Molecular Biology and Human Genetics
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Browsing Molecular Biology and Human Genetics by browse.metadata.advisor "Allie, Nasiema"
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- ItemAntimycobacterial activity of ascidian fungal symbionts(Stellenbosch : Stellenbosch University, 2022-12) Tapfuma, Kudzanai Ian; Mavumengwana, Vuyo; Malgas-Enus, Rehana; Loxton, Andre Gerhard; Allie, Nasiema; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Tuberculosis (TB) is an infectious disease which primarily affects the lungs. Treatment of TB is complicated because the causative agent, Mycobacterium tuberculosis, is an intracellular pathogen which infects and kills cells of the innate immune system, while exhibiting intrinsic and extrinsic resistance to many of the currently available antimicrobial agents. A sizeable percentage of TB patients in the world-population are infected by M. tuberculosis strains which are resistant to currently utilized first- and second-line anti-TB drugs. Drug-discovery studies bioprospecting for compounds with novel anti-TB activities are therefore essential in order to control the spread of TB and to prevent a catastrophic pandemic. In this study, extracts from marine fungi were considered for antimycobacterial activity bioprospecting as they are largely underexplored. A total of 46 cultivable fungi were isolated from ascidians and 32 of these fungal isolates were sequenced and consequently identified. Among these fungi, the methanol crude extract from Clonostachys rogersoniana MGK33 was found to possess the highest antimycobacterial activity with minimum inhibitory concentrations of 0.125 and 0.200 µg/mL against Mycobacterium smegmatis mc2 155 and M. tuberculosis H37Rv, respectively. Untargeted metabolite profiling of the crude extract from C. rogersoniana MGK33 revealed the presence of bionectin F (among other compounds) which has previously been shown to possess antimicrobial activity in other studies. In silico molecular docking and simulation experiments in this study showed that bionectin F is a potential inhibitor of M. tuberculosis β-ketoacyl-ACP reductase (MabA). An attempt was then made to generate novel agents that would be composed of nanoparticles surface functionalized with the bioactive fungal extract from C. rogersoniana MGK33. In particular, mono-metallic SPIONs were synthesized using the co-precipitation method and then surface modified to produce bi-metallic superparamagnetic iron oxide nanoparticles (SPIONs) using nickel, zinc, gold, copper and silver, to produce Ni-SPIONs, Zn-SPIONs, Au-SPIONs, Cu-SPIONs and Ag-SPIONs. Functionalization was then performed using the MGK33 extract to produce Ni-SPIONs@MGK33, Zn-SPIONs@MGK33, Au-SPIONs@MGK33, Cu SPIONs@MGK33 and Ag-SPIONs@MGK33. Among these agents, Cu-SPIONs and Ag SPIONs were found to exhibit the strongest antimycobacterial activity, comparatively stronger than that of the counterparts, Cu-SPIONs@MGK33 and Ag-SPIONs@MGK33. In an experiment involving the treatment of RAW 264.7 macrophage cells infected with M. smegmatis mc2 155, the MGK33 extract exhibited the highest early apoptosis activity (9.61%), followed by Cu-SPIONs@MGK33 (3.34%), both agents tested at 1.96 µg/mL for 24 hours. The MGK33 extract further showed strong antimycobacterial activity against intracellular M. smegmatis mc2 155, compared with the nanoparticles synthesized in this study. Results in this study led to the conclusion that the marine fungus, C. rogersoniana MGK33 is a prolific source of compounds with antimycobacterial and immunomodulatory activity, and that further studies should be done to develop Cu-SPIONs and Ag-SPIONs into lead agents for anti-TB drug development.
- ItemThe 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.
- ItemEvaluation of mycobacterium smegmatis infected d THP-1 macrophages as a model to assess host directed therapy: using apoptotic agents as repurposed anti-tuberculosis drug leads.(Stellenbosch : Stellenbosch University, 2023-03) Ymele Soko, Vivette; Mavumengwana, Vuyo; Loxton, Andre G.; Smith, Liezel; Allie, Nasiema; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Apoptosis is a natural immune protective mechanism that allows the host to clean up nonfunctional cells. Not surprisingly, blocking apoptosis is presented as one of the strategies Mycobacterium tuberculosis (M.tb) uses to avoid host immune defence. In the absence of apoptosis, infected cells are taken captive to support the growth of their invader and eventually progress to an uncontrolled death (necrosis), which in the case of M.tb infection is called caseous necrosis and is characteristic of pathology observed in tuberculosis patients. In this project, host-directed therapy (HDT) was exploited and involved using pro-apoptotic drugs to induce and restore the normal process of controlled cell death (apoptosis) in infected macrophages. The advantage of this strategy is that, unlike current tuberculosis (TB) therapy which is not specific to certain forms of bacteria. Including dormant and multi-resistant M.tb, HDT could affect all forms of bacteria because it does not directly target the pathogen. This project used Mycobacterium smegmatis mc2 155 (M. smegmatis)-infected macrophages as a model to assess the efficacy of selected apoptotic inducer (API) drugs in controlling the intracellular growth of M. smegmatis. The model has been structured so that its product can be cultured on agar plates to evaluate the growth of bacteria representative of the inhibition or growth of intracellular bacteria. Preliminary tests were performed to evaluate the antimicrobial susceptibility of mycobacterium against the selected (API) drugs. Amongst the 12 drugs that were assessed, cepharanthine, CP31398 dihydrochloride