Pyrazinoic Acid Inhibits a Bifunctional Enzyme in Mycobacterium tuberculosis

Abstract

Pyrazinamide (PZA), an indispensable component of modern tuberculosis treatment, acts as a key sterilizing drug. While the mechanism of activation of this prodrug into pyrazinoic acid (POA) by Mycobacterium tuberculosis has been extensively studied, not all molecular determinants that confer resistance to this mysterious drug have been identified. Here, we report how a new PZA resistance determinant, the Asp67Asn substitution in Rv2783, confers M. tuberculosis resistance to PZA. Expression of the mutant allele but not the wild-type allele in M. tuberculosis recapitulates the PZA resistance observed in clinical isolates. In addition to catalyzing the metabolism of RNA and single-stranded DNA, Rv2783 also metabolized ppGpp, an important signal transducer involved in the stringent response in bacteria. All catalytic activities of the wild-type Rv2783 but not the mutant were significantly inhibited by POA. These results, which indicate that Rv2783 is a target of PZA, provide new insight into the molecular mechanism of the sterilizing activity of this drug and a basis for improving the molecular diagnosis of PZA resistance and developing evolved PZA derivatives to enhance its antituberculosis activity.

Keywords: PNPase; antibiotic resistance; drug target; ppGpp; pyrazinamide; pyrazinoic acid.

FIG 1

Interaction of POA with wild-type Rv2783, mutant Rv2783_Asp67Asn, and M. smegmatis PNPase. (A) Representative results of an isothermal titration calorimetry (ITC) binding study indicate that POA binds to wild-type Rv2783 at high concentrations. (Top) Raw data. The data on the y axis indicate the amount of heat released per second during wild-type Rv2783 and POA binding. (Bottom) Integrated heat in each injection of POA together with the fit. The data on the y axis are expressed as the amount of heat released per mole in each injection. The association constants were obtained from fits of POA binding with wild-type Rv2783. (B) Representative results of an ITC binding study indicate that PZA did not bind to the wild-type Rv2783 protein. (Top) No heat was released during the wild-type Rv2783 and PZA interaction; (bottom) there was no integrated heat following each injection of PZA, and thus, there was no curve of the fit. (C and D) Representative results of an ITC binding study indicate that POA did not bind to the mutant Rv2783_Asp67Asn protein (C) or to the M. smegmatis PNPase protein (D). (Top) Raw data indicating that no heat was released during the interaction between POA and the two proteins; (bottom) there was no integrated heat following each injection of POA, and thus, there was no curve of the fit.

FIG 2

Capillary electrograms and graphical presentations of the ssDNA polymerization activities of Rv2783. (A to C) Representative capillary electrograms showing the uninhibited ssDNA polymerization activity of wild-type Rv2783 (A) and inhibition of the ssDNA polymerization activity of wild-type Rv2783 by PZA (B) and POA (C). (D) Comparison of the effects of POA and PZA on the ssDNA polymerization activity of the wild-type Rv2783 protein. (E to G) Representative capillary electrograms showing the uninhibited ssDNA polymerization activity of mutant Rv2783_Asp67Asn (E) and inhibition of the ssDNA polymerization activity of mutant Rv2783_Asp67Asn by PZA (F) and POA (G). (H) Comparison of the effects of POA and PZA on the ssDNA polymerization activity of the mutant Rv2783_Asp67Asn protein. For panels D and H, data are presented as means ± SEMs. Student's t test was used for the statistical analysis. ns, not significant; RFU, relative fluorescence units.

FIG 3

Capillary electrograms and graphical presentations of the ssDNA phosphorolysis activities of Rv2783. (A to C) Representative capillary electrograms showing the uninhibited ssDNA phosphorolysis activity of wild-type Rv2783 (A) and inhibition of the ssDNA phosphorolysis activity of wild-type Rv2783 by PZA (B) and POA (C). (D) Comparison of the effects of POA and PZA on the ssDNA phosphorolysis activity of the wild-type Rv2783 protein. (E to G) Representative capillary electrograms showing the uninhibited ssDNA phosphorolysis activity of mutant Rv2783_Asp67Asn (E) and inhibition of the ssDNA phosphorolysis activity of mutant Rv2783_Asp67Asn by PZA (F) and POA (G). (H) Comparison of the effects of POA and PZA on the ssDNA phosphorolysis activity of the mutant Rv2783_Asp67Asn protein. For panels D and H, data are presented as means ± SEMs. Student's t test was used for the statistical analysis.

FIG 4

Capillary electrograms and graphical presentations of RNA metabolism activities of Rv2783. (A and B) Representative capillary electrograms showing the uninhibited RNA polymerization activity of wild-type Rv2783 (A) and inhibition of RNA polymerization activity of wild-type Rv2783 by POA (B). (C) Comparison of the effects of POA and PZA on the RNA polymerization activity of the wild-type Rv2783 protein. (D and E) Representative capillary electrograms showing the uninhibited RNA polymerization activity of mutant Rv2783_Asp67Asn (D) and inhibition of the RNA polymerization activity of mutant Rv2783_Asp67Asn by POA (E). (F) Comparison of the effects of POA and PZA on the RNA polymerization activity of the mutant Rv2783_Asp67Asn protein. (G and H) Representative capillary electrograms showing the uninhibited RNA phosphorolysis activity of wild-type Rv2783 (G) and inhibition of the RNA phosphorolysis activity of wild-type Rv2783 by POA (H). (I) Comparison of the effects of POA and PZA on the RNA phosphorolysis activity of the wild-type Rv2783 protein. For panels C, F, and I, the data are presented as means ± SEMs. Student's t test was used for the statistical analysis.

FIG 5

ppGpp synthetase assays. Representative HPLC separation of ppGpp synthesis reaction products catalyzed by the wild-type Rv2783 protein (A) and the wild-type M. tuberculosis Rel protein (positive control) (B).

FIG 6

ppGpp hydrolase assays. Representative HPLC separation of ppGpp hydrolysis reaction products catalyzed by the wild-type Rv2783 protein in the absence (A) and presence (B) of POA and the mutant Rv2783_Asp67Asn protein in the absence (C) and presence (D) of POA.