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Published on: June 9, 2022
Pyrazinamide resistance of novel mutations in pncA and their dynamic behavior
Arif Ali1, Muhammad Tahir Khan2, Abbas Khan1
1State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, and Joint Laboratory of International Cooperation in Metabolic and Developmental Sciences, Ministry of Education, Shanghai Jiao Tong University 800 Dongchuan Road Shanghai, Minhang District Shanghai 200240 China arifali@sjtu.edu.cn sathishimb@gmail.com Abbaskhan@sjtu.edu.cn Atharshafiq@sjtu.edu.cn dqwei@sjtu.edu.cn +86-21-3420-4573.
Abstract:
Pyrazinamide (PZA) is one of the essential anti-mycobacterium drugs, active against non-replicating Mycobacterium tuberculosis (MTB) isolates. PZA is converted into its active state, called pyrazinoic acid (POA), by action of pncA encoding pyrazinamidase (PZase). In the majority of PZA-resistance isolates, pncA harbored 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 PZAR mechanism 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 PZAR samples were screened for mutations in pncA along 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 PZAR for 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 PZAR TB. The results may be used as alternative diagnostic tools to infer PZA resistance at a structural dynamics level.
Insights
Pyrazinamide resistance in Mycobacterium tuberculosis is often linked to mutations in the pncA gene. Molecular dynamics simulations revealed how specific pncA mutations (R123P, T76P, G150A, H71R) disrupt pyrazinamidase activity, impacting pyrazinamide drug efficacy.
Area of Science:
- Microbiology
- Structural Biology
- Computational Biology
Background:
- Pyrazinamide (PZA) is a crucial drug for treating Mycobacterium tuberculosis (MTB) infections, particularly effective against non-replicating bacilli.
- Resistance to PZA is a growing concern, frequently associated with mutations in the pncA gene, which encodes pyrazinamidase (PZase).
- The precise resistance mechanisms for several novel pncA variants identified in PZA-resistant (PZA^R) MTB isolates remain undetermined.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying PZA resistance caused by specific pncA mutations (R123P, T76P, G150A, and H71R).
- To investigate the impact of these mutations on the structure and dynamics of the pyrazinamidase enzyme.
- To provide insights for improved management strategies and potential diagnostic tools for PZA-resistant tuberculosis.
Main Methods:
- Susceptibility testing of MTB isolates to PZA using WHO-recommended concentrations.
- Screening for pncA mutations in PZA-resistant and sensitive isolates via polymerase chain reaction and sequencing.
- Molecular dynamics (MD) simulations of wild-type (WT) and mutant pyrazinamidase (PZase) structures over 100 ns.
- Comparative analysis of thermodynamic properties, structural activity, folding effects, and pocket volume variations between WT and mutant PZase.
Main Results:
- Identified novel pncA variants, including R123P, T76P, G150A, and H71R, in over 70% of PZA^R MTB isolates.
- MD simulations revealed significant alterations in folding and pocket volume for the mutant PZase structures compared to WT.
- Geometric matching confirmed that these mutations affect PZase dynamics, impairing its ability to activate the pro-drug PZA into its active form, pyrazinoic acid (POA).
Conclusions:
- The R123P, T76P, G150A, and H71R mutations in pncA confer PZA resistance by destabilizing pyrazinamidase structure and function.
- Understanding these structural dynamics provides a basis for developing alternative diagnostic methods for PZA resistance.
- This research contributes to better management of PZA-resistant tuberculosis by clarifying resistance mechanisms at a molecular level.
