Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007. / The infectious disease tuberculosis (TB) is one of the leading causes of death worldwide. The idea that infectious diseases are the most important driving force in natural selection and that they sustain frequent polymorphisms in the human genome was formally suggested by Haldane in 1949. This hypothesis implicated the human genetic component in the response to infectious disease. Today the involvement of host genetics in TB has been proven unequivocally and, together with environmental factors (e.g. nutrition and crowding) and the causative bacterium, Mycobacterium tuberculosis (M.tuberculosis), may influence the outcome of disease. As is evident, TB is a complex disease and the implication for studying genetic susceptibility is that a number of genes will be involved. Interferon gamma (IFN-7) is the major macrophage-activating cytokine during infection with M.tuberculosis and its role has been well established in animal models and in humans. This cytokine is produced by activated T helper 1 (Th1) cells. These Th1 responses can best deal with intracellular pathogens such as M.tuberculosis. We selected twelve candidate genes based on the hypothesis that genes which regulate the production of IFN-7 may influence TB susceptibility. We also selected polymorphisms from 27 other candidate genes, which may affect immunological pathways involved in TB, to investigate as susceptibility factors based on the following hypotheses: 1) granulomatous diseases can share susceptibility genes; 2) gene expression studies done by DNA-array analysis experiments may reveal TB susceptibility genes; 3) genomewide linkage studies in TB can determine susceptibility loci and genes in this region are possibly susceptibility factors; and 4) functional susceptibility polymorphisms in genes involved in immune-mediated diseases other than TB may contribute to susceptibility to TB. This research tested the association of 136 genetic polymorphisms in 39 potentially important genes with TB in the South African Coloured population. Well-designed case-control association studies were used and we attempted to replicate these findings in an independent sample set using family-based case-control designs (transmission disequilibrium tests (TDTs)). In addition, haplotypes and linkage disequilibrium (LD) in the candidate genes were also investigated. During the case-control analyses we found significant associations for 6 single nucleotide polymorphisms (SNPs) in the following genes: SH2 domain protein 1A, tolllike receptor 2, class II major histocompatibility complex transactivator, interleukin 1 receptor antagonist, runt-related transcription factor 1 and tumour necrosis factor superfamily, member 1B. Discrepant results were obtained during the TDT analyses. The number of families available was small and for this reason we cannot conclude that the case-control results were spurious. We also tested the association of haplotypes with TB. Haplotypes in the interleukin 12, beta (IL12B) and toll-like receptor 4 genes were nominally associated with TB in both the case-control and TDT analyses. We observed strong LD for the genes in the South African Coloured population. In total 17 novel SNPs were identified and one novel allele was found for a microsatellite in IL12B. This research contributes to the increasing amount of information available on genes involved in TB susceptibility, which in the future may help to predict high risk individuals.

Thesis (PhD (Med))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The nitrogen metabolic pathway is essential for growth and survival of all living organisms including prokaryotes. Certain components of the pathway, such as the enzyme glutamine synthetase (GS), have been studied; however, little information is available regarding the pathway in the mycobacteria. Our in silico studies revealed that many of the components and mechanisms involved in the pathway appear to be conserved between closely related Actinomycetales. Therefore, we investigated three aspects of nitrogen metabolic control in Mycobacterium smegmatis; namely, transcriptional regulation of nitrogen metabolism-related genes, control of enzyme activity and the signalling cascade governing the nitrogen metabolic response. At the transcriptional level, it was found that nitrogen metabolism-related genes were regulated in response to ammonium availability. Two possible transcriptional regulators, AmtR and GlnR, which are the regulators responsible for control of nitrogen-related gene transcription in Streptomyces coelicolor and Corynebacterium glutamicum respectively, were identified in M. smegmatis. Through generation of amtR and glnR deletion mutants, we found that both potential regulators played a role in the control of nitrogen-related gene expression in M. smegmatis. GlnR acted as both an activator and repressor of gene transcription whilst AmtR appeared to activate gene expression which is different to the role its homolog plays in C. glutamicum. On a protein level we found that both GS and glutamate dehydrogenase (GDH) were responsible for ammonium assimilation in M. smegmatis and were regulated in response to ammonium availability. Two GDH isoforms (NAD+- and NADP+-specific) were identified in M. smegmatis and whereas only an NAD+-GDH was detected in M. tuberculosis. The M. tuberculosis GDH also played a largely anabolic role with regard to ammonium assimilation which is in contrast to the belief that ammonium can only be assimilated via GS in this pathogen. The signaling cascade was investigated through generation of a glnD deletion mutant in M. smegmatis. We were able to show that this pivotal protein (GlnD) was able to relay the cellular nitrogen status to the transcriptional machinery as well as to GS. The data presented in this study has advanced our understanding of the nitrogen metabolic pathway in the mycobacteria. Through elucidation of such pathways, our knowledge of mycobacterial physiology and thus infection and survival improves, which could ultimately lead to the discovery of novel mechanisms to aid in the eradication of the disease. / AFRIKAANSE OPSOMMING: Stikstof metabolisme is noodsaaklik vir die oorlewing en groei van alle organismes, prokariote ingesluit. Sekere sellulêre komponente, soos die ensiem glutamine sintetase (GS), is al tevore bestudeer, maar baie min verdere inligting is beskikbaar oor stikstof metabolisme in die mycobacteria. Ons in silico studies het gewys dat baie van die komponente en meganismes gekonserveerd gebly het tussen nou-verwante Actinomycetales. Dus het ons drie aspekte in die beheer van stikstof metabolisme ondersoek; naamlik, die transkriptionele regulering van stikstof metabolisme-verwante gene, die beheer van ensiem aktiwiteit en die sein-meganisme wat die reaksie op stikstof konsentrasie reageer. Op transkripsionele vlak het ons gevind dat stikstof metabolisme-verwante gene gereguleer word in reaksie op stikstof beskikbaarheid. AmtR en GlnR is twee moontlike transkripsie reguleerders wat verantwoordelik is vir transkripsionele beheer in onderskeidelik Streptomyces coelicolor en Corynebacterium glutamicum. Beide hierdie proteïene is geïdentifiseer in M. smegmatis. Deur die konstruksie van amtR en glnR mutante, het ons gevind dat beide potensiële reguleerders ‘n rol gespeel het in die beheer van stikstof-verwante transkripsie in M. smegmatis. GlnR het opgetree as beide ‘n aktiveerder en ‘n onderdrukker van transkripsie terwyl AmtR net ‘n aktiverende rol gespeel het. Die funksie van AmtR in M. smegmatis is dus verskillend van sy homoloog in C. glutamicum. Op proteïen-vlak het ons gevind dat beide GS en glutamaat dehidrogenase (GDH) verantwoordelik was vir die assimilasie van ammonium in M. smegmatis en albei was gereguleer in reaksie op ammonium beskikbaarheid. Twee vorme van GDH (NAD+- spesifieke- en NADP+-spesifieke GDH) was geïdentifiseer in M. smegmatis terwyl net ‘n NAD+- spesifieke GDH in M. tuberculosis gevind is. Die M. tuberculosis GDH het ook ‘n anaboliesie rol gespeel met betrekking tot ammonium assimilasie wat in teenstelling is met die huidige opvatting dat ammonium alleenlik deur GS ge-assimileer kan word. Die sein-meganisme is ondersoek deur ‘n glnD M. smegmatis mutant te konstrueer. Ons het bewys dat hierdie deurslaggewende proteïen (GlnD) die sellulêre stikstof status aan die transkripsionele masjinerie, en aan GS kon oordra. Die data wat in hierdie studie voorgelê word, het ons kennis van stikstof metabolisme in die mycobacteria gevorder. Sodanige metaboliese studies verbreed ons kennis van mycobacteriële fisiologie en dus M. tuberculosis infeksie en oorlewing en kan uiteindelik lei tot die ontdekking van unieke teiken meganismes om te help met die beheer van die siekte en nuwe middelontwikkeling.

