Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
Targeting MEP pathway for the development of antitubercular drugs
Thesis (MSc)--Stellenbosch University, 2022.
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | en_ZA |
| Published: |
Stellenbosch : Stellenbosch University
2022
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867613907935297536 |
|---|---|
| access_status_str | Open Access |
| author | Maila, Tumelo |
| author2 | Mashabela, Gabriel Tshwahla |
| author_browse | Maila, Tumelo Mashabela, Gabriel Tshwahla |
| author_facet | Mashabela, Gabriel Tshwahla Maila, Tumelo |
| author_sort | Maila, Tumelo |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/125109 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:43:36.943Z |
| 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:10019.1/125109 Targeting MEP pathway for the development of antitubercular drugs Maila, Tumelo Mashabela, Gabriel Tshwahla Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Antitubercular agents -- South Africa Tuberculosis -- Mortality -- South Africa Intracellular pathogens -- South Africa Metabolites -- South Africa MEP pathway Thesis (MSc)--Stellenbosch University, 2022. ENGLISH ABSTRACT: Background: Tuberculosis (TB) still remains a global health burden with an estimated 1.4 million mortalities in 2019. Despite availability of drug regimens aimed at eradication of TB, the cases of drug-resistant TB continue to increase, reflecting the urgent need for development of new drugs with novel mechanisms of action. To this end, DXS, the first enzyme of the MEP pathway, a unique metabolic pathway used to produce essential isoprenoids in Mycobacteria, was investigated as potential drug target. Methodology: In the recent study, we utilized CRISPR interference technology to deplete intracellular levels of dxs gene (MSM_2776) in Mycobacterium smegmatis to generate a CRISPRi strain, dxs(-), performed quantitative and spot assays to evaluate the gene essentiality and vulnerability. The dxs hypomorph was treated with different compounds to reveal potential chemical-genetic interaction phenotypes. Ability of the mutant to grow under unfavourable pH conditions was also investigated. Results: Activation of the CRISPR interference system through addition of anhydrotetracycline (ATc) resulted in more than 65-fold intracellular dxs depletion, which led to strong growth inhibition in Mycobacterium smegmatis. The hypomorph was also found to be more susceptible to acidic conditions. The mutants were more hypersensitive to nitric oxide than hydrogen peroxide. Additionally, the loss of dxs significantly potentiated antimycobacterial effect of ethambutol while did not change no sensitivity to isoniazid and rifampicin. Most importantly, dxs(-) growth phenotype could not be rescued by exogenous MEP pathway metabolites, with only minimal growth rescue observed with addition of menaquinone derivative. Conclusion: The results showed that intracellular depletion of dxs leads to Mycobacterium growth arrest and the enzyme showed to be essential and vulnerable for bacterial growth. AFRIKAANSE OPSOMMING: Agtergrond: Tuberkulose (TB) bly steeds 'n wêreldwye gesondheidslas met 'n geraamde 1,4 miljoen sterftes in 2019. Ten spyte van die beskikbaarheid van dwelmregimes wat daarop gemik is om TB uit te wis, neem die gevalle van middelweerstandige TB steeds toe, wat die dringende behoefte aan die ontwikkeling van nuwe middels met nuwe meganismes van aksie weerspieël. Vir hierdie doel, DXS, die eerste ensiem van die MEP pad, 'n unieke metaboliese pad wat gebruik word om noodsaaklike isoprenoïede in Mycobacteria te produseer, is ondersoek as potensiële dwelm teiken. Metodologie: In die onlangse studie het ons CRISPR-inmengingstegnologie gebruik om intrasellulêre vlakke van dxs-geen (MSM_2776) in Mycobacterium smegmatis uit te put om 'n CRISPRi-stam, dxs(-), uitgevoerde kwantitatiewe en plek-assays te genereer om die geennoodsaaklikheid en kwesbaarheid te evalueer. Die dxs hipomorf is behandel met verskillende verbindings om potensiële chemiese-genetiese interaksie fenotipes te openbaar. Die vermoë van die mutant om onder ongunstige pH-toestande te groei, is ook ondersoek. Resultate: Aktivering van die CRISPR inmenging stelsel deur die toevoeging van anhydrotetracycline (ATc) het gelei tot meer as 65-vou intrasellulêre dxs uitputting, wat gelei het tot sterk groei inhibisie in Mycobacterium smegmatis. Daar is ook gevind dat die hipomorf meer vatbaar is vir suur toestande. Die mutante was meer hipersensitief vir stikstofoksied as waterstofperoksied. Daarbenewens het die verlies dxs aansienlik versterk mycobacterial effek ethambutol terwyl die verandering van geen sensitiwiteit vir isoniazid en rifampicin. Die belangrikste is dat dxs(-) groei fenotipe nie gered kon word deur eksogene MEP pad metaboliete, met slegs minimale groei redding waargeneem met toevoeging van menaquinone afgeleide. Gevolgtrekking: Die resultate het getoon dat intrasellulêre uitputting van dxs lei tot Mycobacterium groei arrestasie en die ensiem het getoon dat dit noodsaaklik en kwesbaar is vir bakteriese groei. Masters 2022-02-23T12:26:05Z 2022-04-29T12:54:21Z 2022-08-23T03:00:11Z 2022-02 Thesis http://hdl.handle.net/10019.1/125109 en_ZA Stellenbosch University iv, 87 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Antitubercular agents -- South Africa Tuberculosis -- Mortality -- South Africa Intracellular pathogens -- South Africa Metabolites -- South Africa MEP pathway Maila, Tumelo Targeting MEP pathway for the development of antitubercular drugs |
| title | Targeting MEP pathway for the development of antitubercular drugs |
| title_full | Targeting MEP pathway for the development of antitubercular drugs |
| title_fullStr | Targeting MEP pathway for the development of antitubercular drugs |
| title_full_unstemmed | Targeting MEP pathway for the development of antitubercular drugs |
| title_short | Targeting MEP pathway for the development of antitubercular drugs |
| title_sort | targeting mep pathway for the development of antitubercular drugs |
| topic | Antitubercular agents -- South Africa Tuberculosis -- Mortality -- South Africa Intracellular pathogens -- South Africa Metabolites -- South Africa MEP pathway |
| url | http://hdl.handle.net/10019.1/125109 |
| work_keys_str_mv | AT mailatumelo targetingmeppathwayforthedevelopmentofantituberculardrugs |