Masters Degrees (Molecular Biology and Human Genetics)

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Browsing Masters Degrees (Molecular Biology and Human Genetics) by Subject "Antibiotics"

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    CRISPR interference screening model organisms for TB drugs development.
    (Stellenbosch : Stellenbosch University, 2022-11) Masikantsi, Nenekazi; Mashabela, Gabriel; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Tuberculosis remains a global health burden despite the impetus in drug development mainly due to increasing Mycobacterium tuberculosis (M. tb) resistance against the available treatment regimens. There remains an urgent need for development of new antibiotics with distinct mechanisms of action. In the present study, 10 essential gene products, mediating reactions in ten different M. tb metabolic pathways were investigated as potential drug targets for development of antitubercular drugs. CRISPR interference was utilised to deplete intracellular levels of the targets (aceA, argB, asK, folE, ilvA, inhA, panB, ribD, rpoB and thiC) in M. smegmatis as a surrogate for M. tb to produce knockdown strains. The growth phenotype was investigated to evaluate essentiality and vulnerability of these strains. The hypomorphic strains were also treated with clinical antitubercular drugs to investigate their susceptibility and possible chemical-genetic interaction phenotypes. Plant extracts as a potential source for antimicrobial compounds were tested against the hypormophic CRISPRi strains. Activation of the CRISPRi system, through addition of anhydrotetracycline inducer resulted into intracellular depletion of the target genes, which was measured by RT-qPCR. The CRISPRi strains targeting argB, inhA, rpoB, ilvA, asK, and aceA were found to be significantly impaired for growth, demonstrating their requirement for bacterial growth. Most importantly, these genes appeared to be vulnerable, as their transcriptional knockdowns were around 76%, which were in line with previously reported levels for more vulnerable genes. The aspartate kinase (asK) deficient strain was particularly impressive as it had growth impairment despite having only 34% transcriptional gene knockdown. In contrast panB, folE, ribD and thiC CRISPRi strains showed insignificant growth impairment, despite some, such as thiC having the highest transcriptional knockdown at 87%. All the CRISPRi strains were hypersensitive to growth inhibition by selected antitubercular drugs; isoniazid, ethambutol, and rifampicin, suggesting that inhibitors of these enzymes might be compatible with current TB regimen. Hypomorphs of argB, inhA, rpoB and aceA displayed exceptional hypersensitivity to the drugs upon target depletion. Disc diffusion as a screening essay for antimicrobial activity of plant extracts was, however, inconclusive, and required further optimization. Overall, results of this study demonstrated ArgB, InhA, RpoB, IlvA, AsK and AceA as attractive drug targets against which drugs could be developed and evaluated potential new therapeutics to treat TB. CRISPRi strains reported here could therefore be used as screening model in primary drug screening efforts to elucidate mechanism of action of new bioactive compounds.