Evolution of XDR-TB and the associated proteome

McGrath, Marieta (2016-12)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

ENGLISH SUMMARY: Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis, carries a substantial health burden worldwide and in South Africa. Efforts towards prevention and effective treatment of this disease is essential to meet the END TB goals. The disease is exacerbated by HIV co-infection and drug resistance. Multidrug-resistant (MDR) TB (resistance to at least rifampicin and isoniazid) places enormous resource constraints on TB control programs with poorer treatment outcomes. More recently, extensively drug-resistant (XDR) TB (MDR-TB with additional resistance to a fluoroquinolone and an injectable) has become a global concern. XDR-TB develops through the acquisition of mutations in the gyrA and rrs genes. The aim of this work was to investigate aspects of the evolution and physiology of XDR-TB, in particular acquisition of mutations and their impact on protein abundance. Assessment of the impact of nucleoside reverse transcriptase inhibitors on the mutation rate of Mycobacterium smegmatis indicated no significant effect, suggesting that antiretroviral treatment does not contribute to the overlap of HIV infection and drug-resistant TB. Analysis of spontaneous ofloxacin-resistant mutants indicated that a Beijing clinical isolate acquired high level drug resistance mutations more readily than H37Rv, providing a possible reason for the association of Beijing with drug resistance. Analysis of spontaneous moxifloxacin resistant mutants showed that gyrA mutations at codons 88 and 94 were associated with resistance (defined as minimum inhibitory concentration (MIC) of ≥ 2 μg/ml). Despite the presence of gyrA mutations, moxifloxacin significantly impeded bacterial growth, supporting its continued use for the treatment of ofloxacin-resistant M. tuberculosis. In an attempt to determine whether the fluoroquinolone MIC could be increased we selected spontaneous mutants from a fluoroquinolone resistant clone on higher concentrations of the fluoroquinolone. Sequencing of gyrA and gyrB identified additional mutations suggesting that double mutations are responsible for increasing the MIC. We could not find any involvement of efflux pump activity in modulating the MIC. To determine the influence of the gyrA Asp94Gly and rrs A1401G mutation on the physiology of the pathogen we assessed their effect on protein abundance. A strong signature of differentially abundant proteins common to both clones, expressed from the ESX-5 cluster, suggested either the presence of a common unknown genetic variant (in unmapped genomic regions) or common physiological changes related to drug resistance. The gyrA mutant uniquely demonstrated decreased abundance in transport proteins, suggesting decreased cell wall permeability and increased drug tolerance. Changed abundance of proteins involved in transcription/translation was also observed, suggesting impaired functionality of the mutated gyrase. The rrs mutant displayed lowered abundance of stress proteins belonging to the DosR/DevR regulon, potentially impacting the mutant‘s response to dormancy. Ofloxacin treatment of the gyrA mutant resulted in increased abundance of proteins involved in iron acquisition and differential abundance of proteins indicating decreased cell division and growth. We hypothesize that increased abundance of iron acquisition proteins relates to chelation of iron by ofloxacin. Amikacin treatment of the rrs mutant decreased ribosomal protein abundance and increased proteins involved in tRNA-related processes. We hypothesise that this relates to increased degradation of ribosomes and a mechanism to compensate for reduced translational fidelity. This study improved our understanding of the physiological factors contributing to the emergence of XDR-TB. Furthermore, our results suggest that the physiology of XDR M. tuberculosis differs from susceptible strains. These changes in physiology could inform further research on drug targets and optimal treatment regimens.

