Defining the metabolic phenotype of intracellular Mycobacterium tuberculosis

Tuberculosis (TB) is a disease which plagued ancient Egyptians and is still one of the most significant human diseases thousands of years later. A key to the success of Mycobacterium tuberculosis, the bacterium which causes TB is the ability to survive and grow in white blood cells, the very cells which are equipped to eliminate bacteria from the body. In order to do this M. tuberculosis must be able to acquire nutrients and energy from this isolated niche. Several studies have highlighted that targeting nutrient utilisation as a potentially productive route for drug development yet the nutrients consumed by intracellular M. tuberculosis are currently unknown. The aim of this project is to use labelled nutrient sources to directly measure the metabolism of M. tuberculosis growing in white blood cells. This research will allow us to identify the major energy (carbon) source consumed by M. tuberculosis in the host cell. We will also in parallel investigate the metabolism of M. tuberculosis in defined environmental conditions in the laboratory in order to establish the metabolic pathways used to break down different combinations of nutrients that have been identified as being consumed intracellularly. All of the data will be incorporated into mathematical models of TB in order to generate a reconstruction of the intracellular metabolism of this pathogen. This information will be vitally important for new drug and vaccine development, as well as advancing our knowledge of the pathogenesis of this globally important pathogen.

Despite decades of research many aspects of the biology of Mycobacterium tuberculosis remain unclear and this is reflected in the antiquated tools available to treat and prevent tuberculosis and consequently this disease remains a serious public health problem. Important discoveries linking the ability to metabolise specific substrates intracellularly within a macrophage to virulence have identified energy metabolism as a theoretically very powerful therapeutic target for tuberculosis. However the identity of the carbon sources and major metabolic pathways used by intracellular M. tuberculosis is poorly defined. The goal of this research is to identify the key carbon substrates and pathways ustilised by M. tuberculosis during intraphagosomal growth. We will perform comprehensive 13C-isotopologue profiling analysis of wild type and mutant strains of this pathogen in order to identify the different carbon sources which are contributing to the intracellular nutrition of M. tuberculosis. In parallel we will investigate the metabolic pathways required for consumption of these carbon sources under defined conditions in vitro using 13C-metabolic flux analysis. We will also perform transcriptomic analysis of intracellular M. tuberculosis in order to indirectly monitor metabolism. All of this data will then be used to constrain genome scale metabolic networks in order to define the metabolic phenotype of intracellular M. tuberculosis.

Impact summary
Who will benefit?
This research will investigate the intracellular metabolism of one of the most significant human pathogens, M. tuberculosis. Although this research is primarily a basic research project In addition to the academic beneficiaries that are discussed elsewhere this research will also lead to new insights of interest to wider group of beneficiaries:
1. Pharmaceutical industries and charities such as the Global Alliance for TB Drug Development which have an interest in the development of novel anti-tuberculosis drugs.
2. The UK trained workforce will benefit from this proposal through the training of a PDRA who will acquire new skills in systems biology, chemostat cultivation, 13C-metabolic flux analysis and molecular biology from the combined expertise of the applicants.
3. Undergraduate and postgraduate students taught by the applicants
4. As more than one third of the world are estimated to be infected with M. tuberculosis the general public in the UK and the rest of the world could ultimately benefit in terms of new drugs to treat TB

How will they benefit?
1. The market for anti-tuberculous drugs is estimated to be USD 612-670 million annually. In the UK there are several pharmaceutical companies that that have an interest in development of TB drugs, such GSK and AstraZeneka. The metabolic analysis of intracellular pathogens in their host cell is of immense importance in drug design and therefore in the long term this research could help develop the anti-TB drug market in the UK. Within the three years of this research project we will have generated extensive data about the carbon substrates and metabolic pathways required for the intracellular growth of this globally important pathogen. This data can then be exploited by those in industry to guide drug development particularly targeting transporters. By incorporating the data generated during this research into the available genome scale model of M. tuberculosis growing within its host macrophage we will also create a very valuable tool for those working in TB drug development. This new constrained model will be able to test large numbers of multiple target inactivations, which would be otherwise impossible to do by visual examination alone.
2. By developing skills in 13C isotopologue profiling/chemostat/systems biology the PDRA will mature into a highly trained researcher able to pursue a career in academic or industrial research.
3. The knowledge obtained through this project will contribute to fundamental theories and concepts underlying the metabolism of intracellular pathogens. We will impart this knowledge to undergraduate and post graduate students via teaching and research supervision.
4. Studying the metabolism of Mtb directly within its host macrophage has never been performed before and therefore the outcomes of this research will be of interest to the media and 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.