title: pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis.
abstract: Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance.
authors.affiliation: Unidad de Bioinformática y Biología Molecular, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, SMP, Lima, Peru.
authors.forename: Patricia
authors.initials: P
authors.lastname: Sheen
authors.forename: Katherine
authors.initials: K
authors.lastname: Lozano
authors.forename: Robert H
authors.initials: RH
authors.lastname: Gilman
authors.forename: Hugo J
authors.initials: HJ
authors.lastname: Valencia
authors.forename: Sebastian
authors.initials: S
authors.lastname: Loli
authors.forename: Patricia
authors.initials: P
authors.lastname: Fuentes
authors.forename: Louis
authors.initials: L
authors.lastname: Grandjean
authors.forename: Mirko
authors.initials: M
authors.lastname: Zimic
mesh_terms: D000581:Amidohydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000995:Antitubercular Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics / Q000502:physiology; D005798:Genes, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011718:Pyrazinamide / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D000995:Antitubercular Agents; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011718:Pyrazinamide; C005296:pyrazinoic acid; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis; C022490:pyrazinamide deamidase
keywords: Mycobacterium tuberculosis; POA efflux rate; PZA resistance; PZAse activity; pncA expression level