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Determination of the transcriptomic effects and compensatory role ofF INHA C-15T promoter mutation in mycobacterium tuberculosis.
Thesis (MSc)--Stellenbosch University, 2023.
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| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867613815173021696 |
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| access_status_str | Open Access |
| author | Mwendwa, Leah Kalekye |
| author2 | Klopper, Marisa |
| author_browse | Klopper, Marisa Mwendwa, Leah Kalekye |
| author_facet | Klopper, Marisa Mwendwa, Leah Kalekye |
| author_sort | Mwendwa, Leah Kalekye |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description |
Thesis (MSc)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/128384 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:42:07.859Z |
| 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:10019.1/128384 Determination of the transcriptomic effects and compensatory role ofF INHA C-15T promoter mutation in mycobacterium tuberculosis. Mwendwa, Leah Kalekye Klopper, Marisa Barnard, Monique Mavumengwana, Vuyo Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Mycobacterium tuberculosis -- Genetics Promoters (Genetics) Microbial mutation Gene expression UCTD Thesis (MSc)--Stellenbosch University, 2023. ENGLISH ABSTRACT: InhA C-15T promoter mutation confers resistance to both isoniazid (INH) and ethionamide (ETH) in Mycobacterium tuberculosis, the causative agent for human tuberculosis (TB). The physiological role of inhA C-15T promoter mutation in M. tuberculosis is unclear. This study hypothesized that the inhA C15T promoter mutation upregulates all three inhA operon genes (mabA, inhA and hemZ) in addition to differential regulation of related genes, and that it also confers a fitness advantage over M. tuberculosis H37Rv in the presence or absence of additional drug-resistance mutations. Aims The study aimed to explore gene expression profiles between inhApromoter mutants (IPMs) and H37Rv using RNA sequencing (RNAseq). We further aimed to elucidate fitness advantage of inhA promoter mutation using competition assays, comparing wild-type, single (inhA promoter or rpoB) and double mutants. Methods RNA of M. tuberculosis H37Rv and two IPMs (IPM C5 and IPM C7), treated with INH; 0.2µg/ml for 4hrs, and untreated, were extracted at mid-log phase in 6 technical repeats per strain per condition. cDNA libraries were prepared of high-quality RNA using Zymo-Seq RiboFree Total RNA Library kit and sequenced on IlluminaTM NextSeq 550 Series platform. Differential gene expression (DGE) (Padj< 0.05; LFC > 0.5 or < -0.5) was determined on DESeq2 v1.36 in R v4.2.2. A modified Luria Delbrück assay using IPMs was used to generate a double mutant (inhA C-15T promoter/rpoB S531L) by selection on RIF (10µg/ml) plates. Single colonies with RIF resistance were selected and Sanger sequenced to identify colonies that have acquired the rpoB S531L mutation. Whole Genome Sequencing was carried out to identify possible off-target mutations. Individual growth for H37Rv, IPM C7, rpoBmutant and the double mutant was established in supplemented 7H9 media, then compared the growth curves using Student’s ttest. Results and discussion A minimum of 20 million reads were generated for each RNA sample and 100% of reads were mapped to the refence genome (M. tuberculosis H37Rv, NC_000962.3). Differential gene expression analysis showed the upregulation of the inhA operon genes in untreated IPM vs untreated H37Rv, kas operon genes were upregulated in treated H37Rv vs untreated H37Rv but downregulated in treated IPM vs treated H37Rv (corresponding to upregulation of the same genes in treated H37Rv vs treated IPM). There were no significantly differentially expressed genes in treated IPM vs untreated IPM. There was no significant difference in growth in H37Rv, IPM C7, rpoB S531L mutant and the double mutant in supplemented 7H9 media. Conclusion This was the first study to carry out RNAseq using an in vitro-generated IPM. The inhA C-15T promoter mutation upregulates all three of the inhA operon genes(mabA, inhA and hemZ), without any additional transcriptomic effects under standard culturing conditions. INH induces the expression of kas operon genes in M. tuberculosis H37Rv. There is no difference between the growth rates of individual strains investigated in this study, however, future studies will include competition assays where different strains are co-cultured to determine fitness advantage. Keywords: M. tuberculosis, inhA C-15T promoter mutation, tuberculosis, RNAseq, Luria Delbrück assay, WGS, differential gene expression. AFRIKAANSE OPSOMMING: nhA C-15T promotor mutasies veroorsaak weerstandigheid teen beide isoniasied (INH) en ethionamied (ETH) in Mycobacterium tuberculosis, die organisme wat tuberkulose (TB) in mense veroorsaak. Die fisiologiese rol van die inhA C-15T promotor mutasie is egter onseker. Hierdie studie hipotese is dat die inhA