hydrate, marinopyrrole A, and nutlin-3a showed activity against M. tuberculosis and M. smegmatis mc2 155. These four drugs have a minimum inhibitory concentration (MIC) of 3.1 - 6.2 µg/mL, 6.2 – 12.5 µg/mL, 25 – 50 µg/mL and 50–100 µg/mL, respectively, against M. smegmatis mc2 155. Also, when tested against M.tb H37Rv, the MIC value of these drugs was one-fold lower than their respective MIC values observed on M. smegmatis. Furthermore, the minimum bactericidal concentration (MBC) test revealed that cepharanthine or CP-31398 dihydrochloride hydrate could kill M. smegmatis at 12.5 µg/mL or 25 µg/mL, respectively. Ex-vivo treatment of M. smegmatis-infected macrophages with tolerated concentrations of API drugs indicated there was some limitations in this host model due to spontaneous inhibition of M. smegmatis growth in THP-1 macrophages. Nonetheless, it was possible to test the efficacy of drugs intended for HDT using the difference in the relative survival of intracellular bacteria as indicators. Cepharanthine and CP-31398 dihydrochloride hydrate inhibited the intracellular growth of the bacteria after 12hrs treatments at a concentration representing half of their MIC value or one-quarter of their MBC value. Higher concentrations of these drugs, for instance, 6.2 µg/mL of cepharanthine; or 9.2 µg/mL of CP-31398 dihydrochloride hydrate, resulted in the absence of growth of intracellular bacteria after only 6 hrs treatment. Multi-cytokines analysis on the cell culture supernatants sampled during the treatment of macrophages indicated that mild expression of IFN-gamma, TNF-alpha, IL-1beta, IL-5, combined with the inhibition of IL-6 and IL-22 was required to eliminate intracellular M. smegmatis mc2 155. However, high levels of TNF-alpha, IL-1beta, IL-6, and IL-22 coupled with the absence of expression of IFN-gamma and IL-5 correlated with a high burden of intracellular bacteria.
- ItemInvestigation of the antimycobacterial activity of fungal endophytes and their magnetic dendrimer stabilized nanoparticles against mycobacteria(Stellenbosch : Stellenbosch University, 2024-03) Hussan, Raeesa Hoosen; Mavumengwana, Vuyo; Malgas-Enus, Rehana; Loxton, Andre Gareth; 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 disease caused by a single infectious agent Mtb, remains a global health threat that claimed the lives of 1.4 million people worldwide between 2020- 2021. Despite current anti tuberculosis treatment, TB remains one of the leading causes of infection highlighting the imminent need for drug discovery of alternative and novel treatments with distinct modes of action against Mycobacteria. In this context, the present study investigated the antimycobacterial activities of fungal endophyte metabolites functionalized on magnetic dendrimer stabilized nanoparticles (DSNPs) against mycobacterial models. To achieve the aim, fungal endophytes were isolated from four Fynbos families (Asteraceae, Lamiaceae, Ericaceae, and Droseraceae), sequenced and identified by phylogeny. Methanolic crude extracts obtained from cultivated fungi were tested for antimycobacterial activity against Mycobacterium tuberculosis H37Rv (Mtb H37Rv) and Mycobacterium smegmatis mc2155 (Msmeg mc2155). Metabolites within the bioactive fungal extracts were identified by untargeted liquid chromatography-mass spectrometry (LC-QTOF-MS/MS). The infection of THP-1 macrophage cells with Msmeg mc2155 was performed to determine the intracellular antimycobacterial activity of fungal crude extracts. The DSNPs were synthesized from modified magnetic iron oxide nanoparticles (MIONs) and G3-dendrimer micelles. Microscopic and spectral techniques (SEM, FTIR, UV-Vis, and ICP-AES) were used to characterize nanoparticles, followed by antimycobacterial activity assays. Twenty unique fungal isolates were identified with Penicillium being the abundant genera. Penicillium thomii, Diaporthe leucospermi, Penicillium rubens, Cadophora sp. and Penicillium sp. possess antimycobacterial activity. Cadophora sp. demonstrated the most significant inhibition of Msmeg mc2155 and the bioactivity was enhanced in the host-directed approach against intracellular Msmeg mc2155. The metabolites N-benzyl-1-tetradecanamine, N-benzyl-1-hexadecanamine, asperthecin, meleagrin, roquerfortine F, chlamydocin and neoxaline, (-)5-methylmellein, and 9-octadecenamide were identified with the latter two possessing antimycobacterial activity. The nanoparticles (G3- dendrimer micelles, MIONs and DSNPs) were successfully synthesized. G3-dendrimer micelles and DNSPs inhibited Msmeg mc2155 and the former Mtb H37Rv. MIONs possessed enhanced bioactivities against intracellular Msmeg mc2155. Functionalization of DSNPs with bioactive metabolites (nano metabolites) demonstrated no antimycobacterial activity against Msmeg mc2155 directly but antimycobacterial activity was observed intracellularly. Enhanced activity of nano-metabolites was observed directly against Mtb H37RV. Fungal extracts, nanoparticles and functionalized nanoparticles have distinct mechanisms of antimycobacterial activities against the strains of mycobacteria (Msmeg mc2155 and Mtb H37Rv). This study highlighted that the properties of nanoparticles greatly influence their antimycobacterial activities i.e., the ferrous content of a naked nanoparticle opposed to a modified nanoparticle. Functionalization of a nanoparticle contributes to its modification which has an impact on the antimycobacterial activity. Hence, this necessitates further investigations about nanoparticles, their properties and modifications against mycobacterial models. In addition, the nanoparticles have the potential to be modified to accommodate or scaffold existing anti-Tb drugs and to potentiate antimycobacterial effects through pathogen- and host directed approaches.