title: Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis.
abstract: Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug that has been in clinical use for 60 years yet still has an unresolved mechanism of action. Based upon the observation that the minimum concentration of PZA required to inhibit the growth of Mycobacterium tuberculosis is approximately 1,000-fold higher than that of other first-line drugs, we hypothesized that M. tuberculosis expresses factors that mediate intrinsic resistance to PZA. To identify genes associated with intrinsic PZA resistance, a library of transposon-mutagenized Mycobacterium bovis BCG strains was screened for strains showing hypersusceptibility to the active form of PZA, pyrazinoic acid (POA). Disruption of the long-chain fatty acyl coenzyme A (CoA) ligase FadD2 enhanced POA susceptibility by 16-fold on agar medium, and the wild-type level of susceptibility was restored upon expression of fadD2 from an integrating mycobacterial vector. Consistent with the recent observation that POA perturbs mycobacterial CoA metabolism, the fadD2 mutant strain was more vulnerable to POA-mediated CoA depletion than the wild-type strain. Ectopic expression of the M. tuberculosis pyrazinamidase PncA, necessary for conversion of PZA to POA, in the fadD2 transposon insertion mutant conferred at least a 16-fold increase in PZA susceptibility under active growth conditions in liquid culture at neutral pH. Importantly, deletion of fadD2 in M. tuberculosis strain H37Rv also resulted in enhanced susceptibility to POA. These results indicate that FadD2 is associated with intrinsic PZA and POA resistance and provide a proof of concept for the target-based potentiation of PZA activity in M. tuberculosis.
authors.affiliation: Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
authors.forename: Brandon C
authors.initials: BC
authors.lastname: Rosen
authors.affiliation: Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Dillon
authors.affiliation: Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Peterson
authors.affiliation: Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Minato
authors.affiliation: Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA abaughn@umn.edu.
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Baughn
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D003066:Coenzyme A Ligases / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D011718:Pyrazinamide / Q000494:pharmacology*; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
chemical_list: D000995:Antitubercular Agents; D004251:DNA Transposable Elements; D011718:Pyrazinamide; D003066:Coenzyme A Ligases; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
keywords: Mycobacterium tuberculosis; antimicrobial agents; coenzyme A; fatty acids; metabolism; pyrazinamide