Identifying vulnerable steps in the coenzyme A biosynthesis pathway of M. tuberculosis

Abstract

Enzymes in the pantothenate and coenzyme A (CoA) biosynthesis pathways have attracted considerable interest as potential targets for the development of drugs against a number of human pathogens, including M. tuberculosis. While potent inhibitors have been developed against M. tuberculosis pantothenate synthase and pantothenate kinase, these have failed to translate into compounds with significant whole‐cell activity. These findings illustrate the challenges inherent in target‐led approaches to tuberculosis drug discovery, which is confounded by issues of permeability, metabolism and efflux, by a lack of understanding of target vulnerability.To identify vulnerable steps in the pantothenate and CoA biosynthesis pathway in M. tuberculosis, a set of anhydrotetracycline (ATc)‐regulated mutants of M. tuberculosis was constructed using a transcriptional silencing system. The mutants were used to assess the impact of depletion of specific targets in the pantothenate and CoA pathway on growth and survival of the organism. Mutants with inducer‐dependent growth phenotypes were obtained for panB, panC, coaBC, coaD and coaE whereas those in panB, panE and panK were refractory to ATc‐regulated growth. While transcriptional silencing of panB, panC or coaE was bacteriostatic, coaBC silencing was apparently bactericidal in vitro, as determined by CFU enumeration. CoaBC was likewise shown to be required for growth and persistence of M. tuberculosis in vivo based on enumeration of bacillary loads in the lungs of mice in which coaBC was silenced immediately post‐infection, during acute infection, or during chronic infection. However, by taking advantage of the fact that M. tuberculosis is capable of CoaBC bypass via CoA salvage, coaBC silencing was shown to result in a transient, ‘non‐growing but metabolically active’ state from which non‐culturable bacilli can be rescued by uptake and assimilation of pantethine. These findings highlight the limitations of colony‐forming ability as a proxy for viability in M. tuberculosis. In further studies, the impact of silencing of these genes is being investigated by analysing changes in the metabolomic profile of M. tuberculosis with the aim of identifying similarities and differences in the metabolic response to target depletion at different steps of the pathway.Support or Funding InformationThis work was supported by grants from the TB Drug Accelerator program of the Bill and Melinda Gates Foundation (to C.E.B. III, D.S., K.R. and V.M.), the South African Medical Research Council, the National Research Foundation of South Africa and an International Research Scholar's grant from the HHMI (to V.M.) and, in part, by the Intramural Research Program of NIAID (H.B. and C.E.B. III).