ABSTRACT
Recombinant wild-pyrazinamidase from H37Rv Mycobacterium tuberculosis was analyzed by gel electrophoresis under differential reducing conditions to evaluate its quaternary structure. PZAse was fractionated by size exclusion chromatography under non-reducing conditions. PZAse activity was measured and mass spectrometry analysis was performed to determine the identity of proteins by de novo sequencing and to determine the presence of disulfide bonds. This study confirmed that M. tuberculosis wild type PZAse was able to form homo-dimers in vitro. Homo-dimers showed a slightly lower specific PZAse activity compared to monomeric PZAse. PZAse dimers were dissociated into monomers in response to reducing conditions. Mass spectrometry analysis confirmed the existence of disulfide bonds (C72-C138 and C138-C138) stabilizing the quaternary structure of the PZAse homo-dimer.
Subject(s)
Amidohydrolases/metabolism , Mycobacterium tuberculosis/enzymology , Nicotinamidase/metabolism , Binding Sites , Chromatography, Gel/methods , Disulfides/metabolism , Humans , Mass Spectrometry/methods , Models, Molecular , Mycobacterium tuberculosis/metabolismABSTRACT
The pyrazinamidase is a fast and easy to perform assay, recommended by the World Health Organization as an alternative technique to determine pyrazinamide resistant Mycobacterium tuberculosis strains. This study aimed to assess the reproducibility of negative results on pyrazinamidase assay using both primary cultures and respective subcultures, and to correlate them with original cultures quality, as well as their susceptibility profile to streptomycin (S), isoniazid (I), rifampin (R) and ethambutol (E). A total of 115 Mycobacterium tuberculosis cultures were analyzed, which the original growth produced negative results on pyrazinamidase assay, implying a resistance to pyrazinamide. The cultures quality was assessed and recorded; a second testing was performed using recent and abundant subcultures. Results from the first and the second tests demonstrated an agreement rate of 72.2%, and the cultures quality showed correlation with the results (p 0.001). Pyrazinamidase testing is useful when it is combined with other techniques for analyzing mycobacteria susceptibility to S, I, R, E since it is performed with high quality cultures which allow the use of abundant inocula.
A Organização Mundial da Saúde recomenda o uso do teste da pirazinamidase (PZAse) como método alternativo para determinação da resistência do Mycobacterium tuberculosis à pirazinamida, por ser um teste rápido e de fácil execução. Foram objetivos deste estudo: verificar a reprodutibilidade dos resultados negativos do teste da pirazinamidase quando realizado a partir das culturas originais e de seus subcultivos e relacioná-los com a qualidade das culturas originais e com os perfis de suscetibilidade à estreptomicina (S), isoniazida (I), rifampicina (R) e etambutol (E). Foram analisadas 115 culturas de Mycobacterium tuberculosis cujos cultivos originais apresentaram resultados negativos no teste da PZAse, o que representa resistência à pirazinamida. A qualidade das culturas foi avaliada, anotada e um segundo teste foi realizado a partir de subcultivos jovens e abundantes. A concordância entre os resultados do primeiro e do segundo teste foi de 72,2% e a qualidade das culturas mostrou correlação com os resultados (p 0,001). O teste da pirazinamidase é útil quando utilizado juntamente com técnicas de detecção de suscetibilidade às drogas S, I, R, E, desde que seja realizado a partir de cultivos com boa qualidade, que permitam a utilização de inóculo abundante.