Clinically relevant mutations contributing to drug resistance in Mycobacterium tuberculosis

Du Plessis, Juanelle (2017-12)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Single nucleotide variants are the underlying driver of drug resistance, strain fitness and adaptation in Mycobacterium tuberculosis and investigating the mechanistic and physiological aspects of these mutations is key to our understanding of the biology of this pathogen. Rifampicin, one of the most powerful first line drugs used to treat tuberculosis, inhibits transcription in M. tuberculosis by binding to the β subunit of RNA polymerase (RNAP). However, mycobacteria are able to evade binding of this drug by acquiring mutations in the rpoB gene. These resistance-conferring mutations are essential for the survival of M. tuberculosis in the presence of rifampicin, however they impart a fitness cost to the bacterium due to a presumed reduction in transcription efficiency and subsequent changes in gene expression. One of the mechanisms M. tuberculosis uses to buffer the effects of this fitness cost is to acquire compensatory mutations in rpoC and rpoA. As RNAP is at the core of all mechanisms of gene regulation in M. tuberculosis, it is not surprising that mutations within this enzyme led to pleiotropic effects. As this remains a poorly understood area of mycobacterial physiology, the current work encompasses a triad of studies which aims to better understand functional aspects of M. tuberculosis RNAP, and the role of rpoB and rpoC mutations in drug resistance. First, the effect of a bacteriophage protein, Gp2, was investigated to determine whether it is able to inhibit RNAP in M. tuberculosis. As Gp2 is known to bind to the β’ subunit of RNAP in Escherichia coli, positive findings from this work would provide a framework for the identification of novel compounds that inhibit transcription in the presence of rpoB mutations, affecting the β subunit. By way of in vitro and in silico analysis, it was found that Gp2 binds to and inhibits RNAP in M. tuberculosis, however to a much lesser degree than it does in its de facto host, E. coli. Nonetheless, future studies can build on our findings as in silico modification of Gp2 could identify a structure which allows for stronger binding affinity of the protein. Secondly, the effect of rpoB and rpoC mutations on the function of mycobacterial RNAP was investigated using a series of in vitro transcription assays. Radioactivity-based assays were performed using purified wildtype and mutant versions of the RNAP complex, to assess enzyme activity and promoter affinity. Furthermore, the use of a fluorescence-based assay was trialled to develop a comparable method without the use of radiolabelled nucleotides. Lastly, we undertook a study to understand the effect of rpoC mutations on the transcriptome of M. tuberculosis. For this purpose, serial clinical isolates were selected where the acquisition of an rpoC mutation was observed. These samples were used for whole genome sequencing and gene expression analysis, which revealed a potential link between the rpoC V483G mutation and upregulation of Rv2416c (eis) and Rv1258c (tap). Serendipitously, genomic data also revealed that an ald mutation was acquired alongside the rpoC mutation. Recently, ald has been described as a novel gene linked to D-cycloserine resistance in M. tuberculosis, however, to date, the mechanism of drug resistance has not been determined. Given the unique opportunity to study the effect of ald mutations on gene expression in our study, we investigated two genes which were found to be differentially expressed in a clinical isolate with an ald mutation. A turbidity-based microdilution assay revealed that upregulation of Rv0577, a putative glyoxalase, led to an increase in the minimum inhibitory concentration of D-cycloserine, a finding which provides novel insight into the mechanism of D-cycloserine resistance in M. tuberculosis. In summary, this body of work has contributed to existing knowledge surrounding drug resistance and compensatory adaptation in M. tuberculosis.

