Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: INTRODUCTION Mycobacterium tuberculosis (MTB) and Cryptococcus neoformans are the most common causes of chronic meningitis in South Africa. Conventional microbiology has limited utility in diagnosing these pathogens due to the paucibacillary nature of cerebrospinal fluid (CSF) and the diagnostic delay associated with culturing methods. This study aimed to evaluate the utility of an in-house polymerase chain reaction (PCR) method for the detection of the etiological agent of chronic meningitis. METHODS CSF samples (where volume exceeded 5ml) were submitted to the Medical Microbiology diagnostic laboratory of the Tygerberg Hospital from patients with suspected tuberculosis meningitis (TBM). Following routine bacteriology, the sample was used to inoculate two mycobacterial growth indicator tubes (MGIT A and B) and subsequently incubated in the BACTEC 960 automated system. MGIT A followed standard operating procedures and the time to culture positivity was noted. Weekly aliquots (up to 6 weeks) were removed from MGIT B. These samples were boiled to inactivate the bacteria and then the DNA was extracted using the Promega Wizard SV Genomic DNA kit. The DNA was then speciated by PCR and high-resolution melting analysis (HRM) by using primers specific to either the RD9 region of MTB complex or primers specific to the partial internal transcribed spacer 1 (ITS1), 5.8S rRNA gene and partial ITS2 sequence of C. neoformans. RESULTS Routine CSF microscopy indicated that 14 of the 78 patients (17.9%) had typical CSF findings of TBM (lymphocytes predominant, increased protein levels and decreased glucose levels). IV Ziehl-Neelsen (ZN) stains were positive for 12 (15.4%) samples, and MTB was cultured from 19 samples (24.4%). Our optimized PCR and HRM method was able to detect M. tuberculosis in 17 of the 19 culture positive specimens with a sensitivity of 89.5% and a specificity of 62.7%. The sensitivity of this method was higher than that of direct microscopy. In all of the PCR positive samples, the time to detection, compared to culture, could be shortened by 1 to 2 weeks. Only one sample was positive for Cryptococcus culture and another sample was positive with a Cryptococcus latex test. PCR for Cryptococcus was positive in 2 cases (n=78), sensitivities and specificities could not be reported due to the low number of positive cases. CONCLUSION We demonstrated that a short culture period and the use of commercial DNA extraction kit on CSF samples increases the sensitivity of molecular tests to diagnose tuberculosis. Furthermore, the molecular techniques could significantly reduce the time to positivity of results, when compared to culture. Due to the low occurrence of Cryptococcus in the samples included in our study, we could not comment on the diagnostic utility of PCR in the diagnosis of Cryptococcal meningitis, when compared to the conventional methods. / AFRIKAANSE OPSOMMING: INLEIDING Mycobacterium tuberculosis (MTB) en Cryptococcus neoformans is die mees algemeenste oorsake van kroniese meningitis in Suid-Afrika. Routine mikroskopie dra beperkte waarde in die diagnose van hierdie patogene as gevolg van die klein hoeveelhede organismes wat in die SSV (serobrospinale vog) voorkom en die lang tyd wat dit benodig om hierdie organisms te kweek. Hierdie studie beoog om die diagnostiese waarde van ‘n polymerase ketting reaksie (PKR) metode wat intern ontwerp is te evalueer vir die identifikasie van patogene verantwoordelik vir kroniese meningitis. METODES SSV monsters (waarvan die volume 5ml oorskry) en waar daar ‘n kliniese vermoede van tuberkulose meningitis (TBM) was, is na die diagnostiese Mediese Mikrobiologie laboratorium van Tygerberg hospitaal gestuur vir roetine bakteriologiese ontleding. Die oorblywende monsters is gebruik om twee mikobakteriële groei-indikasiebuise (MGIT A en B) te innokuleer en hulle is geïnkubeer in ‘n BACTEC 960 geautomatiseerde sisteem. MGIT A is volgens roetine diagnostiese metodes geanaliseer en die tyd tot ‘n positiewe resultaat is aangeteken Weeklikse monsters (tot en met week 6) is uit MGIT B verwyder en die monsters is gekook om sodoende die bakterië te inaktiveer. Die Promega Wizard SV Genomiese DNS ekstraksiemetode is gebruik om die DNS te versuiwer. Spesiëring van die DNS is deur middel van ‘n intern ontwerpte PKR en hoëresolusiesmeltingsmetode (HRS) gedoen met inleiers wat spesifiek is tot die RD9 gedeelte van die MTB kompleks en inleiers spesifiek tot die gedeeltelike interne getranskribeerde spasieerder 1 (ITS1), 5.8S rRNS geen en die gedeeltelike ITS2 DNS volgorde van C. neoformans. VI RESULTATE Roetine SSV mikroskopie het aangedui dat 14 uit 78 (17.9%) pasiënte tipiese SSV bevindings van TBM (oorwegend limfosiete, verhoogde proteïene en verlaagde glukose) gehad het. Ziehl- Neelsen (ZN) kleurings was positief vir 12 (15.4%) monsters, en MTB is gekweek in 19 (24.4%) van hierdie monsters. Ons geoptimaliseerde PKR en HRS metode het daarin geslaag om M. tuberculosis in 17 van die 