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.

Pyrazinoic Acid Inhibits the Bifunctional Enzyme (Rv2783) in Mycobacterium tuberculosis by Competing with tmRNA

“…POA binds to the Rv2783 protein, and its enzymatic activity is consequently obstructed by conformational changes. The D67N mutation inhibits POA binding to the mutant protein, so the mutant protein fulfills its enzymatic activities [19]. In this study, we discovered another mutation (A2104C) in Rv2783c from two clinical PZA-resistant strains lacking mutations in genes already reported to confer PZA resistance.…”

“…In our previous study, we identified four M. tuberculosis proteins (Rv2731, Rv2783, Rv3169, and Rv3601c) that bind to POA [6]; however, the role of these proteins in PZA action and resistance was not properly addressed. Recently, Rv2783 was shown to be involved in PZA resistance [19], where Asp67 of Rv2783 is indispensable for POA binding, but is inessential and adaptable for catalytic activity. POA binds to the Rv2783 protein, and its enzymatic activity is consequently obstructed by conformational changes.…”

“…Based on our previous report showing that selected proteins encoded by Rv2731, Rv2783c, Rv3169, and Rv3601c could bind to a POA derivative, 5-hydroxyl-2-pyrazinecarboxylic acid [6], and another report demonstrating Rv2783 as a possible target of POA [19], we performed Sanger sequencing of the target gene Rv2783c from a panel of 56 PZA-resistant clinical isolates of M. tuberculosis and identified the same A2104C mutation in Rv2783c in two clinical strains. In addition, we found no Rv2783c mutations in 42 PZA-susceptible extensively drug-resistant tuberculosis (XDR-TB) clinical isolates without pncA and rpsA mutations ( Table 1).…”

“…Recently, a bifunctional enzyme, guanosine pentaphosphate synthetase (GpsI)/polyribonucleotide nucleotidyltransferase (PNPase) (Rv2783), involved in RNA degradation, which was originally identified as a protein that binds POA [6], was found to be associated with PZA resistance and was identified as another target of POA in M. tuberculosis [19]. However, how POA inhibits Rv2783 and how mutations in Rv2783c cause PZA resistance are not yet known.…”

Genotypic characterization of pyrazinamide resistance in Mycobacterium tuberculosis isolated from Lusaka, Zambia

“…After filtering out the two SNPs, there remained one PZA resistant isolate with wild type pncA gene. The lack of mutations in pncA gene of these isolates indicates an alternative mechanism of resistance such as efflux of the drug or the newly described bifunctional enzyme Rv2783c [36] but not rspA and panD because no mutations were seen in these mutations in this study.…”