It is estimated that approximately 2 billion people worldwide are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Immunocompetent individuals infected with M. tuberculosis have a 10% lifetime risk of developing postprimary, or reactivation, TB. Subclinically infected individuals compose a massive reservoir for the development of future disease. In high-incidence countries in sub-Saharan Africa, the TB problem is exacerbated by a high prevalence of HIV co-infection, which greatly increases the risk of progression to disease. Further compounding the already daunting challenge of controlling drug-susceptible TB with the current armamentarium of tools is the emergence and global spread of multi- and extensively drug-resistant strains of M. tuberculosis that are resistant to first- and second-line drugs. We urgently need new treatment-shortening therapies for TB that have novel mechanisms of action and can be coadministered with antiretrovirals. The scientific challenges associated with meeting this need are considerable and, at the very least, demand an understanding of the bacillary factors that mitigate drug efficacy and identification of vulnerabilities that can be exploited for chemotherapeutic intervention. The metabolism and physiology of M. tuberculosis are inextricably linked to pathogenesis; therefore, defining this organism's metabolic capacity and flexibility, and the associated mechanisms of its physiologic adaptation, has formed a central theme of mycobacterial research. My laboratory investigates specific aspects of the physiology and metabolism of M. tuberculosis of relevance to drug discovery and drug resistance through an integrated suite of projects on cofactor biosynthesis, transport and function, DNA metabolism, and nucleotide metabolism.
Our work on cofactor metabolism focuses on the biosynthesis of vitamin B12, coenzyme A, and molybdenum cofactor and on the function of certain enzymes that depend on these cofactors for activity. M. tuberculosis has three vitamin B12–dependent enzymes, which act in nucleotide metabolism, amino acid biosynthesis, and central carbon metabolism. Two of these enzymes catalyze reactions that can be catalyzed by alternate B12-independent enzymes present in M. tuberculosis. The third does not have a B12-independent counterpart and catalyzes a step in the methylmalonyl pathway that has been implicated in the detoxification of propionate, a key metabolite produced by the catabolism of cholesterol, methyl-branched amino acids and fatty acids. Although M. tuberculosis contains an apparently complete complement of vitamin B12 biosynthesis genes, this vitamin is not produced under standard culture conditions. However, we have shown that M. tuberculosis is able to take up and assimilate vitamin B12 from the extracellular milieu even though it lacks the canonical high-affinity B12 transport system found in other bacteria. We undertook forward genetic screens based on B12-dependent growth phenotypes to identify genes involved in the uptake and assimilation of vitamin B12. These screens have yielded candidates that are the subject of ongoing investigation.
In a related study, we are investigating the coenzyme A biosynthetic pathway, which provides a rich source of potential targets for drug discovery. The aim of this work is to compare the vulnerabilities of the enzymes of the biosynthetic pathway. In this and related studies, we are generating sensitized strains of M. tuberculosis by conditional gene silencing for use in target-based whole-cell screening, a method that combines the power of phenotypic and target-led approaches to hit identification and lead generation and that we have shown to hold considerable promise for TB drug discovery.
Our interests in DNA metabolism are geared toward understanding mechanisms of DNA repair, replication, and mutagenesis in M. tuberculosis as they apply to survival and genetic adaptation to the genotoxic conditions encountered in the human host and upon exposure to antitubercular drug action. We identified a novel system for damage tolerance in M. tuberculosis and other mycobacteria that plays a key role in mutation induction in response to DNA damage. This "mutasome" comprises a C-family DNA polymerase, DnaE2; a Pol IV–like pseudopolymerase, ImuB; and a RecA-like conserved hypothetical protein, ImuA. The characteristics of this system suggest that it is the nonorthologous replacement of the Pol V–based system found in other bacteria. We showed that all three proteins are essential for functionality of the novel mutasome, with DnaE2 providing the catalytic polymerase activity for translesion synthesis across sites of DNA damage. Preliminary findings have implicated ImuB' as an adaptor protein that plays an anchoring role in a protein interaction network that involves the mutasome and components of the replication machinery, including the β clamp. However, key questions regarding the mechanism of lesion bypass and the temporal and spatial relationship between the mutasomal components and other proteins remain. For example, the molecular basis underlying the functional differentiation between the nonessential translesion polymerase DnaE2 and the essential replicative polymerase homolog, DnaE1 is unclear. In addition, the specific role of ImuA' in lesion bypass, and the timing and functional significance of interactions of ImuB with ImuA', DnaE2, and the β clamp are unclear and are the subject of ongoing investigation.
The dominant role of the DnaE2-based system in DNA damage tolerance in mycobacteria has also raised fundamental questions about the cellular function(s) served by Y-family DNA polymerases in these and other bacteria that also have a DnaE2 polymerase. Our work has begun to reveal redundancy and differentiation of function of the various specialized DNA polymerases in mycobacteria and has also provided new insights into the functional and/or regulatory interdependence among the specialized DNA polymerases and other proteins, which are being further investigated. In related work, we are investigating the mechanisms of nucleotide metabolism in mycobacteria, with emphasis on the de novo biosynthesis and salvage of pyrimidines. This study also underpins a component of our drug discovery program that aims to identify and validate new drug targets in DNA metabolism by exposing and exploiting vulnerabilities in biosynthetic and/or salvage pathways that arise from the absence of key enzymes.
Our work is also supported by grants from the National Research Foundation of South Africa, the South African Medical Research Council, the Technology Innovation Agency (South Africa), the Seventh Framework program for research of the European Commission, the Bill & Melinda Gates Foundation (via subcontract from the Foundation for the National Institutes of Health), and the University of Cape Town.
As of September 26, 2012