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Overexpression of mmpS5/mmpL5 in Mycobacterium tuberculosis reduces susceptibility to anti-tuberculosis drugs
Thesis (MSc) -- Stellenbosch University, 2022.
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| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867613775434088448 |
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| access_status_str | Open Access |
| author | Scott, Leere Ann |
| author2 | Grobbelaar, Melanie |
| author_browse | Grobbelaar, Melanie Scott, Leere Ann |
| author_facet | Grobbelaar, Melanie Scott, Leere Ann |
| author_sort | Scott, Leere Ann |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc) -- Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/126198 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:41:30.564Z |
| 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:10019.1/126198 Overexpression of mmpS5/mmpL5 in Mycobacterium tuberculosis reduces susceptibility to anti-tuberculosis drugs Scott, Leere Ann Grobbelaar, Melanie Dippenaar, Anzaan Kriel, Nastassja Ismail, Nabila Warren, Robin Van Rie, Annelies Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Mycobacterium tuberculosis Multidrug resistance Rifampin Antitubercular agents Minimum inhibitory concentration UCTD Bedaquiline Thesis (MSc) -- Stellenbosch University, 2022. ENGLISH ABSTRACT: Background: Bedaquiline (BDQ) is the first anti-tuberculosis (anti-TB) drug in recent years with a novel mode of action recommended for the treatment of multi-drug resistant-TB. Rv0678 is the most common genetic determinant of BDQ resistance. It encodes for a regulatory protein (MmpR5) of the MmpS5/MmpL5 efflux system of Mycobacterium tuberculosis. Mutations in Rv0678 lead to the overexpression of the mmpS5/mmpL5 genes and increased efflux of BDQ, resulting in an elevated minimum inhibitory concentration (MIC) or phenotypic resistance. Mutations in Rv0678 are also associated with cross-resistance to clofazimine (CFZ) by the same mechanism of MmpS5/MmpL5 export. As this efflux pump is responsible for hydrophobic siderophore export, we hypothesised that additional drugs are exported, and that drug hydrophobicity may be an important determinant. We hypothesised that a rpoB mutation may exacerbate the effect of a Rv0678 mutation on the expression of mmpS5/mmpL5 as their gene products are both involved in gene transcription. This may be reflected in further alterations in the MICs of drugs exported by the pump. Lastly, due to the overexpression of mmpS5/mmpL5, it was hypothesised that Rv0678 mutations have a fitness cost. Methodology: M. tuberculosis progenitor isolates (a M. tuberculosis H37Rv reference isolate and a Rv0678 mutant harbouring a 193 G deletion) were used to generate rpoB mutants containing a Ser531Leu mutation, using a spontaneous mutagenesis approach. Whole genome sequencing was employed to confirm a clean genetic background of progenitor and rpoB mutant isolates, whereafter MIC assays were performed. Finally, comparative growth curve analysis was conducted between the M. tuberculosis H37Rv and Rv0678 mutant isolates by measuring optical density over time. Results: Spontaneous in vitro rpoB mutants were successfully generated and the mutations of interest were detected with WGS. The Rv0678 mutant showed resistance to BDQ and CFZ, and a slight increase in delamanid MIC within 1-2 doubling dilutions compared to M. tuberculosis H37Rv. The H37Rv rpoB mutant showed resistance to rifampicin (RIF) and rifabutin (RFB). The Rv0678/rpoB mutant showed resistance to BDQ, CFZ, RIF and RFB. This mutant had a higher CFZ MIC within 1- 2 doubling dilutions compared to the Rv0678 mutant. The MICs of other drugs were altered below the epidemiological cut-off (ECOFF) value in the Rv0678 and rpoB mutant isolates and were either raised or decreased, similarly within 1-2 doubling dilutions. The Rv0678 mutant exhibited a similar growth rate to its wildtype M. tuberculosis H37Rv progenitor. Conclusion: There was a clear distinction in resistance levels of BDQ/CFZ, and RIF/RFB in isolates that harboured the Rv0678 and rpoB mutations, respectively. This is supported by literature which shows that a 193 G deletion in Rv0678 is associated with BDQ and CFZ resistance, and that a Ser531Leu rpoB mutation causes high-level RIF and RFB resistance. The remaining drugs showed altered MICs which were below the ECOFF and could not be confirmed as clinically meaningful. The growth curve analysis showed that protein truncating due to the Rv0678 mutation had no significant effect on growth rate and no association with a fitness cost. Further investigation is required to test if a clinical background, different Rv0678 mutations or alternative testing methods would affect the current results. AFRIKAANS OPSOMMING: Agtergrond: Bedaquilien (BDQ) is die eerste tuberkulose middel met ‘n nuwe werkswyse wat in die laaste jare ontwikkel is vir die behandeling van multiweerstandige TB. Die mees algemene genetiese bepaler van BDQ weerstandigheid is