E84G mutation in dihydrofolate reductase from drug resistant strains of Mycobacterium tuberculosis (Mumbai, India) leads to increased interaction with Trimethoprim

Abstract

Background: Dihydrofolate reductase (DHFR) (dfrA gene) is an essential enzyme for cell survival and an unexplored target in Mycobacterium tuberculosis (Mtb). This study was carried out to analyze mutations in the dfrA gene amongst 20 clinical DNA samples from Mtb isolates obtained from Mumbai, India. Methods: Sequencing of the PCR amplified dfrA gene from these DNA isolates revealed a point mutation in one strain, leading to a glutamic acid to glycine change. in silico simulation studies revealed a surface alteration in the enzyme due to this E84G mutation. The amplified mutant gene was cloned and expressed. The mutant protein was assessed against known DHFR inhibitors: Methotrexate and Trimethoprim. Results: An increased affinity for inhibitor Trimethoprim and native substrate dihydrofolate was observed with the mutant. Methotrexate did not vary in its activity with both the enzyme forms. Conclusions: The Glu84Gly point mutation may lead to a variation in the strain which may cause resistance in the future.

Keywords

• Mycobacterium tuberculosis
• Dihydrofolate reductase
• Point mutation
• Molecular docking
• Enzyme assay
• Trimethoprim