly understood, but are likely to resemble the adaptive mechanisms used by other bacteria. Nutrient starvation and chemical stress are potential inducers of latency. Many bacteria respond to starvation and stress by expressing alternate sigma factors which redirect the cellular RNA polymerase to genes preceded by special promoters and thereby induce dozens of genes essential for survival in the new environment. Sigmas which mediate processes resembling latency, such as the gram-positive sporulation sigmas and the recently identified E. coli stationary phase sigma, are members of the omega70-family for which regions of interspecies conservation have been well-defined. I have shown that degenerate oligonucleotides against four of these conserved regions recognize multiple different fragments of M. tuberculosis chromosomal DNA--suggesting the presence of multiple omega70-hemologues. Moreover, these oligonucleotides amplify fragments of the expected size when used as PCR primers. A computer search of the one-quarter of the M. leprae genome that has already been sequenced reveals at least three potential transcription regulators (two of which are sigma factor homologues). One of these potential regulators shares significant homology with differentiation sigmas from Streptomyces species (close phylogenetic relatives of the mycobacteria) and sporulation sigmas from Bacillus species. This gene might well be a mycobacterial """"""""latency"""""""" sigma. I propose to clone as many mycobacterial sigma-like genes as can be found by a combination of hybridization, PCP, and homology searches of the Mycobacterial Genome Project databank. Northern analysis and gene fusion studies following nutrient depletion in culture will be used to characterize the functions of these sigmas. Complementation of known sigma mutants from other organisms will also be used to better define their functions. Candidate genes which appear to play a role in stationary phase survival will be used as probes for in situ hybridization and RT-PCR of the organisms found in both solid caseous (stationary phase) and liquefied (active growth phase) pulmonary lesions isolated from rabbits infected by the aerosol route. Knockout mutations or alterations of genes controlling latency in the tubercle bacilli might lead to potential vaccine strains which would proliferate at the outset, induce an immune response, but fail to establish persistent infection.
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