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Effect of biological oxidants on the functioning of MEP pathway’s [Fe-S] cluster-dependent enzymes
Tukur, Nuhu Ibrahim. 2023. Effect of biological oxidants on the functioning of MEP pathway’s [Fe-S] cluster-dependent enzymes. Unpublished masters dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/ce673701-e16c-416e-9dde-722184fe8b85
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867614068915830784 |
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| access_status_str | Open Access |
| author | Tukur, Nuhu Ibrahim |
| author2 | Mashabela, Gabriel Tshwahla
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| author_browse | Mashabela, Gabriel Tshwahla
Tukur, Nuhu Ibrahim |
| author_facet | Mashabela, Gabriel Tshwahla
Tukur, Nuhu Ibrahim |
| author_sort | Tukur, Nuhu Ibrahim |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Tukur, Nuhu Ibrahim. 2023. Effect of biological oxidants on the functioning of MEP pathway’s [Fe-S] cluster-dependent enzymes. Unpublished masters dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/ce673701-e16c-416e-9dde-722184fe8b85 |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/129401 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:46:10.315Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/129401 Effect of biological oxidants on the functioning of MEP pathway’s [Fe-S] cluster-dependent enzymes Tukur, Nuhu Ibrahim Mashabela, Gabriel Tshwahla Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Multidrug-resistant tuberculosis Oxidation, Physiological Drug resistance UCTD Tukur, Nuhu Ibrahim. 2023. Effect of biological oxidants on the functioning of MEP pathway’s [Fe-S] cluster-dependent enzymes. Unpublished masters dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/ce673701-e16c-416e-9dde-722184fe8b85 Thesis (MSc)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Deleterious setbacks such as multi-drug resistant tuberculosis (TB) have retarded the world-TB eradication strategy, hence the need for emphasis on the development of novel anti-TB drugs. Using a target-based screening approach, our study was aimed at determining whether biological oxidants disrupt the functioning of the MEP pathway [Fe-S] cluster-dependent enzymes (IspG and IspH). Potential growth rescue of ispG and ispH hypomorphs with MEP pathway downstream metabolites was assessed and intracellular and extracellular levels of MEP pathway intermediates were quantified in cultures treated with nitric oxide, H₂O₂, and cadmium. In addition, we assessed the ability of ispG and ispH hypomorphs to survive in activated macrophages. Our first results validated the previous observation that intracellular depletion of IspG and IspH led to a strong growth repression, thereby confirming the essentiality and vulnerability of the encoded enzymes, IspG, IspH, respectively. Importantly, we established that growth inhibition in ispG and ispH hypomorphs corresponded with a 4-fold reduction in intracellular levels of isopentenyl pyrophosphate (IPP), which could be further exacerbated by the addition of hydrogen peroxide (H₂O₂). Moreover, our result showed that MEP pathway product, IPP, and immediate downstream products, geranyl pyrophosphate and farnesyl pyrophosphate failed to rescue the hypomorphs’ growth. Although the hypomorphs showed modest sensitivity to NO source, they displayed high sensitivity to reactive oxygen species. Comparatively, ispH hypomorph was 4 times more susceptible to H₂O₂ and cadmium (superoxide source), than ispG hypomorph. Furthermore, our findings indicated that macrophages survived better when infected with ispG and ispH hypomorphs treated with ATc compared to the untreated, with IFN-γ activated macrophages (M1) exhibiting even greater survival rates. Importantly, intracellular depletion of IspG and IspH rendered the mutants more susceptible to killing by activated macrophages. Finally, we showed that the M. tuberculosis-produced MEP pathway metabolite, HMBPP, is cytotoxic to THP-1 macrophages. In summary, our study offered valuable insights into the intricate connection between