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007. / Tuberculosis (TB) is a global health problem and demands rigorous control management efforts. A dramatic increase in the acquisition and spread of drug resistant TB globally has been observed in recent years. A grim picture has emerged for the control program with the discovery of extreme drug-resistant TB, which is virtually untreatable and is of immense concern for the future of TB control. In the last decade strain-specific genetic markers have been identified to examine the molecular epidemiology and spread of TB, including IS6110 DNA-fingerprinting and spoligotyping. Although spoligotyping has less discriminatory power than the gold standard, IS6110 DNA-fingerprinting, it is simpler, faster and less expensive, as it is PCR-based. Spoligotyping has been applied to enhance our understanding of the dynamics of drug susceptible and drug resistant strains of Mycobacterium tuberculosis in high incidence communities, by studying 3 aspects of the TB epidemic: molecular epidemiology of drug resistant TB, recurrent TB and the evolution of M. tuberculosis. By using spoligotyping and other genotypic and phenotypic analysis of drug-resistant M. tuberculosis isolates from the Western Cape Province of South Africa showed that drug resistance is widespread and recently transmitted. An emerging drug resistant M. tuberculosis outbreak has been identified, termed DRF150, which has specific genotypic characteristics and is resistant to 5 first-line drugs in 45% of the cases. Inappropriate chemotherapy; poor adherence to treatment and prolonged periods of infectiousness due to the delay in susceptibility testing has led to the development and spread of this drug resistant genotype. The study demonstrates the ability of the spoligotyping technique to accurately determine the pathogenic mechanism of recurrent disease by spoligotyping, making it useful in large-scale intervention studies. Application of spoligotyping and a newly developed PCR-method showed that the occurrence of multiple infections was higher than what was previously assumed and also more frequent in retreatment cases than new cases. These findings have important implications for the understanding of protective immunity, and the development and testing of new vaccines and drugs. Various different molecular markers including spoligotyping has been used to reconstruct the evolutionary history of isolates with less than 6 copies of IS6110 element (termed Low Copy Clade (LCC)), which were previously poor defined. It was also shown that LCC is widely disseminated and play an important role in the global tuberculosis epidemic. Reconstruction of the evolutionary relationship of M. tuberculosis Principal Genetic Group 2 strains, identified previously unknown genetic relationships between strain families and laid the foundation to establish correlations between genotype and phenotype. Spoligotyping signatures, created by evolution of the Direct Repeat region in M. tuberculosis, were identified, which will enable the analysis of the strain population structure in different settings and will also enable the rapid identification of strain families that acquire drug-resistance or escape protective immunity in drug and vaccine trials. This study contributed to our understanding of the molecular epidemiology of drug resistant TB, recurrent TB and the evolution of M. tuberculosis in high incidence communities.

Thesis (PhD ) -- Stellenbosch University, 2011. / Bibliography / ENGLISH ABSTRACT: The regulation of cardiac contractility is dependent on cooperative interaction between the thick and thin filaments, as well as their accessory proteins, within the cardiac sarcomere. Alteration in cardiac contractility due to a defective sarcomere typically results in cardiomyopathies, such as hypertrophic cardiomyopathy (HCM). One of the sarcomeric genes frequently mutated and which accounts for the second most common form of HCM encodes cardiac myosin binding protein C (cMyBPC), a thick filament accessory protein whose physiological function is poorly understood. However, studies have implicated cMyBPC in thick filament structure and function as well as in the regulation of contractility. The N-terminal region of cMyBPC houses the cMyBPC-motif, which contains three phosphorylation sites, between domains C1 and C2. The hierarchical phosphorylation of this motif, by first calcium/calmodulin kinase II (CamKII) and then by cyclic AMP-activated protein kinase (PKA), is cardinal in the role of cMyBPC in the regulation of cardiac contractility in response to ß-adrenergic stimulation. Moreover, phosphorylation of this motif is inversely correlated to cMyBPC proteolysis and has been shown to be cardioprotective. Thus, proteins that have an effect on cMyBPC function or turnover may also influence filament structure and hence affect contractility, which, in turn, affects the structure of the cardiac muscle. One such protein is the Copper metabolism MURR1-domain containing protein 4 (COMMD4), which was previously identified as a novel interactor of cMyBPC during a yeast two-hybrid (Y2H) library screen in our laboratory. COMMD4 binds specifically to the cMyBPC motif in a phosphorylation-dependent manner. The exact function of COMMD4 is unknown; however, it is a member of the COMM family of proteins that has been linked to copper metabolism as well as to the ubiquitin-proteasome pathway (UPS). Intriguingly, recent studies have shown that the UPS plays a role in cMyBPC-derived HCM, while dietary copper depletion is also known to cause cardiac hypertrophy. Based on these findings, COMMD4 was considered an interesting candidate regulator of sarcomeric function and contractility, and by extension, a candidate modifier of cardiac hypertrophy. Thus, the aim of the present study was two-fold. Firstly, COMMD4 was used as bait in a Y2H library screen to determine its distal ligands, with a view to further elucidate its function, particularly in the context of MyBPC functioning, and identified interactors were subjected to further in vitro and in vivo verification studies. Also, the phosphorylation-dependent nature of the interaction between COMMD4 and cMyBPC was further investigated using a domain/phosphorylation assay. Secondly, COMMD4 and its Y2H-identified putative interactors were assessed as possible modifiers of hypertrophy in a family-based association study, using three cohorts of South African HCM-families in which one of three founder mutations segregate. Six putative interactors, viz. cardiac actin (ACTC1), Down syndrome critical region 3 (DSCR3), enolase 1 (ENO1), F-box and leucine rich repeat protein 10 (FBXL10), legumain (LGMN) and sorting nexin3 (SNX3) were identified and confirmed as COMMD4 interactors using Y2H analyses, followed by in vitro and in vivo co-immunoprecipitation and 3D co-localisation assays. Moreover, as some COMMD protein family members and the newly-identified interactors of COMMD4 have previously been linked to the UPS, the functional effect of siRNA-mediated knockdown of COMMD4 on cMyBPC turnover was also investigated. Data revealed accumulation of cMyBPC in the endosomes upon