19 kultuurpositiewe monsters aan te toon met ‘n sensitiviteit van 89.5% en ‘n spesifisitiet van 62.7%. Die sensitiwiteit van die direkte PKR was hoër in vergelyking met mikroskopie. In al die PKR positiewe monsters was die tyd tot aantoning, in vergelyking met kultuur, verkort met 1 tot 2 weke. Slegs een monster het C. neoformans gekweek en ‘n ander monster was positief met die kriptokokkale latekstoets. PKR vir C. neoformans was positief in 2 gevalle (n=78). Die sensitiwiteit en spesifisiteit van die C. neoformans PKR kon nie bepaal word nie weens te min gevalle. GEVOLGTREKKINGS Ons het aangetoon dat ‘n verkorte inkubasieperiode en die gebruik van ‘n kommersiële DNS ekstraksiemetode op SSV monsters die sensitiwiteit van die molekulêre tegniek vir die diagnose van tuberkulose verhoog en dat hierdie metode die tyd na positiwiteit aansienlik verkort in vergelyking met kultuur. Weens die lae getalle van kriptokokkale meningitis in ons studie kon ons nie kommentaar lewer op die akkuraatheid van PKR in die diagnose van kriptokokkale meningitis, in vergelyking met meer konvensionele metodes, 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: Mycobacterium tuberculosis is a pathogenic organism that infects a third of the world’s population and causes approximately 2 million deaths per year. Extensive research has been done on this pathogen, however our knowledge of the mechanisms of pathogenicity remain limited. The M. tuberculosis genome contains five ESAT-6 gene cluster regions, ESX-1 to 5, which encode specialized type VII secretion systems. These secretion systems are known to secrete members of the ESAT-6/CFP-10 and PE/PPE protein families, some of which contribute to the pathogenicity and phagosomal escape of the pathogen. ESX-3 has been shown to be essential for in vitro growth and survival of M. tuberculosis. The expression of ESX-3 in M. tuberculosis is regulated by IdeR and Zur, in response to intracellular iron and zinc concentrations, respectively. Interestingly, ESX-3 is not essential for the growth and survival of the saprophytic organism M. smegmatis. In this study, we aimed to identify the subcellular localisation of the individual components of the ESX-3 secretion system in the non-pathogenic, fast-growing organism M. smegmatis. The esx conserved component (ecc) genes from ESX-3 were expressed from the episomal expression vector pDMNI as fusion proteins with green fluorescent protein (GFP). MSMEG_0615 (eccA3), MSMEG_0616 (eccB3), MSMEG_0623 (eccD3) and MSMEG_0626 (eccE3) were successfully cloned into pDMNI and expression of fusion proteins was confirmed by Western blotting for MSMEG_0615-GFP, MSMEG_0616-GFP and MSMEG_0626-GFP in M. smegmatis. In the M. smegmatis ESX-3 knock-out (with MSMEG_0615 to MSMEG_0626 deleted) expression was confirmed for MSMEG_0615-GFP and MSMEG0626-GFP. Fluorescent microscopy determined that MSMEG_0615-GFP localised to a single mycobacterial pole in both strains. MSMEG_0616-GFP and MSMEG_0626-GFP were found to be membrane associated in M. smegmatis, while MSMEG_0626-GFP was found to be membrane associated in the M. smegmatis ESX-3 knock-out. The unipolar localisation of MSMEG_0615-GFP suggests that the assembled ESX-3 secretion system apparatus is situated at a single pole in M. smegmatis. Therefore, we hypothesize that MSMEG_0615 might act as a recruiter protein that is involved in the assembly of ESX-3 at the mycobacterial pole. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is ‘n patogene organisme wat ‘n derde van die wêreld se bevolking infekteer en eis jaarliks 2 miljoen lewens deur tuberkulose. Ten spyte van uitgebreide navorsing, is daar min kennis oor die meganismes van patogenisiteit van hierdie organisme. Die M. tuberculosis genoom bevat vyf duplikasies van die ESAT-6 geen groep gebiede, ESX-1 tot 5, wat kodeer vir gespesialiseerde Tipe VII sekresie sisteme. Hierdie sekresie sisteme is bekend vir die sekresie van lede van die ESAT-6/CFP-10 en PE/PPE proteïen families, waarvan sommige bydra tot die patogenisieit en fagosomale ontsnapping van hierdie organisme. ESX-3 is noodsaaklik vir die in vitro groei en oorlewing van M. tuberculosis. Die uitdrukking van ESX-3 in M. tuberculosis word gereguleer deur IdeR en Zur in reaksie op intrasellulêre yster en sink konsentrasies, onderskeidelik. ESX-3 word nie benodig vir die groei en oorlewing van die saprofitiese organisme M. smegmatis nie. Hierdie studie was gemik om die sub-sellulêre lokalisering van ESX-3 te identifiseer in die niepatogeniese en vinnig-groeiende organisme, M. smegmatis. Die “esx conserved component” (ecc) gene van ESX-3 is uitgedruk vanaf die episomale uitdrukkingsvektor pDMNI as gekombineerde proteïene met die groen fluoreserende proteïen (GFP). MSMEG_0615 (eccA3), MSMEG_0616 (eccB3), MSMEG_0623 (eccD3) en MSMEG_0626 (eccE3) is suksesvol gekloneer en die uitdrukking van die gekombineerde proteïene is bevestig deur Western oordrag vir MSMEG_0615-GFP, MSMEG_0616-GFP en MSMEG_0626-GFP in M. smegmatis. In die M. smegmatis ESX-3 uitklopmutant (met