Thesis (MScMedSc)--Stellenbosch University, 2015. / 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.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Tuberculosis (TB) has become a global health epidemic affecting millions of people worldwide with a high incidence in third-world countries. The emergence of multi-drug and extremely-drug resistant M. tuberculosis strains together with the HIV/AIDS pandemic warrants the need for new drugs or new drug combinations. The folic acid synthesis pathway is one of the key pathways that are essential for the survival of bacteria in general. Sulfonamides are a group of compounds that target folic acid synthesis, particularly dihydropteroate synthetase, the first enzyme in the folate pathway. Some of these sulfonamides were used during the introduction of chemotherapy for the treatment of TB in the 1930s, but had toxic side effects. Newer derivatives became safer, but were not employed again for TB treatment. In a recent case study it was reported that the combination of trimethoprim-sulfamethoxazole (Bactrim), which is used to treat various bacterial infections, such as urinary tract infections, had activity against M. tuberculosis. In light of this and the fact that trimethoprim-sulfamethoxazole is well tolerated by humans, we have investigated their antimycobacterial activity with particular interest in the combinational effect of sulfamethoxazole and trimethoprim with the first-line anti-TB drugs, Isoniazid, Rifampicin and Ethambutol against M. tuberculosis. Since sulfonamides are known to produce oxidative stress, we also investigated the contribution of this factor to the efficacy of sulfamethoxazole using a mycothiol deficient strain of M. tuberculosis, ΔmshA. Though trimethoprim-sulfamethoxazole targets the folic acid pathway, we also investigated the possibility that trimethoprim-sulfamethoxazole may have other cellular targets and applied proteomic analysis. We have found that Trimethoprim-Sulfamethoxazole has activity against M. tuberculosis and that Sulfamethoxazole is the active compound. However, our observation was that not all sulfonamides are active against M. tuberculosis. In addition we observed that sulfamethoxazole enhances the activity of Rifampicin against M. tuberculosis in a synergistic way. We also observed that a mycothiol deletion mutant was more susceptible to Sulfamethoxazole compared to the wild type strain CDC 1551. Through global protein expression profiling (Proteomics) we were also able to show that sulfamethoxazole could also kill M. tuberculosis by oxidative stress production as we identified oxidative stress responsive proteins that were differentially regulated upon exposure to sulfamethoxazole. As trimethoprim-sulfamethoxazole is a registered drug combination, inexpensive and widely available, we propose that this regimen could be used in our fight against M. tuberculosis infection. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is ‘n globale gesondheidsprobleem wat miljoene mense wêreldwyd affekteer met ‘n besoderse hoë voorkoms in die derdewêreld lande. Die voorkoms van multi-middel weerstandige en uitersweerstandige M. tuberculosis stamme, tesame met die HIV/VIGS pandemie, steun die erns vir die ontwikkeling van nuwe middels teen M.tuberculosis . Die foliensuur sintesepad is essensieël tot die oorlewing van bakterieë in die algemeen. Vir daardie rede is daar vele middels ontwerp om hierdie metaboliese pad te teiken. Die sulfonamiedes is ‘n groep antibiotika wat foliensuursintese, spesifiek dihidropteroaatsintese, die eerste ensiem in die foliensuursintese pad, teiken. Van hierdie sulfonamiedes is voorheen in die 1930’s gebruik vir die behandeling van tuberkulose, maar het toksiese newe-effekte getoon. Nuwe, minder toksiese derivate, is later ontwikkel maar is nooit vir TB behandeling weer aangewend nie. In ‘n onlangse gevallestudie is daar gerapporteer dat die kombinasie trimethoprim-sulfamethoxazole (TMP/SMX. Handelsnaam: Bactrim), wat normaalweg gebruik word vir die behandeling van algemene bakteriële infeksies soos blaasinfeksies, aktiwiteit teen M. tuberculosis getoon het. Na aanleiding hiervan en dat Bactrim veilig in mense gebruik kan word, het ons die aktiwiteit van Bactrim komponente teen M. tuberculosis bepaal en in die besonder die aktiwiteite van SMX en TMP in kombinasie met die eerstelinie anti-tuberkulose middels