Identification of mechanisms regulating the intra cellular concentration of rifampicin in Mycobacterium Tuberculosis

De Vos, Margaretha (2013-03)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Rifampicin resistance in clinical isolates of Mycobacterium tuberculosis develops through selection of bacterial variants harbouring mutations in the rpoB gene. These mutations infer a fitness-cost in the absence of antibiotic pressure, however, fitness-levels of rifampicin-resistant strains can be restored by compensatory mutations in rpoA and rpoC. This study was the first to investigate the epidemiological relevance of these compensatory mutations in clinical M. tuberculosis isolates collected in South Africa. Through targeted DNA sequencing, we demonstrated a strong association between rpoC mutations and transmission, and the rpoB S531L mutation. Our study emphasises the epidemiological relevance of compensatory evolution in response to the emergence of rifampicin resistance, and illustrates how compensatory mutations may be selected as a function of epistatic interactions. Recently a hypothesis has been developed which suggests that the activation of efflux systems through exposure to rifampicin may explain the observed spectrum of rifampicin resistance phenotypes. To elucidate whether rifampicin dependent activation of efflux systems also increases energy production, the RNA expression profiles of candidate energy metabolism genes were investigated. This study demonstrated that rifampicin exposure induced an overall increase in the expression of energy metabolism genes. Our findings suggest that the response to rifampicin is not universal and may depend on other genomic mutations. From these results we conclude that the stress response induced by exposure to rifampicin increases the energy production which fuels efflux activity thereby enabling the cell to extrude rifampicin in an energy dependent manner. This also provides a platform to explain the mechanism by which the newly developed drug, TMC207, increases the rate of culture conversion when used in combination with second-line anti-TB drugs. We propose that inhibition of ATP synthesis by TMC207 will deprive the efflux pumps and transporter genes of energy, which will result in the accumulation of second-line anti-TB drugs within the bacilli, leading to more efficient binding of the second-line drugs to their targets and ultimately to cell death. To identify the genetic basis governing the level of rifampicin resistance, we sequenced the genomes of MDR clinical isolates and in vitro generated rifampicin resistant mutants. Only minor genetic changes in addition to the rpoB mutation were identified in the genomes of in vitro rifampicin resistant mutants which displayed varying levels of resistance. This suggests that these mutants may either use alternative regulatory mechanisms or have acquired SNPs outside the genetic regions investigated in this study to modulate rifampicin resistance levels. In contrast, the genomes of clinical MDR isolates from the Low Copy Clade showed considerable variability in genes involved in cell wall, cellular processes and lipid metabolism, while the genomes from the Beijing Clade displayed variability in genes known to confer drug resistance and compensatory mechanisms. These results suggest that the structure and processes of the cell wall, as well as lipid metabolism plays a critical role in determining the intra-cellular concentration of rifampicin. Finally, this study illustrated the complexity in the physiology of M. tuberculosis resistant to rifampicin, whereby multiple mechanisms are employed by the bacteria to modulate its resistance levels.

AFRIKAANSE OPSOMMING: Rifampisien weerstandigheid in kliniese isolate van Mycobacterium tuberculosis ontwikkel deur die seleksie van bakteriële variante wat mutasies in die rpoB geen het. Alhoewel hierdie mutasies lei tot „n afname in fiksheid van die bakterieë in die teenwoordigheid van antibiotika, kan die fiksheids vlakke van rifampisien weerstandige stamme herstel word deur vergoedende mutasies in rpoA en rpoC. Hierdie is die eerste studie wat die epidemiologiese relevansie van hierdie vergoedende mutasies in kliniese M. tuberculosis isolate wat in Suid-Afrika versamel is, ondersoek. Deur middel van doelgerigte DNA volgordebepaling het ons „n sterk assosiasie tussen rpoC mutasies en transmissie, en die rpoB S31L mutasie getoon. Hierdie studie beklemtoon die epidemiologiese relevansie van regstellende evolusie na aanleiding van die ontwikkeling van rifampisien weerstandigheid en illustreer hoe regstellende mutasies geselekteer mag word as „n funksie van epistatiese interaksies. „n Hipotese is onlangs ontwikkel wat voorstel dat blootstelling aan rifampisien uitvloei sisteme in die bakterium aktiveer, wat moontlik die waargenome spektrum van rifampisien weerstandige fenotipes kan verklaar. Ons het die RNA uitdrukkingsprofiele van kandidaat-energiemetabolisme gene ondersoek om te bepaal of rifampisien afhanklike aktivering van uitvloei sisteme ook energieproduksie verhoog. Hierdie studie demonstreer dat rifampisien-blootstelling „n algehele verhoging in die uitdrukking van energiemetabolisme gene induseer. Ons bevindinge stel voor dat die reaksie van die sel op rifampisien blootstelling nie universeel is nie, en moontlik ook afhanklik is van ander genomiese mutasies. Uit hierdie resultate kan ons aflei dat die stres respons wat geïnduseer word deur rifampisien-blootstelling energieproduksie verhoog, wat weer die uitvloei aktiwiteit aanvuur, en gevolglik die sel in staat stel om rifampisien op „n energie-afhanklike wyse uit te dryf. Dit bied ook „n basis om die meganisme te verklaar waardeur die nuwe middel, TMC207, die tempo van kultuuromskakeling verhoog wanneer dit saam met tweede-linie anti-TB middels gebruik word. Ons stel voor dat die inhibisie van ATP sintese deur TMC207 die uitvloeipompe en transporteerder gene van energie ontneem. Gevolglik veroorsaak dit „n ophoping van tweedelinie anti-TB middels binne-in die bakterium, wat geleentheid bied vir meer effektiewe binding tussen die middels en hulle teikens en uiteindelik seldood veroorsaak. Ons het DNA volgordes bepaal van die genome van MDR kliniese isolate en in vitro selekteerde rifampisienweerstandige mutante om sodoende die genetiese grondslag waarop die vlak van rifampisienweerstandigheid beheer word, te identifiseer. Slegs klein verskille, bo en behalwe die rpoB mutasie, is geïdentifiseer in die genome van in vitro rifampisien weerstandige mutante wat verskillende vlakke van weerstandigheid getoon het. Dit dui aan dat hierdie mutante of ander regulatoriese meganismes gebruik, of hulle het enkelnukleotied polimorfismes buite die genetiese area wat in hierdie studie ondersoek is, waarmee rifampisien weerstandigheid gemoduleer word. In teenstelling hiermee het die genome van kliniese MDR isolate van die “Low Copy Clade” aansienlike variasie getoon in gene wat betrokke is by die selwand, sellulêre prosesse en lipiedmetabolisme. Verder het die genome van die Beijing genotipe variasie in gene getoon wat betrokke is by middelweerstandigheid en regstellende meganismes. Hierdie resultate dui aan dat die struktuur en prosesse van die selwand, asook lipiedmetabolisme, „n kritiese rol speel in die bepaling van die intrasellulêre konsentrasie van rifampisien. Opsommend, hierdie studie toon verskeie meganismes aan wat deur die bakterieë gebruik word om weerstandigheidsvlakke te moduleer en die kompleksiteit van die fisiologie van M. tuberculosis wat weerstandig is teen rifampisien.

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