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CRISPR interference screening model organisms for TB drugs development.
Thesis (MSc) -- Stellenbosch University, 2022.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867613951504678912 |
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| access_status_str | Open Access |
| author | Masikantsi, Nenekazi |
| author2 | Mashabela, Gabriel |
| author_browse | Mashabela, Gabriel Masikantsi, Nenekazi |
| author_facet | Mashabela, Gabriel Masikantsi, Nenekazi |
| author_sort | Masikantsi, Nenekazi |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc) -- Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/126069 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:18.274Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/126069 CRISPR interference screening model organisms for TB drugs development. Masikantsi, Nenekazi Mashabela, Gabriel Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Tuberculosis -- Prevention Drug development CRISPR activation Antibiotics UCTD Thesis (MSc) -- Stellenbosch University, 2022. ENGLISH ABSTRACT: Tuberculosis remains a global health burden despite the impetus in drug development mainly due to increasing Mycobacterium tuberculosis (M. tb) resistance against the available treatment regimens. There remains an urgent need for development of new antibiotics with distinct mechanisms of action. In the present study, 10 essential gene products, mediating reactions in ten different M. tb metabolic pathways were investigated as potential drug targets for development of antitubercular drugs. CRISPR interference was utilised to deplete intracellular levels of the targets (aceA, argB, asK, folE, ilvA, inhA, panB, ribD, rpoB and thiC) in M. smegmatis as a surrogate for M. tb to produce knockdown strains. The growth phenotype was investigated to evaluate essentiality and vulnerability of these strains. The hypomorphic strains were also treated with clinical antitubercular drugs to investigate their susceptibility and possible chemical-genetic interaction phenotypes. Plant extracts as a potential source for antimicrobial compounds were tested against the hypormophic CRISPRi strains. Activation of the CRISPRi system, through addition of anhydrotetracycline inducer resulted into intracellular depletion of the target genes, which was measured by RT-qPCR. The CRISPRi strains targeting argB, inhA, rpoB, ilvA, asK, and aceA were found to be significantly impaired for growth, demonstrating their requirement for bacterial growth. Most importantly, these genes appeared to be vulnerable, as their transcriptional knockdowns were around 76%, which were in line with previously reported levels for more vulnerable genes. The aspartate kinase (asK) deficient strain was particularly impressive as it had growth impairment despite having only 34% transcriptional gene knockdown. In contrast panB, folE, ribD and thiC CRISPRi strains showed insignificant growth impairment, despite some, such as thiC having the highest transcriptional knockdown at 87%. All the CRISPRi strains were hypersensitive to growth inhibition by selected antitubercular drugs; isoniazid, ethambutol, and rifampicin, suggesting that inhibitors of these enzymes might be compatible with current TB regimen. Hypomorphs of argB, inhA, rpoB and aceA displayed exceptional hypersensitivity to the drugs upon target depletion. Disc diffusion as a screening essay for antimicrobial activity of plant extracts was, however, inconclusive, and required further optimization. Overall, results of this study demonstrated ArgB, InhA, RpoB, IlvA, AsK and AceA as attractive drug targets against which drugs could be developed and evaluated potential new therapeutics to treat TB. CRISPRi strains reported here could therefore be used as screening model in primary drug screening efforts to elucidate mechanism of action of new bioactive compounds. AFRIKAANS OPSOMMING: Tuberkulose bly 'n globale gesondheidslas ten spyte van die stukrag in geneesmiddelontwikkeling hoofsaaklik