COMMD4 knockdown, suggesting a functional role for COMMD4 in the turnover of cMyBPC. In addition, association analysis revealed strong evidence of association between various single nucleotide polymorphisms (SNPs) in SNX3 and a number of hypertrophy traits, thus suggesting a role for SNX3 as a candidate modifier of hypertrophy in HCM. No evidence of association was observed for any of the genes encoding the other COMMD4 interactors implicated in protein turnover. The present study demonstrates that COMMD4, a little understood member of the COMM family of proteins, binds to the cMyBPC motif of cMyBPC in a phosphorylation-dependent manner. Furthermore, based on the functions of its protein interactions, we hypothesise that COMMD4 plays a role in protein trafficking and turnover. More specifically, COMMD4 seems to help to facilitate formation of protein complexes with the Skp1-Cul1-Fbxl (SCF) E3 ubiquitin ligase and probably helps to stabilise the target substrate for subsequent ubiquitin-conjugation. As COMMD4 seems to affect the protein turnover of cMyBPC and possibly other sarcomeric proteins, such as actin, these results establish a novel association between the sarcomere, HCM and the UPS. In addition, identification of SNX3 as a hypertrophy modifier will allow for the improved understanding of HCM patho-aetiology. SNX3 thus adds to the growing body of sarcomeric modifier genes, which, eventually, may improve risk profiling in HCM. Furthermore, as genetic modifiers appear sufficient to completely prevent disease expression in some HCM carriers, the identification of SNX3 may point to the protein turnover pathway as a potential new target for intervention. / AFRIKAANSE OPSOMMING: Die regulering van kardiale kontraktiliteit is afhanklik van die koöperatiewe interaksie tussen die dik en dun filamente, asook hul geassosieerde proteïene, in die kardiale sarkomeer. Veranderinge in kardiale kontraktiliteit as gevolg van 'n defektiewe sarkomeer lei tot kardiomiopatieë soos hipertrofiese kardiomiopatie (HKM). Een van die sarkomeriese gene wat dikwels gemuteer is en wat verantwoordelik is vir die tweede algmeenste vorm van HKM,is dié van kardiale miosien-bindingsproteïen C (cMyBPC),'n proteïen geassosieer met die dik filament waarvan die fisiologiese funksie nog nie goed bekend is nie. Studies betrek cMyBPC in dik filament struktuur en funksie asook in die regulering van kontraktiliteit. Die N-terminale gebied van cMyBPC huisves die cMyBPC-motief, wat drie fosforilerings-setels tussen domeine C1 en C2 bevat. Die hiërargiese fosforilering van hierdie motief, eerstens deur kalsium/kalmodulien-gereguleerde kinase II (CamKII), gevolg deur siklies AMP-geaktiveerde proteïen kinase (PKA), is kardinaal in die rol van cMyBPC in die regulering van kardiale kontraktiliteit in reaksie op ß-adrenergiese stimulasie. Verder, fosforilering van hierdie motief is omgekeerd gekorreleer aan cMyBPC proteolise en is ook bewys om kardiobeskermend te wees. Dus, proteïene wat 'n uitwerking het op die funksie van cMyBPC mag ook filament struktuur en kontraktiliteit beïnvloed, wat op hul beurt die struktuur van die kardiale spier affekteer. Die koper metabolisme MURR1-domein bevattende protein 4 (COMMD4), was voorheen geïdentifiseer as 'n nuwe bindingsgenoot van cMyBPC tydens gis twee-hibried (G2H) analise in ons laboratorium. COMMD4 bind spesifiek aan die cMyBPC motief in 'n fosforilasie afhanklike wyse. Die presiese funksie van COMMD4 is onbekend; maar dit is 'n lid van die COMM domein familie van proteine wat geassosieerd is met koper metabolisme sowel as die “ubiquitin” proteosoom pad (UPP). Interesant genoeg, onlangse studies het bewys dat die UPP 'n rol speel in cMyBPC-afgeleide HKM, terwyl koper uitputting in die dieet ook bekend is om kardiale hipertrofie te veroorsaak. Gebaseer op hierdie bevindinge was COMMD4 oorweeg as 'n interessante kandidaat reguleerder van sarkomeries funksie en kontraktiliteit, asook 'n kandidaat modifiseerder van kardiale hipertrofie. Dus, die doel van die huidige studie was tweeledig. Eerstens, was COMMD4 as aas gebruik in 'n G2H biblioteek sifting om sy distale ligande te bepaal, met die oog om verdere lig te werp op sy funksie, veral in die konteks van MyBPC funksionering, en geïdentifiseerde bindingsgenote was onderwerp aan verdere 'in vitro’ en 'in vivo’ verifikasie studies. Daarbenewens was die fosforilering-afhanklike aard van die interaksie tussen COMMD4 en cMyBPC verder ondersoek met behulp van 'n domein/fosforilasie toets. Tweedens, COMMD4 en sy G2H-geïdentifiseerde vermeende bindingsgenote was geassesseer as moontlik modifiseerders van hipertrofie in 'n familie-gebaseerde assosiasie studie, met behulp van drie kohorte van Suid-Afrikaanse HKM-families waarin een van die drie stigter mutasies segregeer. Ses vermeende interaktors, nl. kardiale aktien (ACTC1), Down-sindroom kritiese streek 3 (DSCR3), enolase 1 (ENO1), F-boks en leusien ryke herhalings proteïen 10 (FBXL10), legumain (LGMN) en sorteer nexin3 (SNX3) is geïdentifiseer en bevestig as COMMD4 bindingsgenote deur G2H analises, gevolg deur in vitro en in vivo ko-immunopresipitasie en 3D ko-lokalisasie toetse. Die funksionele effek van siRNA-bemiddelde uitklop van COMMD4 op cMyBPC omset was ook ondersoek omdat 'n paar COMMD proteïen familielede, asook die nuut-geïdentifiseerde bindingsgenote van COMMD4, geassosieerd is met die UPP. Data toon ophoping van cMyBPC in die endosome by COMMD4 uitklop, wat dus aandui op 'n funksionele rol vir COMMD4 in die omset van cMyBPC. Daarbenewens, toon assosiasie analise sterk bewyse van assosiasie tussen die verskillende enkele nukleotied polimorfismes (SNPs) in SNX3 en 'n aantal hipertrofiese kenmerke,wat aandui op 'n rol vir SNX3 as 'n kandidaat modifiseerder van hipertrofie in HKM. Geen bewyse van assosiasie was waargeneem vir enige van die gene wat kodeer vir die ander COMMD4 bindingsgenote wat geïmpliseer word in die proteïen omset. Die huidige studie toon dat COMMD4, 'n min verstaande lid van die COMM familie van proteïene, aan die cMyBPC motief van cMyBPC in'n fosforilasie-afhanklike wyse bind. Verder, gebasseer op die funksies van die proteïen interaksies, hipotiseer ons dat COMMD4 'n rol speel in proteïen vervoer en omset. Meer spesifiek, COMMD4 blyk om die vorming van proteïene komplekse met die Skp1-Cul1-Fbxl (SCF) E3 "ubiquiti". ligase te fasiliteer en help waarskynlik om die teiken-substraat vir die daaropvolgende ubiquitin-konjugasie te stabiliseer. Omdat dit lyk asof COMMD4 die proteïen-omset van cMyBPC en moontlik ander sarkomeriese proteïene, soos aktien, ook beïnvloed, vestig die resultate dus 'n nuwe assosiasie tussen die sarkomeer, HKM en die UPP. Daarbenewens sal die identifisering van SNX3 as 'n hipertrofie modifiseerder voorsiening maak vir die verbeterde begrip van HKM pato-etiologie. SNX3 voeg dus by tot die groeiende ?getal van sarkomeriese modifiseerende gene, wat uiteindelik, die risiko-ontleding in HKM mag verbeter. Verder, omdat dit blyk dat genetiese modifiseerders voldoende is om die siekte-uitdrukking heeltemal te verhoed in sekere HKM draers, kan die identifikasie van SNX3 na die proteïen-omset roete dui as 'n potensiële nuwe teiken vir intervensie.

