Background Neisseria gonorrhoeae, a major STD causing pathogens, tends to pose high burden of morbidity that is borne disproportionately by women and infants with approximately 2/3rd of cases from developing countries. In the absence of appropriate vaccine and rapid, easy, economical test, antibiotic therapy is recommended for treatment on the basis of clinical symptoms. This has led to the emergence of antibiotic resistant strains. Since increasing antimicrobial resistance makes Neisseria as super bug, we have tried to elucidate the mechanism of development of antibiotic resistance.
Methods Mutational analysis of mtrR gene and its DNA binding site was carried out for 28 clinical isolates resistant to multiple drugs. Wild type and mutant mtrR were cloned, expressed and purified. Fluorescence assay and electrophoretic mobility shift assay (EMSA) were carried out to study the effect of mutations in MtrR on its biological activity. Using discovery studio, structure of MtrR was modelled in-silico to understand how mutations affect its interaction with DNA.
Results Mutations in DNA binding domain (G45D) and dimerization domain of MtrR (H105Y) as well as in promoter region of MtrR (A/T deletion) were observed in clinical isolates (n = 28). EMSA and fluorimetric results suggest decreased binding of mutant MtrR with its promoter. In silico modelled structure of wild type and mutant MtrR proteins suggest altered conformation of the mutant protein. Altered conformation leads to difference in the posture of homodimer formed and increased centre to centre distance of helix 1 and helix 1’ in two monomers of mtrR. In silico analysis of protein-DNA complex suggest that this increased distance cause altered binding of the mutant with DNA.
Conclusions Mutations in mtrR result is altered conformation of the protein leading to its decrease binding to DNA. This leads to enhanced expression of MtrCDE efflux pump resulting in increased efflux of drug.
- Neisseria gonorrhoeae