Isoniasied, Rifampisien en Ethambutol. Aangesien sulfonamiedes ook oksidatiewe stres intrasellulêr genereer, het ons ook die bydrae van hiervan tot die doeltreffendheid van SMX bepaal deur gebruik te maak van ‘n mycothiol-gemuteerde M. tuberculosis stam ( mshA). Omdat TMP/SMX die foliensuur-pad hoofsaaklik teiken het ons ook die moontlikheid ondersoek dat SMX ander sellulêre teikens het en het ons proteomiese (Proteomics) tegnieke hiervoor aangewend. Ons het gevind dat TMP/SMX aktiwiteit teen M. tuberculosis toon en dat SMX die aktiewe komponent van Bactrim is teen M. tuberculosis . Ons wys ook dat sulfonamiedes in die algemeen nie noodwendig ook aktiwiteit teen M. tuberculosis toon nie. Ons het ook waargeneem dat SMX die aktiwiteit van rifampisien bevorder en dat die twee middels saamwerk op ‘n sinergistiese wyse. Ons het ook getoon dat oksidatiewe stres ‘n rol speel deurdat‘n mycothiol delesie-mutant meer vatbaar was vir SMX in vergelyking met die wilde-tipe stam van M. tuberculosis (CDC1551). Met globale proteïenkartering (Proteomics) het ons ook getoon dat SMX M. tuberculosis kan doodmaak deur oksidatiewe stres te genereer omdat ons oksidatiewe stres reaktiewe proteïne geïdentifiseer het wat differensieël gereguleer is gedurende blootstelling aan SMX. Aangesien Bactrim ‘n reeds geregistreerde middel is, goedkoop is en geredelik beskikbaar is, stel ons voor dat Bactrim moontlik geïnkorporeer kan word in die huidige behandeling van Tuberkulose.

Thesis (MSc. (Medical Sciences)) -- University of Limpopo, 2015 / Introduction: Multidrug-resistant tuberculosis (MDR-TB) caused by resistance to at least rifampicin (RIF) and isoniazid (INH) drugs is a growing public health concern in South Africa. The detection of MDR-TB still relies on culture despite advancement in molecular diagnostic technology. Currently MTBDRplus and GeneXpert are the only available assays used in rapid diagnosis of MDR-TB using chromosomal mutations in drug target regions. Some strains are missed by these assays due to their limitation in mutational detection profile. Novel Seegene Anyplex assays simultaneously detect TB and resistance to RIF and INH using fifteen and six mutational probes, respectively within 3 hours. Limpopo Province has limited information on the circulating strains of TB. Aim: To determine drug-resistant Mycobacterium tuberculosis (M. tuberculosis) mutations using Anyplex™ MTB/NTM/MDR-TB real time assay and characterise the drug-resistant strains. Methods: We prospectively collected 204 clinical samples at Modimolle MDR-TB unit and retrospectively used 104 culture isolates from MRC laboratory in Pretoria. The MTBDRplus assay was used to screen for M. tuberculosis and drug resistant mutations to RIF and INH drugs. Anyplex™ MTB/NTM/MDR-TB assay was used for rapid detection of M. tuberculosis and drug resistance to RIF and INH within 3 hours. The discordance between phenotypic and genotypic assays was resolved by sequencing and the Anyplex™ resistant profiles were spoligotyped. Diagnostic data was collected from NHLS and MRC databases and analysed using the Microsoft excel and Epi Info version 3.5. Descriptive statistics (percentages and frequencies) were used to explain proportions. Results: The Anyplex™ MTB/NTM assay detected M. tuberculosis in 69/111(62%) and 100/104 (96%) of clinical and culture samples respectively. The sensitivities, specificity, PPV and NPV obtained for both RIF and INH resistance by Anyplex™ MDR-TB assay were 67%, 59%, 67%, 55% and 15%, 100%, 100% and 17%, respectively. Anyplex™ MTB/NTM/MDR-TB resolved 23/45 (51%) of discordant vi samples. Sequencing of remaining discordant isolates revealed L511P, L533P and D516Y mutations within rpoB gene. A novel R385W mutation within katG was also detected. Spoligotyping of Anyplex™ MDR-TB resistant clinical isolates revealed Euro American clade with 20% followed by 15% Manu2, 5% East African Indian, 5% H37Rv, 5% atypical and 50% were orphans. Conclusion: The novel Anyplex™ MTB/NTM/MDR-TB assay is a rapid and valid technique for detecting M. tuberculosis and most common mutations conferring resistance to RIF and INH. However further investigations are required, as the assay has a lower sensitivity as compared to already endorsed techniques. / National Research Foundation (NRF) and University of Limpopo TB Grant

Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2017. / According to the Namibia National Tuberculosis Control Programme (NTCP) report of 2008, Namibia has one of the highest TB infection rates in the world with a case notification rate of 748/100,000. Rapid, specific and sensitive diagnosis of Mycobacterium tuberculosis (MTB) is needed for correct TB patient management. One of the aims of this study was thus to compare direct microscopy with two rapid molecular diagnostic tools (viz. GeneXpert MTB/RIF and Hain Genotype® MTBDR plus assay) for the identification of MTB from samples collected from the Khomas Region, Windhoek, Namibia. Only patients with positive TB sputum collected at the clinics and health facilities in the Khomas Region, Windhoek were eligible for the study. Three hundred and eighty-four samples were confirmed acid-fast positive by utilising the auramine staining method. The rifampicin (RIF) resistance profile detected by both molecular techniques was then compared for characterisation of the samples as drug resistant. Lastly, participants completed a survey, which included questions related to demographic and epidemiological data. Demographic data included patient age, gender, region of residence and history of treatment. The data was collected using a structured questionnaire and was captured in an Excel spreadsheet. It was then imported into Statistical Package for Social Sciences (SPSS) Version 25 for data analysis. A memorandum of understanding was also signed with the Namibia Institute of Pathology (NIP) to obtain permission to use their samples and the equipment at their site.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Setting This study was conducted in the Tygerberg district, Cape Town, in the Western Cape, South Africa Background The evaluation of early tuberculosis (TB) treatment response is based on month 2 sputum culture status. This method of evaluation has a number of limitations: the test requires relatively advanced laboratory infrastructure and procedures, it takes several weeks to obtain results and is a relatively a poor marker at predicting treatment response. The discovery of potential host markers which reflect the efficacy of early treatment would be of great importance for clinical management of individual patients. The treatment failure would be detectable earlier than at week 8 of treatment. The duration of clinical trials of new anti-tuberculosis drugs may also be substantially reduced by such markers if these would be measurable earlier than at week 8 of therapy. Objectives 1) To evaluate diluted, 7-day whole blood cultures stimulated with live Mycobacterium tuberculosis (M.tb) for the presence of host markers of early TB treatment response 2) To evaluate an overnight, undiluted, M.tb antigen stimulated whole blood culture Quantiferon Gold In Tube (QFT-GIT) supernatants for host markers of early TB treatment response The study designs were as follows: In study one, baseline samples and samples from week 1, week 2 and week 4 of treatment from 30 cured TB patients were selected from a larger biomarker study, in which whole blood was stimulated with live M.tb or left unstimulated. Fifty seven host markers were measured in supernatants by multiplex cytokine arrays. In study two, baseline samples and samples from week 2 and week 8 of treatment from 19 cured TB patients were randomly selected from the placebo group in a micronutrient supplement study. QFT-GIT supernatants from these participants were assessed through multiplex cytokine arrays for levels of fifty seven host markers. All of the participants in both studies were Human Immunodeficiency Virus (HIV) negative. Changes in marker expression over time and between fast and slow responders to treatment were evaluated. Comparability between the two culture methods was assessed for markers that were evaluated in both studies. Results In study one, the majority of host markers showed significant changes over time in the unstimulated supernatants. Only GRO and IL-1beta changed significantly in an antigen-specific manner (background levels subtracted). No significant changes were observed between fast and slow responders. In study two, the majority of host markers showed significant changes over time in the unstimulated supernatants whereas only MDC and IL-4 changed during the observation period in antigen stimulated levels. Significant differences were observed between fast and slow responders at pre-treatment