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Defining mechanisms that determine the levels of drug resistance in Mycobacterium tuberculosis

Bester, Margaretha (2009-12)

Thesis (MSc (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009.


ENGLISH ABSTRACT: Varying levels of Rifampicin (RIF) resistance in closely related clinical Mycobacterium tuberculosis isolates and in vitro generated mutants question the dogma that non-synonymous single nucleotide polymorphisms in the rpoB gene are the only mechanism explaining RIF resistance. This study aimed to identify biological mechanisms that define the level of RIF resistance in two closely related clinical M. tuberculosis isolates using proteomic, transcriptomic and genomic approaches. Two dimensional electrophoresis revealed an increase in the abundance of numerous membrane proteins in response to RIF at the critical concentration of 2g/ml. Fourty-one of these proteins were identified by mass spectrometry and could be grouped according to their cellular function (Energy metabolism, degradation, biosynthesis of cofactors, metabolic groups and carriers, lipid biosynthesis, central intermediate metabolism, synthesis and modification of macromolecules, chaperone/heat shock proteins). The identification of proteins responsible for ATP synthesis (atpA and atpH) suggests an ATP requirement to combat the toxic effect of RIF. These proteins are components of the FoF1 ATP synthase an enzyme which is involved in the oxidative phosphorylation pathway that generates ATP in the cell. QRT-PCR confirmed the up regulation of the transcription of the atpA and atpH genes in response to RIF, while DNA sequencing failed to identify mutations that could define the rate of transcription. To explain our findings we proposed that RIF induces a toxic response leading to the up regulation of a number of genes. The induction of metabolic enzymes, such as the FoF1 ATP synthase provides energy to activate ATP dependant mechanisms, including membrane ABC transporters. These ABC transporters actively pump RIF out of the cell thereby lowering the intracellular concentration of RIF to below its binding concentration with the rpoB protein leading to RIF resistance. Inhibition of efflux by the efflux pump inhibitors reserpine and verapamil leads to an accumulation of RIF within the cell and concurrent binding of RIF to rpoB, leading to inhibition of transcription and cell death (ongoing research in our laboratory). Similarly, we propose that the recently identified diarylquinoline compound (TMC207) inhibit ATP synthesis, thereby depleting the energy source necessary for active efflux. This will lead to an accumulation of anti-TB drug within the cell and subsequent cell death. In summary, this study provides the first evidence to suggest that the evolution of RIF resistance is a dynamic process involving a cascade of adaptive events which leads to a bacterial growth state where hydrophobic compounds are actively extruded from the cell. This has important ramifications for the treatment of RIF resistant TB and supports the need for the development of anti-TB drugs that target both efflux and ATP synthesis to improve the treatment outcome of MDR-TB and XDR-TB.

AFRIKAANSE OPSOMMING: Verskillende vlakke van Rifampisien (RIF) weerstandigheid, in naby verwante Mycobacterium tuberculosis kliniese isolate en in vitro mutante, bevraagteken die dogma dat nie-sinonieme enkel nukleotied polimorfismes in die rpoB geen die enigste verklaarbare meganisme vir RIF weerstandigheid is. Die doel van hierdie studie was om deur 'n proteomiese, transkriptomiese en genomiese benadering, biologiese meganismes te identifiseer wat die vlakke van RIF weerstandigheid in twee naby verwante kliniese M. tuberculosis isolate bepaal. Twee dimensionele elektroferese het gevind dat daar 'n verhoging in die hoeveelheid van verskeie proteïne is wanneer die isolate aan RIF by die 'n kritiese konsentrasie van 2μg/ml blootgestel is. Massa spektrometrie het 41 van hierdie proteine geïdentifiseer en die proteïne kan gegroepeer word in verskeie sellulêre funksies (Energie metabolism, degradering, biosintese van kofaktore, metaboliese groepe en draers, lipied biosintese, sentrale intemediêre metabolisme, sintese en modifisering van makromolekules, en “chaperone/heat shock” proteine). Die identifisering van proteïne verantwoordlik vir ATP sintese (atpA en atpH) stel voor dat ATP belangrik is om die toksiese effek van RIF te ontwyk. Hierdie proteïne is komponente van die FoF1 ATP sintase ensiem wat betrokke is in die oksidatiewe fosforilerings pad en wat lei tot die generering van ATP in die sel. Kwantitatiewe QRT-PCR het bevestig dat hierdie gene, atpA en atpH, opgereguleer word nadat die bakterium aan RIF blootgestel is. In teen deel kon DNA volgorde bepaling nie mutasies identifiseer wat die verandering in geen transkripsie kon verklaar nie. Om ons bevindings te verduidelik, stel ons voor dat RIF 'n toksiese effek in die sel induseer wat lei tot die opregulering van verskeie gene. Die indusering van metaboliese ensieme, soos die FoF1 ATP sintase, voorsien energie om ATP afhanklike meganismes, insluitende membraan ABC transporters, te aktiveer. Hierdie ABC transporters pomp RIF aktief uit die sel, wat daarvolgens die intrasellulêre konsentrasie van RIF verlaag tot 'n konsentrasie laer as die bindings konsentrasie met die rpoB protein en gevolglik lei tot weerstandigheid. Die onderdrukking van membraan pompe wat RIF uit die sel pomp deur middels soos reserpine en verapamil sal aanleiding gee lei tot akkumulering van RIF in die sel. Die verhoogde RIF in die sel versoorsaak dat RIF aan die rpoB protein gebind bly sodat dit transkripsie inhibeer, wat dan aanleiding gee tot seldood. (voortgesette navorsing in ons laboratorium). Soortgelyk, stel ons voor dat die onlangs geïdentifiseerde dairylquinoline verbinding (TMC207) ATP sintese inhibeer en daarvolgens die energie bron uitput wat noodsaaklik is vir aktiewe uitpomp van RIF. Dit sal aanleiding gee tot die ophoping van RIF in die sel en gevolglik lei tot seldood. In opsomming, hierdie studie voorsien die eerste bewys wat voorstel dat die evolusie van RIF weerstandighied 'n dinamiese proses is. Dit sluit 'n kaskade van aanpasbare gebeurtenisse in wat lei tot 'n bakteriële groei fase waar hidrofobiese verbindings aktief uit die sel gedryf word. Dit het rampspoedige gevolge vir die behandeling van RIF weerstandige TB en ondersteun die noodsaaklikheid om teen-TB middels te ontwikkel wat beide effluks pompe en ATP sintese teiken om die uikoms van behandeling vir MDR-TB en XDR-TB te verbeter.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/4610
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