Antimycobacterial activity of ascidian fungal symbionts

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dc.contributor.advisorMavumengwana, Vuyoen_ZA
dc.contributor.advisorMalgas-Enus, Rehanaen_ZA
dc.contributor.advisorLoxton, Andre Gerharden_ZA
dc.contributor.advisorAllie, Nasiemaen_ZA
dc.contributor.authorTapfuma, Kudzanai Ianen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.en_ZA
dc.date.accessioned2022-10-18T17:32:52Zen_ZA
dc.date.accessioned2023-01-16T12:43:11Zen_ZA
dc.date.available2022-10-18T17:32:52Zen_ZA
dc.date.available2023-01-16T12:43:11Zen_ZA
dc.date.issued2022-12en_ZA
dc.descriptionThesis (PhD) -- Stellenbosch University, 2022.en_ZA
dc.description.abstractENGLISH 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.en_ZA
dc.description.abstractAFRIKAANS OPSOMMING: Tuberkulose (TB) is 'n aansteeklike siekte wat hoofsaaklik die longe affekteer. Behandeling van TB is ingewikkeld omdat die veroorsakende middel, Mycobacterium tuberculosis (M. Tuberculosis), 'n intrasellulêre patogeen is wat selle van die aangebore immuunstelsel infekteer en doodmaak, terwyl dit intrinsieke en ekstrinsieke weerstand teen baie van die tans beskikbare antimikrobiese middels toon. 'n Aansienlike persentasie TB-pasiënte in die wêreldbevolking is besmet met M. tuberculosis-stamme wat weerstand bied teen tans gebruikte eerste- en tweedelyn-anti-TB-middels. Geneesmiddel-ontdekkingstudies bioprospektering vir verbindings met nuwe anti-TB-aktiwiteite is dus noodsaaklik om die verspreiding van TB te beheer en 'n katastrofiese pandemie te voorkom. In hierdie studie is ekstrakte van mariene swamme met antimikobakteriële aktiwiteit oorweeg vir bioprospektering aangesien hulle grootliks onderontgin is. 'n Totaal van 46 kweekbare swamme is uit ascidians geïsoleer en 32 van hierdie swam-isolate is gevolglik geïdentifiseer. Onder hierdie swamme het die metanol ru-ekstrak van Clonostachys rogersoniana (C. Rogersoniana) MGK33 die hoogste antimikobakteriese aktiwiteit met minimum inhiberende konsentrasies van 0.125 en 0.200 µg/mL teen Mycobacterium smegmatis mc2 155 en M. tuberculosis H37Rv onderskeidelik getoon. Ongeteikende metabolietprofilering van die ru ekstrak van C. rogersoniana MGK33 het die teenwoordigheid van bionektien F (onder ander verbindings) bevestig wat in vorige studies getoon het om antimikrobiese aktiwiteit te besit. In silico molekulêre dok- en simulasie-eksperimente in hierdie studie het getoon dat bionektien F 'n potensiële inhibeerder van M. tuberculosis β-ketoacyl-ACP reduktase (MabA) is. 'n Poging is aangewend om nuwe middels te genereer wat bestaan uit nanopartikels wat met die bioaktiewe swamekstrak van C. rogersoniana MGK33 gefunksionaliseer is. ‘n Mono metaal SPIONs is gesintetiseer deur gebruik te maak van die mede-presipitasie metode. Die oppervlak is gemodifiseer om bi-metaal SPIONs te produseer deur nikkel, sink, goud, koper en silwer te gebruik om Ni-SPIONs, Zn-SPIONs, Au-SPIONs, Cu-SPIONs en Ag-SPIONs te produseer. Funksionalisering is dan uitgevoer met die MGK33-ekstrak om Ni SPIONs@MGK33, Zn-SPIONs@MGK33, Au-SPIONs@MGK33, Cu-SPIONs@MGK33 en Ag-SPIONs@MGK33 te produseer. Onder hierdie middels is gevind dat Cu-SPIONs en Ag SPIONs die sterkste antimikobakteriële aktiwiteit toon, betreklik sterker as dié van die eweknieë, Cu-SPIONs@MGK33 en Ag-SPIONs@MGK33. In 'n eksperiment waar M. smegmatis mc2 155 geïnfekteerde RAW 264.7 makrofaagselle M. smegmatis mc2 155 behandel was het die MGK33-ekstrak die hoogste vroeë apoptose-aktiwiteit (9.61%) getoon, gevolg deur Cu-SPIONs@MGK33 (3.34%), albei middels getoets teen 1.96 µg /mL vir 24 uur. Die MGK33-ekstrak het verder sterk antimikobakteriële aktiwiteit teen intrasellulêre M. smegmatis mc2 155 getoon, in vergelyking met die nanopartikels wat in hierdie studie gesintetiseer is. Resultate in hierdie studie het gelei tot die gevolgtrekking dat die mariene swam, C. rogersoniana MGK33 'n produktiewe bron van verbindings met antimikobakteriese en immunomodulerende aktiwiteit is, en dat verdere studies gedoen behoort te word om Cu SPIONs en Ag-SPIONs te ontwikkel tot hoofmiddels vir anti -TB geneesmiddel ontwikkelin.af_ZA
dc.description.versionDoctoralen_ZA
dc.format.extentxix, 160 pages : illustrationsen_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/125961en_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subjectTuberculosis -- Treatmenten_ZA
dc.subjectMarine fungien_ZA
dc.subjectNanoparticlesen_ZA
dc.subjectSecondary metabolitesen_ZA
dc.subjectAnti-infective agentsen_ZA
dc.subjectNatural immunityen_ZA
dc.subjectAntimycobacterial activityen_ZA
dc.subjectUCTDen_ZA
dc.titleAntimycobacterial activity of ascidian fungal symbiontsen_ZA
dc.typeThesisen_ZA
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Doctoral Degrees (Molecular Biology and Human Genetics)