Molecular characterization of the Pyrazinamidase (PZase) drug target protein in multidrug-resistant mycobacterium tuberculosis clinical isolates

Abstract

Pyrazinamide (PZA) is a first-line anti-tuberculosis (TB) drug whose activity depends on the pyrazinamidase (PZase) enzyme encoded by the pncA gene in Mycobacterium tuberculosis. Mutations in pncA that impair PZase function are strongly associated with PZA resistance. In this study, 20 multidrug-resistant TB (MDR-TB) clinical isolates were analyzed to assess the molecular diversity of the PZA target protein. The pncA gene was successfully amplified by PCR (0.6 kb) and sequenced using the Sanger method. Mutations in pncA were detected in 85% of the isolates, consisting of substitutions, insertions, deletions, and stop-codon alterations. Many of the mutations led to amino acid changes or frameshift events in the PZase protein. Seven MDR-TB isolates carried substitution mutations that altered the PZase amino acid sequence, while three isolates harbored insertion mutations and three had deletion mutations, leading to frameshifts in the protein. In addition, four isolates exhibited stop-codon mutations that were converted into nonstop codons. Five substitution mutations (C175T, G289A, C83A, T173G, and G356T) were identified as novel and resulted in the amino acid changes Leu59Phe, Gly97Ser, Ala28Asp, Phe58Cys, and Trp119Leu, respectively. These findings underscore the strong association between pncA mutations and PZA resistance and highlight the need for further studies to elucidate the structural and functional impacts of these mutations on PZase.