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

Lead Research Organisation: University of Leicester
Department Name: Infection Immunity and Inflammation

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50

publication icon
Commandeur S (2020) Zebrafish Embryo Model for Assessment of Drug Efficacy on Mycobacterial Persisters. in Antimicrobial agents and chemotherapy

publication icon
O'Connor BD (2015) Can resuscitation-promoting factors be used to improve culture rates of extra-pulmonary tuberculosis? in The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease

publication icon
Rosser A (2018) Differentially Culturable Tubercule Bacilli Are Generated during Nonpulmonary Tuberculosis Infection. in American journal of respiratory and critical care medicine

 
Description We have developed a model for generation of dormant Mycobacterium tuberculosis by treatment of mycobacteria with nitric oxide donors. The model has being currently refined for validation of several mycobacterial strains and investigation of molecular mechanisms underlying non-replicating persistence in mycobacteria.

We have established the role of Rv1675c in mycobacterial response to nitrosative stress and dormancy.
Exploitation Route Our model can be used for validation of drugs targeting non-replicating mycobacteria.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description Application of Resuscitation-promoting factor for improvement of extra pulmonary tuberculosis diagnosis.
Amount £4,252 (GBP)
Organisation The Dowager Countess Eleanor Peel Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2015 
End 10/2016
 
Description MIBTP iCASE
Amount £21,400 (GBP)
Organisation Public Health England 
Sector Public
Country United Kingdom
Start 09/2018 
End 09/2022
 
Description Molecular basis of PknB essentiality in mycobacteria
Amount £396,391 (GBP)
Funding ID BB/P001513/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2016 
End 10/2019
 
Description PhD studentship
Amount £34,814 (GBP)
Funding ID DSTL/AGR/00287/01 
Organisation Defence Science & Technology Laboratory (DSTL) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2016
 
Title Methods for cryopreservation of Rpf-dependent mycobacteria 
Description We developed a procedure which enabling long-term storage of Rpf-dependent mycobacteria isolated from patients and generated in vitro. 
Type Of Material Biological samples 
Year Produced 2015 
Provided To Others? Yes  
Impact Characterisation of mycobacteria from clinical samples requires a robust procedure for preservation of phenotypic features. Our protocol will allow scientists collection and preservation of mycobacterial samples at field sites and further characterisation and use these samples for research. Our procedure will enables multiple tests and check of reproducibility of results. 
 
Title Models for generation of Rpf-dependent mycobacteria 
Description Treatment of mycobacteria with nitric oxide donors or cell wall biosynthesis inhibitors. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact The model can be used for validation of drug candidates 
 
Description Collaboration with DSTL 2013 
Organisation Defence Science & Technology Laboratory (DSTL)
Country United Kingdom 
Sector Public 
PI Contribution Studentship jointly funded by the University of Leicester and DSTL to investigate resuscitation of dormant Burkholderia pseudomallei. Leicester offered expertise in dormancy, genetic manipulations and protein work.
Collaborator Contribution Expertise in handling Burkholdria pseudomallei and infection models.
Impact PhD thesis by Christopher Jenkins.
Start Year 2013
 
Description Collaboration with Kyrgyzstan 
Organisation National Center of Phthisiology
Country Kyrgyzstan 
Sector Public 
PI Contribution We provide expertise in analysis of clinical samples, growth, resuscitation and antimicrobial tolerance assays.
Collaborator Contribution Kyrgyzstan provide clinical samples and expertise in management of multi-drug resistant TB.
Impact Developed protocols for preservation of Mtb populations in clinical samples for further analysis. Initiated discussions about measures for controlling MDTB and search of biomarkers of Rpf-dependent Mtb.
Start Year 2013
 
Description Dr Makarov provision of NO donors 
Organisation Russian Academy of Sciences
Country Russian Federation 
Sector Public 
PI Contribution My group provided expertise in mycobacterial dormancy and resuscitation and established a novel experimental model for generation of Resuscitation-promoting factor (Rpf) dependent mycobacteria.
Collaborator Contribution Dr Makarov synthesised novel nitric oxide donors (inducers of Rpf-dependency) and Rpf inhibitors.
Impact Establishment of experimental model for validation of drugs targeting persisting and Rpf-depdent Mycobacterium tuberculosis.
Start Year 2013
 
Description UCL Dr Kristine Arnvig 
Organisation University College London
Department Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided strains and plasmids for genetic manipulations in mycobacteria. We provided expertise in mycobacterial physiology and Resuscitation-promoting factors.
Collaborator Contribution Our collaborator provided us expertise in non-coding RNA and gene transcription.
Impact Grant application to BBSRC.
Start Year 2013
 
Description Leicester TB Research Group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Regular meetings of academic scientists and clinicians to discuss TB challenges and research impact on TB control in Leicester and worldwide.
Year(s) Of Engagement Activity 2016,2017,2018,2019,2020
 
Description Open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Open day activities and discussions with potential candidates about research and study in Leicester.
Year(s) Of Engagement Activity 2016,2017,2018,2019
 
Description World tuberculosis day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact 24th March is the World Tuberculosis day. We organised a special event to mark this day and increase public awareness on tuberculosis.
Year(s) Of Engagement Activity 2019
URL http://le.ac.uk/news/2019/march/22-world-tb-day