MSMEG_0615 tot MSMEG_0626 uitgeslaan) is uitdrukking bevestig vir MSMEG_0615-GFP en MSMEG0626-GFP. Fluoresensie mikroskopie het bepaal dat MSMEG_0615-GFP gelokaliseer is by ‘n enkele mikobakteriese pool in beide stamme. MSMEG_0616-GFP en MSMEG_0626-GFP was membraan-geassosieerd in M. smegmatis, terwyl en MSMEG_0626-GFP geassosieer het met die membraan in die M. smegmatis uitklopmutant. MSMEG_0615 het gelokaliseer by ‘n enkele pool in M. smegmatis en dit dui aan dat die saamgestelde ESX-3 sekresie sisteem apparaat slegs by ‘n enkele pool voorkom in M. smegmatis. Ons hipotiseer dat MSMEG_0615 dalk mag optree as ‘n werwer proteïen wat betrokke is by die samestelling van die ESX-3 sekresie sisteem by die mikrobakteriese pool. / Stellenbosch University

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: BACKGROUND: The emergence of mycobacterium tuberculosis resistance to first line drugs has renewed interest in second-line anti-tuberculosis drugs. Generally, Paraaminosalicylic acid (PAS) is less potent and frequently more toxic than the first line drugs. Furthermore, the pharmacokinetics of PAS in children has not been well characterized. AIMS: The aims of the present study were (1) to determine the pharmacokinetics of PAS in pediatric patients, (2) to describe the discrepancy between children and adult pharmacokinetics and the appropriate dosing regimen of PAS and (3) to investigate the potential of the second-line anti-tuberculosis drugs PAS, terizidone and ethionamide (often used as first-line drug in children) to inhibit the catalytic activities of CYP450 1A2 and 2C9. PATIENTS: Twenty two patients with drug resistant tuberculosis were included in the study. Ten patients were children with mean age of 4.2 years (range: 1 to 12 years). Twelve patients were adults with mean age of 31.3 years (range: 18 to 53). 4 children (40%) and 4 adults (33.3%) were HIV positive and were on ART. METHODS: Children received 75 mg/kg twice daily on the first visit and after two weeks they received 150 mg/kg once. Adults received a standard 4 g twice daily. Blood samples were taken at different time points after the dose. In the additional study, the inhibitory effects of PAS, ethionamide and terizidone on phenacetin O-deethylation, a marker substrate of CYP1A2 and diclofenac 4’-hydroxylation, a marker substrate of CYP2C9, were studied using human liver microsomes. RESULTS: For the 75 mg/kg dose, the mean AUC was 233.3 =g•h/ml and the mean CL was 10.4 l/h/kg. The mean of the observed Cmax of the drug was 45.4 =g/ml and the mean Tmax was 4.8 hrs. For the 150 mg/kg dose, the mean AUC of PAS was 277.9 =g•h/ml and the mean CL was 47.1 l/h/kg. The mean of the observed Cmax of the drug was 56.5 =g/ml and the mean Tmax was 4.8 hrs. On the first visit the mean AUC was 368 =g•h/ml and the mean CL was 13.2 l/h/kg. The mean of the observed Cmax of PAS was 51.3 =g/ml and the mean Tmax was 5.2 hrs. On the second visit the mean AUC was 230 =g•h/ml and the mean CL was 23.9 l/h/kg. The mean of the observed Cmax of PAS was 37.6 =g/ml and the mean Tmax was 5.2 hrs. The comparisons between pharmacokinetics profile of PAS and patients characteristics e.g. age, indicated no statistically significant differences between children (both treatment regimens) and adult patients as well as HIV positive and negative patients. In the in vitro study, all drugs demonstrated no inhibition potency towards the investigated CYP450 enzymes. CONCLUSIONS:The dose of 75 mg/kg twice daily in children appears to be appropriate to achieve serum concentration above the PAS minimum inhibitory concentration of approximately 1 =g/ml. PAS, ethionamide and terizidone are unlikely to affect the metabolism of concomitantly administered medications that are metabolized by either CYP450 1A2 and/or 2C9 isoenzymes. / AFRIKAANSE OPSOMMING: AGTERGROND: Die opkoming van eersteliniemiddel-weerstandige mycobacterium tuberculosis het opnuut belangstelling in tweedelinie-antituberkulosemiddels aangewakker. Oor die algemeen is para-aminosalisielsuur (PAS) minder kragtig en dikwels ook meer toksies. Verder is die farmakokinetika van PAS in kinders nog nie goed vasgestel nie. DOELSTELLINGS: Die doelstellings van hierdie studie was (1) om die farmakokinetika van PAS in pediatriese pasiënte vas te stel, (2) om die diskrepansie tussen kinder- en volwasse-farmakokinetika, sowel as die toepaslike doseringskedule, van PAS te beskryf en (3) om die potensiaal van die tweedeline-antituberkulosemiddels PAS, terisidoon en etioonamied (gereeld gebruik as eerste linie middels in kinders) te ondersoek wat betref hul vermoë om die katalitiese werking van CYP450 1A2 en 2C9 te inhibeer. PASIËNTE: Twee-en-twintig pasiënte met middelweerstandige tuberkulose is in hierdie studie ingesluit. Tien pasiënte was kinders met ‘n gemiddelde ouderdom van 4.2 jaar (reeks: 1 tot 12 jaar). Twaalf pasiënte was volwassenes met ‘n gemiddelde ouderdom van 31.3 jaar (reeks: 18 tot 53 jaar). 