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Abstract
Resistance to key first-line drugs is a major hurdle to achieve the global end tuberculosis (TB) targets. A prodrug, pyrazinamide (PZA) is the only drug, effective in latent TB, recommended in drug resistance and susceptible Mycobacterium tuberculosis (MTB) isolates. The prodrug conversion into active form, pyrazinoic acid (POA), required the activity of pncA gene encoded pyrazinamidase (PZase). Although pncA mutations have been commonly associated with PZA resistance but a small number of resistance cases have been associated with mutationss in RpsA protein. Here in this study a total of 69 PZA resistance isolates have been sequenced for pncA mutations. However, samples that were found PZA resistant but pncA wild type (pncAWT), have been sequenced for rpsA and panD genes mutation. We repeated a drug susceptibility testing according to the WHO guidelines on 18 pncAWT MTB isolates. The rpsA and panD genes were sequenced. Out of total 69 PZA resistant isolates, 51 harbored 36 mutations in pncA gene (GeneBank Accession No. MH46111) while, fifteen different mutations including seven novel, were detected in the fourth S1 domain of RpsA known as C-terminal (MtRpsACTD) end. We did not detect any mutations in panD gene. Among the rpsA mutations, we investigated the molecular mechanism of resistance behind mutations, D342N, D343N, A344P, and I351F, present in the MtRpsACTD through molecular dynamic simulations (MD). WT showed a good drug binding affinity as compared to mutants (MTs), D342N, D343N, A344P, and I351F. Binding pocket volume, stability, and fluctuations have been altered whereas the total energy, protein folding, and geometric shape analysis further explored a significant variation between WT and MTs. In conclusion, mutations in MtRpsACTD might be involved to alter the RpsA activity, resulting in drug resistance. Such molecular mechanism behind resistance may provide a better insight into the resistance mechanism to achieve the global TB control targets.
Highlights
According to the World Health Organization report 2018, 23% of the world’s population (1.7 billion), are infected with latent TB1
Molecular dynamic (MD) simulations is a method of choice that have been applied widely in exploring the mechanisms of conformational changes in protein, especially in drug resistance mechanisms caused by mutations
We identified different mutations in pncA gene[22] (GeneBank Accession No MH46111) and RpsA23, whose molecular mechanism of resistance have been investigated through MD simulation[24,25,26]
Summary
According to the World Health Organization report 2018, 23% of the world’s population (1.7 billion), are infected with latent TB1. PZA resistance emerges due to mutations at MtRpsACTD of mycobacterial species, causing conformational changes in the POA binding site[3]. Molecular dynamic (MD) simulations is a method of choice that have been applied widely in exploring the mechanisms of conformational changes in protein, especially in drug resistance mechanisms caused by mutations. MD simulation studies of ligand-protein interactions are widely applied approach to explain the mechanisms of drug resistance due to mutations especially in target protein, which is one of the major causes behind resistance. We identified different mutations in pncA gene[22] (GeneBank Accession No MH46111) and RpsA23, whose molecular mechanism of resistance have been investigated through MD simulation[24,25,26]. We have investigated the possible changes in the RpsA dynamics, that results due to mutations in MtRpsACTD which may provide useful information behind the drug resistance
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