Identification of nitrogen source and metabolism of Mycobacterium tuberculosis during intracellular replication.

M. tuberculosis (Mtb), the etiological agent of TB, is presently the most devastating infectious agent of mortality worldwide, responsible for 8.8 million cases of TB each year resulting in 1.4 million deaths. Co-infection with the human immunodeficiency virus (HIV), along with the emergence of multi- and extensively- drug resistant (MDR and XDR) strains of TB, has reaffirmed Mtb as a primary public health threat throughout the world. The limited number of drugs available that have activity against Mtb, and the prolonged multi-drug regimen needed to eradicate the infection, are the fundamental problems of TB treatment. New drugs active against Mtb are urgently needed. Intracellular metabolism of Mtb is an attractive target for development of novel anti-tuberculosis drugs; however most studies have focussed on carbon metabolism. Nitrogen is also an essential nutrient of Mtb but few studies have attempted to elucidate fundamental questions such as the nature of the nitrogen source of the pathogen when it grows inside the host. Our previous studies have identified amino acids as putative sources of nitrogen for Mtb when growing inside host cells. The hypothesis to be investigated in this project is that Mtb obtains nitrogen from a diverse range of intracellular nutrients including amino acids.

To address these fundamental questions we will first perform genetic analysis of Mtb to identify genes involved in uptake and assimilation of a range of possible nitrogen sources, such as amino acids. We will then inactivate these genes in Mtb and measure the ability of the mutated strains (lacking the ability, for example, to uptake a particular amino acid) to replicate in human cells. By examining the intracellular replication efficiency of a range of mutants that have specific defects in uptake of specific nutrients we will identify which nitrogen nutrients are important for intracellular replication. The next question is how the pathogen assimilates nitrogen. To tackle this question we in parallel we will directly analyse the nitrogen metabolic pathways used to assimilate nitrogen using cutting-edge systems-based metabolomics techniques that we have spearheaded at the University of Surrey. This will involve development of entirely novel systems to measure the nitrogen metabolism of the pathogen simultaneously with its carbon metabolism. Finally, we will develop a computer model of nitrogen and carbon metabolism in Mtb. The model will be used both to integrate data generated in the project but also to generate predictions that can test the hypothesis underpinning the project. In this way we will elucidate the role of nitrogen metabolism in the biology and virulence of this important pathogen. The study is also likely to generate novel targets for development of antituberculous drugs.

M. tuberculosis (Mtb), the etiological agent of TB, is presently the most devastating infectious agent of mortality worldwide, responsible for 1.4 million deaths. New drugs active against Mtb are urgently needed. Intracellular metabolism of Mtb is an attractive drug target but most studies have focussed on carbon metabolism. Nitrogen is also an essential nutrient of M. tuberculosis (Mtb) but the nature of its nitrogen source and metabolic pathways involved during intracellular replication are unknown.

Our previous studies have identified amino acids as putative sources of intracellular nitrogen and suggested the hypothesis that Mtb obtains nitrogen from a diverse range of intracellular nutrients including amino acids. To test this hypothesis we will first perform transposon mutagenesis (Tn-seq) to identify Mtb genes involved in uptake and assimilation of a range of possible nitrogen sources, including amino acids. We will generate KO mutants of these genes and measure their ability to replicate in the THP-1 macrophage-like cell line, thereby identifying nitrogen nutrients that are obtained by Mtb from its host cell. To elucidate mechanisms and pathways involved in nitrogen assimilation we will directly analyse both the carbon and nitrogen metabolism of Mtb using 13C and 15N-labelled substrates and cutting-edge metabolomics techniques that we have spearheaded at the University of Surrey. This will involve development of novel mass spectrometry techniques to distinguish between 13C and 15N-labelled metabolites. Finally, we will generate a computer model of nitrogen and carbon metabolism in Mtb. The model will be used both to integrate data generated in the project but also to generate predictions that can test the hypothesis underpinning the project. In this way we will elucidate the role of nitrogen metabolism in the biology and virulence of this important pathogen. The study is also likely to generate novel targets for development of antituberculous drugs.

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 PDRAs 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. This was recently exemplified by an extensive chemical/genetic screen performed in vitro which identified compounds that were active in the in vitro environment with glycerol as carbon source but had no activity in vivo. The study concluded that 'the importance of understanding central bacterial metabolism in vivo ... for the rational discovery of new antibiotics'(Pethe et al., 2010). Within the three years of this research project we will have generated extensive data about the nitrogen 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 in activations, which would be otherwise impossible to do by visual examination alone.
2. Pathways for nitrogen uptake and assimilation have only been elucidated for a small number of organisms so this study will widen our knowledge of this vital metabolic process. The importance of central metabolism including nitrogen pathways for novel drug development has recently been exemplified by the launch of the first new TB drug in 50 years, which is an ATPase inhibitor (Avorn, 2013).
3. By developing skills in 13C/15N isotopologue profiling/chemostat/systems biology the PDRAs will mature into highly trained researchers able to pursue a career in academic or industrial research.
4. 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.
5. Studying the metabolism of Mtb directly within its host macrophage has seldom 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.

Avorn,J. (2013). JAMA 309, 1349-1350.
Pethe,K., et al. (2010). Nat. Commun. 1, 57.