4 kinders (40%) en 4 volwassenes (33.3%) was MIV positief en was op TRM’s. METODES: Kinders het 75 mg/kg twee maal daaliks gedurende die eerste besoek ontvang en 150 mg/kg een maal ná twee weke ontvang. Volwassenes het ‘n standaarddosis van 4 g twee maal daagliks ontvang. Bloedmonsters is op verskillende tye ná die dosering geneem. In die addisionele studie is in die inhiberende effekte van PAS, etioonamied en terisidoon op fenasetien-O-deëtilering, ‘n merkersubstraat van CYP1A2 en diklofenak-4’-hidroksilasie, ‘n merkersubstraat van CYP2C9, ondersoek deur gebruik te maak van menslike lewermikrosome. RESULTATE: Vir die 75 mg/kg dosis was die gemiddelde area-onder-die-kurwe (AOK) 233.3 =g•h/ml en die gemiddelde middelopruiming (CL) 10.4 l/h/kg. Die gemiddelde geobserveerde Cmaks van die middel was 45.4 =g/ml en die gemiddelde Tmaks was 4.8 h. Vir die 150 mg/kg dosering was die gemiddelde AOK van PAS 277.9 =g•h/ml en die gemiddelde CL 47.1 l/h/kg. Die gemiddelde geobserveerde Cmaks van die middel was 56.5 =g/ml en die gemiddelde Tmaks was 4.8 h. Gedurende die eerste besoek was die AOK 368 =g•h/ml en die gemiddelde CL was 13.2 l/h/kg. Die gemiddelde geobserveerde Cmaks van PAS was 51.3 =g/ml en die gemiddelde Tmaks was 5.2 h. Gedurende die tweede besoek was die gemiddelde AOK 230 =g•h/ml en die gemiddelde CL 23.9 l/h/kg. Die gemiddelde geobserveerde Cmaks van PAS was 37.6 =g/ml en die gemiddelde Tmaks was 5.2 h. Die vergelyking van PAS-farmakokinetika en eienskappe van die pasiënte het geen statisties beduidende verskille in die gemiddelde AOK tussen kinders (op albei doserings) en volwassenes getoon nie. Met die in vitrostudie het geen van die middels inhibisie-werking teenoor die CYP450-ensieme wat ondersoek is, getoon nie. GEVOLGTREKKINGS: Die gevolgtrekking kan gemaak word dat die dosering van 75 mg/kg twee maal daagliks voldoende is om serumkonsentrasies wat bo PAS se minimum inhiberende konsentrasie van 1 =g/ml te bereik. Dit is onwaarskynlik dat PAS, etioonamied en terisidoon die metabolisme van gelyktydig-toegediende medikasies, wat op hul beurt deur die CYP240-isoënsieme 1A2 en/of 2C9 gemetaboliseer word, sal affekteer. / Division of Pharmacology, Stellenbosch University / National Research Foundation (NRF) grant generously offered by Professor Donald Grant

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007. / Despite the ongoing global tuberculosis (TB) problem and extensive research into protective immunity against this intracellular pathogen, mechanisms of protective immunity against Mycobacterium tuberculosis (Mtb) in humans have not been fully clarified. Numerous reports have addressed the potential immunological defect(s) in infected individuals that have developed active TB in comparison to those who have remained healthy in spite of infection. Markers of treatment response phenotypes are still elusive. The aims of this study were to define lymphocyte subsets in the peripheral blood of TB patients and controls, to determine intracellular interferon-γ (IFN-γ) and interleukin-4 (IL-4) production and to find correlations of these data with microbiologically-defined treatment response. Methods Whole blood tests were done on 30 HIV-negative, smear-positive pulmonary TB patients and 18 healthy skin test positive volunteers resident in the same community. Immunophenotyping was performed by flow cytometry, combined with routine haematology, for the enumeration of peripheral blood immune cell subtypes. Whole blood was also stimulated in vitro with anti-CD3 monoclonal antibody and intracellular IFN-γ and IL-4 determined by flow cytometry. Lymphocyte proliferation in response to heat-killed Mtb was determined by tritiated thymidine incorporation. Routine microbiological monitoring by sputum smears and culture was done throughout the patients’ 26 weeks of treatment. Results Compared to healthy controls, absolute numbers of peripheral blood lymphocytes and lymphocyte subsets were significantly depressed in patients at diagnosis but normalized during treatment with the exception of natural killer (NK) cells and natural killer T (NKT) cells. A novel subset of the latter was found to correlate significantly with treatment response. IFN-γ-producing T cells after a 4-hour T cell receptor stimulation were significantly higher in patients at diagnosis and normalized during treatment. Supplementary kinetic experiments showed that IFN-γ production in patients at diagnosis seemed to be accelerated. Lymphocyte proliferation was lower in patients at diagnosis and normalized during treatment. Neither IFN-γ production nor lymphocyte proliferation correlated with treatment response. Low intracellular IL-4 production was constitutive in patients and controls, was insignificantly lower in patients at diagnosis than in controls and, in the slow responder patient group, it was significantly lower than in the fast responder group. High IL-4 expression was found in low numbers of T cells in patients and controls and supplementary experiments showed co-expression of active caspase-3 in these cells, which signified apoptosis. Conclusions Lymphocyte subset phenotypes associated with TB are largely abnormal only during active infection and only a novel subset of NKT cells showed correlation with treatment response. Intracellular IFN-γ production and lymphocyte proliferation is increased and decreased, respectively, only during active infection and does not correlate with treatment response. The T helper 1/T helper 2 (Th1/Th2) hypothesis could not be confirmed in the context of tuberculosis but instead constitutive IL-4 production may play a role as a growth factor.