Masters Degrees (Molecular Biology and Human Genetics)

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Browsing Masters Degrees (Molecular Biology and Human Genetics) by Subject "Anti-infective agents"

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    The antimycobacterial activity of phytocannabinoids
    (Stellenbosch : Stellenbosch University, 2023-02) Williams, Ricquelle Daphne; Mavumengwana, Vuyo; Loxton, Andre; Smith, Liezel; Allie, Nasiema; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Tuberculosis (TB) is a deadly and communicable disease that is caused by the bacterium, Mycobacterium tuberculosis (M.tb). M.tb is skilled at manipulating and evading hostdefense mechanisms by alveolar macrophages, allowing its survival and replication intracellularly. Since the current treatment regimen makes use of antibiotics, the everincreasing number of multi-drug resistant (MDR) M.tb strains has resulted in a need for research into alternative options. Although most anti-TB drug discovery strategies target the actual pathogen, slow pace in discovering new antimycobacterials is testament to the need to change strategies. Host directed therapy (HDT) is an intervention where the eradication of the intracellular pathogen is mediated by the host immune response modulated by small molecules. Cannabis sativa L. (C. sativa) is valued for its psychoactive potentials and varied ethnobotanical medicinal properties due to a plethora of bioactive constituents. In addition to the plant’s utility as a treasure trove for medicinal applications, this research study aims to present a case for the use of small molecules derived from C. sativa as an alternative HDT against TB. We aimed to evaluate the antimycobacterial effect of crude extracts of two C. sativa plants, one grown outdoor (C. sativa plant 1 or P1) and one grown under controlled indoor conditions (C. sativa plant 2 or P2) and evaluated their bioactive organic extracts activity in THP-1 macrophages infected with mycobacteria. In addition, we isolated endophytic fungi from C. sativa and evaluated their antimycobacterial activity and whether they produce similar compounds to the host plant. Herein, it was demonstrated that the dichloromethane (DCM) extract of C. sativa plant 1 (DP1) and the methanol and ethyl acetate extracts of C. sativa plant 2 (MP2 and EP2) stimulated THP-1 macrophages in the killing of Mycobacterium smegmatis (M. smegmatis) mc2155 compared to untreated macrophages. In addition, the methanol extract of C. sativa plant 2 (MP2) displayed the best activity with a percentage survival of 14.31% (p = 0.000009) at 6-hours post treatment; 2.63% at 12-hours post treatment (p = 0.0001) and 0% survival at 24-hours post treatment (p = 0.0005). The metabolite profile showed that these three extracts (DP1, MP2 and EP2) share three compounds, cannabinol (CBN), cannabigerol (CBG), and cannabielsoin (CBE), which could be the cause of macrophage stimulation. However, MP2, which showed the best activity, contains cannabidiol (CBD), which could be the cannabinoid causing the increased activity. Although the actual bioactivities of CBN, CBG, CBE and CBD remain speculative until further purification, characterization and reevaluations are carried out, a cautious judgement can be made that these compounds likely play a beneficial role. Fungal endophytes Alternaria alternata (A. alternata), Alternaria infectoria (A. infectoria), Fusarium incarnatum (F. incarnatum), and Fusarium chlamydosporum (F. chlamydosporum) were isolated from surface sterilized buds of C. sativa and identified using molecular and phylogenetic methods. A. alternata showed some compounds (via LC-QTOF-MS) which were previously isolated in C. sativa, with its extracts exhibiting immunomodulatory activity against THP-1 macrophages infected with M. smegmatis mc2155. The percentage survival for treated THP-1 cells was found to be 51.81% at 6 hours (p = 0.0003) compared to 81.91% untreated. Overall, these results establish that cannabinoids are able to influence THP-1 infected cells’ ability to clear mycobacterial infection albeit a low percentage cell survival. To develop specialized HDT strategies targeting macrophages, a deeper comprehension of the mutual interaction between cannabis and immunity is required. Future studies to isolate and characterize compound(s) from the endophytic fungi and plant extracts could result in lead development of naturally sourced drugs for host-directed TB treatment.
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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.