Pyrazinamide (PZA) is a first-line antitubercular drug for which the mode of action remains unresolved. Mycobacterium tuberculosis lacks measurable susceptibility to PZA under standard laboratory growth conditions. However, susceptibility to this drug can be induced by cultivation of the bacilli in an acidified growth medium. Previous reports suggested that the active form of PZA, pyrazinoic acid (POA), operates as a proton ionophore that confers cytoplasmic acidification when M. tuberculosis is exposed to an acidic environment. In this study, we demonstrate that overexpression of the PZA-activating enzyme PncA can confer PZA susceptibility to M. tuberculosis under neutral and even alkaline growth conditions. Furthermore, we find that wild-type M. tuberculosis displays increased susceptibility to POA relative to PZA in neutral and alkaline media. Utilizing a strain of M. tuberculosis that expresses a pH-sensitive green fluorescent protein (GFP), we find that unlike the bona fide ionophores monensin and carbonyl cyanide 3-chlorophenylhydrazone, PZA and POA do not induce rapid uncoupling or cytoplasmic acidification under conditions that promote susceptibility. Thus, based on these observations, we conclude that the antitubercular action of POA is independent of environmental pH and intrabacterial acidification.
Bibliographical noteFunding Information:
This work was supported by a grant from the NIAID (7UM1 AI068636-07) and institutional startup funds from the University of Minnesota to A.D.B. B.C.R. was supported by an ASM undergraduate research fellowship and a UROP fellowship from the University of Minnesota. N.A.D. was supported by an institutional training grant from the NHLBI (T32 HL07741). We thank Sabine Ehrt for providing plasmid pUV15-pHGFP and William R. Jacobs, Jr., for providing M. tuberculosis strains H37Ra and H37Rv. We thank Dean Crick for helpful comments and thoughtful discussions.