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Regulatory responses to rifampicin exposure in Mycobacterium tuberculosis

Black, Philippa Anne (2015-03)

Thesis (PhD)--Stellenbosch University, 2015

Thesis

ENGLISH ABSTRACT : There is increasing evidence that the mechanisms of drug resistance in Mycobacterium tuberculosis are not limited to mutations in the known drug resistance causing genes. Classically a mutation in one gene confers resistance to a drug, for example a mutation in the rpoB gene confers rifampicin resistance. However varying levels of resistance and fitness observed, as well as the lack of mutations in some clinically resistant isolates has guided researchers to focus on alternate genetic and physiological factors influencing drug resistance in M. tuberculosis. Whole genome sequencing (WGS) has previously identified polymorphisms in the rpoC gene of M. tuberculosis which were later confirmed to compensate for the loss of fitness occurring with the acquisition of rpoB mutations. Similarly, numerous mutations have been identified to be ancillary to drug resistance. Additionally, WGS has been used to investigate the evolution of drug resistance in M. tuberculosis. In addition to genetic factors, the activity of energy dependent efflux pumps has been associated with drug resistance and has been demonstrated to provide low levels of drug resistance to anti-TB drugs to allow for the development of drug resistance causing mutations. This study aimed to further investigate both genetic and efflux based mechanisms of resistance in M. tuberculosis, as well the response of M. tuberculosis to rifampicin exposure. It was therefore hypothesised that M. tuberculosis acquires additional genomic mutations during the evolution of rifampicin mono-resistance to multidrug resistant (MDR). In addition this study hypothesised that efflux pump activity contributes to the level of rifampicin resistance in M. tuberculosis, and this activity is dependent on the presence of specific rpoB mutations. Lastly, this study hypothesised that rifampicin exposure induces the expression of energy metabolism genes and efflux pumps in M. tuberculosis. The Department of Biomedical Sciences strain bank was interrogated and a total of 6 clinical isolates originating from 3 patients representing evolution from rifampicin mono-resistance to MDR were selected for WGS analysis. WGS identified novel genetic variants occurring during the evolution of drug resistance. Numerous variants were only present in a proportion of the population and were observed to change (emerge or disappear) during the course of treatment. This heterogeneity suggests that M. tuberculosis populations are dynamic during the evolution of drug resistance, accumulating numerous genetic changes. Experiments investigating the role of efflux pump activity in rifampicin resistance demonstrated that efflux pump activity differs in M. tuberculosis isolates with an rpoB Ser531Leu mutation compared to those with an rpoB His526Leu mutation. In addition, the efficacy of verapamil as an efflux pump inhibitor differed between these isolates. This finding may have implications for the consideration of the inclusion of efflux pump inhibitors in treatment regimens. Lastly, this study is the first to identify mmpL5 expression in response to rifampicin exposure; however the consequence of this association remains unknown. An additional finding of the investigation into the response of M. tuberculosis to rifampicin exposure is the identification of a proposed transcriptional regulator responding to rifampicin exposure. Together these findings demonstrate the numerous genetic and physiological mechanisms contributing to drug resistance in M. tuberculosis.

AFRIKAANSE OPSOMMING : Toenemde bewyse stel voor dat meganismes van weerstandigheid in Mycobacterium tuberculosis is nie beperk tot slegs mutasies in gene wat bekend is vir die oorsaak van middelweerstandigheid nie. Dit word algemeen aanvaar dat ‘n mutasie in een geen weerstandigheid teen n middel veroorsaak, byvoorbeeld ‘n mutasie in die rpoB geen veroorsaak rifampisien weerstandigheid. Daar is egter al waargeneem dat verskillende vlakke van weerstandigheid en fiksheid voorkom, asook die afwesigheid van mutasies in sommige kliniese middelweerstandige isolate. Dit het navorsers gelei om te fokus op alternatiewe genetiese en fisiologiese faktore wat weerstandigheid in M. tuberculosis kan beЇnvloed. Mutasies in rpoC is voorheen geïdentifiseer deur heelgenoom volgorde bepaling en is later bevestig om vergoedend te wees vir die afname in fiksheid as gevolg van die verwerwing van rpoB mutasies. Soortgelyk is dit ook gevind dat verskeie mutasies aanvullend is tot middel weerstandigheid. Verder is heelgenoom volgorde bepaling ook gebruik om die evolusie van middelweerstandighein in M. tuberculosis te bestudeer. Ten spyte van genetiese faktore, is daar ook gevind dat die aktiwiteit van energie afhanklike uitvloei pompe geassosieer is met middel weerstandigheid en daar is gewys dat dit n verlaagde vlak van weerstandigheid voorsien sodat middel weerstandige mutasies kan ontwikkel. Hierdie studie stel ten doel om beide die genetiese en uitvloei pomp meganismes van weerstandigheid in M. tuberculosis verder te ondersoek, asook die reaksie van M. tuberulosis op rifampisien blootstelling. Hierdie studie het uit drie aparte navorsing vrae bestaan om die volgende hipoteses aantespreek: M. tuberculosis verkry additionele genomiese mutasies gedurende die evolusie van rifampisien enkel weerstandigheid na meervoudige middelweerstandigheid (MDR); dat uitvloei pompe bydra tot die vlakke van rifampisien weerstandigheid in M. tuberculosis en dat hierdie aktiwiteit afhanklik is van spesifieke rpoB mutasies; dat die blootstelling van M. tuberculosis aan rifampisien lei tot die verhoging in geen uidrukking van energie metabolisme en uitvloei pompe. Die departement van Biomediese Wetenskappe se isolaat versameling is ondersoek en n totaal van ses kliniese isolate afkomstig van drie pasiënte wat die evolusie van rifampisien enkel weerstandigheid na MDR voorstel is met heelgenoom volgorde bepaling ondersoek. Heelgenoom volgorde bepaling het nuwe genetiese variante geïndentifiseer tydens die evolusie van weerstandigheid. Verskeie variante was slegs teenwoordig in n gedeelte van die populasie wat verander het (verskyn of verdwyn) tydens die behandelingskursus. Hierdie heterogenetiese eienskappe stel voor dat M. tuberculosis populasies dinamies is gedurende die evolusie van middel weerstandigheid deur die akkumulering van verskeie genetiese verandering. Eksperimente wat die rol van uitvloei pomp aktiwiteit in rifampisien weerstandiheid ondersoek het gewys dat uitvloei pomp aktiwiteit verskil tussen M. tuberculosis isolate met rpoB Ser531Leu en rpoB His526Leu mutasies. Verder was die effektiwiteit van verapamil as uitvloei pomp inhibeerder verskillend tussen hierdie isolate. Hierdie bevinding mag dalk implikasies inhou vir die oorweging om uitvloei pomp inhibeerders intesluit by behandelingskursusse.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96599
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