Regulation of Mycobacterium tuberculosis intracellular gene expression

A relatively large genome, predicted to encode more than 4200 bacterial factors functions to allow Mycobacterium tuberculosis to survive and grow within a variety of different environments encountered during the progressive course of human infection. Among the different environments potentially encountered during human infection is that within phagosomes of resident macrophages. Initial adaptation to such environment is of central relevance to the ability of bacilli to cause and or initiate infection. Bacterial survival inside macrophages and modulation of host cell function required coordinated and regulated changes in bacterial gene expression to ensure survival.;The complexity of the master regulator cpsA regulation network was also investigated at the down stream response regulator. Experiments identified the Rv0195 transcriptional regulator as a differentially expressed gene in response to an artificial elevation of cpsA RNA. I confirmed the increased RNA levels of Rv0195 in response to an artificial elevation of cpsA RNA level as well as during infection. The Rv0195 deleted strain was greatly reduced in intracellular proliferation relative to parent or complemented strains. Also the RNA levels of an orphan two component transcriptional response were increased in response to both in vitro osmotic and acidic shocks. Rv0195 putative regulated genes were identified using qPCR and chromatin immunoprecipitation where direct regulation of Rv1958c, Rv1129c, and Rv1130 by Rv0195 transcriptional regulator was confirmed. Additionally, the contribution of some Rv0195 regulated genes, which are regulated by cpsA through Rv0195; in M. tuberculosis intracellular survival was investigated independently.;Moreover, initial observation using subtractive hybridization indicated M. tuberculosis tm-RNA ssr as a differentially expressed gene in response to phagocytosis. I have found that the ssr RNA level is greatly increased in response to phagocytosis by human macrophages as well as in response to different in vitro stress conditions. Genetic DNA manipulations revealed that ssr-Rv3099c-smpB genes are not essential for growth in laboratory broth media. H37RvDelta ssr-Rv3099c-smpB strain exhibits a dramatic growth defect in infected macrophages relative to H37Rv and Rvs20 strains. Also the mutant strain was more sensitive to nitric oxide, which along with reactive nitrogen intermediates, represents an important means through which macrophages can partially control M. tuberculosis infection. The mutant strain was also more sensitive to other hostile environment including heat shock and SDS treatment relative to H37Rv. Findings presented here indicate an important role of ssr-smpB system in M. tuberculosis intracellular survival and resistance to oxidative stress.;Screening for bacterial RNAs produced in response to interaction with host resulted in a list of candidates that includes transcriptional regulator cpsA. Given the important role of transcriptional regulators in response to changing environment, I investigated the role of cpsA transcriptional regulator in adaptation to phagosomal environments and global gene expression. Steady-state levels of cpsA RNA were increased in response to phagocytosis by human macrophages as well as by growing in murine lung tissue. Similarly the transcript of cpsA was enhanced in response to osmotic and acidic shock suggesting a critical role of the gene product in adaptation to such environments. An H37Rv cpsA antisense expressing strain and an H37RvDeltacpsA strain both were impaired for intercellular growth and were less virulent in a mice model of infection relative to the parent H37Rv strain. Putative cpsA regulated RNAs, that belongs to various functional categories, were identified using both array hybridization and qPCR. Then, direct regulation of cpsA, sigE, icl, and Rv0195 by the M. tuberculosis cpsA transcriptional regulator was demonstrated by chromatin immuno-precipitation assay.