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

Tuberculosis (TB) is often considered as a disease of the past, but it remains an endemic disease in many developing countries. One third of the world's population some 2 billion people are infected with the causative agent of TB, the bacterium Mycobacterium tuberculosis. Most infected individuals are asymptomatic and the bacterium resides within their bodies in a dormant state. However, at some point during their lifetimes about 10% of these infected individuals will develop active TB in which conditions that compromise their immune systems allow the bacteria to 'wake up' and begin to grow and multiply. Consequently about 1.8 million deaths are caused by TB every year, making it the greatest cause of death due to a single infectious agent. This is despite the availability of an effective drug treatment. However, the treatment regimen is prolonged, taking at least 6 months, and is threatened by the emergence of multi-drug resistant strains. Moreover, the BCG vaccine, although very safe, is of variable efficacy. Thus, TB is a major worldwide healthcare problem and the quest for new and better drugs and vaccines is a pressing research goal. To open up new opportunities for interventions to control TB we need a more detailed understanding of the fundamental biology of M. tuberculosis. It is clear that its ability to exist for decades in the lungs of infected individuals in a dormant, non-replicating state only to emerge later and cause active TB (reactivation TB) is a central feature of the disease. If we had a better understanding of how the bacterium enters and exits from the dormant state this could offer the prospect of identifying new drug targets and vaccine components. Our previous work has established that two gene regulators play key roles in controlling this central feature of TB pathogenesis. A protein known as CRPMT is required for M. tuberculosis virulence and it regulates a suite of genes in response to changes in the concentration of a small signalling molecule cAMP, which the bacterium produces to promote growth by compromising host signalling pathways. Amongst the genes regulated by CRPMT is whiB1. WhiB1 is a gene regulator that responds to nitric oxide. Nitric oxide is produced by host lung macrophages to kill M. tuberculosis, but low levels of nitric oxide push the bacterium into the dormant state. CRPMT and WhiB1 together regulate the function of a bacterial protein secretion system that releases specific bacterial proteins into host cells to promote bacterial growth. Hence, we believe that CRPMT and WhiB1 are key players in determining whether M. tuberculosis will grow and cause active TB or whether it will enter the dormant non-replicating state (latent TB). Our goal is to obtain a detailed mechanistic understanding of how CRPMT (and a related protein Cmr) and WhiB1 work together to optimize gene expression to control central features of M. tuberculosis virulence. In doing so we will provide the scientific underpinning to aid the quest to identify and develop new drug targets and vaccine components.

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.