AFRIKAANSE OPSOMMING: Enkel-nukleotiedvariante is die onderliggende dryfveer van middelweerstandigheid, fiksheid en aanpassing in Mycobacterium tuberculosis en die ondersoek van die meganistiese en fisiologiese aspekte van hierdie mutasies is die sleutel tot ons begrip omtrent die biologie van hierdie patogeen. Rifampisien, een van die mees kragtigste eerste linie medikasies wat gebruik word om tuberkulose te behandel, inhibeer transkripsie in M. tuberculosis deur te bind aan die β subeenheid van RNA polimerase (RNAP). Mikobakterieë kan egter die binding van hierdie geneesmiddel ontduik deur mutasies in die rpoB geen te ontwikkel. Hierdie weerstandigheidsmutasies is noodsaaklik vir die oorlewing van M. tuberculosis in die teenwoordigheid van rifampisien, maar as gevolg van ‘n vermoedelike vermindering in transkripsie doeltreffendheid en daaropvolgende veranderinge in geenuitdrukking, gee hierdie mutasies ‘n fiksheidskoste aan die bakterieë. Een van die meganismes wat M. tuberculosis gebruik om die effekte van hierdie fiksheidskoste te buffer, is om kompenserende mutasies in rpoC en rpoA te bekom. Aangesien RNAP die kern van alle meganismes van geen-regulering in M. tuberculosis is, is dit nie verbasend dat mutasies in hierdie ensiem tot pleiotropiese effekte lei nie. Aangesien dit 'n gebied van mikobakteriese fisiologie is wat steeds swak verstaan word, sluit die huidige werk 'n triade van studies in wat poog om die funksionele aspekte van M. tuberculosis RNAP en die rol van rpoB en rpoC mutasies in middelweerstandigheid beter te verstaan. Eerstens is die effek van 'n bakteriofaag proteïen, Gp2, ondersoek om te bepaal of dit RNAP in M. tuberculosis kan inhibeer. Dit is bekend dat Gp2 aan die β'-subeenheid van RNAP in Escherichia coli bind, daarom sal positiewe bevindings uit hierdie werk 'n raamwerk verskaf vir die identifisering van nuwe verbindings wat transkripsie inhibeer in die teenwoordigheid van rpoB mutasies, wat die β subeenheid beïnvloed. By wyse van in vitro en in silico analise is gevind dat Gp2 aan RNAP in M. tuberculosis bind en transkripsie inhibeer, maar tot 'n veel mindere mate as wat die geval is in sy de facto gasheer, E. coli. Nietemin kan toekomstige studies op ons bevindinge voortbou, aangesien in silico modifikasie van Gp2 'n struktuur kan identifiseer wat ’n sterker bindende affiniteit van die proteïen moontlik maak. Tweedens is die effek van rpoB en rpoC mutasies op die funksie van mikobakteriese RNAP met behulp van 'n reeks in vitro transkripsie toetse ondersoek. Radioaktiwiteits-gebaseerde toetse is uitgevoer met behulp van gesuiwerde normale en mutante weergawes van die RNAP kompleks om ensiem aktiwiteit en promotor affiniteit te assesseer. Verder is fluoressensie-gebaseerde toetse gebruik om 'n vergelykbare metode te ontwikkel sonder die gebruik van radioaktief-gemerkte nukleotiede. Laastens het ons 'n studie onderneem om die effek van rpoC mutasies op die transkriptoom van M. tuberculosis te verstaan. Vir hierdie doel is ’n reeks opeenvolgende kliniese isolate gekies waar die ontwikkeling van die rpoC mutasie waargeneem is. Hierdie monsters is gebruik vir die heel genoom volgordebepaling en geenuitdrukking analise, wat 'n moontlike verband tussen die rpoC V483G mutasie en opregulering van Rv2416c (eis) en Rv1258c (tap) onthul het. Genomiese data het ook per toeval aan die lig gebring dat 'n ald mutasie saam met die rpoC mutasie ontwikkel het. Ald is onlangs beskryf as 'n nuutgevonde geen wat gekoppel is aan D-sikloserienweerstandigheid in M. tuberculosis, maar die meganisme van weerstand is egter tot op hede nog nie bepaal nie. Gegewe die unieke geleentheid om die effek van ald mutasies op geenuitdrukking in ons studie te bestudeer, het ons twee gene ondersoek wat differensiëel uitgedruk is in 'n kliniese isolaat met 'n ald mutasie. 'n Turbiditeits-gebaseerde mikroverdunningstoets het getoon dat opregulering van Rv0577, 'n vermeende glioksilase, gelei het tot 'n toename in die minimum inhibitiewe konsentrasie van D-sikloserien, 'n bevinding wat nuwe insig bied in die meganisme van D-sikloserienweerstandigheid in M. tuberculosis. In samevatting, hierdie werk het bygedra tot bestaande kennis rondom middelweerstandigheid en kompenserende aanpassing in M. tuberculosis.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/102613
This item appears in the following collections: