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CRISPRi mediated investigation of the vulnerability of two essential cell wall biosynthetic gene products in mycobacteria.
Thesis (MSc)--Stellenbosch University, 2023.
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| Format: | Thesis |
| Language: | en_ZA en_ZA |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867613750516776960 |
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| access_status_str | Open Access |
| author | Mahlakwana, Gugulethu Boreadi |
| author2 | Mashabela, Gabriel Tshwahla |
| author_browse | Mahlakwana, Gugulethu Boreadi Mashabela, Gabriel Tshwahla |
| author_facet | Mashabela, Gabriel Tshwahla Mahlakwana, Gugulethu Boreadi |
| author_sort | Mahlakwana, Gugulethu Boreadi |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/126959 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA en_ZA |
| last_indexed | 2026-06-10T12:41:06.301Z |
| 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:10019.1/126959 CRISPRi mediated investigation of the vulnerability of two essential cell wall biosynthetic gene products in mycobacteria. Mahlakwana, Gugulethu Boreadi Mashabela, Gabriel Tshwahla Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. CRISPR (Genetics) Drug resistance Anti-infective agents Mycobacterial diseases -- Molecular aspects Tuberculosis -- Prevention Thesis (MSc)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Tuberculosis (TB) is a global health burden and claimed an estimated 1.6 million lives in 2021. Increase in drug resistant TB cases hampers efforts to eradicate the disease, despite availability of treatment regimen. This urges the need for development of new, novel, and effective antimicrobial agents. Polyisoprenyl-phosphate Nacetylaminosugar-1-phosphate transferases (PNPTs), involved in mycobacterial cell wall biosynthesis, are attractive but difficult enzymes to characterize using conventional biochemical assays. In this drug target prioritization study, we aimed to utilize CRISPRi technique to determine and compare vulnerability of two genes encoding PNPTs enzymes, MurX and WecA. The ∆murX (MSMEG_4230) and ∆wecA (MSMEG_4947) knockdown strains were generated using CRISPR interference (CRISPRi) technology in Mycobacterium smegmatis. Mutant characterization was performed by spot and quantitative RT-qPCR assays and morphological analysis was performed by scanning electron microscopy (SEM). The knockdown strains were also exposed to a range of antibiotics for chemical-genetic interaction investigation as well as hostile environment survival such as acidic pH. It was found that exposure of the CRISPRi strains to anhydrotetracycline (ATc) activated CRISPRi system leading to intracellular depletion of murX and wecA by 37% and 99%, respectively, and subsequent loss of fitness. Both mutants displayed similar lag phase, which lasted for 9 hours after activation of the CRISPRi system. However, ∆murX failed to grow beyond 9 hours, instead rapidly lysing from 12 to 18 hours, until OD600 was below detection limit. In contrast, ∆wecA strain continued to grow, albeit lower that the control and only stopped growth 48 hours after activation of the CRISPRi system, and there were no signs of cell lysis. Intracellular depletion of both murX and wecA led to drastic morphological changes, which included cell swelling, incomplete cell division and surface debris, with ∆murX clearly showing signs of cell lysis. The compromised cell wall increased overall hypersensitivity of the ΔmurX and ΔwecA strains to ethambutol and rifampicin. Overall, the study showed that intracellular depletion of murX elicited more fitness cost to the mutants, even when depleted much less, than wecA, thereby making MurX more vulnerable drug target that should be prioritized ahead of its paralog WecA for development of new antimycobacterial drugs. AFRIKAANS OPSOMMING: Tuberkulose (TB) is 'n wêreldwye gesondheidslas en het 'n geraamde 1,6 miljoen lewens geëis in 2021. Toename in dwelmweerstandige TB-gevalle belemmer pogings om die siekte uit te roei, ondanks die beskikbaarheid van behandelingsregime. Dit