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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 (pncA WT ), have been sequenced for rpsA and panD genes mutation. We repeated a drug susceptibility testing according to the WHO guidelines on 18 pncA WT 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 (MtRpsA CTD ) 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 MtRpsA CTD 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 MtRpsA CTD 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.

Pyrazinamide (PZA) is an essential first line antitubercular drug, which plays a crucial role in tuberculosis treatment. The PZA, which is considered as a pro-drug needs an enzyme of mycobacterial pyrazinamidase (PZase) for its conversion into an active form pyrazinoic acid. Further, this active form of PZA inhibits the ribosomal proteins S1, which facilitates the transfer-mRNA complex formation throughout the translation. The spontaneous mutations in RpsA have been found to be associated with PZA drug resistance. However, the drug resistance mechanism is still unclear. Furthermore, there is no such information available about the structural dynamics of RpsA protein because of mutations that confer Pyrazinoic acid resistance. Moreover, a total of 18 clinical PZA-resistant isolates were investigated and found to be pncA WT , which allowed exploration of the resistance mechanism of RpsA in the mutated state. Samples were repeated for the drug susceptibility testing followed by RpsA gene sequencing. A total of 11 clinical isolates harbored a total of 15 mutations. Almost half of the total strains (7/15) were observed to be in the conserved region of RpsA and known as Mycobacterium tuberculosis C-terminal domain. In the current study, (2/7) mutation T370P (mutant 1) and W403G (mutant 2) were explored to ensure the RpsA resistance mechanism through essential dynamics simulation. The essential dynamics study results revealed that the distal loop mutations drastically altered the conformation of RpsA both in the absence (-) and presence (+) of pyrazinoic acid drug for two reasons: (1) dramatic alteration or reduction in the binding pattern of pyrazinoic acid with active site residues observed and (2) a clear image of the opening and closing switching mechanism was seen upon the distal site mutation on nearby 3 10 -helixes beside the pyrazinoic acid binding site. This switch was found to consistently remain closed only in wild type systems, while it was open in the mutant systems. We called such distance impact an "allosteric effect." The overall mechanistic investigations will provide useful information behind drug resistance for better understanding to manage tuberculosis.

Background: Pyrazinamide (PZA) is an important component of first-line drugs because of its distinctive capability to kill subpopulations of persistent Mycobacterium tuberculosis (MTB). The prodrug (PZA) is converted to its active form, pyrazinoic acid (POA) by MTB pncA-encoded pyrazinamidase (PZase). Mutation in pncA is the most common and primary cause of PZA resistance. The aim of the present study was to explore the molecular characterization of PZA resistance in a Pashtun-dominated region of Khyber Pakhtunkhwa, Pakistan., Methods: We performed drug susceptibility testing (DST) on 753 culture-positive isolates collected from the Provincial Tuberculosis Control Program Khyber Pakhtunkhwa using the BACTEC MGIT 960 PZA method. In addition, the pncA gene was sequenced in PZA-resistant isolates, and PZA susceptibility testing results were used to determine the sensitivity and specificity of pncA gene mutations., Results: A total of 69 isolates were PZA resistant (14.8%). Mutations were investigated in 69 resistant, 26 susceptible and one H37Rv isolates by sequencing. Thirty-six different mutations were identified in PZA-resistant isolates, with fifteen mutations, including 194_203delCCTCGTCGTG and 317_318delTC, that have not been reported in TBDRM and GMTV Databases and previous studies. Mutations Lys96Thr and Ser179Gly were found in the maximum number of isolates (n = 4 each). We did not detect mutations in sensitive isolates, except for the synonymous mutation 195C > T (Ser65Ser). The sensitivity and specificity of the pncA sequencing method were 79.31% (95% CI, 69.29 to 87.25%) and 86.67% (95% CI, 69.28 to 96.24%)., Conclusion: Mutations in the pncA gene in circulating isolates of geographically distinct regions, especially in high-burden countries, should be investigated for better control and management of drug-resistant TB. Molecular methods for the investigation of PZA resistance are better than DST.

In an effort to discover the mechanism of resistance offered by Mycobacterium tuberculosis (Mtb) toward the pyrazinamide (PZA) drug, an extensive molecular dynamics strategy was employed. PZA is a first-line prodrug that effectively cuts therapy time by 33% (from 9 to 6 months). Pyrazinamidase enzyme (PZase), encoded by the pncA gene, is responsible for the activation of prodrug PZA into pyrazinoic acid (POA). POA is toxic and potently inhibits the growth of latent Mtb even at low pH values. PZA resistance is caused by three genes pncA, rpsA, and panD. Among them, the pncA gene contributes 72-99% to the resistance. Hence, the present study focused on the novel mutations N11K, P69T, and D126N in the pncA gene. In the present study, the possible mechanism of these three mutations was studied through molecular dynamics simulation and docking techniques. Our in-depth analysis and results are in strong agreement with our experimental observation. The binding pocket analysis showed that mutations decrease the volume of the active site and hinder the correct orientation of PZA drug in the active site. Moreover, the Patchdock score was found to be low as compared to WT showing the disturbance of shape complementarity between PZase and PZA drug. These mutations were found to disturb the position of the Fe 2+ ion. Among the mutations, D126N allosterically disturbed the position of the Fe 2+ ion. MMGBSA analyses showed that these mutations decrease the binding affinity toward the PZA drug. In conclusion, mutations N11K, P69T, and D126N result in weak binding affinity with PZA and also cause significant structural deformations that lead to PZA resistance. This study provides useful information that mutations in other than active parts may also cause protein folding and ligand displacement effects, altering the biological functions.