Integrating cAMP- and nitric oxide- signalling in Mycobacterium tuberculosis: novel regulatory networks that challenge established paradigms

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Mycobacterium tuberculosis causes ~2 million deaths per annum. There are ~2 billion carriers in which the bacterium is present in a non-replicating dormant state. Of these individuals ~10% will suffer from active TB infections when bacterial growth is restored. We have identified the cAMP- and NO- responsive regulators, CRPMT and WhiB1, as key components in TB pathogenesis. We have shown that CRPMT controls the expression of WhiB1, which is an essential transcription factor with an iron-sulphur cluster that is sensitive to NO. Furthermore, CRPMT and WhiB1 co-ordinate the regulation of the espA-Rv3612c oepron that encodes proteins that are required for function of the essential virulence factor ESX-1, which exports the major effector protein ESAT-6. Thus, we have established roles for cAMP-CRPMT and NO-WhiB1 in controlling processes relevant to M. tuberculosis dormancy (signalled by NO) and reactivation/growth (signalled by cAMP). We now propose to obtain a deeper understanding of these processes and the mechanisms by which they are controlled by applying a multidisciplinary combination of in vivo and in vitro analyses to establish:
1. the nature of the relationship between CRPMT and cAMP;
2. the function of Cmr (a second CRP-family regulator in M. tuberculosis) that is proposed to be linked to but does not respond to cAMP signalling
3. the structure-function relationships of the essential NO-sensing transcription factor WhiB1 and how the WhiB1 iron-sulphur cluster reacts with NO
4. the breadth of the WhiB1 regulon
5. how WhiB1 regulates gene expression
6. how cAMP-CRP and NO-WhiB1 work with EspR to regulate the virulence critical espA operon
7. how Cmr works with WhiB1 to regulate the expression of the essential chaperonin groEL2
Thus, our overall aim is to elucidate novel, fundamental aspects of the biology of a bacterial pathogen and thereby provide the scientific underpinning for the development of new therapies.

This research will establish a deep understanding of the roles of cyclic AMP- and nitric oxide-responsive processes in Mycobacterium tuberculosis. The new insights provided by this project will provide opportunities for developing new control strategies. The foreseeable impacts on the UK and internationally include:
1. high quality training of early-career bio-scientists;
2. engaging with the public to highlight the importance of fundamental underpinning science in advancing medicine
3. publishing quality science in high impact peer-reviewed scientific journals;
4. establishing new paradigms in signal perception and gene regulation in bacteria;
4. establishing generic experimental tools and bacterial strains essential for deepening our understanding of pathogenesis;
5. "induced" impacts, in which the employment of an individual or stimulating an area of research subsequently results in either economic and social impact;
6. providing underpinning knowledge and tools that may be applied to other systems, such as other bacterial pathogens that infect humans and animals (e.g. M. bovis) that employ cAMP and nitric oxide based signalling systems;
7. encouraging multidisciplinary and collaborative research by building upon our successful previous collaborations;
8. identifying targets in the form of regulatory circuits that could be used to control bacterial infections.