Browsing by Author "Mahlakwana, Gugulethu Boreadi"

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    ​​CRISPRi mediated investigation of the vulnerability of two essential cell wall biosynthetic gene products in mycobacteria.
    (Stellenbosch : Stellenbosch University, 2022-12-01) Mahlakwana, Gugulethu Boreadi; Mashabela, Gabriel Tshwahla; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
    ENGLISH ABSTRACT: Tuberculosis (TB) is a global health burden and claimed an estimated 1.6 million lives in 2021. Increase in drug resistant TB cases hampers efforts to eradicate the disease, despite availability of treatment regimen. This urges the need for development of new, novel, and effective antimicrobial agents. Polyisoprenyl-phosphate Nacetylaminosugar-1-phosphate transferases (PNPTs), involved in mycobacterial cell wall biosynthesis, are attractive but difficult enzymes to characterize using conventional biochemical assays. In this drug target prioritization study, we aimed to utilize CRISPRi technique to determine and compare vulnerability of two genes encoding PNPTs enzymes, MurX and WecA. The ∆murX (MSMEG_4230) and ∆wecA (MSMEG_4947) knockdown strains were generated using CRISPR interference (CRISPRi) technology in Mycobacterium smegmatis. Mutant characterization was performed by spot and quantitative RT-qPCR assays and morphological analysis was performed by scanning electron microscopy (SEM). The knockdown strains were also exposed to a range of antibiotics for chemical-genetic interaction investigation as well as hostile environment survival such as acidic pH. It was found that exposure of the CRISPRi strains to anhydrotetracycline (ATc) activated CRISPRi system leading to intracellular depletion of murX and wecA by 37% and 99%, respectively, and subsequent loss of fitness. Both mutants displayed similar lag phase, which lasted for 9 hours after activation of the CRISPRi system. However, ∆murX failed to grow beyond 9 hours, instead rapidly lysing from 12 to 18 hours, until OD600 was below detection limit. In contrast, ∆wecA strain continued to grow, albeit lower that the control and only stopped growth 48 hours after activation of the CRISPRi system, and there were no signs of cell lysis. Intracellular depletion of both murX and wecA led to drastic morphological changes, which included cell swelling, incomplete cell division and surface debris, with ∆murX clearly showing signs of cell lysis. The compromised cell wall increased overall hypersensitivity of the ΔmurX and ΔwecA strains to ethambutol and rifampicin. Overall, the study showed that intracellular depletion of murX elicited more fitness cost to the mutants, even when depleted much less, than wecA, thereby making MurX more vulnerable drug target that should be prioritized ahead of its paralog WecA for development of new antimycobacterial drugs.