Elsevier

Tuberculosis

Volume 89, Issue 2, March 2009, Pages 109-113
Tuberculosis

Effect of pyrazinamidase activity on pyrazinamide resistance in Mycobacterium tuberculosis

https://doi.org/10.1016/j.tube.2009.01.004Get rights and content

Summary

Resistance of Mycobacterium tuberculosis to pyrazinamide is associated with mutations in the pncA gene, which codes for pyrazinamidase. The association between the enzymatic activity of mutated pyrazinamidases and the level of pyrazinamide resistance remains poorly understood.

Twelve M. tuberculosis clinical isolates resistant to pyrazinamide were selected based on Wayne activity and localization of pyrazinamidase mutation. Recombinant pyrazinamidases were expressed and tested for their kinetic parameters (activity, kcat, Km, and efficiency). Pyrazinamide resistance level was measured by Bactec-460TB and 7H9 culture. The linear correlation between the resistance level and the kinetic parameters of the corresponding mutated pyrazinamidase was calculated.

The enzymatic activity and efficiency of the mutated pyrazinamidases varied with the site of mutation and ranged widely from low to high levels close to the corresponding of the wild type enzyme. The level of resistance was significantly associated with pyrazinamidase activity and efficiency, but only 27.3% of its statistical variability was explained.

Although pyrazinamidase mutations are indeed associated with resistance, the loss of pyrazinamidase activity and efficiency as assessed in the recombinant mutated enzymes is not sufficient to explain a high variability of the level of pyrazinamide resistance, suggesting that complementary mechanisms for pyrazinamide resistance in M. tuberculosis with mutations in pncA are more important than currently thought.

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

References (36)

  • D.A. Mitchison

    The action of antituberculosis drugs in short-course chemotherapy

    Tubercle

    (1985)
  • M.A. Steele et al.

    The role of pyrazinamide in tuberculosis chemotherapy

    Chest

    (1988)
  • S.S. Trivedi et al.

    Pyrazinamidase activity of Mycobacterium tuberculosis—a test of sensitivity to pyrazinamide

    Tubercle

    (1987)
  • Y. Hu et al.

    Sterilising action of pyrazinamide in models of dormant and rifampicin-tolerant Mycobacterium tuberculosis

    Int J Tuberc Lung Dis

    (2006)
  • Ministerio de Salud. Magnitud del problema de TB MDR y cobertura de los tratamientos para TB MDR. In: Bonilla C, Jave...
  • M. Zignol et al.

    Global incidence of multidrug-resistant tuberculosis

    J Infect Dis

    (2006)
  • L. Vasquez-Campos et al.

    Drug resistance trends among previously treated tuberculosis patients in a national registry in Peru, 1994–2001

    Int J Tuberc Lung Dis

    (2004)
  • Y. Zhang et al.

    The curious characteristics of pyrazinamide: a review

    Int J Tuberc Lung Dis

    (2003)
  • S.J. Cheng et al.

    pncA mutations as a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis: spread of a monoresistant strain in Quebec, Canada

    Antimicrob Agents Chemother

    (2000)
  • K. Konno et al.

    Pyrazinamide susceptibility and amidase activity of tubercle bacilli

    Am Rev Respir Dis

    (1967)
  • Y. Zhang et al.

    Mode of action of pyrazinamide: disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid

    J Antimicrob Chemother

    (2003)
  • M.M. Wade et al.

    Mechanisms of drug resistance in Mycobacterium tuberculosis

    Front Biosci

    (2004)
  • X. Du et al.

    Crystal structure and mechanism of catalysis of a pyrazinamidase from Pyrococcus horikoshii

    Biochemistry

    (2001)
  • A. Scorpio et al.

    Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis

    Antimicrob Agents Chemother

    (1997)
  • N. Lemaitre et al.

    Characterization of new mutations in pyrazinamide-resistant strains of Mycobacterium tuberculosis and identification of conserved regions important for the catalytic activity of the pyrazinamidase PncA

    Antimicrob Agents Chemother

    (1999)
  • H. Zhang et al.

    Characterization of Mycobacterium tuberculosis nicotinamidase/pyrazinamidase

    Febs J

    (2008)
  • N. Lemaitre et al.

    Study of the structure-activity relationships for the pyrazinamidase (PncA) from Mycobacterium tuberculosis

    Biochem J

    (2001)
  • Y. Suzuki et al.

    Rapid detection of pyrazinamide-resistant Mycobacterium tuberculosis by a PCR-based in vitro system

    J Clin Microbiol

    (2002)
  • Cited by (64)

    • Computational modeling and bioinformatic analyses of functional mutations in drug target genes in Mycobacterium tuberculosis

      2021, Computational and Structural Biotechnology Journal
      Citation Excerpt :

      M.tb acquires these less favorable mutations which completely or partially eliminate the activity of the associated protein but in turn, make the bacteria more fit to survive against the anti-TB drugs. Similar observations were made for I335T, T262R and T76P mutations of KatG and PncA where the mutations led to substantial deprivation in activity of proteins but in turn, provided resistance to bacteria against two most potent anti-TB drugs namely INH and PZA [58,59]. Therefore, the secondary mutations identified to be resistance conferring in this work could be declared as suspects that despite being less favorable by evolution hold great importance in the survival of the pathogen in stress conditions.

    • Crystal structure of the nicotinamidase/pyrazinamidase PncA from Bacillus subtilis

      2018, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      PZA resistance is another important factor in the control of these bacteria [11]. PZA resistance is associated with mutations in the pncA gene [12,13]. Therefore, an understanding of the chemical mechanisms underlying the transition state of the reaction catalyzed by this enzyme is crucial for the development of new and effective agents to combat anti-PZA resistance in pathogenic bacteria.

    View all citing articles on Scopus
    View full text