title: Effect of pyrazinamidase activity on pyrazinamide resistance in Mycobacterium tuberculosis.
abstract: 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, k(cat), K(m), 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.
authors.affiliation: Laboratorio de Enfermedades Infecciosas, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias, Universidad Peruana Cayetano Heredia, San Martín de Porres, Lima, Peru. psheen@jhsph.edu
authors.forename: Patricia
authors.initials: P
authors.lastname: Sheen
authors.forename: Patricia
authors.initials: P
authors.lastname: Ferrer
authors.forename: Robert H
authors.initials: RH
authors.lastname: Gilman
authors.forename: Jon
authors.initials: J
authors.lastname: López-Llano
authors.forename: Patricia
authors.initials: P
authors.lastname: Fuentes
authors.forename: Eddy
authors.initials: E
authors.lastname: Valencia
authors.forename: Mirko J
authors.initials: MJ
authors.lastname: Zimic
mesh_terms: D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D000995:Antitubercular Agents / Q000494:pharmacology*; D003001:Cloning, Molecular; D024881:Drug Resistance, Bacterial / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011718:Pyrazinamide / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D013183:Sputum / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D011994:Recombinant Proteins; D011718:Pyrazinamide; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis; C022490:pyrazinamide deamidase