Towards an understanding of the regulation of the oxidative stress responses of Mycobacterium tuberculosis
Knowledge of the mechanisms by which Mycobacterium tuberculosis regulates its defence against oxidative stress will aid in the development of drugs and vaccines to combat the extensive morbidity and mortality caused by tuberculosis. In this thesis I have investigated the SenX3-RegX3 two component signal transduction system, one of 12 such systems in M. tuberculosis , which is essential for virulence in a mouse model and is similar to systems in other organisms that are involved in sensing oxygen. By complementing the attenuated growth of the senX3 and regX3 null mutants in mice I have shown that this phenotype is truly due to the targeted mutations in these strains. In vitro assays of oxygen stress have shown the senXS and regX3 null mutants to be less sensitive than the wild-type to superoxide and organic hydroperoxide stresses. Microarray analysis of mutants in the SenX3-RegX3 system after growth in microaerobic conditions suggested some functions for RegX3 but there was little overlap between strains. However, microarray comparison of a strain overproducing regX3 transcript demonstrated control of genes such as ahpC and cydB, indicating that this system may indeed play a role in the resistance of M. tuberculosis to oxidative stress. RegX3 was expressed and purified in E. coli and used to examine the interaction of this protein to different promoters, including that of senX3. Two further genes, which are probable one component regulators of oxidative stress in M. tuberculosis, Rv0465c and Rvl049, as judged by similarity to proteins in other organisms, were analysed through the isolation of null mutations. In vitro analysis suggests that Rvl049 is necessary for resistance to organic hydroperoxide stress, although neither the Rv0465c nor the Rvl049 null mutants were attenuated in macrophage or murine intravenous injection models of infection.