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009. / Dissertation presented for the degree of Doctor of Philosophy in Medical Biochemistry at Stellenbosch University. / ENGLISH ABSTRACT: The infectious disease tuberculosis (TB) still represents a global threat due to its devastating effect on health and the subsequent high mortality rate. Previous studies have indicated that host genetic factors are implicated in host susceptibility to TB. Since TB is a complex disease, it can be assumed that susceptibility to M. tuberculosis has multiple genetic causative factors (as well as environmental causes). The current study focussed on a number of South African Coloured (SAC) individuals, some of whom were TB cases and others controls. Population substructure was tested in the admixed SAC population as it can be a strong confounding factor for association studies. Our results using the programme STRUCTURE indicated no population substructure in the SAC population. We further investigated the population structure of the SAC group using Affymetrix 500k SNP chip data which showed that the SAC population group has 4 major ancestral components: the Khoesan, European, African and Asian (Indian). A number of candidate polymorphisms in eight genes, previously indicated to play an important role in TB susceptibility, were tested in case-control associations studies. We found statistically significant associations between IFNGR1, IL-8, IL-1Ra and NRAMP1 polymorphisms and TB susceptibility in the SAC population. It has become increasingly evident that gene-gene interactions play a far more important part in an individual’s susceptibility to a complex disease than single polymorphisms would on their own. The importance of epistasis was clearly identifiable in this study with only four associations found between the individual variants and TB susceptibility, but eight instances of statistically significant gene-gene interactions. A combined data set consisting of 106 variants constructed from our database and also used for gene-gene interaction analysis yielded numerous statistically significant interactions. The interaction between the genotype of the human host and the bacterial strain genotype was also investigated and yielded interesting results. Owing to various polymorphisms in several cytokine genes, the protein levels of the main modulators of the immune system, cytokines and chemokines, are changed in several diseases such as infectious diseases and may affect susceptibility or resistance to TB. The functional polymorphisms or haplotype patterns in some of these cytokine genes might be vital for protective immune responses and may serve as biomarkers of protection or susceptibility to TB. The present study investigated 18 cytokines including pro-inflammatory, anti-inflammatory and chemokine factors in healthy (mantoux positive or negative) children using the Linco-plex immunoassay, and investigated potential interactions. The basic research will one day contribute to personalised genetics which may benefit infectious diseases such as TB. If individuals can be identified as potentially more vulnerable, they may require different vaccination strategies, a higher index of suspicion if exposed to TB, and prophylactic treatment. / AFRIKAANSE OPSOMMING: Die infektiewe siekte tuberkulose (TB) is steeds ‘n gevaar wat die hele wêreld bedreig weens die groot impak op gesondheid en die gevolglike hoë mortaliteit. Vorige studies het bevind dat die gasheer se genetiese faktore wel betrokke mag wees by die gasheer se vatbaarheid vir TB. Aangesien TB ‘n komplekse siekte is, kan dit aanvaar word dat vatbaarheid tot M. tuberculosis veelvuldige genetiese oorsaaklike faktore (sowel as omgewingsoorsake) het. Hierdie studie het gefokus op ‘n aantal Suid-Afrikaanse Kleurling (SAC) individue, waarvan sommige TB pasiënte en ander kontroles was. Die gemengde SAC populasie is getoets vir populasie-stratifikasie, aangesien stratifikasie ‘n sterk verwarrende invloed op pasiënt-kontrole studies kan hê. Ons resultate is verkry met behulp van die program STRUCTURE en het aangedui dat daar geen populasie sub-struktuur tussen die pasiënte en kontroles was nie. Ons het ook die populasiesamestelling van die SAC groep ondersoek met data verkrygbaar van die Affymetrix 500k enkel nukleotied polimorfisme mikroskyfie. Hierdie data het getoon dat die SAC populasie uit 4 hoof voorouerlike komponente bestaan naamlik die Khoesan, Europeërs, Afrikane en Asiate (Indiërs). ‘n Aantal kandidaat polimorfismes in agt gene, wat volgens vorige studies ‘n belangrike rol in TB vatbaarheid te speel, was in hierdie pasiënt-kontrole assosiasie studie bestudeer. Ons het statistiese beduidende verwantskappe tussen IFNGR1, IL-8, IL-1Ra en NRAMP1 polimorfismes en TB vatbaarheid in die SAC populasie gevind. Dit het al hoe meer duidelik geword dat geen-geen interaksies ‘n baie belangriker rol in ‘n individu se vatbaarheid vir ‘n komplekse siekte speel as enkel polimorfismes op hul eie. Die belang van epistase kon duidelik in hierdie studie geïdentifiseer word met slegs vier assosiasies wat tussen die individuele variante en TB vatbaarheid gevind is, in vergelyking met agt statisties beduidende geen-geen interaksies. ‘n Gekombineerde datastel wat uit ons databasis saamgestel is en wat 106 variante bevat is ook in ‘n aparte geen-geen interaksie analise gebruik, wat verskeie statisties beduidende interaksies getoon het. Die interaksie tussen die menslike gasheer genotipe en die bakteriese stam genotipe is ook in hierdie studie ondersoek en het interessante resultate opgelewer. Veranderde proteïen uitdrukking van die hoofmoduleerders van die immuunsisteem, sitokine en chemokine, kom voor in verskeie siektes soos infektiewe siektes weens verskillende polimorfismes in verskeie sitokien-gene. Sulke polimorfismes kan ook vatbaarheid vir of weerstandigheid teen TB beïnvloed. Die funksionele polimorfismes of haplotipe patrone in sommige van hierdie sitokien-gene mag noodsaaklik wees vir beskermende immuunresponse en mag ook as biomerkers vir beskerming teen of vatbaarheid vir TB dien. Hierdie studie het 18 sitokiene (insluitend pro-inflammatoriese-, anti-inflammatoriese- en chemokiene faktore), sowel as potensiële interaksies in gesonde (mantoux positiewe of negatiewe) kinders, ondersoek met behulp van die Linco-plex immuno-analise. Hierdie basiese navorsing sal eendag in die toekoms bydrae tot persoonlike genetiese analises wat tot voordeel kan wees vir infektiewe siektes soos TB. Indien individue as potensieël meer vatbaar vir TB geïdentifiseer kan word, kan sulke persone ander vaksineringstrategieë sowel as voorkomende behandeling vereis.