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The tuberculosis (TB) incidence has increased in Southern Africa and the situation is worsened by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular biological techniques have been used to understand the disease dynamics of TB. In a series of studies we describe the use of these techniques to understand the disease dynamics of TB in Southern Africa. Using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) to characterize M. tuberculosis strains from TB patients in Zimbabwe, we identified a genotype causing a disproportionate number of TB cases. The genotype belonged to the Latin American Mediterranean (LAM) lineage and we named it the Southern Africa1 (SAF1) family and later renamed it SAF1/RDRio, also reflecting its predominance in South America. To establish if this family of strains was predominant elsewhere in Southern Africa, genotypes were compared to those from Western Cape, South Africa and Zambia. The SAF1/RDRio strains were highly prevalent in Zambia but were only a minor fraction of the strains in South Africa. The geographical distribution of SAF1/RDRio strains was determined in Gweru, Zimbabwe, and was found to be spread in high incidence areas. From these two studies it was hypothesized that certain host and bacterial factors were associated with disease due to SAF1/RDRio. Subsequently potential risk factors and clinical outcomes of disease due to SAF1/RDRio strains were explored. An association was found with smoking and cavitary pulmonary disease suggesting that SAF1/RDRio caused a more severe and highly transmissible formof TB Using IS6110-RFLP, principal genetic grouping, spoligotyping, IS6110 insertion-site mapping and variable-number tandem repeats (VNTR) typing, low IS6110 copy clade (LCC) identified in Zimbabwe were characterized and compared to the strains from Cape Town, South Africa and other regions. The LCC strains from Cape Town, South Africa, were found to have close evolutionary relationship with strains from Zimbabwe and other regions and were widely distributed suggesting they play an important role in the global TB epidemic. Observations from these studies and those from other studies led to the hypothesis that specific genotypes of M. tuberculosis predominate in regions of Southern Africa. To gain an insight on the population structure of M. tuberculosis strains in Southern Africa, spoligotyping and/or IS6110-RFLP data from eight countries were compared. This is the first study to describe the M. tuberculosis population structure in Southern Africa. Distinct genotypes were associated with specific geographic regions. These findings have important implications for TB diagnostics, anti-TB drug and vaccine development. The population structure of multidrug-resistant (MDR), pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) M. tuberculosis isolates from provinces in South Africa was also determined. This is again the first study to describe the population structure of drug-resistant M. tuberculosis in South Africa. The results also showed geographic localization of genotypes and an association with resistance class. However, decreasing strain diversity was observed as the isolates evolved from MDR-TB to XDR-TB suggesting selection for the specific genotypes. These findings highlight the importance of identifying genetic markers in drug-resistant strains, to enhance early detection of those at risk of developing XDR-TB. / AFIKAANSE OPSOMMING: Die voorkoms van tuberkulose (TB) in Suider Afrika word vererger deur stamme van Mycobacterium tuberculosis wat weerstandig is teen die beskikbare anti-tuberkulose middels. Molekulêre tegnieke word gebruik om in hierdie reeks studies die dinamika van TB in Suider Afrika te ondersoek Deur spoligotipering en IS6110 restriksie fragment lengte polimorfisme (RFLP) tegnieke te gebruik om M. tuberculosis stamme van pasiente in Zimbabwe te beskryf, het ons ‘n genotipe gevind wat ‘n buitengewone aantal TB gevalle veroorsaak het. Hierdie genotipe is deel van die internasionaal beskryfde Latyns Amerikaase en Meditereense (LAM) stam familie. Ons het dit die Suider Afrikaanse Familie1 (SAF1) genoem, maar later hernoem na SAF1/RDRio, omdat dieselfde genotipe in ook volop is in Suid Amerika. Om vas te stel of hierdie familie ook oorheesend is in die res van Suider Afrika, is dit vergelyk met beskikbare databasisse van die Wes-Kaap, Suid-Afrika en Zambië. Alhoewel SAF1/RDRio in die Wes-Kaap gevind is, dra dit slegs tot ‘n mindere mate by tot die plaaslike TB epidemie. Aan die anderkant kom SAF1/RDRio baie algemeen in Zambië voor. ‘n Verdere studie wys ook dat die SAF1/RDRio familie eweredig en wyd verspreid voorkom in hoë insidensie gebiede in Gweru, Zimbabwe. Vanuit die bevindings van hierdie 2 studies, kan ons aflei dat sekere gasheer- en bakteriële eienskappe geassosieer is met SAF1/RDRio-TB-infeksie. Hierna is potensiële risiko faktore en kliniese uitkomste van siekte as gevolg van infeksie met SAF1/RDRio ondersoek. ‘n Assosiasie met rook en kaviterende pulmonale infeksie is gevind,wat daarop dui dat SAF1/RDRio erger vorm van TB veroorsaak en hoogs oordraagbaar is. Deur gebruik te maak van IS6110- (RFLP), hoof groep groepering, spoligotipering, IS6110 invoegings kaartering en veranderlike getal tandem herhaling (VNTR) tipering kon lae IS6110 invoeginsgetal (LCC) stamme van Kaapstad, Zimbabwe en ander gebiede vergelyk word. Al die LCC stamme in die studie is evolusionêr naby verwant aan mekaar en is wyd verspreid, wat dui op hulle belangrike rol in die wêreldwye TB epidemie. Waarnemings in hierdie asook ander studies het tot die hipotese gely dat spesifieke genotipes van M. tuberculosis dominant is in verskillende gebiede van Suider Afrika. Om meer insig tot die populasie samestelling van M. tuberculosis stamme in Suider Afrika in te win is spoligotipes en RFLP-data van 8 lande vergelyk. Hierdie is die eerste studie om die populasie samestelling van M. tuberculosis in Suider Afrika te beskryf en is belangrike fir toekomstige ontwikkeling van nuwe TB diagnose tegnieke, anti-TB middels en TB entstowwe. Die populasie samestelling van multiweerstandige (MDR), pre-ekstreme weerstandige (pre-XDR) en ekstreme weerstandige (XDR) M. tuberculosis van verskillende provinsies in Suid-Afrika is ook bepaal. Hierdie studie is ook die eerste wat die populasie samestelling van weerstandige M. tuberculosis in Suid-Afrika beskryf. Die resultate wys geografiese lokalisering van genotipes en ‘n assosiasie met weerstandigheidsklas. ‘n Afname in stam diversiteit soos die isolate van MDR-TB tot XDR-TB ontwikkel, dui op seleksie van spesifieke genotipes. Hierdie bevinding lê die klem op die belangrikheid van die identifisering van genetiese merkers in weerstandige stamme om die risiko vir die ontwikkeling van XDR-TB te verminder deur vroë deteksie.

Thesis (PhD)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative organism of tuberculosis (TB), is a major cause for mortality and morbidity world-wide with a death toll only second to HIV among infectious diseases. Drug resistance is widespread and cases of multiple drug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB) have emerged in several countries. Drug treatment is problematic and new drugs are not developed rapidly enough to offset the rapid drug resistance mutation rate of M. tuberculosis. Simple and effective diagnostics are required to contain the spread of the disease as current routine diagnostics are not fulfilling this role. Additionally, current rapid TB diagnostics are out of reach to resource poor settings due to infrastructure, cost and skill requirements. Novel TB diagnostics are thus required that meet these requirements. Mycobacteriophages are phages that infect mycobacteria and could offer a viable and cost effective alternative rapid TB diagnostics. In this study, an affinity-tagged fluorescent reporter mycobacteriophage is described, which was engineered to act as a TB diagnostic. Its performance proved favourable and superior to current existing mycobacteriophage-based TB diagnostics. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme verantwoordelik vir tuberkulose (TB), is `n groot bron van mortaliteit en morbiditeit wêreldwyd en slegs HIV is verantwoordelik vir groter getalle sterftes as gevolg van n aansteeklike siekte. Middelweerstandigheid is algemeen en gevalle van meervoudigemiddelweerstandige tuberkulose (MDR-TB) en uiters weerstandige tuberkulose (XDR-TB) kom in verskeie lande voor. Antibiotika behandeling is problematies en nuwe anti-TB middels word nie vinnig genoeg ontwikkel om die antibiotika weerstandigheid mutasie spoed van M. tuberculosis te bekamp nie. Doeltreffende diagnostiese toetse word benodig om die verspreiding van die siekte te beheer en bestaande roetine diagnostiese toetse voldoen tans nie aan hierdie vereiste nie. Behalwe hiervoor, is huidige vinnige TB diagnostiese toetse buite bereik van arm instansies weens vereistes aan infrastruktuur, meegaande kostes en werknemervaardigheid. Nuwe TB diagnostiese toetse is dus nodig om aan hierdie vereistes te voldoen. Mikobacteriofaage is fage wat mikobacteria infekteer en kan moontlik 'n lewensvatbare en koste-effektiewe alternatief bied vir vinnige TB diagnostiese toetse. In hierdie studie word 'n affiniteitgekoppelde fluoreserende rapporteringsmikobakteriofaag beskryf wat ontwerp is om op te tree as `n nuwe vinnige TB diagnostiese toets. Die werking hiervan vertoon gunstige en beter resultate as die huidige, mikobacteriofaaggebaseerde TB-diagnostiese toetse.

Thesis (MScMedSc)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: Evidence suggests that biological mechanisms, such as energy dependant efflux pumps, in addition to the rpoB gene mutations, define the level of rifampicin (RIF) resistance in drug resistant Mycobacterium tuberculosis (M. tuberculosis) strains with similar genetic backgrounds. Additionally, proteomic studies showed up-regulation of components of F1F0-ATP synthase enzyme, encoded by the atpBEFHAGDC operon which is responsible for ATP production, in response to RIF. The hypothesis of the current study is that the exposure of a multi-drug resistant (MDR) M. tuberculosis strain to RIF leads to the initiation of a signalling cascade of events. This results in the increased expression of F1F0-ATP synthase leading to an increase in energy production and subsequent activation of efflux pumps. RIF will thus be actively extruded from the cell, increasing the level of RIF resistance. This study aims to identify genetic regions responsible for the regulation of expression of the atpBEFHAGDC operon. Additionally we aim to identify other novel mechanisms contributing to the level of RIF resistance in M. tuberculosis. A specialised reporter vector was constructed to monitor the expression of atpBEFHAGDC, with the use of a fluorescent protein. Subsequently a library of random knockouts was created by transposon mutagenesis in order to identify possible regulators, as well as novel mechanisms contributing to RIF resistance. Two hypothetical proteins, Rv2005c and Rv2417c, were identified in M. tuberculosis transposon mutants showing decreased fluorescence correlating to decreased expression of atpBEFHAGDC. Rv2005c encodes a universal stress protein, suggesting its potential role in a signalling cascade initiated upon RIF exposure. In a model pathway of regulation we propose that the product of Rv2005c is responsible for releasing a repressor protein, Rv1049, thereby stimulating a cascade of signalling events resulting in the up-regulation of atpBEFHAGDC. This increase in ATP production thereby fuels the extrusion of RIF from the cell via efflux pumps. In addition, it was found that disruptions in Rv2524c (fatty acid synthase), Rv1048c (hypothetical protein) and Rv3163c (probable conserved secreted protein) resulted in an increase in the level of RIF resistance in a RIF resistant clinical isolate. Interestingly, Rv2524c also showed to have a potential role in regulation of atpBEFHAGDC, whereby it ensures the repression of atpBEFHAGDC. Another gene identified to be involved in the increase in RIF resistance, Rv0260c, is annotated as a possible transcriptional regulator. This study was successful in identifying possible regulatory proteins involved in regulation of the F1F0-ATP synthase in response to RIF, and highlights the complexity of the regulatory events that occur in response to RIF in a MDR M. tuberculosis strain. This study was also successful in identifying candidates for functional analysis to determine novel mechanisms contributing to the level of RIF resistance in M. tuberculosis. Together these findings demonstrate that RIF resistance in M. tuberculosis is more complex than originally thought. Considering that anti-Tuberculosis (TB) drug TMC207 targets the F1F0-ATP synthase, a key enzyme in the production of energy in mycobacteria, the newly identified regulatory genes of F1F0-ATP synthase may represent ideal targets for novel anti-TB drug design. / AFRIKAANSE OPSOMMING: Huidige dogma toon dat mutasies in die rpoB geen vir rifampicin (RIF) weerstandigheid in Mycobacterium tuberculosis (M. tuberculosis) verantwoordelik is. Onlangs is egter bevind dat ander biologiese meganismes, soos energie afhanklike membraanpompe, saam met mutasies in hierdie geen, die verskillende vlakke van RIF weerstandigheid in M. tuberculosis isolate met soortgelyke genetiese agtergrond kan verklaar. Addisionele proteïen studies het gewys dat die komponente van die F1F0-ATP sintase ensiem, wat verantwoordelik vir ATP sintese is en gekodeer word deur die atpBEFHAGDC operon, opgereguleer word na RIF blootstelling. Die hipotese van hierdie studie is dat blootstelling van ‘n multi-middelweerstandige M. tuberculosis isolaat aan RIF aanleiding sal gee tot ʼn aanvanklike sein wat dan verskeie ander biologiese paaie sal aanskakel. Hierdie gebeure sal dan lei tot ‘n verhoging in geenuitdrukking van die F1F0-ATP sintase operon met gevolglike verhoging in energie produksie, wat uiteindelik energie afhanklike membraan pompe sal aanskakel. Die aktiewe uitpomp van RIF uit die sel sal dan ʼn verhoging in die vlak van RIF weerstandigheid veroorsaak. Die eerste doel van hierdie studie is om genetiese areas te identifiseer wat verantwoordelik is vir die regulering van geenuitdrukking van die atpBEFHAGDC operon. Die tweede doel is om nuwe meganismes te identifiseer wat verskille in die vlakke van RIF weerstandigheid in verskillende nou verwante kliniese isolate sal verklaar. ʼn Gespesialiseerde vektor wat die geenuitdrukking van die atpBEFHAGDC operon sal monitor is suksesvol ontwikkel met die gebruik van ʼn fluoresserende proteïen. Daarna is van die transposon mutagenese metode gebruik gemaak om ʼn biblioteek van ewekansige geenuitlatings te maak en hierdie biblioteek is dan gebruik om nuwe meganismes van RIF weerstandigheid te ondersoek. Hierdie studie het twee hipotetiese proteïene, Rv2005c en Rv2417c, in M. tuberculosis transposon mutante geïdentifiseer wat verantwoordelik is vir verlaagde fluoressensie. Dit korreleer met die verwagte verlaagde geenuitdrukking van atpBEFHAGDC. Die geen Rv2005c kodeer vir ʼn universele spanningsproteïen en die resultaat voorspel dat Rv2005c ʼn potensiële rol het om ʼn netwerk van seine in die bakterium aan te skakel direk na blootstelling aan RIF. In ʼn voorgestelde model van regulerende paaie voorspel ons dat die produk van Rv2005c verantwoordelik is vir die vrystelling van ʼn onderdrukker proteïen, Rv1049. Dit lei dan tot die stimulering van ʼn netwerk van intrasellulêre seine wat aanleiding gee tot die opregulering van atpBEFHAFDC. Die opregulering van atpBEFHAFDC sal dan aanleiding gee tot ʼn verhoging in ATP produksie wat die uitpomp van RIF uit die sel sal versnel met die gebruik van energie afhanklike membraan pompe. Dit is verder gevind dat uitskakeling van die gene Rv2524c (vetsuur sintase), Rv1048c (hipotetiese proteïen) en Rv3163c (moontlike konserwatiewe uitskei proteïen) aanleiding gegee het tot die verhoging in die vlakke van RIF weerstandigheid in ʼn RIF weerstandige kliniese isolaat. In die studie is ook bewys dat Rv2524c ʼn potensiële rol in die regulering van atpBEFHAGDC het deurdat dit die onderdrukking van atpBEFHAGDC verseker. Rv0260c is voorheen gelys as ʼn moontlike transkripsionele reguleerder wat betrokke is by die verhoging van RIF weerstandigheid. Hierdie studie was suksesvol in die identifisering van moontlike gene en proteïne wat betrokke is in die regulering van die F1F0-ATP sintase in reaksie tot RIF blootstelling. Dit beklemtoon die kompleksiteit van die regulerende gebeurtenisse wat plaasvind in reaksie tot RIF blootstelling in ʼn multi-middelweerstandige M. tuberculosis isolaat. Verder was daar suksesvol kandidaat gene en ʼn reguleerder geïdentifiseer wat in toekomstige studies ondersoek kan word vir hulle funksionele bydrae om nuwe meganismes te vind wat die varierende vlakke van RIF weerstandigheid in M. tuberculosis sal verklaar. Opsommend demonstreer hierdie studie dat RIF weerstandigheid meer kompleks is as wat voorheen aangeneem was. Die nuwe baie belowende teen-Tuberkulose middel, TMC207, se aanslag is gemik op die belangrike ensiem (F1F0-ATP sintase) wat in hierdie studie ondersoek was. Dus kan nuut geïdentifiseerde proteïene wat betrokke is by die regulering van hierdie ensiem beskou word as ideale kandidate vir die ontwikkeling van nuwe teen -Tuberkulosemiddels. / The National Research Foundation and the Department of Biomedical Sciences

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.

The efficacy of Bacille-Calmette Guerin (BCG) vaccination in protection against pulmonary tuberculosis (TB) is highly variable between populations. One possible explanation is increased exposure of certain populations to non-tuberculous mycobacteria (NTM). Given the variable efficacy of BCG an improved vaccine against TB is required. The novel TB vaccine MVA85A has shown promising results, however, the immunogenicity of the vaccine is reduced when it is administered in the Expanded Programme on Immunisation (EPI) schedule. This thesis aims to explore: (A) the effect of exposure to NTM on the level of protection afforded by BCG vaccination against Mycobacterium tuberculosis (M. tb) and (B) the immunological mechanisms behind EPI interference with MVA85A. The effect of M. avium (MA) exposure via systemic and oral routes on the efficacy of BCG was tested using M. tb aerosol infection in a mouse model. The adaptive immune response was profiled in BCG vaccinated mice with and without exposure to MA pre- and post- M. tb infection. The results showed BCG efficacy could be enhanced by exposure to dead MA by a systemic route; T helper 1 and T helper 17 responses were associated with increased protection. In contrast, BCG efficacy may have been reduced by exposure to live MA by the oral route; T helper 2 and regulatory T cells were associated with reduced protection. To answer the second aim MVA85A was co-administered to mice with aluminium adjuvants or aluminium-containing vaccines to replicate the effect of co-administration in the EPI schedule; the adaptive immune response was profiled. T helper 2 and regulatory T cell responses induced by aluminium-containing vaccines were associated with a reduction in the immunogenicity of MVA85A.