Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
Validation of the MEP pathway’s [Fe-S] cluster enzymes as drug targets in mycobacteria
Thesis (MSc)--Stellenbosch University, 2021.
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | en_ZA |
| Published: |
Stellenbosch : Stellenbosch University
2021
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867613954705981440 |
|---|---|
| access_status_str | Open Access |
| author | Nicolaai, Mischke |
| author2 | Mashabela, Gabriel |
| author_browse | Mashabela, Gabriel Nicolaai, Mischke |
| author_facet | Mashabela, Gabriel Nicolaai, Mischke |
| author_sort | Nicolaai, Mischke |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2021. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/124247 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:21.236Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/124247 Validation of the MEP pathway’s [Fe-S] cluster enzymes as drug targets in mycobacteria Nicolaai, Mischke Mashabela, Gabriel Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Tuberculosis -- Pathogenesis Scanning electron microscopy Drug targeting UCTD Thesis (MSc)--Stellenbosch University, 2021. ENGLISH ABSTRACT: Tuberculosis is the leading cause of mortality because of a single microorganism. In part, this is due to the increase in drug-resistant TB. Thus, new drugs with novel mechanisms of action are needed. In a quest to find new drug targets, CRISPRi technology was used to investigate the vulnerability of IspG and IspH, the two [Fe-S] cluster-dependent enzymes found in the methylerythritol phosphate pathway, which display a high level of homology amongst mycobacterial species. Activation of the CRISPRi system for 24 hours in Mycobacterium smegmatis strains, resulted in over 96 % gene knockdown for both genes. The intracellular gene depletion led to severe growth impairment of the hypomorphs, thereby confirming the essentiality of both gene products in standard growth medium. Vulnerability of IspG and IspH was also sustained in growth medium supplemented with different carbon sources, with partial growth rescue only observed on ispG and ispH hypomorphs when alanine and succinate were used as the main carbon sources, respectively. Extensive clumping was observed in the liquid cultures of the ispG hypomorph and scanning electron microscopy (SEM) subsequently revealed a large number of particles on the surfaces of the cells. Notably, the ispG and ispH hypomorphs were hypersensitive to nitric oxide, a product of an activated human immune system. Although gene depletion failed to potentiate the antimycobacterial effect of the two first-line TB antibiotics, isoniazid and rifampicin, both hypomorphs were highly sensitive to the sub-lethal concentration of ethambutol and the ispH hypomorph potentiated the effect of tunicamycin. This suggests that potential inhibitors of either enzyme could be used to improve the efficacy of the standard TB treatment regimen. The results of this study, therefore, validated the products of ispG and ispH as novel targets for the development of antimycobacterial drugs, whose effectiveness could be enhanced by host activated immune systems and not reduced by different carbon sources available in host macrophages. AFRIKAANSE OPSOMMING: Tuberkulose is die hoofoorsaak van sterftes as gevolg van 'n enkele mikro -organisme. Dit is deels te wyte aan die toename in geneesmiddelweerstandige TB. Nuwe middels met nuwe werkingsmeganismes is dus nodig. In 'n poging om nuwe geneesmiddeldoelwitte te vind, is CRISPRi-tegnologie gebruik om die kwesbaarheid van IspG en IspH, die twee [Fe-S] groepafhanklike ensieme wat in die metielerytritolfosfaatweg voorkom, te ondersoek, wat 'n hoë homologie onder mykobakteriese spesies toon . Die aktivering van die CRISPRi -stelsel vir 24 uur in Mycobacterium smegmatis -stamme, het meer as 96 % gene -afslag vir beide gene tot gevolg gehad. Die intrasellulêre genuitputting het gelei tot ernstige groei -verswakking van die hipomorfe, waardeur die noodsaaklikheid van beide geenprodukte in standaard groeimedium bevestig is. Die kwesbaarheid van IspG en IspH is ook volgehou in groeimedium aangevul met verskillende koolstofbronne, met gedeeltelike groei -redding wat slegs op ispG- en ispH -hypomorfe waargeneem word wanneer alanien en suksinaat onderskeidelik as die belangrikste koolstofbronne gebruik is. Uitgebreide klonte is waargeneem in die vloeibare kulture van die ispG hypomorph en skandeerelektronmikroskopie (SEM) het daarna 'n groot aantal deeltjies op die oppervlak van die selle onthul. Die ispG en ispH hipomorfe was veral sensitief vir stikstofoksied, die produk van 'n geaktiveerde menslike immuunstelsel. Alhoewel geenuitputting die antimikobakteriese effek van die twee eerste-lyn TB-antibiotika, isoniazid en rifampisien, versterk het, was beide hipomorfe hoogs sensitief vir die sub-dodelike konsentrasie van ethambutol en die ispH-hypomorf het die effek van tunicamycin versterk. Dit dui daarop dat potensiële remmers van enige van die ensieme gebruik kan word om die doeltreffendheid van die standaard TB - behandelingsregime te verbeter. Die resultate van hierdie studie bekragtig dus die produkte van ispG en ispH as nuwe teikens vir die ontwikkeling van antimikobakteriese middels, waarvan die doeltreffendheid verbeter kan word deur gasheer geaktiveerde immuunstelsels en nie verminder word deur verskillende koolstofbronne wat in gasheer makrofage beskikbaar is nie. Masters 2021-12-01T16:55:23Z 2022-02-22T10:19:53Z 2022-12-22T03:00:15Z 2021-12 Thesis http://hdl.handle.net/10019.1/124247 en_ZA Stellenbosch University xvii, 101 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Tuberculosis -- Pathogenesis Scanning electron microscopy Drug targeting UCTD Nicolaai, Mischke Validation of the MEP pathway’s [Fe-S] cluster enzymes as drug targets in mycobacteria |
| title | Validation of the MEP pathway’s [Fe-S] cluster enzymes as drug targets in mycobacteria |
| title_full | Validation of the MEP pathway’s [Fe-S] cluster enzymes as drug targets in mycobacteria |
| title_fullStr | Validation of the MEP pathway’s [Fe-S] cluster enzymes as drug targets in mycobacteria |
| title_full_unstemmed | Validation of the MEP pathway’s [Fe-S] cluster enzymes as drug targets in mycobacteria |
| title_short | Validation of the MEP pathway’s [Fe-S] cluster enzymes as drug targets in mycobacteria |
| title_sort | validation of the mep pathway s fe s cluster enzymes as drug targets in mycobacteria |
| topic | Tuberculosis -- Pathogenesis Scanning electron microscopy Drug targeting UCTD |
| url | http://hdl.handle.net/10019.1/124247 |
| work_keys_str_mv | AT nicolaaimischke validationofthemeppathwaysfesclusterenzymesasdrugtargetsinmycobacteria |