Rickman, Lisa; (2003) Global regulators of gene expression in Mycobacterium tuberculosis: Functional analysis using DNA microarrays. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

There is a pressing need to identify virulence genes of Mycobacterium tuberculosis to develop new vaccine strains and antibiotic therapies. I have used post-genomic approaches to generate M. tuberculosis global regulatory mutants to identify such virulence determinants. Using DNA microarrays, global gene transcription has been analysed in the mutants, and after exposure to oxidative stress. The ability to survive oxidative stress is thought to be important for intracellular pathogens. M. tuberculosis is challenged by this type of stress when it is phagocytosed by the macrophage during the initial stages of infection but the importance of this stress in infection has been called into question by the natural mutation of the M. tuberculosis oxyR gene. I have discovered a two-component signal transduction system that responds to peroxide stress. The sensor protein, SenX3, contains an atypical PAS domain, found in other oxygen and redox sensors. A null mutation of the accompanying regulator gene regX3 results in reduced transcription of ahpC and ahpD alkyl hydroperoxidase reductases in vitro, and attenuation of virulence in mice. The transcription of these and other genes is up-regulated in the wild type after peroxide stress. These results suggest that oxidative stress is indeed important in the macrophage response to M. tuberculosis infection, and implicates the SenX3 and RegX3 proteins as important players in the bacterium's defence to this stress. I have also identified the M. tuberculosis orthologue of the Crp protein that in E. coli, in complex with cAMP, regulates the expression of catabolite-sensitive operons. A null mutation of crp results in very poor growth in vitro and attenuation of virulence in mice. This gene may have functions other than catabolite repression in M. tuberculosis and significantly I have found that transcription of two genes coding for resuscitation promoting factors (Rpf) was altered in this mutant.

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