Thesis (PhD (MedSc))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Fumonisins are carcinogenic mycotoxins which occur world-wide in maize and maize-based products intended for human consumption. Consumption of fumonisin contaminated maize as a staple diet has been associated with oesophageal and liver cancer incidence as well as neural tube defects. This study has confirmed the State of Santa Catarina, Brazil as another region where the consumption of maize contaminated with fumonisins and high oesophageal cancer incidence co-occur. Since fumonisins exert their main biochemical effect by disruption of the sphingolipid biosynthetic pathway and are implicated in cancer, the role of fumonisin B1 (FB1) in FB1–induced rat hepatocyte nodules was investigated. The current study showed that FB1 exposure activated sphingosine accumulation in the nodules which could induce the bio-active sphingosine 1-phosphate to provide a selective growth stimulus on subsequent FB1 exposure. Since the FB1-induced hepatocyte nodules were not resistant to the disruption of sphingolipid biosynthesis, it was not the mechanism whereby the altered hepatocytes escaped the mitoinhibition of FB1 and selectively proliferated into hepatocyte nodules. A study in maize subsistence farming communities investigated the sphingosine and sphinganine levels in blood and urine of participants. Fumonisin exposure was assessed in these communities based on fumonisin levels in maize that was concurrently collected from the areas where the participants resided. Subsequently fumonisin exposure was assessed in individuals based on the fumonisin levels in maize collected from each household and by acquiring weighed food records for each member of the household. It was confirmed in both these studies that communities are chronically exposed to fumonisin levels well above the provisional maximum tolerable daily intake determined by the Joint FAO/WHO Expert Committee on Food Additives. Since the sphinganine and sphingosine levels in blood and urine of the participants exposed to various levels of fumonisin were not significantly different, the sphingoid bases and their ratios could not be established as a biomarker of fumonisin exposure. Therefore, an alternative biomarker of exposure was investigated during studies into a practical cost effective method to reduce fumonisin. The customary maize food preparation practices were assessed in a maize subsistence farming community and subsequently optimised to reduce the fumonisin levels in the maize under laboratory-controlled conditions. Implementation of this optimised and culturally acceptable intervention method of sorting and washing maize in a rural community reduced fumonisin contamination in home-grown maize by 84%. The intervention study attained a 62% reduction in fumonisin exposure based on fumonisin levels in maize-based food and consumption as assessed by 24-h dietary recall questionnaires. The alternative biomarker of fumonisin exposure, urinary FB1, was investigated during the intervention study. The FB1 urinary biomarker measured fumonisin intake at the individual level and confirmed the reduction achieved as assessed by food analysis and food intake data. The biomarker was thus well correlated with fumonisin exposure and confirmed the efficacy of the simple and culturally acceptable intervention method. Utilisation of the urinary FB1 biomarker and the customised hand-sorting and washing of maize to reduce fumonisin exposures has the potential to improve food safety and health in subsistence maize farming communities. / AFRIKAANSE OPSOMMING: Fumonisien is kankerverwekkende mikotoksiene wat wêreldwyd voorkom op mielies en mielie-verwante produkte bestem vir menslike verbruik. Daar is ‘n verband tussen die voorkoms van slukderm en lewer kanker, sowel as neuraalbuisdefekte, in gemeenskappe waar fumonisien-gekontamineerde mielies die stapel voedsel is. Die Brasiliaanse Staat, Santa Catarina is uitgewys as nog 'n area waar hoë voorkoms van slukdermkanker en hoë fumonisin vlakke in mielies gesamentlik voorkom. Aangesien fumonisien verbind word met van kanker en die hoof biochemiese effek die ontwrigting van die sfingolipiedbiosintese weg is, is die rol van fumonisien B1 (FB1) in FB1-geinduseerde rot hepatosietnodules ondersoek. Die studie het getoon dat FB1 blootstelling aktiveer sfingosien ophoping in die hepatosietnodules wat moontlik die bio-aktiewe sfingosien 1-fosfaat aktiveer om op daaropvolgende FB1 blootstellings geselekteerde groei stimulasie te ondergaan. Die FB1-geïnduseerde hepatosietnodules was nie bestand teen die inhibisie van die sfingolipied biosintese nie en dus nie die meganisme waardeur die veranderde hepatosiete mito- inhibisie van FB1 vryspring, en selektief ontwikkel in hepatosietnodules nie. ‘n Studie in bestaansboerdery gemeenskappe het die sfingosien en sfinganien vlakke in bloed en uriene vergelyk met individuele fumonisien blootstelling. Laasgenoemde is gebaseer op fumonisien vlakke in gekolleekterde mielies vanuit die deelnemers se huise en aannames vanuit die literatuur. Die opvolg studie in die areas het individuele fumonisien blootstelling bepaal gebaseer op fumonisien vlakke in die mielies van elke huishouding en die inname van mielies deur die voedsel van elke individu te weeg. Albei hierdie studies het bevestig dat die gemeenskappe blootgestel is aan kroniese fumonisien vlakke wat die maksimum toelaatbare daaglikse inname wat deur die gesamentlike FAO/WHO deskundige komitee op voedsel toevoegsels vasgestel is, oorskei. Aangesien die sfingosien en sfinganien vlakke nie beduidend verskil in bloed of uriene van mense wat aan verskillende fumonisien-kontaminasie vlakke blootgestel is nie, kan die lipiedbasisse en hul verhouding nie as ‘n biologiese merker vir fumonisien blootstelling bevestig word nie. Dus is ‘n alternatiewe biologiese merker vir fumonisien blootstelling ondersoek gedurende ‘n studie oor praktiese bekostigbare maniere om fumonisin blootstelling te verlaag. Die tradisionele voedsel voorbereidingspraktyke in ‘n bestaansboerdery gemeenskap is bestudeer en onder laboratorium-gekontroleerde toestande aangepas om fumonisien vlakke in die mielies optimaal te verlaag. Die kultureel aanvaarbare intervensie metode, sortering en was van die mielies, is in ‘n bestaansboerdery gemeenskap toegepas waar ‘n 84% verlaging in fumonisien vlakke van die mielies verkry is. Die intervensie metode het ‘n 62% verlaging in fumonisien blootstelling te weeggebring deur fumonisien vlakke in die mieliegebasserde disse te meet en inname daarvan deur die deelnemers met 24-h diëetkundige vraelyste vas t e stel. Gedurende die intervensie studie is urienêre FB1, die alternatiwe biologiese merker van fumonisien blootstelling, ondersoek. Individuele fumonisien blootstelling data, bepaal met die urienêre FB1 biomerker, het goed ooreengestem met die voedsel analise en voedsel inname data en het dus die doeltreffendheid van die praktiese kultuur aanvaarbare intervensie metode bevestig. Benutting van die FB1 urienêre biologies merker en die optimale sortering en was van die mielies om die fumonisien blootstelling te verlaag het die potensiaal om voedselveiligheid en gesondheid in hierdie bestaansboerdery gemeenskappe aansienlik te verbeter.