TY - JOUR
T1 - Long-chain fatty acyl coenzyme A ligase FadD2 mediates intrinsic pyrazinamide resistance in Mycobacterium tuberculosis
AU - Rosen, Brandon C.
AU - Dillon, Nicholas A.
AU - Peterson, Nicholas D.
AU - Minato, Yusuke
AU - Baughn, Anthony D.
N1 - Funding Information:
This study was supported by NIH grant R01 AI123146 and institutional start-up funds from the University of Minnesota Department of Microbiology and Immunology to A.D.B. B.C.R. was supported by an American Society for Microbiology undergraduate research fellowship and the Undergraduate Research Opportunities Program at the University of Minnesota. N.A.D. was supported by NIH institutional training grant T32 HL07741. We thank William R. Jacobs, Jr., for providing M. smegmatis mc2155, M. bovis BCG, M. tuberculosis H37Rv, phAE159, and phAE180; Joshua M. Thiede for constructing the pJT6a vector; and Shannon Lynn Kordus for her assistance with FadD2 enzymology.
Publisher Copyright:
© 2017 American Society for Microbiology. All Rights Reserved.
PY - 2017/2/1
Y1 - 2017/2/1
N2 - 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 POAmediated 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.
AB - 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 POAmediated 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.
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U2 - 10.1128/AAC.02130-16
DO - 10.1128/AAC.02130-16
M3 - Article
C2 - 27855077
AN - SCOPUS:85010966315
VL - 61
JO - Antimicrobial Agents and Chemotherapy
JF - Antimicrobial Agents and Chemotherapy
SN - 0066-4804
IS - 2
M1 - e02130
ER -