The role of the anaplerotic node in redox homeostasis and pathogenesis of Mycobacterium tuberculosis and its exploitation as a therapeutic target

Summary. The role of the anaplerotic node in redox homeostasis and pathogenesis of Mycobacterium tuberculosis and its exploitation as a therapeutic target
We desperately need new treatments to control the tuberculosis pandemic, which is fuelled by the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb, the causative agent of tuberculosis) as well as a catastrophic synergy with HIV. Mtb is an unusual bacterial pathogen, which has the remarkable ability to cause both acute life threatening disease and also symptomless latent tuberculosis infections that can persist for the lifetime of the human host. It is estimated that 23% of the world has latent tuberculosis, and in 2017 alone, there were 10.0 million cases of tuberculosis and 1.5 million tuberculosis deaths, making TB once again the leading infectious disease globally. A key to the success of tuberculosis as a human disease is ascribed to the extraordinary metabolic flexibility of Mtb. This promotes survival in the variable and harsh environments within the human host, which include exposure to REDuctive and OXidative (redox) stresses. Mtb is able to monitor these stresses in the human host and co-ordinate an appropriate response in order to survive. Maintenance of redox homeostasis is critical to the ability of Mtb to cause disease however many questions in this area of research remain unanswered. A fundamental question is what is the role of metabolism in maintaining redox balance?
We have preliminary evidence that a central metabolic hub (the ANA node) consisting of four enzymes is involved in maintaining this balance and have identified a drug which targets this node and kills Mtb. Our previous work showed that this hub is required for the survival of Mtb within its human host cell and we now want to test our hypothesis that this node is essential for the maintenance of redox balance, the ability to cause disease and therefore represents a drug target.
We propose to characterise the function of the node in redox control using our mutant strains of Mtb which lack a complete ANA node. Firstly we will investigate the effect of having an incomplete ANA node on the survival, redox balance and immediate metabolic responses of Mtb during exposure to reductive or oxidative stresses. For this work we will use a bespoke system which reports on the redox status of Mtb. The next question is how does the ANA node reconfigure metabolism during redox stress? To tackle this question we will directly analyse the metabolic pathways involved in mediating redox stress using cutting-edge systems-based metabolomics techniques that we have spearheaded at the University of Surrey. By utilising a human cell and mouse model of tuberculosis infection we will probe the essentiality of the ANA node to maintaining redox homeostasis and virulence in the host environment. Finally we will chemically modify a drug which we have shown in preliminary work targets the ANA node and kills Mtb in order to improve the antimicrobial activity. By exploring the mode of action of these tool compounds in the context of redox homeostasis we will validate the ANA node as a drug target. In this way we will elucidate the role of the ANA node in redox control and virulence of this important pathogen and synthesise compounds which can potentially be developed into future treatments for tuberculosis.

The urgent need for new drugs to treat TB, particularly multidrug-resistant strains of the causative agent Mycobacterium tuberculosis (Mtb) is well established. The unique ability of Mtb to survive the redox stress manifested by the human host is central to its success as a pathogen. We already know that Mtb has enzymes, redox buffers and regulators dedicated to maintaining redox homeostasis. A fundamental outstanding question is how does metabolism rewire in order to maintain redox homeostasis? This is important as mechanisms maintaining redox homeostasis represent a major target for drug interventions. Previously we demonstrated that the anaplerotic (ANA) node was essential for the intracellular survival of Mtb and in preliminary work we have shown that this node is also required for the maintenance of redox balance during oxidative stress. This led to our hypothesis that the ANA node has a pivotal role in maintaining the redox homeostasis of Mtb which critically influences pathogenicity and represents a potential drug target. To test our hypothesis we will use bespoke redox reporter strains, lipidomics and 13C isotopomer analysis to establish how each ANA enzyme contributes to redox homeostasis. To elucidate how the ANA node rewires metabolism during redox stress we will directly analyse the carbon metabolism of Mtb using cutting-edge metabolomics techniques that we have spearheaded at the University of Surrey. This will define the metabolic signature of Mtb under redox stress for the first time identifying enzymes and pathways involved in redox homeostasis. Using macrophage and murine models of TB we will probe the role of the ANA node in virulence and redox homeostasis in the host environment. Finally, we will explore whether the ANA node represents a potential target for drug development. This project will advance our understanding of redox biology which is fundamental to all of life and provide new possibilities for the development of novel anti-TB therapies.

IMPACT SUMMARY
Who will benefit?
Although primarily a basic research project focused on understanding how a metabolic hub contributes to redox homeostasis this work will be of interest not just to academics working on TB, infectious diseases and redox metabolism (discussed elsewhere) but to a wider group of beneficiaries.
1. TB charities and also drug developers such as AstraZeneca and GlaxoSmithKline which have a commitment to developing drugs against TB will be interested in this research. This position has already been endorsed by the TB Alliance (see letter of support) who have been intent in developing drugs which target critical metabolic pathways in Mtb for several years.
2. Pharmaceuticals developing antiparasitics may benefit from this work as PPDK has been proposed as a therapeutic target for drug design against protozoal diseases (e.g. Entamoeba histolytica).
4. As inhibition of pyruvate phosphate dikinase (PPDK) significantly hinders C4 plant growth agricultural companies working on developing herbicides will be interested in this work.
5. This research project will contribute to the UK's workforce, facilitating the career development of scientists, including two PDRAs, who will benefit by acquiring new skills from the combined expertise of the applicants.
6. Undergraduate and postgraduate students taught by the applicants.
7. As infectious disease and antibiotics have recently captured the interest of the public the outcomes of this research will be of interest to the media.
8. As TB represents a major global threat to human health ultimately, the beneficiaries will be public health and social and economic benefits to the general public in the UK and the rest of the world.
How will they benefit?
1. After three years of this research project the data generated about the interaction between metabolism and redox and will be beneficial in the development of potent drug combinations for the treatment of tuberculosis infections.
2. At a historic United Nations General Assembly on TB in 2018 the UK committed £7.5 million to the TB Alliance to develop shorter and less toxic TB treatments that are affordable to the poorest countries in the world. Repurposing drugs such as the nonsteroidal anti-inflammatory and analgesic drug studied in this research project offers a cheap low-risk strategy to achieve this goal. The analogues generated in this study will be beneficial to the TB Alliance who are committed to taking promising lead compounds from this project forward as fast as possible (see supporting letter).
3. Any PPDK inhibitors identified in this study this work can be exploited by pharmaceutical companies and agriculture.
4. How metabolism rewires during redox stress has never been studied in Mtb so this research will expand our knowledge on this vital biological process. These studies can be exploited by drug companies interested in developing metabolic adjuvants to enhance the killing efficacy of existing antibiotics.
5.The knowledge obtained through this project will contribute to fundamental theories and concepts underlying redox homeostasis and the metabolism of intracellular pathogens. We will impart this knowledge to undergraduate and postgraduate students via teaching and also research projects aligned with this research.
6.Improved skills and technical understanding for PDRAs working in this multidisciplinary project. The PDRAs will mature into highly trained researchers able to pursue a career in academic or industrial research.
7.As this research will contribute to our knowledge about TB the outcomes of this research will be of interest to the general public. In the longer term this research could lead to new drugs for treating TB and therefore impacting in the area of public health and societal issues.
8.The project will catalyse new activities between the project partners, extended academic sector and/or SMEs. At least 1 follow-on funding application will be submitted.