Effect of pyrazinamidase activity on pyrazinamide resistance in Mycobacterium tuberculosis
Introduction
Pyrazinamide (PZA) is an important first-line drug for tuberculosis (TB) and appears to be the most important drug in killing latent Mycobacterium tuberculosis.1, 2, 3 The emergence of strains resistant to PZA represents an important public health problem, as both primary and secondary line treatment schemes include PZA. Multidrug resistant (MDR) tuberculosis, defined as isoniazid and rifampin resistant, is increasing globally;4, 5 and more than 30% of Peruvian MDR TB strains are also resistant to PZA.6
PZA-susceptible M. tuberculosis isolates possess a pyrazinamidase (PZAse) that is constitutively expressed7, 8 and hydrolyzes PZA to pyrazinoic acid (POA) which is the lethal molecule.9, 10, 11 A defective POA efflux pump is required to accumulate intracellular POA.7
Mutations in the PZAse coding gene (pncA) are scattered throughout its sequence with some degree of clustering in the regions that contain the catalytic residues of PZAse.12, 13, 14 The catalytic residues comprise the active site (D8, K96, A134 and C138) and the metal-binding site (D49, H51 and H71).12 According to previous studies, the ion likely to bind the metal coordination site would be zinc or iron.12, 15 The specific activity of recombinant mutated PZAses varies as much as 1000 fold depending on the site of the mutation.15, 16 It is suggested that mutations causing significant loss of PZAse activity are those that produce a physical-chemical alteration of the active site or the metal-binding site. Mutations located farther away are thought to have less effect on PZAse activity.12, 16
Only one study has addressed the correlation of PZAse activity with the ‘yes/no’ microbiological resistance.17 The study showed that low levels of PZAse activity are found in resistant isolates with pncA mutations, however it did not examine other kinetic parameters or its association with the quantitative level of resistance.
In this study we will examine the correlation between the kinetic parameters of recombinant mutated PZAses cloned from PZA resistant M. tuberculosis clinical strains and the microbiological PZA resistance level.
Section snippets
Selection of M. tuberculosis sputum isolates
In a recent study, we identified 26 pncA unique sequences with single missense mutation in M. tuberculosis clinical isolates.18 We selected 12 of these strains based upon the Wayne activity and type of mutations. We also included the PZA-susceptible wild type reference strain H37Rv. The selected strains displayed negative, weak, and positive Wayne activities. Mutations were of three types: mutations of the metal-binding residues (D49N, H51R), mutations close to the metal-binding or active site
Cloning, expression and purification of M. tuberculosis PZAse
The PZAses were cloned, expressed and purified at a final concentration of 2 mg/ml. The purified protein was stored at −70°C for further analysis. The purity of the recombinant PZAse was confirmed as a single band in a Coomassie blue stained SDS-PAGE (Figure 1).
PZA-susceptibility parameters in the M. tuberculosis sputum isolates
The BZRL, the 7H9 PZA MIC, and the Wayne activity of the 13 strains are shown in Table 1. All the mutant isolates were resistant according to Bactec except for the strain K48T, which was susceptible according to the 7H9 culture with a PZA
Discussion
In this study, we have investigated the kinetic parameters of mutated PZAses from M. tuberculosis clinical isolates resistant to PZA. We demonstrated that only 27.3% of the statistical variability of resistance is explained by the PZAse activity and there is a wide variation in the enzymatic activity in recombinant proteins associated with mutations in pncA. In some cases these proteins had enzymatic levels similar to the wild type PZAse of the susceptible strain (H37Rv). Confirming previous
Acknowledgements
We acknowledge Drs. Mark A. Swancutt, Carlton Evans, David Moore, Alberto Mendoza, Laurie Baumann and Marjory Bravard for their advice and review of the manuscript. The National Institute of Allergy and Infectious Diseases, National Institutes of Health US, funded this research under the terms of Award No # 1 R03 AI067608-0. P. Sheen and M.J. Zimic received some support from TMRC New Tools to Understand and Control Endemic Parasites # 1 P01 AI51976. J. López-Llano received an award from the
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