C-15T promotor mutasie al drie die gene in die inhA operon (mabA, inhA en hemZ) opreguleer, en dat dat dit ‘n invloed het op die transkripsie van verwante gene. Verder hipotetiseer ons dat die mutasie ‘n fiksheidsvoordeel veroorsaak bo M. tuberculosis H37Rv in die teenwoordigheid of afwesigheid van addisionele weerstandigheidsmutasies. Doelwitte Die studie doel was om die geenuitdrukkingsprofiele van inhA promotor mutante (IPMs) en H37Rv te vergelyk met RNA volgordebepaling (RNAseq). Ons het ook verder ten doel gehad om enige fiksheidsvoordeel wat die inhA promotor mutasie mag oordra, te demonstreer deur middle van kompetisie toetse, waar die oorspronklike genotipe (H37Rv) vergelyk word met enkel- (inhA promotor of rpoB) en dubbel mutante. Metodes RNA is geëkstraeer van M. tuberculosisH37Rv en twee IPMs (IPM C5 en IPM C7) by mid-log fase, met en sonder blootstelling aan 0.2µg/ml INH vir 4 ure, in 6 tegniese replikate per stam per kondisie. cDNA biblioteke is voorberei van hoë kwaliteit RNA met die Zymo-Seq RiboFree Total RNA Library pakket, en volgordebepaling is gedoen op die IlluminaTM NextSeq 550 Series platform. Differensiële geenuitdrukking (Padj 0.5 of < -0.5) is bepaal met behulp van DESeq2 v1.36 in R v4.2.2. Ons het ‘n aangepaste Luria Delbrück toets gebruik om ‘n dubbelmutant (inhA C-15T promotor/rpoB S531L) te genereer van ‘n IPM stam, deur seleksie op RIF plate (10µg/ml). Enkelkolonies wat RIF weerstandig was, is geselekteer en Sanger volgordebepaling is gebruik om kolonies met die gewenste rpoBS531L mutasie te identifiseer. Heelgenoom volgordebepaling is gebruik om potensiële ongewenste mutasies te identifiseer. Ons het ook die groei van individuele H37Rv, IPM C7, rpoB mutant en dubbelmutante kulture geassesseer in verrykte 7H9 media, en die groeikurwes vergelyk met ‘n Student T-toets. Resultate en bespreking Ten minste 20 miljoen fragmentlesings is vir elke RNA monster gegenereer, en 100% van die fragmentlesings kon op die naslaangenoom (M. tuberculosis H37Rv, NC_000962.3) gepas word. Differensiële geenuitdrukkingsanalise het getoon dat die inhA operon gene upgereguleer is in IPM (onbehandel) teenoor H37Rv (onbehandel); kas operon gene is opgereguleer in H37Rv (behandel) teenoor H73Rv (onbehandel), maar afgereguleer is in behandelde IPM teenoor behandelde H37Rv (wat gelykstaande is aan opregulering van die gene in behandelde H37Rv teenoor behandelde IPM). Daar was geen beduidende verskil in geenuitdrukking tussen behandelde en onbehandelde IPM nie. Daar was geen verskil in die groei van H37Rv, IPM C7, die rpoBmutant of die dubbelmutant in verrykte 7H9 media nie. Gevolgtrekkings Hierdie is die eerste studie wat RNAseq uitvoer op in vitro gegenereerde IPM. Die inhA C-15T promotor mutasie veroorsaak opregulering van al drie inhA operon gene (mabA, inhA en hemZ), sonder enige addisionele transkriptomiese effekte by standaard kultuurtoestande. INH induseer die uitdrukking van kas operon gene in M. tuberculosis H37Rv. Daar is geen verskil tussen die groeitempo van die individuele stamme wat in hierdie studie ondersoek is nie, maar in ‘n opvolgstudie sal kompetisietoetse gedoen word waarin die verskillende stamme bymekaar gevoeg sal word om ‘n fiksheidsvoordeel uit te lig. 2023-03-01T20:41:03Z 2023-08-30T13:12:30Z 2023-03 2023-03-01T20:41:03Z 2023-08-31T09:18:33Z 2023-03-01T20:41:03Z 2023-08-31T09:18:33Z 2023-03 Thesis https://scholar.sun.ac.za/handle/10019.1/128384 en_ZA Stellenbosch University application/pdf xxiii, 134 : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Mycobacterium tuberculosis -- Genetics Promoters (Genetics) Microbial mutation Gene expression UCTD Mwendwa, Leah Kalekye Determination of the transcriptomic effects and compensatory role ofF INHA C-15T promoter mutation in mycobacterium tuberculosis. |
| title | Determination of the transcriptomic effects and compensatory role ofF INHA C-15T promoter mutation in mycobacterium tuberculosis. |
| title_full | Determination of the transcriptomic effects and compensatory role ofF INHA C-15T promoter mutation in mycobacterium tuberculosis. |
| title_fullStr | Determination of the transcriptomic effects and compensatory role ofF INHA C-15T promoter mutation in mycobacterium tuberculosis. |
| title_full_unstemmed | Determination of the transcriptomic effects and compensatory role ofF INHA C-15T promoter mutation in mycobacterium tuberculosis. |
| title_short | Determination of the transcriptomic effects and compensatory role ofF INHA C-15T promoter mutation in mycobacterium tuberculosis. |
| title_sort | determination of the transcriptomic effects and compensatory role off inha c 15t promoter mutation in mycobacterium tuberculosis |
| topic | Mycobacterium tuberculosis -- Genetics Promoters (Genetics) Microbial mutation Gene expression UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/128384 |
| work_keys_str_mv | AT mwendwaleahkalekye determinationofthetranscriptomiceffectsandcompensatoryroleoffinhac15tpromotermutationinmycobacteriumtuberculosis |