Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis

Abstract

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug-susceptible and multidrug-resistant tuberculosis (MDR-TB). PZA is a prodrug that is converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. β-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.

Genetic Determinants of Drug Resistance in Mycobacterium tuberculosis and Their Diagnostic Value

“…Eis had not been identified as a resistance-associated gene at the time our study began, but mutations in this locus have since been found to explain up to 20% of KAN resistance (43). Other recently identified resistance genes in MTB include panD and rpsA, which was reported to confer PZA resistance in isolates that lack pncA mutations (12,44,45). Previous studies have also focused exclusively on either MDR (39) or XDR (38) isolates, which may have a narrower range of resistance mutations.…”

Pyrazinamide Is Active against Mycobacterium tuberculosis Cultures at Neutral pH and Low Temperature

“…Later, Zhang and Mitchison (4) described a model for the antimycobacterial effect of PZA, in which extracellular protonation of POA facilitates its reentry into the mycobacteria, causing membrane damage and acidification of the cytoplasm. This model is not regarded as complete, and alternative mechanisms and targets, e.g., ATP depletion (19), inhibition of ribosomal protein S1 (20), aspartate decarboxylase (21), and quinolinic acid phosphoribosyltransferase (22) have been proposed, but the requirement for low pH has hardly ever been questioned. The data presented here, together with the historical data (18) and those of the recent study of Peterson et al (17), where antimicrobial activity of PZA at neutral pH was detected in starved cultures in vitro, clearly demonstrate that an extracellular acidic pH is not a prerequisite.…”

Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis

“…While the basis for this disparity remains unclear, the PZA susceptibility of M. tuberculosis can be enhanced by stresses that are known to be encountered during infection, such as low pH (6), nutrient limitation (7,8), and exposure to hypoxia (9). On the basis of these and other findings, a number of models have been proposed to explain the antimycobacterial action of PZA and POA, including direct inhibition of fatty acid synthase I (10), disruption of the membrane potential and cytoplasmic pH homeostasis via a protonophore activity (11), inhibition of transtranslation (12), and inhibition of the aspartate decarboxylase PanD, involved in pantothenate and coenzyme A (CoA) biosynthesis (13). While the bases for these models provide clues toward understanding PZA and POA susceptibility, fundamental aspects of these models have been challenged by recent reports (7,(14)(15)(16)(17)(18)(19).…”