Background: Tuberculosis is the 13th leading cause of death and the second leading infectious killer after COVID-19 (above HIV/AIDS) around the world. In 2020, an estimated 10 million people fell ill with tuberculosis (TB) worldwide. Extensively drug-resistant TB (XDR TB) is a rare type of MDR TB that is resistant to isoniazid and rifampin, plus any fluoroquinolone and at least one of three injectable second-line drugs (CDC). To avoid another pandemic from happening after covid-19 we must work on drug discovery and vaccines to prevent TB from spreading.
Objective: This work is done on mycobacterium tuberculosis, the causative agent of tuberculosis, using laboratory experiments and computational biological tools to characterize methyltransferase proteins of Mtb. This study is targeted to find out any potential drug or vaccine target in Mtb.
Method: With the help of bioinformatic tools a hypothetical methyltransferse protein was selected from previously reported variety of 121 methyltransferses according to the domain configuration. The protein was verified by the thermal stability, Protein conformational changes, localization, signal peptide chain, structure prediction and function, mutational analysis and molecular docking.
Results: Rv3366 gene was selected based on its secretory characteristic and localisation in mitochondria among 121 methyltransferases. SignalP4.1 and Mitoprot ll were used for the cleavage site prediction. Protein modelled by online server has 95.6% allowed region and validated by Ramachandran Plot Statistics from Richardsons lab.
The biophysical characteristics of the gene were also identified using methyltransferase assay, thermal denaturation assay, spectrophotometry, fluorescence assay. Alarm assay was done for quantitation of viable cell number in proliferation and cytotoxicity in the presence of the target protein. MitoTracker Red CMXRos kit was used to verify the localisation of the protein in mitochondria.
Conclusion: This study covers essential identification of hypothetical methyltransferase that will help in targeting dataset for future studies leading to drug target and TB control of MTB or probably even MDRTB.
TWAS- the world academy of science amp;amp; DBT- Department of Biotechnology