dring aan op die behoefte aan die ontwikkeling van nuwe, nuwe en doeltreffende antimikrobiese middels. Poliisopreniel-fosfaat N-asetielaminosuiker-1- fosfaattransferases (PNPTs), betrokke by mikobakteriële selwandbiosintese, is aantreklike maar moeilike ensieme om te karakteriseer deur gebruik te maak van konvensionele biochemiese toetse. In hierdie geneesmiddelteikenprioritiseringstudie het ons daarop gemik om CRISPRI-tegniek te gebruik om die kwesbaarheid van twee gene wat vir PNPTs-ensieme, MurX en WecA kodeer, te bepaal en te vergelyk. Die ∆murX (MSMEG_4230) en ∆wecA (MSMEG_4947) knockdown stamme is gegenereer deur gebruik te maak van CRISPR interferensie (CRISPRi) tegnologie in Mycobacterium smegmatis. Mutantkarakterisering is uitgevoer deur kol- en kwantitatiewe RT-qPCR-toetse en morfologiese analise is uitgevoer deur skandeerelektronmikroskopie (SEM). Die knockdown-stamme is ook blootgestel aan 'n reeks antibiotika vir chemiese-genetiese interaksie-ondersoek sowel as vyandige omgewingsoorlewing soos suur pH. Daar is gevind dat blootstelling van die CRISPRistamme aan anhidrotetrasiklien (ATc) die CRISPRi-stelsel geaktiveer het wat gelei het tot intrasellulêre uitputting van murX en wecA met onderskeidelik 37% en 99%, en daaropvolgende verlies aan fiksheid. Beide mutante het soortgelyke vertragingsfase getoon, wat vir 9 uur geduur het na aktivering van die CRISPRI-stelsel. ∆murX kon egter nie langer as 9 uur groei nie, maar het vinnig van 12 tot 18 uur gelys, totdat OD600nm onder die opsporingslimiet was. Daarteenoor het ∆wecA-stam voortgegaan om te groei, alhoewel laer as die kontrole en groei eers 48 uur na aktivering van die CRISPRi-stelsel gestop, en daar was geen tekens van sellise nie. Intrasellulêre uitputting van beide murX en wecA het gelei tot drastiese morfologiese veranderinge, wat sel swelling, onvolledige seldeling en oppervlak puin ingesluit het, met ∆murX wat duidelik tekens van sellise toon. Die gekompromitteerde selwand het algehele hipersensitiwiteit van die ΔmurX- en ΔwecA-stamme vir etambutol en rifampisien verhoog. In die algemeen het die studie getoon dat intrasellulêre uitputting van murX meer fiksheidskoste vir die mutante veroorsaak het, selfs wanneer dit baie minder uitgeput is, as wecA, wat MurX meer kwesbare middelteiken maak wat voor sy paralog WecA geprioritiseer moet word vir die ontwikkeling van nuwe antimikobakteriële middels. Masters 2023-02-20T11:29:36Z 2023-05-18T06:57:38Z 2023-02-20T11:29:36Z 2023-05-18T06:57:38Z 2022-12-01 Thesis http://hdl.handle.net/10019.1/126959 en_ZA en_ZA Stellenbosch University xv, 94 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | CRISPR (Genetics) Drug resistance Anti-infective agents Mycobacterial diseases -- Molecular aspects Tuberculosis -- Prevention Mahlakwana, Gugulethu Boreadi CRISPRi mediated investigation of the vulnerability of two essential cell wall biosynthetic gene products in mycobacteria. |
| title | CRISPRi mediated investigation of the vulnerability of two essential cell wall biosynthetic gene products in mycobacteria. |
| title_full | CRISPRi mediated investigation of the vulnerability of two essential cell wall biosynthetic gene products in mycobacteria. |
| title_fullStr | CRISPRi mediated investigation of the vulnerability of two essential cell wall biosynthetic gene products in mycobacteria. |
| title_full_unstemmed | CRISPRi mediated investigation of the vulnerability of two essential cell wall biosynthetic gene products in mycobacteria. |
| title_short | CRISPRi mediated investigation of the vulnerability of two essential cell wall biosynthetic gene products in mycobacteria. |
| title_sort | crispri mediated investigation of the vulnerability of two essential cell wall biosynthetic gene products in mycobacteria |
| topic | CRISPR (Genetics) Drug resistance Anti-infective agents Mycobacterial diseases -- Molecular aspects Tuberculosis -- Prevention |
| url | http://hdl.handle.net/10019.1/126959 |
| work_keys_str_mv | AT mahlakwanagugulethuboreadi crisprimediatedinvestigationofthevulnerabilityoftwoessentialcellwallbiosyntheticgeneproductsinmycobacteria |