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

Pooja Gopal,Jickky Sarathy,Véronique Dartois,Jian Liang Low,Martin Gengenbacher,Jansy P Sarathy,Firat Kaya,Michelle Yee,Thomas Dick

doi:10.1021/acsinfecdis.6b00070

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.

Highlights

PZA is a critical component of the current first-line regimen for treating TB
To determine whether pyrazinoic acid (POA) resistance mutations confer resistance to prodrug PZA, we introduced a functional copy of the pncA gene from M. tuberculosis H37Rv into the five representative mutants and wild-type M. bovis BCG using an integrative plasmid construct
The results indicate that the three carboxylic acids share a phthiocerol dimycocerosate (PDIM) synthesis-related mechanism of action whereas the coenzyme A (CoA)-related mechanism of action is unique to POA

Summary

Introduction

PZA is a critical component of the current first-line regimen for treating TB. Its inclusion in the regimen in the 1980s resulted in dramatically shortening the duration of therapy from 12 to 6 months; i.e., PZA is a key sterilizing drug.[1]. By targeting RpsA, POA may impair trans−translation, a rescue mechanism that frees ribosomes stuck in translation.[11] gene sequencing of PZA-resistant clinical isolates did not support these biochemical findings.[12−14] Multiple investigators failed to isolate POA-resistant mutants in vitro on acidic agar, with. Mutations either in fatty acid synthase I, ribosomal protein RpsA, or any other target.[9,15] During the course of this work, Zhang and colleagues identified mutations in the aspartate decarboxylase PanD associated with PZA resistance[16,17] and proposed this enzyme as a direct target of POA. PanD as a target was called into question by Dillon et al.[18]

Methods

Results

Conclusion