for IL-13 Ag-Nil and IL-1betaAg-Nil . Conclusion This study revealed, antigen-specific responses showed only limited potential for early TB treatment response monitoring, but may have potential in differentiating between treatment outcomes. Future investigations may have to include later time points during treatment as these were not included in the present assessment. The QFT-GIT samples do not appear to be equivalent to live M.tb stimulated 7-day whole blood assays. / AFRIKAANSE OPSOMMING: Instelling Die studie is uitgevoer in die Tygerbergdistrik, Kaapstad, Wes-Kaap, Suid-Afrika. Agtergrond Die evaluering van die respons op vroeë tuberkulose (TB) behandeling word gebaseer op die status van maand 2 sputum kulture. Hierdie evalueringsmetode het ‘n paar beperkinge: die toets benodig relatief gevorderde laboratorium infrastruktuur en prosedures, die toetsuitslae is eers na ‘n paar weke beskikbaar en dit is n relatiewe swak merker om repons op behandeling te voorspel. Die ontdekking van potensiële selfmerkers wat die doeltreffendheid van vroeë behandeling weerspieël sal van groot belang wees vir die kliniese bestuur van individuele pasiënte. Mislukking van die behandeling sal sodoende voor week 8 van behandeling waargeneem kan word. Die tydsduur van kliniese proewe van nuwe anti-tuberkulose medikasie mag ook baie verkort word met sulke merkers as dit voor week 8 van behandeling gemeet kan word. Doelwitte 1) Om verdunde, 7-dae oue volbloedkulture, met lewende Mikobakterium tuberkulosis (M.tb) gestimuleer, te evalueer vir die teenwoordigheid van vroeë TB behandeling respons selfmerkers. 2) Om die supernatant van oornag, onverdunde, M.tb antigeen gestimuleerde volbloedkulture Quantiferon Gold In Tube (QFT-GIT) vir vroeë behandeling respons selfmerkers te evalueer. Die studie-ontwerpe was soos volg: Met studie een is basislynmonsters en monsters verkry na week 1, week 2 en week 4 van behandeling van 30 geneesde TB-pasiënte geselekteer uit ‘n groter biomerkerstudie waarin die volbloed met lewende M.tb gestimuleer is of ongestimuleer gelaat is. Sewe-en-vyftig selfmerkers is in die supernatante gemeet deur middel van multipleks sitokine arrays. Met studie twee is basislynmonsters en monsters verkry na week 2 en week 8 van behandeling van 19 geneesde TB-pasiënte lukraak uit die plasebo-groep in ‘n mikrovoedingstowwe-aanvullingstudie geselekteer. Vlakke van 57 selfmerkers is in die QFT-GIT supernatante van hierdie deelnemers, deur middel van die multipleks sitokine arrays, bepaal. Al die deelnemers van beide studies was HIV negatief. Veranderinge in merker-uitdrukking oor tyd, asook tussen vinnige en stadige respons tot behandeling, is ge-evalueer. Die vergelykbaarheid van die twee kultuurmetodes is geassesseer ten opsigte van die ge-evalueerde merkers in albei studies. Resultate Met studie een het die meerderheid van die selfmerkers in die ongestimuleerde supernatante kenmerkende verandering oor tyd gewys. Slegs GRO en IL-1beta het aansienlik verander in die antigeenspesifieke wyse (agtergrond vlakke afgetrek). Geen kenmerkende veranderinge was waargeneem tussen die vinnige en stadige respons pasiënte nie. Met studie twee het die meerderheid van die selfmerkers aansienlike veranderinge oor tyd in die ongestimuleerde supernatante gewys, in vergelyking waar net die MDC en IL-4 veranderinge gedurende die observasie periode in antigeen gestimuleerde vlakke getoon het. Kenmerkende verskille is tussen die vinnige en stadige respons pasiënte in voorbehandeling vir IL-13 Ag-Nil en IL-1betaAg-Nil waargeneem. Gevolgtrekking Die studie bewys dat antigeenspesifieke response slegs beperkte potensiaal vir vroeë TB behandeling respons monitering het, maar mag die potensiaall vir onderskeidende behandeling uitkomste hê. Toekomstige ondersoeke sal dalk latere tydpunte gedurende die behandeling moet insluit aangesien dit nie in hierdie evaluasie ingesluit is nie. Die QFT-IT monsters verskyn nie as gelykwaardig met die lewendig M.tb gestimuleerde 7-dae volbloed toetse nie.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Isoniazid forms part of the first line anti-tuberculosis therapy and it is generally used to treat latent Mycobacterium tuberculosis infection. Isoniazid inhibits synthesis of long chain mycolic acids found in cell wall of Mycobacterium tuberculosis, which have proven vital for the survival of the bacterium. Mycolic acids are primarily synthesized by the fatty acid synthase enzyme (FAS) system found in mycobacteria as the FAS-I and FAS-II complex. Isoniazid kills the bacteria by blocking the FAS-II complex, required for extension of mycolates. It does this by entering the tubercle bacilli as a prodrug where isoniazid becomes activated by catalase peroxidase encoded by katG gene. The activated isoniazid then forms a complex with NAD+ which targets InhA (NADH-dependent enoyl-acyl carrier protein reductase) located in the FAS-II complex. Loss of catalase peroxidase, due to gene mutations or a complete katG gene deletion is one of the primary mechanisms conferring resistance to INH in Mycobacterium tuberculosis. In addition, four other genes (inhA, KasA, ndh and ahpC) are also associated with INH resistance. Nonetheless, mutations in these five genes are present in only 70-80% of INH resistant clinical isolates, implying that other mechanisms are involved in resistance of Mycobacterium tuberculosis to isoniazid. This study aims to quantify the expression level of genes induced by isoniazid in the mycolic acid pathway and drug transport in two closely related Mycobacterium tuberculosis Beijing cluster 208 isolates. These are the fully susceptible (K636) and isoniazid mono-resistance strains (R55), with minimum inhibitory concentrations of 0.1 and 4 µg/ml, respectively. Both these isolate had no isoniazid gene associated mutations. The isolates were cultured in the presence and absence of 0.1µg/ml isoniazid for 24 hours after which RNA was extracted followed by QRT-PCR analysis to identify differentially expressed genes. This result has shown that various genes were differentially expressed in response to low level INH exposure. The most significant up-regulation was observed in genes (acpM, fabD, Accd6 and fbpC) encoding the FAS-II complex and genes (efpA, iniA, iniB, and mmpl7) involved in drug transport. In addition, two genes (ndh and fbpC) were significantly down-regulated in the isoniazid mono-resistant isolate. Based on these findings, we propose a model whereby isoniazid exposure in the susceptible isolate inhibits FAS-II complex and with its associated accumulation in mycolates kills the bacterium. In contrast, we propose that in the resistance isolate the bacterium acquires additional resistance by the activation of efflux pumps in combination with disruption in INH-NAD+ complex formation that protect inhibition of InhA located in FAS-II complex. / AFRIKAANSE OPSOMMING: Isoniasied vorm deel van die eerste linie van behandeling teen tuberkulose en word algemeen gebruik om latente Mycobacterium tuberculosis infeksie te behandel. Isoniasied inhibeer die sintese van langketting mikolitiese sure wat in die selwand van Mycobacterium tuberculosis voorkom. Dit is bewys dat hierdie sure essensieel is vir die oorlewing van die bakterie. Mikolitiese sure word hoofsaaklik gesintetiseer deur die vetsuur sintase ensiem (FAS) sisteem wat in mikobakteriee voorkom as die FAS-I en FAS-II komplekse. Isoniasied dood die bakteriee deur die FAS-II kompleks, wat nodig is om die verlenging van mikoliete, te blokkeer. Dit word bewerkstellig deurdat 'n pro-vorm van die middel die tuberkulose bacilli binnedring, waarna isoniasied geaktiveer word deur katalase peroksidase, wat deur die katG geen geenkodeer word. Die geaktiveerde isoniasied vorm 'n kompleks met NAD+, wat InhA (NADH-afhanklike eno.asiel draer prote.enreduktase), gelee in die FAS-II kompleks teiken. Een van die primere meganismes wat weerstandigheid teen isoniasied bewerkstellig, is die verlies van katalase peroksidase weens geenmutasies of algehele delesie van die katG geen. 'n verdere vier gene (inhA, kasA, ndh en ahpC) word ook verbind met isoniasied weerstandigheid. Nietemin is mutasies in hierdie vyf gene teenwoordig in slegs 70-80% van isoniasied weerstandige kliniese isolate, wat impliseer dat ander meganismes ook betrokke is in die weerstandigheid van Mycobacterium tuberculosis teen isoniasied. Die doel van hierdie studie is om die vlak van uitdrukking van gene wat deur isoniasied in die mikolitiese suur biochemiese pad geïnduseer word, asook middel transport te kwantifiseer in twee naby verwante Mycobacterium tuberculosis isolate van Beijing groep 208. Die twee isolate is die volledig sensitiewe (K636) en isoniasied monoweerstandige (R55), met minimum inhiberende konsentrasies van onderskeidelik 0.1 en 