as gevolg van toenemende Mycobacterium tuberculosis (M. tb)-weerstand teen die beskikbare behandelingsregimes. Daar bly 'n dringende behoefte aan die ontwikkeling van nuwe antibiotika met duidelike werkingsmeganismes. In die hedge studie is 10 noodsaaklike geenprodukte, bemiddelende reaksies in tien verskillende M. tb metaboliese weë ondersoek as potensiële geneesmiddelteikens vir die ontwikkeling van antituberkulêre middels. CRISPR interferensie is gebruik om intrasellulêre vlakke van die teikens (aceA, argB, asK, folE, ilvA, inhA, panB, ribD, rpoB en thiC) in M. smegmatis uit te put as 'n surrogaat vir M. tb om afbreekstamme te produseer. Die groeifenotipe is ondersoek om die noodsaaklikheid en kwesbaarheid van hierdie stamme te evalueer. Die hipomorfiese stamme is ook met kliniese antituberkulêre middels behandel om hul vatbaarheid en moontlike chemies-genetiese interaksie fenotipes te ondersoek. Plantekstrakte as 'n potensiële bron vir antimikrobiese verbindings is getoets teen die hipormofiese CRISPRi-stamme. Aktivering van die CRISPRi-stelsel, deur byvoeging van anhidrotetrasiklien-induseerder, het gelei tot intrasellulêre uitputting van die teikengene, wat deur RT-qPCR gemeet is. Daar is gevind dat die CRISPRi-stamme wat argB, inhA, rpoB, ilvA, asK en aceA teiken aansienlik benadeel is vir groei, wat hul behoefte aan bakteriële groei demonstreer. Die belangrikste is dat hierdie gene kwesbaar blyk te wees, aangesien hul transkripsie-afsakkings ongeveer 76% was, wat in lyn was met voorheen gerapporteerde vlakke vir meer kwesbare gene. Die aspartaatkinase (asK) gebrekkige stam was besonder indrukwekkend aangesien dit groei inkorting gehad het ten spyte van slegs 34% transkripsionele geen-afname. In teenstelling hiermee het panB, folE, ribD en hierdie CRISPRI-stamme onbeduidende groeiverlies getoon, ten spyte daarvan dat sommige, soos thiC die hoogste transkripsie-afname teen 87% gehad het. Al die CRISPRi-stamme was hipersensitief vir groei-inhibisie deur geselekteerde antituberkulêre middels; isoniazid, etambutol en rifampisien, wat daarop dui dat inhibeerders van hierdie ensieme versoenbaar kan wees met die huidige TB-regime. Hipomorfe van argB, inhA, rpoB en aceA het buitengewone hipersensitiwiteit vir die middels getoon by teikenuitputting. Skyfdiffusie as 'n siftingsopstel vir antimikrobiese aktiwiteit van plantekstrakte was egter onbeslis, en het verdere optimalisering vereis. Oor die algemeen het resultate van hierdie studie ArgB, InhA, RpoB, IlvA, AsK en AceA gedemonstreer as aantreklike geneesmiddelteikens waarteen middels ontwikkel kan word en potensiële nuwe terapeutika om TB te behandel, geëvalueer kan word. CRISPRi-stamme wat hier gerapporteer word, kan dus as siftingsmodel gebruik word in primêre dwelmsiftingspogings om die werkingsmeganisme van nuwe bioaktiewe verbindings toe te lig. Masters 2022-11-25T09:39:30Z 2023-01-16T12:48:28Z 2022-11-25T09:39:30Z 2023-01-16T12:48:28Z 2022-11 Thesis http://hdl.handle.net/10019.1/126069 en_ZA Stellenbosch University xvi, 95 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Tuberculosis -- Prevention Drug development CRISPR activation Antibiotics UCTD Masikantsi, Nenekazi CRISPR interference screening model organisms for TB drugs development. |
| title | CRISPR interference screening model organisms for TB drugs development. |
| title_full | CRISPR interference screening model organisms for TB drugs development. |
| title_fullStr | CRISPR interference screening model organisms for TB drugs development. |
| title_full_unstemmed | CRISPR interference screening model organisms for TB drugs development. |
| title_short | CRISPR interference screening model organisms for TB drugs development. |
| title_sort | crispr interference screening model organisms for tb drugs development |
| topic | Tuberculosis -- Prevention Drug development CRISPR activation Antibiotics UCTD |
| url | http://hdl.handle.net/10019.1/126069 |
| work_keys_str_mv | AT masikantsinenekazi crisprinterferencescreeningmodelorganismsfortbdrugsdevelopment |