Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Anti-tuberculosis drugs target specific essential cellular processes and structural components. The first line drug, rifampicin (RIF) is a RNA polymerase inhibitor which targets the β-subunit and subsequently inhibits the initiation of transcription. Previous proteomic and transcriptomic analyses have shown that exposure to RIF for 24hrs significantly increased the abundance of proteins involved in energy metabolism in clinical isolates. No studies have been done to describe the transcriptional responses to RIF in an in vitro RIF resistant M. tuberculosis isolate. Application of in vitro mutants is novel since it will exclude most of the confounding factors which may be present in clinical isolates obtained from patients where the bacterium may have been incubated for several weeks or even years. This study aimed to determine the effect of prolonged exposure to RIF and the effect of the rpoB Ser531Leu mutation on the expression of energy metabolism genes, sigma factors and a regulator in RIF mono-resistant in vitro mutants with different levels of RIF resistance (minimum inhibitory concentration (MIC): 40μg/ml and 70μg/ml). RIF mono-resistant in vitro mutants were generated from a pan susceptible Beijing cluster 208 progenitor using the Luria Delbruck assay. In vitro RIF mono-resistant mutants harbouring the Ser531Leu rpoB mutation and which displayed different levels of RIF resistance were selected. To assess the effect of prolonged RIF exposure on the expression of candidate genes, the in vitro mutants were cultured in liquid media and exposed to RIF for 1, 7 and 14 days. High quality RNA was extracted from these cultures at each time point and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) was done on the selected candidate genes. The results indicate that limited expression of energy metabolism genes and sigma factors was observed after prolonged RIF exposure. In addition, the activity of the regulator (Rv1846c) was down-regulated in the presence of RIF explaining the up-regulated state of energy metabolism genes. To assess the effect of the rpoB Ser531Leu mutation on the candidate genes, RNA was extracted from the RIF unexposed culture at mid-log phase. RT-qPCR was done for each in vitro mutant in addition to the wild-type progenitor isolate. These results show that energy metabolism genes and sigma factors were significantly up-regulated in the RIF resistant mutantss harbouring an rpoB Ser531Leu mutation. This suggests that the mutation had a significant effect on the cellular energy cost due to the up-regulated state of the energy metabolism genes. In addition, an increase in the expression of sigma factors may be required to compensate for the rpoB mutation by enforcing the binding of the RNA polymerase and sigma factors to the promoter for transcription to be initiated. It is therefore important to assess these candidate genes for their potential as novel candidates for future drug design as this is an important aspect to influence tuberculosis control. / AFRIKAANSE OPSOMMING: Teen-tuberkulose middels teiken essensiële sellulêre prosesse en strukturele komponente. Die eerste linie teen-tuberkulose middel, rifampisien (RIF) is ʼn RNS polimerase inhibeerder wat die β-subeenheid teiken en daarna die inisiasie van transkripsie onderdruk. Vorige proteomiese en transkriptomiese analises het getoon dat blootstelling aan RIF vir 24 uur beduidende styging in sekere protiene wat verband hou met energie metabolisme in kliniese isolate veroorsaak. Die huidige studie poog om die effek van langdurige RIF blootstelling, asook die effek van die rpoB Ser531Leu mutasie op die uitdrukking van energie metabolisme gene, sigma faktore en reguleerders op RIF-enkel weerstandige in vitro mutante by verskillende vlakke van RIF weerstandigheid (Minimum Inhiberende Konsentrasie (MIK): 40μg/ml en 70μg/ml) te ondersoek. RIF-enkelweerstandige in vitro mutante isolate is gegenereer van ʼn sensitiewe Beijing 208 stamfamilielid deur die Luria Delbruck metode. In vitro RIF enkelweerstandige mutante met die rpoB Ser531Leu mutasie en verskillende vlakke van RIF weerstandigheid is geselekteer. Om die langdurige effek van RIF blootstelling op kandidaat geen uitdrukking te ondersoek, is in vitro mutante isolate gegroei in vloeibare medium en blootgestel aan RIF vir 1, 7 en 14 dae. Goeie kwaliteit RNS is geëkstraheer van hierdie kulture by elke tydpunt om Werklike-tyd Kwantitatiewe Polimerase Ketting Reaksie (RT-qPCR) op die kandidaat gene uit te voer. Die resultate toon dat ʼn beperkte aantal energie metabolisme en sigma faktor gene uitgedruk was na RIF blootstelling. Verder is die uitdrukking van die reguleerder (Rv1846c) af gereguleer in die teenwoordigheid van RIF en dit verduidelik die op gereguleerde energie metaboliese geen patroon. Om die effek van die rpoB Ser531Leu mutasie op die kandidaat gene te evalueer, is RNS geëkstraheer van ʼn weerstandige en RIF sensitiewe kultuur wat nie blootgestel was aan RIF nie. RT-qPCR is uit gevoer op elke in vitro mutante isolaat asook op ʼn sensitiewe isolaat sonder ʼn mutasie. Hierdie resultate toon dat energie metabolisme gene en sigma faktore beduidend opreguleer word in die isolate met ʼn rpoB Ser531Leu mutasie. Dit dui daarop dat die mutasie ʼn beduidende effek op die sellulêre energie koste het, omdat die energie metabolisme gene op gereguleer is. Verder kan ʼn toename in die uitdrukking van sigma faktore benodig word om die effek van die rpoB mutasie te oorkom deur binding van die RNS polimerase en die sigma faktore aan die promotor om transkripsie inisiasie te forseer. Dit is daarom belangrik om hierdie kandidaat gene verder te ondersoek vir toekomstige ontwikkeling van teenmiddels teen tuberkulose.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/20387 |
| Date | 03 1900 |
| Creators | Grobbelaar, Melanie |
| Contributors | Victor, T. C., Warren, R. M., Louw, G. E., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 104 p. : ill. |
| Rights | Stellenbosch University |
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