AFRIKAANSE OPSOMMING: Tuberkulose (TB), wat veroorsaak word deur die bakterie Mycobacterium tuberculosis, plaas aansienlike druk op gesondheidstelsels wêreldwyd en in Suid-Afrika. Pogings tot voorkoming en effektiewe behandeling van hierdie siekte is van kardinale belang om die Wêreld Gesondheidsorganisasie se doelwitte van "End TB― te bereik. Die siekte word vererger deur MIV mede-infeksie en middelweerstandigheid. Multi-middelweerstandige (MDR) TB (weerstandig teen isoniasied en rifampisien) plaas geweldige hulpbronbeperkings op TB behandelingsprogramme met swakker behandelingsuitkomste. Meer onlangs het ekstensiewe middelweerstandige (XDR) TB (MDR TB met bykomende weerstand tot ‗n fluorokinoloon en ‗n tweede-linie middel wat as inspuiting toegedien word) ‗n wêreldwye kommer geword. XDR TB ontwikkel deur die verkryging van mutasies in die gyrA en rrs gene. Die algemene doel van hierdie projek was om aspekte van die evolusie en fisiologie van XDR TB te ondersoek en spesifiek, die verkryging van mutasies en hulle impak op relatiewe kwantitatiewe proteïen-verskille. Assessering van die impak van nukleosied trutranskriptase inhibeerders op die mutasie tempo van Mycobacterium smegmatis het geen beduidende effek gewys nie, wat daarop dui dat antiretrovirale behandeling nie bydra tot die dikwelse oorvleueling van MIV en middelweerstandige TB nie. Analise van spontane ofloksasien-weerstandige mutante het gewys dat ʼn kliniese isolaat van die Beijing stamfamilie van M. tuberculosis meer geredelik mutasies wat hoë-vlak weerstandigheid veroorsaak, verkry het as die laboratorium stam, H37Rv, wat ʼn moontlike rede kan wees waarom die Beijing stamfamilie met middelweerstandigheid geassosieer word. Analise van spontane moxifloksasien-weerstandige mutante het gewys dat gyrA mutasies in kodons 88 en 94 met weerstandigheid (gedefineër as ‗n minimum inhiberende konsentrasie (MIK) van ≥ 2 μg/ml) geassosieer kon word. Ten spyte van die teenwoordigheid van gyrA mutasies het moxifloksasien bakteriële groei beduidend verminder, wat ondersteunende bewyse is vir die voortgaan van gebruik van die middel vir ofloksasien-weerstandige tuberkulose behandeling. In ‗n poging om te bepaal of die fluorokinoloon MIK verhoog kon word, is spontane mutante gekies vanaf ‗n fluorokinoloon-weerstandige kloon met hoër konsentrasie van die middel. Volgordebepaling van gyrA en gyrB het bykomende mutasies geïdentifiseer, wat daarop dui dat dubbele mutasies verantwoordelik is vir die verhoogde MIK. Geen betrokkenheid van effluks pompe by die modulering van die MIK is waargeneem nie. Om die invloed van die gyrA Asp94Gly en rrs A1401G mutasies op die fisiologie van die patogeen te bepaal, is relatiewe kwantitatiewe proteïen-verskille gemeet. Daar was ‗n sterk aanduiding van gemeenskaplike proteïen-verskille in die twee klone, spesifiek uitgedruk vanaf die ESX-5 geengroepgebied, wat moontlik toegeskryf kan word aan ‗n gemene genetiese variant (in genomiese gebiede waarvan die volgorde nie bepaal kon word nie) of ‗n gemene fisiologiese verandering verwant aan middelweerstandigheid. Die gyrA mutant het unieke veranderinge getoon, waaronder verminderde hoeveelhede van vervoer- proteïene, wat op verminderde deurlaatbaarheid van die selwand en potensieël verhoogde middeltoleransie dui. Verskille in proteïene betrokke by transkripsie en proteïen-sintese is ook waargeneem, wat dui op verswakte funksie van die gemuteerde girase. Die rrs mutant het verlaagde hoeveelhede van stres- proteïene gereguleer deur DosR/DevR getoon, wat potensieël die mutant se reaksie op dormansie kan beïnvloed. Behandeling van die gyrA mutant met ofloksasien het ‗n vermeerdering van proteïene betrokke by ysterverkryging tot gevolg gehad, sowel as proteïenverskille wat dui op verminderde selverdeling en groei. Daaruit het die hipotese ontstaan dat vermeerdering van ysterverkrygingsproteïene verwant is aan chelaatvorming van ofloksasien met yster. Behandeling van die rrs mutant met amikasien het gelei tot verminderde vlakke van ribosoom- proteïene en vermeerde vlakke van proteïene betrokke by oordrag-RNA prosesse. Daaruit het die hipotese ontstaan dat die veranderinge verwant is aan verhoogde afbreking van ribosome en ‗n kompenseringsmeganisme vir die verminderde akkuraatheid van proteïensintese. Hierdie studie het gelei tot verbeterde kennis van die fisiologiese faktore wat bydra tot die opkoms van XDR TB. Verder het die resultate daarop gedui dat die fisiologie van XDR M. tuberculosis verskil van middel sensitiewe stamme. Hierdie veranderings in fisiologie kan kennis bydra tot verdere navorsing op teikens vir antibakteriese middels en optimale behandelingsplanne.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100386
This item appears in the following collections: