Pyrazinamide Resistance Is Caused by Two Distinct Mechanisms: Prevention of Coenzyme A Depletion and Loss of Virulence Factor Synthesis

Abstract

Pyrazinamide (PZA) is a critical component of first- and second-line treatments of tuberculosis (TB), yet its mechanism of action largely remains an enigma. We carried out a genetic screen to isolate Mycobacterium bovis BCG mutants resistant to pyrazinoic acid (POA), the bioactive derivative of PZA, followed by whole genome sequencing of 26 POA resistant strains. Rather than finding mutations in the proposed candidate targets fatty acid synthase I and ribosomal protein S1, we found resistance conferring mutations in two pathways: missense mutations in aspartate decarboxylase panD, involved in the synthesis of the essential acyl carrier coenzyme A (CoA), and frameshift mutations in the vitro nonessential polyketide synthase genes mas and ppsA-E, involved in the synthesis of the virulence factor phthiocerol dimycocerosate (PDIM). Probing for cross resistance to two structural analogs of POA, nicotinic acid and benzoic acid, showed that the analogs share the PDIM- but not the CoA-related mechanism of action with POA. We demonstrated that POA depletes CoA in wild-type bacteria, which is prevented by mutations in panD. Sequencing 10 POA-resistant Mycobacterium tuberculosis H37Rv isolates confirmed the presence of at least 2 distinct mechanisms of resistance to the drug. The emergence of resistance through the loss of a virulence factor in vitro may explain the lack of clear molecular patterns in PZA-resistant clinical isolates, other than mutations in the prodrug-converting enzyme. The apparent interference of POA with virulence pathways may contribute to the drug’s excellent in vivo efficacy compared to its modest in vitro potency.

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

“…5A). Consistent with recent findings (20), POA treatment led to a significant reduction in CoA levels in the wild-type strain (Fig. 5B).…”

“…2). Consistent with the recent finding that POA-resistant strains of M. tuberculosis and M. bovis BCG can be isolated at near neutral pH (20,27), the population that emerged following 3 weeks of incubation with POA maintained the ability to grow when subcultured to fresh medium containing the respective concentration of POA. FadD2 loss of function confers hypersusceptibility to PZA.…”

“…Intracellular CoA measurements were performed as previously described with slight modifications (20). Briefly, M. bovis BCG strains were grown until early logarithmic phase (OD 600 ϭ 0.2 to 0.3) and collected by centrifugation at 4,000 rpm (4°C) for 20 min.…”

“…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). Consistent with a role for PanD in PZA action, it has recently been demonstrated that exogenously supplied intermediates of the CoA biosynthetic pathway can antagonize PZA activity, point mutations in panD are associated with in vitro PZA resistance, and PZA treatment leads to the depletion of intrabacterial levels of pantothenate and CoA (13,15,20). However, it is important to note that a strain from which panD was deleted was found to retain susceptibility to PZA when grown with a low concentration of pantetheine (15).…”

Identification of Novel Efflux Proteins Rv0191, Rv3756c, Rv3008, and Rv1667c Involved in Pyrazinamide Resistance in Mycobacterium tuberculosis

“…Mutations in pncA leading to the loss of PZase activity are the major mechanism of PZA resistance (4,10,11). PZA is known to interfere with multiple functions in Mycobacterium tuberculosis, including cytoplasmic acidification (12), disruption of membrane energy and transport function (12,13), inhibition of the protein degradation pathway via RpsA involved in trans-translation (14) and also ClpC1 protease (15,16), and energy production via PanD (17)(18)(19). Although resistance to PZA is mostly caused by pncA mutations and less commonly by rpsA, panD, and clpC1, clinical strains without these mutations such as 9739 (20) are known to exist.…”