Rosen Brandon C, Dillon Nicholas A, Peterson Nicholas D, Minato Yusuke, Baughn Anthony D
Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA
Antimicrob Agents Chemother. 2017 Jan 24;61(2). doi: 10.1128/AAC.02130-16. Print 2017 Feb.
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.
吡嗪酰胺（PZA）是一种一线抗结核药物，已临床使用60年，但作用机制仍未明确。基于观察到抑制结核分枝杆菌生长所需的PZA最低浓度比其他一线药物高约1000倍，我们推测结核分枝杆菌表达介导对PZA内在耐药性的因子。为了鉴定与PZA内在耐药性相关的基因，筛选了转座子诱变的牛分枝杆菌卡介苗菌株文库，以寻找对PZA的活性形式吡嗪酸（POA）超敏感的菌株。长链脂肪酰辅酶A（CoA）连接酶FadD2的破坏使琼脂培养基上的POA敏感性提高了16倍，并且从整合分枝杆菌载体表达fadD2后恢复了野生型敏感性水平。与最近观察到的POA扰乱分枝杆菌CoA代谢一致，fadD2突变菌株比野生型菌株更容易受到POA介导的CoA消耗的影响。在fadD2转座子插入突变体中异位表达PZA转化为POA所需的结核分枝杆菌吡嗪酰胺酶PncA，在中性pH的液体培养中活跃生长条件下，PZA敏感性至少增加了16倍。重要的是，在结核分枝杆菌H37Rv菌株中缺失fadD2也导致对POA的敏感性增强。这些结果表明FadD2与PZA和POA的内在耐药性相关，并为基于靶点增强结核分枝杆菌中PZA活性提供了概念验证。
