RESUMO
In clinical isolates of Mycobacterium tuberculosis (MTB), resistance to pyrazinamide occurs by mutations in any positions of the pncA gene (NC_000962.3) especially in nucleotides 359 and 374. In this study we examined the pncA gene sequence in clinical isolates of MTB. Genomic DNA of 33 clinical isolates of MTB was extracted by the Chelex100 method. The polymerase chain reactions (PCR) were performed using specific primers for amplification of 744 bp amplicon comprising the coding sequences (CDS) of the pncA gene. PCR products were sequenced by an automated sequencing Bioscience system. Additionally, semi Nested-allele specific (sNASP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were carried out for verification of probable mutations in nucleotides 359 and 374. Sequencing results showed that from 33 MTB clinical isolates, nine pyrazinamide-resistant isolates have mutations. Furthermore, no mutation was detected in 24 susceptible strains in the entire 561 bp of the pncA gene. Moreover, new mutations of G→A at position 3 of the pncA gene were identified in some of the resistant isolates. Results showed that the sNASP method could detect mutations in nucleotide 359 and 374 of the pncA gene, but the PCR-RFLP method by the SacII enzyme could not detect these mutations. In conclusion, the identification of new mutations in the pncA gene confirmed the probable occurrence of mutations in any nucleotides of the pncA gene sequence in resistant isolates of MTB.
RESUMO
BACKGROUND: Mutations in pncA and gyrA genes cause pyrazinamide (PZA) and fluroquinolone resistance in Mycobacterium tuberculosis (MTB). In the present study, structures of pyrazinamidase (PZase) and DNA gyrase proteins were studied in resistant and susceptible clinical isolates of MTB. MATERIALS AND METHODS: Sixty clinical isolates of MTB were used in this study. Polymerase chain reaction (PCR) amplification of pncA and gyrA genes was accomplished on purified DNA. Sequence of the fragments was determined by an Applied BiosystemsTM apparatus. Bioinformatic analysis was performed by online software and three-dimensional (3D) structures of proteins was predicted using Molegro Virtual Docker (MVD) Modeler software. RESULTS: Amplified 744 and 194 bp fragments of pncA and gyrA genes, respectively were yielded suitable sequence results. Predicted 3D structures of proteins showed some differences between wild-type and mutant structures. Mutation in amino acid No.31 (T92C) caused an increase in distance from metal ion position to enzyme active site, but it was considered as a polymorphism. Docking results by MVD revealed a relationship in quinolone resistance-determining regions (QRDR) amino acids in interaction with antibiotic. T92C mutation in PZase from non-polar aliphatic amino acid Ile (ATC) to polar aliphatic amino acid threonine (ACC) was a polymorphism. CONCLUSION: Structural changes in two important proteins related to drug resistance were proven in clinical isolates of MTB.
RESUMO
INTRODUCTION: Pyrazinamide is one of the first line four drugs for treatment of tuberculosis. It was proved that mutations in two nucleotides of 359 and 374 pnc genes are highly associated with resistance to pyrazinamide. MATERIALS AND METHODS: In this study, mutations in these two codones in 30 clinical isolates of Mycobacterium tuberculosis were detected by means of sequencing. Protein structures encoded by this gene with and without mutation were investigated in resistant and susceptible isolates to pyrazinamide, respectively. RESULTS: Mutation in the positions 359 and 374 altered some parameters like change in electronic charge, distance change of mutated amino acids to situation of active enzyme and metal connection situation. In these conditions, structure and function of pyrozinamidase enzyme were changed and antibiotic was ineffective and consequently caused resistance to pyrazinamide in M. tuberculosis. CONCLUSION: This work was revealed protein changes in resistance to pyrazinamide in clinical isolates of M. tuberculosis.