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Rifampicin resistance in clinical isolates of Mycobacterium tuberculosis develops through selection of bacterial variants harbouring mutations in the rpoB gene. These mutations infer a fitness-cost in the absence of antibiotic pressure, however, fitness-levels of rifampicin-resistant strains can be restored by compensatory mutations in rpoA and rpoC. This study was the first to investigate the epidemiological relevance of these compensatory mutations in clinical M. tuberculosis isolates collected in South Africa. Through targeted DNA sequencing, we demonstrated a strong association between rpoC mutations and transmission, and the rpoB S531L mutation. Our study emphasises the epidemiological relevance of compensatory evolution in response to the emergence of rifampicin resistance, and illustrates how compensatory mutations may be selected as a function of epistatic interactions. Recently a hypothesis has been developed which suggests that the activation of efflux systems through exposure to rifampicin may explain the observed spectrum of rifampicin resistance phenotypes. To elucidate whether rifampicin dependent activation of efflux systems also increases energy production, the RNA expression profiles of candidate energy metabolism genes were investigated. This study demonstrated that rifampicin exposure induced an overall increase in the expression of energy metabolism genes. Our findings suggest that the response to rifampicin is not universal and may depend on other genomic mutations. From these results we conclude that the stress response induced by exposure to rifampicin increases the energy production which fuels efflux activity thereby enabling the cell to extrude rifampicin in an energy dependent manner. This also provides a platform to explain the mechanism by which the newly developed drug, TMC207, increases the rate of culture conversion when used in combination with second-line anti-TB drugs. We propose that inhibition of ATP synthesis by TMC207 will deprive the efflux pumps and transporter genes of energy, which will result in the accumulation of second-line anti-TB drugs within the bacilli, leading to more efficient binding of the second-line drugs to their targets and ultimately to cell death. To identify the genetic basis governing the level of rifampicin resistance, we sequenced the genomes of MDR clinical isolates and in vitro generated rifampicin resistant mutants. Only minor genetic changes in addition to the rpoB mutation were identified in the genomes of in vitro rifampicin resistant mutants which displayed varying levels of resistance. This suggests that these mutants may either use alternative regulatory mechanisms or have acquired SNPs outside the genetic regions investigated in this study to modulate rifampicin resistance levels. In contrast, the genomes of clinical MDR isolates from the Low Copy Clade showed considerable variability in genes involved in cell wall, cellular processes and lipid metabolism, while the genomes from the Beijing Clade displayed variability in genes known to confer drug resistance and compensatory mechanisms. These results suggest that the structure and processes of the cell wall, as well as lipid metabolism plays a critical role in determining the intra-cellular concentration of rifampicin. Finally, this study illustrated the complexity in the physiology of M. tuberculosis resistant to rifampicin, whereby multiple mechanisms are employed by the bacteria to modulate its resistance levels. / AFRIKAANSE OPSOMMING: Rifampisien weerstandigheid in kliniese isolate van Mycobacterium tuberculosis ontwikkel deur die seleksie van bakteriële variante wat mutasies in die rpoB geen het. Alhoewel hierdie mutasies lei tot „n afname in fiksheid van die bakterieë in die teenwoordigheid van antibiotika, kan die fiksheids vlakke van rifampisien weerstandige stamme herstel word deur vergoedende mutasies in rpoA en rpoC. Hierdie is die eerste studie wat die epidemiologiese relevansie van hierdie vergoedende mutasies in kliniese M. tuberculosis isolate wat in Suid-Afrika versamel is, ondersoek. Deur middel van doelgerigte DNA volgordebepaling het ons „n sterk assosiasie tussen rpoC mutasies en transmissie, en die rpoB S31L mutasie getoon. Hierdie studie beklemtoon die epidemiologiese relevansie van regstellende evolusie na aanleiding van die ontwikkeling van rifampisien weerstandigheid en illustreer hoe regstellende mutasies geselekteer mag word as „n funksie van epistatiese interaksies. „n Hipotese is onlangs ontwikkel wat voorstel dat blootstelling aan rifampisien uitvloei sisteme in die bakterium aktiveer, wat moontlik die waargenome spektrum van rifampisien weerstandige fenotipes kan verklaar. Ons het die RNA uitdrukkingsprofiele van kandidaat-energiemetabolisme gene ondersoek om te bepaal of rifampisien afhanklike aktivering van uitvloei sisteme ook energieproduksie verhoog. Hierdie studie demonstreer dat rifampisien-blootstelling „n algehele verhoging in die uitdrukking van energiemetabolisme gene induseer. Ons bevindinge stel voor dat die reaksie van die sel op rifampisien blootstelling nie universeel is nie, en moontlik ook afhanklik is van ander genomiese mutasies. Uit hierdie resultate kan ons aflei dat die stres respons wat geïnduseer word deur rifampisien-blootstelling energieproduksie verhoog, wat weer die uitvloei aktiwiteit aanvuur, en gevolglik die sel in staat stel om rifampisien op „n energie-afhanklike wyse uit te dryf. Dit bied ook „n basis om die meganisme te verklaar waardeur die nuwe middel, TMC207, die tempo van kultuuromskakeling verhoog wanneer dit saam met tweede-linie anti-TB middels gebruik word. Ons stel voor dat die inhibisie van ATP sintese deur TMC207 die uitvloeipompe en transporteerder gene van energie ontneem. Gevolglik veroorsaak dit „n ophoping van tweedelinie anti-TB middels binne-in die bakterium, wat geleentheid bied vir meer effektiewe binding tussen die middels en hulle teikens en uiteindelik seldood veroorsaak. Ons het DNA volgordes bepaal van die genome van MDR kliniese isolate en in vitro selekteerde rifampisienweerstandige mutante om sodoende die genetiese grondslag waarop die vlak van rifampisienweerstandigheid beheer word, te identifiseer. Slegs klein verskille, bo en behalwe die rpoB mutasie, is geïdentifiseer in die genome van in vitro rifampisien weerstandige mutante wat verskillende vlakke van weerstandigheid getoon het. Dit dui aan dat hierdie mutante of ander regulatoriese meganismes gebruik, of hulle het enkelnukleotied polimorfismes buite die genetiese area wat in hierdie studie ondersoek is, waarmee rifampisien weerstandigheid gemoduleer word. In teenstelling hiermee het die genome van kliniese MDR isolate van die “Low Copy Clade” aansienlike variasie getoon in gene wat betrokke is by die selwand, sellulêre prosesse en lipiedmetabolisme. Verder het die genome van die Beijing genotipe variasie in gene getoon wat betrokke is by middelweerstandigheid en regstellende meganismes. Hierdie resultate dui aan dat die struktuur en prosesse van die selwand, asook lipiedmetabolisme, „n kritiese rol speel in die bepaling van die intrasellulêre konsentrasie van rifampisien. Opsommend, hierdie studie toon verskeie meganismes aan wat deur die bakterieë gebruik word om weerstandigheidsvlakke te moduleer en die kompleksiteit van die fisiologie van M. tuberculosis wat weerstandig is teen rifampisien. / The National Research Foundation (NRF) / South African Medical Research Council (MRC) / Harry Crossley Foundation