4µg/ml. Mutasies wat geassosieer word met isoniasied weerstandigheid was afwesig in beide die isolate. Kulture is van die isolate gemaak met en sonder 0.1µg/ml isoniasied vir 24 uur, waarna RNA geekstraeer is deur middel van QRT-PCR analise om gene te identifiseer wat verskillend uitgedruk word. Die resultate toon dat verskeie gene verskillend uitgedruk is in reaksie op laevlak isoniasied blootstelling. Die mees prominente opregulering is waargeneem in die gene (acpM, fabD, accd6 en fbpC) wat die FAS-II kompleks enkodeer, asook die gene (efpA, iniA, iniB en mmpl7) wat betrokke is in middel transport. Beduidende afregulering van 'n verdere twee gene in die isoniasied monoweerstandige isolate, naamlik ndh en fbpC is waargeneem. Op grond van hierdie waarnemings, stel ons 'n model voor waarvolgens isoniasied blootstelling in die sensitiewe isolaat die FAS-II kompleks inhibeer, en met die gevolglike akkumulasie van mikoliete, dood dit die bakterium. In teenstelling stel ons voor dat addisionele weerstandigheid bekom word in die weerstandige isolaat deur die aktivering van uitvloeipompe, in kombinasie met die ontwrigting van die INH-NAD+ kompleksvorming wat die inhibisie van InhA binne die FAS-II kompleks beskerm.

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Anti-tuberculosis drugs target specific essential cellular processes and structural components. The first line drug, rifampicin (RIF) is a RNA polymerase inhibitor which targets the β-subunit and subsequently inhibits the initiation of transcription. Previous proteomic and transcriptomic analyses have shown that exposure to RIF for 24hrs significantly increased the abundance of proteins involved in energy metabolism in clinical isolates. No studies have been done to describe the transcriptional responses to RIF in an in vitro RIF resistant M. tuberculosis isolate. Application of in vitro mutants is novel since it will exclude most of the confounding factors which may be present in clinical isolates obtained from patients where the bacterium may have been incubated for several weeks or even years. This study aimed to determine the effect of prolonged exposure to RIF and the effect of the rpoB Ser531Leu mutation on the expression of energy metabolism genes, sigma factors and a regulator in RIF mono-resistant in vitro mutants with different levels of RIF resistance (minimum inhibitory concentration (MIC): 40μg/ml and 70μg/ml). RIF mono-resistant in vitro mutants were generated from a pan susceptible Beijing cluster 208 progenitor using the Luria Delbruck assay. In vitro RIF mono-resistant mutants harbouring the Ser531Leu rpoB mutation and which displayed different levels of RIF resistance were selected. To assess the effect of prolonged RIF exposure on the expression of candidate genes, the in vitro mutants were cultured in liquid media and exposed to RIF for 1, 7 and 14 days. High quality RNA was extracted from these cultures at each time point and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) was done on the selected candidate genes. The results indicate that limited expression of energy metabolism genes and sigma factors was observed after prolonged RIF exposure. In addition, the activity of the regulator (Rv1846c) was down-regulated in the presence of RIF explaining the up-regulated state of energy metabolism genes. To assess the effect of the rpoB Ser531Leu mutation on the candidate genes, RNA was extracted from the RIF unexposed culture at mid-log phase. RT-qPCR was done for each in vitro mutant in addition to the wild-type progenitor isolate. These results show that energy metabolism genes and sigma factors were significantly up-regulated in the RIF resistant mutantss harbouring an rpoB Ser531Leu mutation. This suggests that the mutation had a significant effect on the cellular energy cost due to the up-regulated state of the energy metabolism genes. In addition, an increase in the expression of sigma factors may be required to compensate for the rpoB mutation by enforcing the binding of the RNA polymerase and sigma factors to the promoter for transcription to be initiated. It is therefore important to assess these candidate genes for their potential as novel candidates for future drug design as this is an important aspect to influence tuberculosis control. / AFRIKAANSE OPSOMMING: Teen-tuberkulose middels teiken essensiële sellulêre prosesse en strukturele komponente. Die