Pyrazinamide resistance of novel mutations inpncAand their dynamic behavior

Abstract

Pyrazinamide (PZA) is one of the essential anti-mycobacterium drugs, active against non-replicatingMycobacterium tuberculosis(MTB) isolates. PZA is converted into its active state, called pyrazinoic acid (POA), by action ofpncAencoding pyrazinamidase (PZase). In the majority of PZA-resistance isolates,pncAharbored mutations in the coding region. In our recent report, we detected a number of novel variants in PZA-resistance (PZAR) MTB isolates, whose resistance mechanisms were yet to be determined. Here we performed several analyses to unveil the PZARmechanism of R123P, T76P, G150A, and H71R mutants (MTs) through molecular dynamics (MD) simulations. In brief, culture positive MTB isolates were subjected to PZA susceptibility tests using the WHO recommended concentration of PZA (100 μg ml−1). The PZARsamples were screened for mutations inpncAalong sensitive isolates through polymerase chain reactions and sequencing. A large number of variants (GeneBank accession no. MH461111), including R123P, T76P, G150A, and H71R, have been spotted in more than 70% of isolates. However, the mechanism of PZARfor mutants (MTs) R123P, T76P, G150A, and H71R was unknown. For the MTs and native PZase structures (WT), thermodynamic properties were compared using molecular dynamics simulations for 100 ns. The MTs structural activity was compared to the WT. Folding effect and pocket volume variations have been detected when comparing between WT and MTs. Geometric matching further confirmed the effect of R123P, T76P, G150A, and H71R mutations on PZase dynamics, making them vulnerable for activating the pro-drug into POA. This study offers a better understanding for management of PZARTB. The results may be used as alternative diagnostic tools to infer PZA resistance at a structural dynamics level.