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: This study is the first of its kind to highlight the importance of using the latest state of the art technology available in the field of proteomics as a complementary tool to characterize the proteome of members of the Mycobacterium tuberculosis Beijing lineage which have been linked to outbreaks and drug resistance of Tuberculosis (TB). Our label-free comparative analysis of two closely related M. tuberculosis strains with different transmission patterns and levels of virulence highlighted numerous factors that may alter metabolic pathways leading to hyper-virulence whereby the strain was able to rapidly replicate in the host and cause extensive disease. This comparative analysis clearly demonstrated that both instrumentation and analysis software impacts on the number of proteins identified and thereby the interpretation of the proteomic data. These proteomes also served as substrates for the discovery of phosphorylation sites, a field of research that reflects a significant knowledge gap in the field of M. tuberculosis. By using differential separation techniques in combination with the state of the art mass spectrometry we described the phosphorylation sites on 286 proteins. This was the first study to document phosphorylation of tyrosine residues in M. tuberculosis. By this means, our data set further extend and complement previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis. Using advanced mass spectrometry methods we further investigated the impact of the in vivo evolution of rifampicin resistance on the proteome of a rifampicin-resistant strain containing a S531L rpoB mutation. We identified the presence of overabundant proteins which could provide novel insight into potential compensatory mechanisms that the bacillus uses to reduce susceptibility to anti-TB drugs. Our findings suggest that proteins involved in a stress response may relate to an altered physiology enabling the pathogen to tolerate and persist when exposed to anti-TB drugs. Together this suggests that structural changes in the RNA polymerase precipitated a cascade of events leading to alterations of metabolic pathways. In addition, we present the first comprehensive analysis of the effect of rifampicin on the proteome of a rifampicin resistant M. tuberculosis isolate suggesting that rifampicin continues to influence the biology of M. tuberculosis despite the presence of an rpoB mutation. Our analysis showed alterations in the cell envelope composition and allowing the bacterium to survive in a metabolically dormant/persistent growth state. The results presented in this study illustrate the full potential of using a proteomic approach as a complementary molecular technique to select promising candidate molecules and genes for further characterization using the tools of molecular biology. / AFRIKAANSE OPSOMMING: Die huidige studie is ‘n eerste van sy soort, deur die nuutste gevorderde tegnologie in die proteomika veld te gebruik. Die proteoom van lede van die Mycobacterium tuberculosis Beijing stam, wat die oorsaak is van tuberkulose (TB) uitbrake en ook weerstandige TB, is gekarakteriseer. Ons merkervrye vergelykende analise van twee naby verwante M. tuberculosis stamme met verskillende vlakke van oordraagbaarheid en virulensie, beklemtoon verskeie faktore wat metaboliese paaie mag verander, wat kan ly tot hiper-virulensie, wat die TB-stam in staat stel om vinniger te repliseer in die gasheer en ‘n uitgebreide siektetoestand kan veroorsaak. Die analise het duidelik gewys dat die toerusting wat gebruik word, sowel as die sagteware ‘n invloed kan hê op die hoeveelheid proteïne wat geïdentifiseer kan word en daardeur intrepretasie van proteomika data kan beïnvloed. Hierdie proteome dien as substrate vir die ondekking van fosforilasie setels, ‘n veld van navorsing wat dui op ‘n gaping in ons kennis van M. tuberculosis. Deur gebruik te maak van differensiële skeidingstegnieke en moderne spektrometrie beskryf ons fosforileringsetels in 286 proteine. Hierdie is die eerste studie wat fosforilasie van tirosien residue in M. tuberculosis beskryf. Hierdeur komplimenteer en brei ons data die huidige kennis oor gefosforileerde peptiede en fosforilasie setels in M. tuberculosis uit. Deur gebruik te maak van gevorderde massa spektrometriese tegnieke het ons verder ook die impak van in vivo evolusie van rifampicin weerstandigheid op die proteoom van ‘n rifampicin weerstandige TB-stam met die algemene S531L rpoB mutasie ondersoek. Ons het proteïne geïdentifiseer wat in groot hoeveelhede voorkom en kan nuwe insigte gee tot potensiele kompenserende meganismes wat deur die bacillus gebruik word om vatbaarheid vir anti-TB middels te verminder. Ons bevindings dui daarop dat proteïene betrokke in ‘n stresreaksie mag lei tot ‘n verandering in fisologie wat die patogeen in staat stel om anti-TB middels te verdra en te volhard in die teenwoordigheid van sulke middels. Saam impliseer dit dat ‘n ketting van gebeure wat lei tot veranderinge in metaboliese paaie, word vooraf gegaan deur strukturele veranderinge in die RNS polimerase. Tesame hiermee bied ons ook die eerste omvattende analise aan van die effek wat rifampicin op die proteoom van ‘n rifampicin weerstandige M. tuberculosis isolaat het, en wat aan die hand doen dat rifampicin voordurend die biologie van M. tuberculosis beïnvloed, ten spyte van die teenwoordigheid van ‘n rpoB mutasie. Ons analise dui op veranderinge in die samestelling van die selomhulsel wat die bakterie toelaat om te oorleef in ‘n metabolies dormante staat. Die resultate wat in hierdie studie aangebied word illustreer die volle potensiaal van ‘n proteomiese benadering as komplementêre molekulêre tegniek om belowende kandidaat molekules en gene te kies vir verdere karakterisering, deur gebruik te maak van molekulêre tegnieke. / The National Research Foundation (RSA), / Norwegian Research Council (Norway) / National Institute of Health –Forgarty (USA) / Southern Africa Consortium for Research Excellence-Welcome Trust (SACORE) (United Kingdom) / Kwazulu-Natal Research Institute for Tuberculosis and HIV (K-RITH) (USA)

