Molecular targets and mechanisms of action of pyrazinamide in Mycobacterium tuberculosis

Abstract

The global control and management of tuberculosis (TB), caused by Mycobacterium tuberculosis, is faced with the formidable challenge of worsening scenarios of drug-resistant disease. Pyrazinamide (PZA) is one of four first-line drugs used in standard short-course combination therapy for the treatment of both drug-sensitive TB (DS-TB) and drug-resistant TB (DR-TB). It exhibits a preferential sterilizing activity against non-replicating persistent bacilli with low metabolism at acidic pH, and is thus anticipated to be an irreplaceable component of future first-line TB drug regimens. Although the mechanism of PZA activation by the enzyme pyrazinamidase (PZase), encoded by pncA gene, into its active moiety, pyrazinoic acid (POA), and resistance has been characterized, the precise cellular targets and physiological functions in M. tuberculosis that are inhibited by POA remain elusive. The ribosomal protein S1 (RpsA) and the aspartate decarboxylase (panD), involved in trans-translation and the synthesis of the essential metabolic cofactors pantothenate and coenzyme A respectively, have been suggested to be the targets of POA. In this study however, sequencing analysis has identified the same G199A (Asp67Asn) nonsynonymous substitution in Rv2783c of 2 PZA-resistant clinical strains lacking mutations in pncA, rpsA and panD. M. tuberculosis Rv2783c encode a probable bifunctional enzyme: polyribonucleotide nucleotidyltransferase (PNPase), involved in RNA and single stranded- DNA (ss-DNA) metabolism; and guanosine pentaphosphate synthetase (GpsI), involved in Molecular targets and mechanisms of action of PZA in Mycobacterium tuberculosis IV the synthesis and degradation of the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) implicated in the stringent response in bacteria. To gain more insight into a possible new target of PZA, we overexpressed the Rv2783cG199A mutant in M. tuberculosis H37Rv which resulted in PZA resistance in vitro, while overexpression of the wild type Rv2783c did not cause PZA resistance. Using isothermal titration calorimetry (ITC), purified wild type M. tuberculosis Rv2783 protein was found to bind to POA, and not to the prodrug PZA. However, purified M. tuberculosis Rv2783D67N protein and PNPase from naturally PZA-resistant M. smegmatis failed to bind either POA or PZA. In addition, both wild type and the mutant M. tuberculosis Rv2783 proteins catalyzed both template-independent RNA and ss-DNA polymerization and phosphorolysis activities. Interestingly however, the ss-DNA and RNA catalytic activities of the wild type and not the Rv2783D67N mutant protein were significantly inhibited by POA and not the prodrug PZA. Moreover, both wild type and the mutant M. tuberculosis Rv2783 proteins demonstrated strong ppGpp hydrolysis but only weak ppGpp synthesis activities. Similarly, the ppGpp hydrolysis activity of the wild type but not the Rv2783D67N mutant protein was significantly inhibited by POA. Taken together, these results suggest M. tuberculosis Rv2783 as a possible cellular target of POA. Our findings thus have implications for a better understanding of this unique sterilizing drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.