Mechanisms of resistance to new generation anti-TB drugs

Visser, Hanri (2015-04)

Thesis (MScMedSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Drug resistance in Mycobacterium tuberculosis is an increasing global problem. Drug resistance is mostly caused by single nucleotide polymorphisms (SNPs) within the bacterial genome. This observed increase in global incidence of drug resistant tuberculosis (TB) has sparked the search for new anti-TB drugs and the repurposing of drugs that are currently used against other organisms or species of mycobacteria. One such repurposed drug, clofazimine (CFZ), is currently used for the treatment of leprosy, caused by Mycobacterium leprae. The mechanism of action of CFZ is not clear, but it is hypothesized that CFZ is reduced by a mycobacterial type II NADH oxidoreductase (NDH-2). The reduction of CFZ drives the production of reactive oxygen species (ROS) which is toxic to the pathogen. The aim of this study was to elucidate the mechanism of CFZ resistance. Towards this aim, spontaneous in vitro CFZ resistant mutants were selected, characterized and whole genome was used identify SNPs which may cause CFZ resistance. Mutations were identified in a transcriptional regulator encoded by Rv0678, fatty-acid-AMP ligase, or FadD28 (Rv2941) and glycerol kinase or GlpK (Rv3696c). Mutations in Rv0678 have previously been shown to play a role in both CFZ resistance and bedaquiline (BDQ) cross-resistance, while no link has been found between CFZ resistance and mutations in fadD28 and glpK. The novel, non-synonymous SNP identified in Rv0678 resulted in the replacement of an alanine residue with threonine at codon 84, which is located in the DNA binding domain. Virtual modelling of the mutated Rv0678 protein showed that the A84T mutation may influence DNA binding, possibly due to its proximity to the DNA binding domain. This mutation caused a change in hydrophobicity, which may influence binding to DNA. Previous studies showed that mutations in Rv0678 resulted in the upregulation of mmpL5, a putative efflux pump. However, the mechanism whereby CFZ resistance occurs via increased abundance of this efflux pump in the cell wall is not clear and needs further investigation. The cross-resistance between CFZ and BDQ, caused by mutations in Rv0678, is of concern and may influence the planning of anti-TB drug regimens for the future. The roles of the other two mutations identified in this study in CFZ resistance is also not clear and requires further investigation. Finally, the findings of this study support the role of Rv0678 in CFZ resistance thereby suggesting that this gene could be useful as a diagnostic marker to test for CFZ resistance in clinical isolates.

AFRIKAANSE OPSOMMING: Middelweerstandigheid in Mycobacterium tuberculosis is 'n wêreldwye toenemende probleem. Middelweerstandigheid word meestal veroorsaak deur enkel nukleotied polimorfismes (SNPs) in die bakteriële genoom. Hierdie toename in middelweerstandige tuberkulose (TB) het gelei tot die soektog na nuwe anti-TB-middels en die alternatiewe aanwending van middels wat tans teen ander organismes of spesies van mikobakterieë gebruik word. Een so 'n alternatiewe middel, clofazimine (CFZ), word tans gebruik vir die behandeling van melaatsheid wat veroorsaak word deur Mycobacterium leprae. CFZ se meganisme van werking is nie duidelik nie, maar dit word vermoed dat CFZ gereduseer word deur 'n mikobakteriële tipe II NADH oksidoreduktase (NDH-2). Die reduksie van CFZ dryf die produksie van reaktiewe suurstof spesies wat giftig is vir die patogeen. Die doel van hierdie studie was om die meganisme van CFZ weerstandigheid te ondersoek. Om hierdie doel te bereik was spontane in vitro CFZ weerstandige mutante gekies, gekarakteriseer en heel genoom volgorde bepaling is gebruik om SNPs te identifiseer wat CFZ weerstandigheid veroorsaak. Mutasies in Rv0678, 'n transkripsie reguleerder, vetsuur-AMP ligase, of FadD28 (Rv2941) en gliserol kinase of GlpK (Rv3696c) geïdentifiseer. Dit is al voorheen gevind dat mutasies in Rv0678 ‘n rol speel in beide CFZ weerstandigheid en bedaquiline (BDQ) kruis-weerstandigheid, terwyl geen verband gevind is tussen CFZ weerstandigheid en mutasies in fadD28 en glpK nie. Die nuwe, nie-sinonieme SNP, geïdentifiseer in Rv0678 het gelei to die vervanging van 'n alanien aminosuur met treonien by kodon 84, wat geleë is in die DNS bindings domein. Virtuele modellering van die gemuteerde Rv0678 proteïen het getoon dat die A84T mutasie DNS binding moontlik kan beïnvloed, as gevolg van sy nabyheid aan die DNS bindings domein. Hierdie mutasie veroorsaak 'n verandering in die hidrofobiese natuur, wat DNS binding kan beïnvloed. Vorige studies het getoon dat mutasies in Rv0678 lei tot die opregulering van mmpL5, 'n waarskynlike uitvloei pomp. Die meganisme waardeur CFZ weerstandigheid veroorsaak, deur ‘n groot aantal van hierdie uitvloei pompe in die selwand, is nie duidelik nie en moet verder ondersoek word. Die kruis-weerstandigheid tussen CFZ en BDQ, wat veroorsaak word deur mutasies in Rv0678, is van belang en kan die beplanning van anti-TB middel behandeling vir die toekoms beïnvloed. Die rolle van die ander twee mutasies, wat in hierdie studie geïdentifiseer is, in CFZ weerstandigheid is ook nie duidelik nie en vereis verdere ondersoek. Ten slotte, die bevindinge van hierdie studie steun die rol van Rv0678 in CFZ weerstandigheid en dit dui daarop dat hierdie geen gebruik kan word as 'n diagnostiese merker om vir CFZ weerstandigheid te toets in kliniese isolate.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96863
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