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Investigation on the functional role of two mycobacterium smegmatis gene products on heme O biosynthesis and translocation
Mangale, Blessing. 2023. Investigation on the functional role of two mycobacterium smegmatis gene products on heme O biosynthesis and translocation. Unpublished master's dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/4e0b0f5e-6dcf-4bb3-a4...
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867613874140741632 |
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| access_status_str | Open Access |
| author | Mangale, Mpatla Blessing |
| author2 | Mashabela, Gabriel Tshwahla
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| author_browse | Mashabela, Gabriel Tshwahla
Mangale, Mpatla Blessing |
| author_facet | Mashabela, Gabriel Tshwahla
Mangale, Mpatla Blessing |
| author_sort | Mangale, Mpatla Blessing |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Mangale, Blessing. 2023. Investigation on the functional role of two mycobacterium smegmatis gene products on heme O biosynthesis and translocation. Unpublished master's dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/4e0b0f5e-6dcf-4bb3-a4cb-2d2bd70e27ab |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/129384 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:43:03.527Z |
| 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:10019.1/129384 Investigation on the functional role of two mycobacterium smegmatis gene products on heme O biosynthesis and translocation Mangale, Mpatla Blessing Mashabela, Gabriel Tshwahla Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Heme -- Metabolism Mycobacterium -- Genetic aspects Drug resistance in microorganisms -- Genetic aspects UCTD Mangale, Blessing. 2023. Investigation on the functional role of two mycobacterium smegmatis gene products on heme O biosynthesis and translocation. Unpublished master's dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/4e0b0f5e-6dcf-4bb3-a4cb-2d2bd70e27ab Thesis (MSc)--Stellenbosch University, 2023. ENGLISH ABSTRACT: The search for novel therapeutic antitubercular agent has become urgent due to the rapid increase in incidences of drug-resistant Mycobacterium tuberculosis, etiological agent of Tuberculosis. Unexplored bacterial systems such as heme A biosynthetic pathway have recently attracted interest of drug target prioritization studies. In the present study, we aimed to use clustered short palindromic repeats interference (CRISPRi) system to investigate the functional role of cyoE and MSMEG_3117 gene products on heme O biosynthesis and translocation, with the ultimate our drug discovery goal of achieving a complete shut-down of heme A via methyerythriotyl-4-phosphate (MEP) pathway, a system through which heme A production in mycobacteria is heavily dependent on. For the proof of concept, we, therefore, hypothesis that CRISPRi knockdown of cyoE gene will results in heme B accumulation leading to heme toxicity, heme A starvation and bacterial killing. Mycobacterium smegmatis cyoE CRISPRi strain was generated and phenotypically characterized in 7H9 glucose media, and the degree of gene repression was assessed by real-time quantitative polymerase chain reaction (RT-qPCR). Our results showed that cyoE was required for optimal growth in M. smegmatis and the hypomorph growth was severely attenuated on solid medium. The cyoE hypomorph had 1.7 times more membrane heme B and displayed higher susceptible to growth inhibition by exogenous iron, H2O2, and KNO2 than the wild type. Additionally, the depletion of cyoE resulted in about 100 x loss in fitness in reduced oxygen levels, thereby confirming disruption of efficient respiratory mechanism. Strikingly, treatment of mutants with KNO2 increased heme B production 4-fold, which increased further in cyoE hypomorph when the gene was depleted. These results strongly suggest that heme A biosynthesis could serve as a promising drug target in combating M. tb and the data gives more insights in understanding biosynthesis of heme O and heme B translocation. AFRIKAANSE OPSOMMING: Die soektog na nuwe terapeutiese antituberkulêre middel het dringend geword as gevolg van die vinnige toename in die voorkoms van middelweerstandige Mycobacterium tuberculosis, etiologiese middel van tuberkulose. Onontginde bakteriële stelsels soos heem A biosintetiese pad het onlangs belangstelling gelok van medisyne-teikenprioritiseringstudies. In die huidige studie het ons ten doel gehad om gegroepeerde kort palindromiese herhalings (CRISPR) interferensiestelsel te gebruik om die funksionele rol van cyoE geenprodukte op heem O biosintese en translokasie te ondersoek, met die uiteindelike doel van ons geneesmiddelontdekking om 'n volledige sluiting van heem te bereik. B via metieritriotiel-4fosfaat (MEP) pad, 'n stelsel waardeur heem B produksie in mikobakterieë sterk afhanklik is. Vir die bewys van konsep, veronderstel ons dus dat CRISPRi-afbreek van cyoE-geen sal lei tot heem Bakkumulasie wat lei tot heem-toksisiteit, heem A-hongersnood en bakteriële dood. Mycobacterium smegmatis cyoE CRISPRi stam is gegenereer en fenotipies gekarakteriseer in 7H9 glukose media, en die mate van geen onderdrukking is geassesseer deur intydse kwantitatiewe polimerase kettingreaksie (RT-qPCR). Ons resultate het getoon dat cyoE benodig word vir optimale groei in M. smegmatis en die hipomorfe groei is ernstig verswak op soliede medium. Die cyoE-hipomorf het 1.7 keer meer membraanheem B gehad en het hoër vatbaar vir groei-inhibisie deur eksogene yster, H2O2 en KNO2 vertoon as die wilde tipe. Daarbenewens het die uitputting van cyoE gelei tot ongeveer 100 x verlies in fiksheid in hipoksiese toestand, wat sodoende ontwrigting van doeltreffende respiratoriese meganisme bevestig. Opvallend genoeg het behandeling van mutante met KNO2 heem B-produksie 4-voudig verhoog, wat verder toegeneem het in cyoE-hipomorf wanneer die geen uitgeput was. Hierdie resultate dui sterk daarop dat heem A-biosintese as 'n belowende geneesmiddelteiken kan dien in die bekamping van M. tb en die data gee meer insigte in die verstaan van biosintese van heem O- en heem B-translokasie. Masters 2023-11-17T12:41:39Z 2024-02-20T09:40:03Z 2023-11-17T12:41:39Z 2024-02-20T09:40:03Z 2023-11 Thesis https://scholar.sun.ac.za/handle/10019.1/129384 en Stellenbosch University xvi, 63 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Heme -- Metabolism Mycobacterium -- Genetic aspects Drug resistance in microorganisms -- Genetic aspects UCTD Mangale, Mpatla Blessing Investigation on the functional role of two mycobacterium smegmatis gene products on heme O biosynthesis and translocation |
| title | Investigation on the functional role of two mycobacterium smegmatis gene products on heme O biosynthesis and translocation |
| title_full | Investigation on the functional role of two mycobacterium smegmatis gene products on heme O biosynthesis and translocation |
| title_fullStr | Investigation on the functional role of two mycobacterium smegmatis gene products on heme O biosynthesis and translocation |
| title_full_unstemmed | Investigation on the functional role of two mycobacterium smegmatis gene products on heme O biosynthesis and translocation |
| title_short | Investigation on the functional role of two mycobacterium smegmatis gene products on heme O biosynthesis and translocation |
| title_sort | investigation on the functional role of two mycobacterium smegmatis gene products on heme o biosynthesis and translocation |
| topic | Heme -- Metabolism Mycobacterium -- Genetic aspects Drug resistance in microorganisms -- Genetic aspects UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/129384 |
| work_keys_str_mv | AT mangalempatlablessing investigationonthefunctionalroleoftwomycobacteriumsmegmatisgeneproductsonhemeobiosynthesisandtranslocation |