eerste linie teen-tuberkulose middel, rifampisien (RIF) is ʼn RNS polimerase inhibeerder wat die β-subeenheid teiken en daarna die inisiasie van transkripsie onderdruk. Vorige proteomiese en transkriptomiese analises het getoon dat blootstelling aan RIF vir 24 uur beduidende styging in sekere protiene wat verband hou met energie metabolisme in kliniese isolate veroorsaak. Die huidige studie poog om die effek van langdurige RIF blootstelling, asook die effek van die rpoB Ser531Leu mutasie op die uitdrukking van energie metabolisme gene, sigma faktore en reguleerders op RIF-enkel weerstandige in vitro mutante by verskillende vlakke van RIF weerstandigheid (Minimum Inhiberende Konsentrasie (MIK): 40μg/ml en 70μg/ml) te ondersoek. RIF-enkelweerstandige in vitro mutante isolate is gegenereer van ʼn sensitiewe Beijing 208 stamfamilielid deur die Luria Delbruck metode. In vitro RIF enkelweerstandige mutante met die rpoB Ser531Leu mutasie en verskillende vlakke van RIF weerstandigheid is geselekteer. Om die langdurige effek van RIF blootstelling op kandidaat geen uitdrukking te ondersoek, is in vitro mutante isolate gegroei in vloeibare medium en blootgestel aan RIF vir 1, 7 en 14 dae. Goeie kwaliteit RNS is geëkstraheer van hierdie kulture by elke tydpunt om Werklike-tyd Kwantitatiewe Polimerase Ketting Reaksie (RT-qPCR) op die kandidaat gene uit te voer. Die resultate toon dat ʼn beperkte aantal energie metabolisme en sigma faktor gene uitgedruk was na RIF blootstelling. Verder is die uitdrukking van die reguleerder (Rv1846c) af gereguleer in die teenwoordigheid van RIF en dit verduidelik die op gereguleerde energie metaboliese geen patroon. Om die effek van die rpoB Ser531Leu mutasie op die kandidaat gene te evalueer, is RNS geëkstraheer van ʼn weerstandige en RIF sensitiewe kultuur wat nie blootgestel was aan RIF nie. RT-qPCR is uit gevoer op elke in vitro mutante isolaat asook op ʼn sensitiewe isolaat sonder ʼn mutasie. Hierdie resultate toon dat energie metabolisme gene en sigma faktore beduidend opreguleer word in die isolate met ʼn rpoB Ser531Leu mutasie. Dit dui daarop dat die mutasie ʼn beduidende effek op die sellulêre energie koste het, omdat die energie metabolisme gene op gereguleer is. Verder kan ʼn toename in die uitdrukking van sigma faktore benodig word om die effek van die rpoB mutasie te oorkom deur binding van die RNS polimerase en die sigma faktore aan die promotor om transkripsie inisiasie te forseer. Dit is daarom belangrik om hierdie kandidaat gene verder te ondersoek vir toekomstige ontwikkeling van teenmiddels teen tuberkulose.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Multidrug resistant tuberculosis (MDR-TB), defined as having resistance to at least the first-line drugs, isoniazid and rifampicin (RIF), is a global health problem. Mutations in the rpoB gene, encoding the β-subunit of RNA polymerase, are implicated in RIF resistance - with the S531L and H526Y mutations occurring most frequently. The level of RIF resistance varies for strains with identical rpoB mutations, which suggests that other factors play a role in RIF resistance. Efflux has been implicated in determining the intrinsic level of RIF resistance. Increased expression of the multidrug efflux pump, Rv1258c, following RIF exposure was observed in some Mycobacterium tuberculosis MDR clinical isolates and H37Rv RIF mono-resistant mutants, but not others. The factors influencing the induction of Rv1258c are poorly understood. The aim of this study was to investigate the effects of rpoB mutations on expression of Rv1258c and whiB7, a transcriptional regulator of Rv1258c, in M. tuberculosis H37Rv in vitro generated RIF resistant mutants, in the absence and presence of RIF. The promoter region of M. tuberculosis H37Rv Rv1258c was cloned into a position upstream of a lacZ gene (encoding β-galactosidase) in multi-copy episomal and integrating vectors. Vector functioning and the effect of rpoB mutations on Rv1258c promoter activity were initially investigated in the non-pathogenic related species, Mycobacterium smegmatis mc2155 rpoB mutants and subsequently in M. tuberculosis by doing β-galactosidase assays. qRT-PCR was done to investigate the effects of rpoB mutations on native Rv1258c and whiB7 gene expression. Episomal and integrating vectors were functional and the integrating vector system was used for subsequent β-galactosidase