DISCOVERING OF PYRAZINAMIDE RESISTANCE IN LOCAL STRAIN OF Mycobacterium tuberculosis CLINICAL ISOLATES BY MOLECULAR DETECTION OF pncA GENE ENCODING PZase

Pyrazinamide (PZA) is one of the anti-tuberculosis (TB) drugs that have an action on the pncA gene encoding the PZase enzyme. Mycobacterium tuberculosis clinical isolates R14 and R7 showed PZA resistance up to doses 40 and 30 µg/mL, however, the basis of their resistance was unknown. The pncA gene of them was cloned into a pGemT vector, then characterized by sequencing. The pncA measuring 0.6 kb has been inserted into pGemT to form recombinant DNA at 3,6 kb. The pncA nucleotides of R14 and R7 showed four and two mutations toward the PZA-sensitive isolate H37RV. The mutations of G76T; G112C; A403C; G426A changed amino acids A26S; A38P; T135P were found in R14, while mutations G115A and T506C which changed A39T and V169A amino acids found in R7. The mutation triggered the decrease of two hydrophobic amino acids in R14 PZase, but one in R7. An additional polar amino acid was also found in R7 PZase. The PZase structure of both mutants changed conformation from the native structure H37RV with RSMD as 0.5 and 0.3 Å. The pncA mutations that followed by the change in protein properties and structures might induce the emergence of PZA resistance in the isolates.