Masters Degrees (Molecular Biology and Human Genetics)
Permanent URI for this collection
Browse
Browsing Masters Degrees (Molecular Biology and Human Genetics) by Subject "Antitubercular agents -- South Africa"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- ItemTargeting MEP pathway for the development of antitubercular drugs(Stellenbosch : Stellenbosch University, 2022-02) Maila, Tumelo; Mashabela, Gabriel Tshwahla; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Background: Tuberculosis (TB) still remains a global health burden with an estimated 1.4 million mortalities in 2019. Despite availability of drug regimens aimed at eradication of TB, the cases of drug-resistant TB continue to increase, reflecting the urgent need for development of new drugs with novel mechanisms of action. To this end, DXS, the first enzyme of the MEP pathway, a unique metabolic pathway used to produce essential isoprenoids in Mycobacteria, was investigated as potential drug target. Methodology: In the recent study, we utilized CRISPR interference technology to deplete intracellular levels of dxs gene (MSM_2776) in Mycobacterium smegmatis to generate a CRISPRi strain, dxs(-), performed quantitative and spot assays to evaluate the gene essentiality and vulnerability. The dxs hypomorph was treated with different compounds to reveal potential chemical-genetic interaction phenotypes. Ability of the mutant to grow under unfavourable pH conditions was also investigated. Results: Activation of the CRISPR interference system through addition of anhydrotetracycline (ATc) resulted in more than 65-fold intracellular dxs depletion, which led to strong growth inhibition in Mycobacterium smegmatis. The hypomorph was also found to be more susceptible to acidic conditions. The mutants were more hypersensitive to nitric oxide than hydrogen peroxide. Additionally, the loss of dxs significantly potentiated antimycobacterial effect of ethambutol while did not change no sensitivity to isoniazid and rifampicin. Most importantly, dxs(-) growth phenotype could not be rescued by exogenous MEP pathway metabolites, with only minimal growth rescue observed with addition of menaquinone derivative. Conclusion: The results showed that intracellular depletion of dxs leads to Mycobacterium growth arrest and the enzyme showed to be essential and vulnerable for bacterial growth.