oxidative stress, MEP pathway, and mycobacterial pathogenicity. Additionally, it sheds light on the role played by the MEP pathway in mycobacterial survival and highlights potential targets for therapeutic interventions. AFRIKAANSE OPSOMMING: Skadelike terugslae soos multi-middelweerstandige tuberkulose (TB) het die wêreld-TBuitwissingstrategie vertraag, vandaar die behoefte aan klem op die ontwikkeling van nuwe antiTB-middels. Met behulp van 'n teikengebaseerde siftingsbenadering was ons studie daarop gemik om te bepaal of biologiese oksidante die werking van die MEP-pad [Fe-S] clusterafhanklike ensieme (IspG en IspH) ontwrig. Potensiële groeiredding van ispG- en ispH-hipomorfe met MEPpad stroomaf metaboliete is beoordeel en intrasellulêre en ekstrasellulêre vlakke van MEPpadintermediêres is gekwantifiseer in kulture wat met stikstofoksied, H2O2 en kadmium behandel is. Daarbenewens het ons die vermoë van ispG- en ispH-hipomorfe om in geaktiveerde makrofage te oorleef, beoordeel. Ons eerste resultate het die vorige waarneming bekragtig dat intrasellulêre uitputting van ispG en ispH gelei het tot 'n sterk groeionderdrukking, waardeur die noodsaaklikheid en kwesbaarheid van die gekodeerde ensieme, IspG, IspH, onderskeidelik, bevestig is. Wat belangrik is, is dat ons vasgestel het dat groeiremming in ispG- en ispH-hipomorfe ooreenstem met 'n 4- voudige vermindering in intrasellulêre vlakke van isopentenielpyrofosfaat (IPP), wat verder vererger kan word deur die toevoeging van waterstofperoksied (H2O2). Daarbenewens het ons resultaat getoon dat MEP-padproduk, IPP en onmiddellike stroomafprodukte, geranielpyrofosfaat en farnesielpyrofosfaat nie die hipomorfe groei kon red nie. Alhoewel die hipomorfe beskeie sensitiwiteit vir GEEN bron getoon het nie, het hulle 'n hoë sensitiwiteit vir reaktiewe suurstofspesies getoon. Vergelykenderwys was ispH hipomorf 4 keer meer vatbaar vir H2O2 en kadmium (superoksiedbron), as ispG hipomorf. Verder het ons bevindings aangedui dat makrofage beter oorleef het as dit besmet is met ispG- en ispH-hipomorfe as die wilde tipe, met IFN-γ geaktiveerde makrofage (M1) wat selfs groter oorlewingsyfers toon. Wat belangrik is, is dat intrasellulêre uitputting van ispG en ispH die mutante meer vatbaar gemaak het vir doodmaak deur geaktiveerde makrofage. Laastens het ons getoon dat die M. tuberkulose-vervaardigde MEP-padmetaboliet, HMBPP, sitotoksies is vir THP1 makrofage. Samevattend het ons studie waardevolle insigte gebied in die ingewikkelde verband tussen oksidatiewe stres, MEP-pad en mikobakteriese patogenisiteit. Daarbenewens werp dit lig op die rol wat die MEP-pad in mikobakteriese oorlewing speel en beklemtoon potensiële teikens vir terapeutiese intervensies. Masters 2023-11-17T21:54:45Z 2024-02-20T10:17:11Z 2023-11-17T21:54:45Z 2024-02-20T10:17:11Z 2023-11 Thesis https://scholar.sun.ac.za/handle/10019.1/129401 en Stellenbosch University xiii, 59 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Multidrug-resistant tuberculosis Oxidation, Physiological Drug resistance UCTD Tukur, Nuhu Ibrahim Effect of biological oxidants on the functioning of MEP pathway’s [Fe-S] cluster-dependent enzymes |
| title | Effect of biological oxidants on the functioning of MEP pathway’s [Fe-S] cluster-dependent enzymes |
| title_full | Effect of biological oxidants on the functioning of MEP pathway’s [Fe-S] cluster-dependent enzymes |
| title_fullStr | Effect of biological oxidants on the functioning of MEP pathway’s [Fe-S] cluster-dependent enzymes |
| title_full_unstemmed | Effect of biological oxidants on the functioning of MEP pathway’s [Fe-S] cluster-dependent enzymes |
| title_short | Effect of biological oxidants on the functioning of MEP pathway’s [Fe-S] cluster-dependent enzymes |
| title_sort | effect of biological oxidants on the functioning of mep pathway s fe s cluster dependent enzymes |
| topic | Multidrug-resistant tuberculosis Oxidation, Physiological Drug resistance UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/129401 |
| work_keys_str_mv | AT tukurnuhuibrahim effectofbiologicaloxidantsonthefunctioningofmeppathwaysfesclusterdependentenzymes |