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, 2013. / ENGLISH ABSTRACT: Important steps towards the global control of Tuberculosis include the improvement of diagnosis, the development of effective vaccines and the identification of correlates of protection/protective immunity to Mycobacterium tuberculosis. This study has of three objectives: 1. To validate the findings of a previous study that showed increased levels of IL-1β and decreased levels of IL-17 in children who are exposed to tuberculosis but remain uninfected compared to those who are exposed/infected and unexposed/uninfected. 2. To define the protective immunological phenotype in children with negative IGRA’s and TST following exposure to Mycobacterium tuberculosis. 3. To evaluate a number of cytokines in both serum and saliva samples of identified tuberculosis cases and controls for their diagnostic potential and to evaluate saliva as a possible new diagnostic sample type. The study designs were as follows: Objectives1, and 2: Children with documented tuberculosis exposure and with Mycobacterium tuberculosis infection as assessed through interferon gamma release assays, children with exposure but no infection and a control group with no exposure nor infection were investigated. These participants were selected according to their exposure and infection phenotypes from a larger TB household contact study that was conducted in communities in Cape Town. Whole blood was stimulated in QuantiFeron tubes overnight and ten cytokines were measured in antigen stimulated and unstimulated supernatants by Luminex multiplex Immunoassay. Differential production of cytokines in the three groups was evaluated. Objective 3. Saliva and serum samples were collected from thirty eight adults with suspected tuberculosis who were recruited from a community health centre in Cape Town, after which the levels of thirty three host markers were evaluated in the samples using the Luminex platform. The main findings of the studies included: 1. Increased levels of IL-1β and decreased levels of IL-17 in children who are tuberculosis exposed but remain uninfected compared to those who are exposed/infected and unexposed/uninfected could not be confirmed. 2. Immune responses other than IFN-γ are different in children with different exposure and infection phenotypes. Higher IL-23 and IL-33 levels in children with tuberculosis exposure without subsequent Mycobacterium tuberculosis infection compared to children with no exposure were shown. 3. In both the tuberculosis cases and controls, the levels of most markers were above the minimum detectable limit in both serum and saliva, but marker levels were not consistently higher in one sample type. The levels of fractalkine , IL-17, IL-6, IL-9, MIP-1β, CRP, VEGF and IL-5 in saliva, and those of IL-6, IL-2, SAP and SAA in serum, were significantly higher in tuberculosis patients, in comparison to the levels obtained in those without active tuberculosis (p<0.05). The area under the ROC curve was ≥ 0.70 for most of these markers, thereby confirming their diagnostic potential for TB disease. The work presented in this thesis has identified markers that may grant an improved understanding on the mechanisms that are associated with protection against Mycobacterium tuberculosis in children. The preliminary results presented show that the identification of host markers in saliva is possible and the utility of saliva for the development of rapid immune-based tests for active tuberculosis is promising. / AFRIKAANSE OPSOMMING: Noemenswaardige vooruitgang in die globale beheer van Tuberkulose is onderworpe aan verbeterde diagnose, die ontwikkeling van doeltreffende vaksienes en die identifikasie van aanwysers van immuniteit teen Mycobacterium tuberculosis. Die doel van hierdie studie is: 1. Om die bevindinge van ‘n vorige studie te bevestig, waar verhoogde vlakke van IL-1β en verlaagde vlakke van IL-17 waargeneem is in kinders wat aan tuberkulose blootgestel is, maar nie geïnfekteer is nie. Hierdie bevindinge was in vergelyking met geïnfekteerde en nie-blootgestelde kinders. 2. Om ‘n beskermende immunologiese fenotipe te definieer in kinders met negatiewe IGRA’s en TST, na blootstelling aan Mycobacterium tuberculosis. 3. Om sekere sitokines, in beide serum en speeksel monsters van tuberkulose gevalle en kontroles, te evalueer as potensiële diagnosemiddels, asook die moontlikheid dat speeksel kan dien as ‘n nuwe diagnostiese monstertipe. Die studieraamwerk was as volg: Doel 1 &2:Die volgende groepe was onder meer ondersoek – Kinders blootgestel aan tuberkulose en wat gevolglik geïnfekteer is, soos vasgestel deur interferon gamma vrystellingstoetse; kinders wat wel blootgestel is maar nie geïnfekteer is nie en ‘n kontrolegroep wat geen blootstelling aan Mycobacterium tuberculosis gehad het nie. Hierdie individue is geselekteer volgens hul blootstellingsprofiel en infeksiefenotipes, uit ‘n groter blootstellingstudie op Kaapse huishoudings. Heelbloed is oornag gestimuleer en tien sitokiene is gemeet in antigeen-gestimuleerde en ongestimuleerde supernatante, deur middel van Luminex multipleks Immunotoetse. Differensiële produksie van sitokienes in hierdie groepe is gevolglik geëvalueer Doel 3: Speeksel en serummonsters van 38 volwassenes met vermeende tuberkulose, is versamel en die vlakke van drie en dertig gasheermerkers is gemeet deur middel van die Luminex platvorm. Die hoof bevindinge van hierdie studie sluit in: 1.Vehoogde vlakke van IL-1β en verlaagde vlakke van IL-17 kon nie bevestig word in die verskeie kindergroepe (Sien doel 1) nie. 2. Die immuunrespons, uitsluitend die IFN- γ respons, is veskillend in kinders met uiteenlopende blootstelling en infeksiefenotipes. Hoër vlakke van IL-23 en IL-33 is gevind in kinders wat blootgestel is aan tuberkulose, maar nie geïnfekteer is nie, in teenstelling met nie-blootgestelde kinders.. 3. In beide die pasiënte en kontroles was die meeste sitokienvlakke hoër as die minimum meetbare limiet in beide speeksel en serummonsters, hoewel merkervlakke nie konstant hoër was in enige van die twee monstertipes nie. Die vlakke van fractalkine, IL-17, IL-6, IL-9, MIP-1β, CRP, VEGF en IL-5 in speeksel en IL-6, IL-2, SAP en SAA in serum, was merkbaar hoër in tuberkulosepasiënte, in vergelyking met vasgestelde vlakke in individue sonder aktiewe tuberkulose. (p<0.05). Die oppervlak onder die ROC kurwe was ≥ 0.70 vir die meerderheid van die merkers. Dit is ‘n sterk aanduiding dat hierdie merkers potensiaal het as diagnostiese merkers vir tuberkulose. Hierdie navorsing het merkers geïdentifiseer wat die begrip van die megansime waarmee beskerming teen Mycobacterium tuberculosis gebied word in kinders, verbreed. Hierdie voorlopige resultate dui aan dat die identifikasie van gasheermerkers in speeksel moontlik is en dat speeksel moontlik kan dien as ‘n proefkonyn vir die ontwikkeling van immuungebaseerde sneltoetse vir die diagnose van aktiewe tuberkulose. / The EDCTP through the African European Tuberculosis Consortium (AE-TBC, grant number IP_2009_32040) / Trials of Excellence in Southern Africa (TESA, project code CG_cb_07_41700)