assays in M. tuberculosis. Rv1258c promoter activity in the S531L mutant was approximately 1.5 times less and in the H526Y mutant 1.5 times higher than that of the wild-type in M. smegmatis. Similarly, Rv1258c promoter activity in the S531L mutant was approximately half and in the H526Y mutant approximately double that of the wild-type in M. tuberculosis. A similar trend in Rv1258c and whiB7 expression to those observed using β-galactosidase assays were observed when investigating the native Rv1258c and whiB7 gene transcript levels compared to the wild-type using qRT-PCR, although differences were not significant. Exposure of the M. smegmatis and M. tuberculosis rpoB mutants to sub-inhibitory levels of RIF did not affect Rv1258c promoter activity. Mutations in rpoB had a marginal effect on Rv1258c and whiB7 transcript levels, but showed the same trend as that seen for Rv1258c promoter activity. It remains to be determined whether these differences are biologically significant. When considering efflux pumps as new targets for treatment, possible differences in efflux pumps expression due to different rpoB mutations should be considered. / AFRIKAANSE OPSOMMING: Multi-middel weerstandige tuberkulose (MDR-TB) word gedefinieer as weerstandigheid tot ten minste rifampisien (RIF) en isoniasied, wat deel van die eerstelyn anti-tuberkulose behandeling vorm. Mutasies in die rpoB geen, wat die β-subeenheid van die RNA polimerase enkodeer, word geassosieer met RIF weerstandigheid. S531L en H526Y rpoB mutasies kom die algemeenste voor. RIF weerstandigheids vlakke verskil egter tussen isolate met identiese rpoB mutasies, wat impliseer dat ander faktore ook 'n rol in RIF weerstandigheid speel. 'n Toename in transkripsie van die Rv1258c geen, wat 'n multi-middel effluks pomp enkodeer, is waargeneem met blootstelling aan RIF, slegs in sommige M. tuberculosis H37Rv RIF mono-weerstandige mutante and MDR kliniese isolate, maar nie in ander nie. Die faktore wat die induksie van die Rv1258c effluks pomp beïnvloed is nie goed nagevors nie. Die studie ondersoek die effek van die rpoB mutasies op die uitdrukking van die Rv1258c en whiB7,'n transkripsionele regulator van Rv1258c, gene in M. tuberculosis H37Rv in vitro gegenereerde RIF weerstandige mutante, in die teenwoordigheid en afwesigheid van RIF. Die promotor area van die M. tuberculosis H37Rv Rv1258c geen is in 'n posisie stroomop van 'n lacZ geen, wat vir β-galaktosidase enkodeer, in multi-kopie episomale en integreerende vektors ingekloneer. Die funksionaliteit van die vektor en effek van rpoB mutasies op Rv1258c promotor aktiwiteit is ondersoek in die naverwante nie-patogeniese spesies, M. smegmatis en daarna in M. tuberculosis deur β-galaktosidase essais te doen. qRT-PCR is gedoen om die effek van rpoB mutasies op die vlak van transkripsie van die natuurlike Rv1258c geen en die whiB7 geen te bestudeer. Beide die episomale en integreerende vektors was funksioneel en daar is besluit om die integreerende vektor vir daaropeenvolgende β-galaktosidase essais in M. tuberculosis te gebruik. Rv1258c promotor aktiwiteit van die S531L mutant was ongeveer 1.5 keer minder as en die van die H526Y mutant 1.5 keer hoër as die van die ongemuteerde bakterië in M. smegmatis. Soortgelyk was die Rv1258c promoter aktiwiteit van die S531L mutant ongeveer die helfde van en die van H526Y mutant ongeveer dubbel die van die ongemuteerde bakterië in M. tuberculosis 'n Soortgelyke neiging in die vlakke van Rv1258c en whiB7 transkripte van die natuurlike geen is gedurende qRT-PCR waargeneem alhoewel die verskille nie beduidend was nie. Blootstelling aan sub-inhibitoriese konsentrasies van RIF het geen effek op Rv1258c uitdrukking in die M. smegmatis of M. tuberculosis rpoB mutante gehad nie. Die rpoB mutasies het net 'n effense effek op Rv1258c en whiB7 transkrip vlakke in M. tuberculosis rpoB mutante, maar transkrip vlakke het 'n soortgelyke neiging as die Rv1258c promoter aktiwiteit getoon. Of die waargenome verskille biologies betekenisvol is, moet nog bepaal word. Indien effluks pompe as teikens vir bahandeling gebruik sou word, moet in ag geneem word dat effluks pompe moontlik verskillend uitgedruk word in verskillende rpoB mutante. / The DST/NRF Centre of Excellence in Biomedical Tuberculosis Research, Stellenbosch University / DAAD-NRF in Country Scholarship and Ernst and Ethel Eriksen Trust / Harry Crossley Foundation