Rv0678, wat MmpR5 enkodeer, ‘n regulatoriese proteïen van die MmpS5/MmpL5 uitvloeisisteem van Mycobacterium tuberculosis. Mutasies in Rv0678 veroorsaak verhoogde uitdrukking van die mmpS5/mmpL5 gene, sowel as meer uitvloei van BDQ, wat lei tot verhoogde minimum inhibitoriese konsentrasie (MIK) van BDQ, oftewel fenotipiese weerstandigheid. Mutasies in Rv0678 word ook geassosiëer met weerstandigheid teen clofazimine (CFZ) deur dieselfde meganisme van MmpS5/MmpL5 uitvloei. Ons hipotese is dat addisionele middels uitgevoer word deur hierdie uitvloeipomp, aangesien dit verantwoordelik is vir siderofoor uitvoer, en dat hidrofobisiteit ‘n bepalende faktor is. Verder postuleer ons dat ‘n rpoB mutasie die effek van ‘n Rv0678 mutasie op die uitdrukking van mmpS5/mmpL5 vererger, aangesien beide die geenprodukte betrokke is by transkripsie. Dit mag verder duidelik word uit veranderings in die MIKs van middels wat deur die pomp uitgevoer word. Laastens, postuleer ons dat Rv0678 mutasies ‘n fiksheidskoste dra as gevolg van die oor-uitdrukking van mmpS5/mmpL5. Metodiek: M. tuberculosis voorganger isolate (‘n M. tuberculosis H37Rv indeks stam en ‘n Rv0678 mutant met ‘n 193 G uiwissing) is gebruik om rpoB mutante met ‘n Ser531Leu mutasie te maak deur middel van ‘n spontane mutagenese benadering. Heelgenoom volgordebepaling is gebruik om te bevestig dat beide die voorlooper en die rpoB mutant ‘n overanderde genetiese agtergrond het, waarna MIK bepalings gedoen is. Laastens is vergelykende groeikurwe analise gedoen deur die optiese digtheid van die H37Rv- en Rv0678 mutant-isolate te meet oor tyd. Resultate: Spontane in vitro rpoB mutante is suksesvol gegenereer en die teenwoordigheid van mutasies van belang is met heelgenoom volgordebepaling bevestig. Die Rv0678 mutant het weerstandigheid getoon teen BDQ en CFZ, sowel as ‘n geringe toename in delamanied MIK, binne 1-2 verdubbelde verdunnings, in vergelyking met M. tuberculosis H37Rv. Die H37Rv rpoB mutant het weerstandigheid teen rifampisien (RIF) en rifabutien (RFB) getoon. Die Rv0678/rpoB mutant het weerstandigheid teen BDQ, CFZ, RIF en RFB getoon. Hierdie mutant het ‘n hoër CFZ MIK binne 1- 2 verdubbelde verdunnings, in vergelyking met die Rv0678 mutant. Die MIKs van ander middels het verander in die Rv0678 en rpoB mutante isolate, maar by vlakke onder die epidemiologiese afsnypunt (epidemiological cut-off, ECOFF). Hierdie veranderings sluit verhoging en verlaging van MIK in, maar soos voorheen binne 1-2 verdubbelde verdunnings. Die Rv0678 mutant het ‘n soortgelyke groeitempo getoon as die ongemuteerde M. tuberculosis H37Rv voorloper. Gevolgtrekking: Daar is ‘n duidelike onderskeid tussen BDQ/CFZ en RIF/RFB weerstandigheid in isolate wat onderskeidelik die Rv0678 en rpoB mutasies het. Dit word ondersteun deur literatuur wat toon dat ‘n 193 G uitwissing in Rv0678 geassosiëer is met BDQ en CFZ weerstandigheid en dat ‘n Ser531Leu rpoB mutasie hoëvlak RIF en RFB weerstandigheid veroorsaak. Die res van die middels het veranderde MIKs onder die ECOFF getoon, wat nie as klinies beduidend bevestig kon word nie. Die groeikurwe analise toon dat die verkorting van die proteïen as gevolg van die Rv0678 mutasie geen beduidende effek het op die groeitempo nie, en dat daar geen verband is met fiksheidskoste nie. Verdere ondersoek word benodig om te bepaal of ‘n kliniese agtergrond, verskillende Rv0678 mutasies of alternatiewe toetsmetodes die huidige resultate sou affekteer. Masters 2022-11-23T13:15:13Z 2023-01-16T12:55:11Z 2022-11-23T13:15:13Z 2023-01-16T12:55:11Z 2022-12 Thesis http://hdl.handle.net/10019.1/126198 en_ZA Stellenbosch University xviii, 121 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Mycobacterium tuberculosis Multidrug resistance Rifampin Antitubercular agents Minimum inhibitory concentration UCTD Bedaquiline Scott, Leere Ann Overexpression of mmpS5/mmpL5 in Mycobacterium tuberculosis reduces susceptibility to anti-tuberculosis drugs |
| title | Overexpression of mmpS5/mmpL5 in Mycobacterium tuberculosis reduces susceptibility to anti-tuberculosis drugs |
| title_full | Overexpression of mmpS5/mmpL5 in Mycobacterium tuberculosis reduces susceptibility to anti-tuberculosis drugs |
| title_fullStr | Overexpression of mmpS5/mmpL5 in Mycobacterium tuberculosis reduces susceptibility to anti-tuberculosis drugs |
| title_full_unstemmed | Overexpression of mmpS5/mmpL5 in Mycobacterium tuberculosis reduces susceptibility to anti-tuberculosis drugs |
| title_short | Overexpression of mmpS5/mmpL5 in Mycobacterium tuberculosis reduces susceptibility to anti-tuberculosis drugs |
| title_sort | overexpression of mmps5 mmpl5 in mycobacterium tuberculosis reduces susceptibility to anti tuberculosis drugs |
| topic | Mycobacterium tuberculosis Multidrug resistance Rifampin Antitubercular agents Minimum inhibitory concentration UCTD Bedaquiline |
| url | http://hdl.handle.net/10019.1/126198 |
| work_keys_str_mv | AT scottleereann overexpressionofmmps5mmpl5inmycobacteriumtuberculosisreducessusceptibilitytoantituberculosisdrugs |