At the author’s request, the abstract has been removed due to the confidential nature of the thesis. It will be added once the embargo period has passed.

Tuberculosis (TB) is the leading cause of death from a single infectious agent worldwide and HIV-1 co-infection is the leading cause of susceptibility to tuberculosis. Sub-Saharan Africa has a high incidence of TB-HIV-1 coinfection and the risk of TB in HIV-1 infected people is increased at all stages of the infection. Antiretroviral treatment (ART) is the most effective way to reduce the risk of TB in HIV-1 co-infected people. By studying the protective, ART induced immune reconstitution in HIV infected individuals sensitised by Mycobacterium tuberculosis (Mtb), we can identify correlates of protection against tuberculosis in the form of transcriptomic, soluble or cellular biomarkers. This thesis focuses on characterising Mtb-specific reconstituting CD4 T cells as well as soluble and transcriptomic markers in HIV infected persons, sensitised by Mtb, by analysing samples collected longitudinally during 6 months of ART. Analysis of peripheral blood mononuclear cells by 14-colour flow cytometry revealed the proportion and numbers of central memory CD4+ T cells significantly expanded in HIV infected persons on ART, while the proportion and numbers of effector memory and terminally differentiated effector CD4+ T cells decreased significantly. Additionally we noted a significant decrease in the proportion of activated CD4+ T cells, and IL-2 single producing CD4+ T cells in HIV infected persons at 6 months of ART, while polyfunctional Mtb specific CD4+ T cells secreting IFN-γ, IL-2 and TNF-α simultaneously, proportionally increased. Analysis of soluble markers in the plasma of HIV infected persons revealed an overall decrease in pro-inflammatory cytokines during 6 months of ART. A significant decrease in IFN-γ, IL-1α, IL-1β, IL-6, IL-17A and TNF-α was observed, and concentrations of these cytokines fell towards those observed in HIV uninfected persons. Transcriptomic analyses of 30 genes normalized to 3 different housekeeping genes, showed an overall increase in the expression of T cell memory specific genes, illustrating the regeneration of the memory T cell pool in HIV infected adults on ART. Larger number of central memory specific genes showed increased expression when normalised to at least two housekeeping genes, as compared to effector memory specific genes. These results support the reconstitution of central memory CD4 T-cell specific response at 6 months of ART. Our data provides insight into the reconstituting immune response to latent TB infection in the context of HIV infection and identifies potential correlates of decreased susceptibility to TB. We also show decreasing soluble and cellular factors indicative of decreasing immune activation in HIV infected persons receiving ART.

A dissertation submitted to the Faculty of Health science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. Johannesburg 2013 / The causative pathogen of tuberculosis, Mycobacterium tuberculosis (Mtb), is equipped with several DNA repair mechanisms for continued survival within the host. One such mechanism is Base Excision Repair (BER) that repairs DNA damage caused by reactive oxygen and nitrogen species (ROS/RNS) generated by the host immune cells during infection. BER is dependent on DNA glycosylases namely: formamidopyrimidine (Fpg/MutM/Fapy), endonucleaseVIII (Nei) and endonucleaseIII (Nth) with Nei being structurally similar to Fpg but functionally similar to Nth. Bioinformatics analysis of the genome sequences of Mtb and its non-pathogenic relative Mycobacterium smegmatis (Msm) identified a unique duplication of Fpg and Nei glycosylases and a single nth gene in the same chromosomal context in both organisms. Previously, it has been shown that the lack of Fpg/Nei glycosylases in Msm display no differences in growth and survival under normal and oxidative stress conditions with no increase in spontaneous mutation rates as compared to the parental strain, suggesting that nth maybe significant for mycobacterial genome maintenance. Hence, in this study the nth gene was site specifically inactivated by homologous recombination in the parental Msm strain and in selected combinatorial mutant strains deficient in the Fpg/Nei glycosylases. Loss of the nth allele in the panel of mutants was genotypically confirmed by PCR and southern blot analyses. Inactivation of the nth gene did not affect the in vitro growth of the mutant strains under normal culture conditions. Interestingly, UV induced DNA damage of the single nth mutant resulted in a dramatic increase in mutation frequency that was not observed in any of the mutants. The progressive loss of fpg, nei and nth genes showed exaggerated reduced survival under oxidative stress. The subsequent deletion of nth in mutants deficient in fpg/nei resulted in a dramatic increase in spontaneous mutation rates and frequencies, implying that nth is integral for the repair of both spontaneous and induced DNA damage. Undoubtedly, these results indicate that Msm nth encoding the Nth glycosylase is involved in DNA repair and has anti-mutator properties. Furthermore, nth together with fpg and nei is part of a robust DNA repair system that maintains the integrity of the mycobacterial genome.

L’hémoglobine tronquée N de Mycobacterium tuberculosis (TrHbN) protège la respiration aérobie de Mycobacterium bovis BCG contre l’inhibition causée par le •NO. De plus, TrHbN catalyse efficacement la dioxygénation du •NO en NO3- (« réaction NOD », TrHbN-Fe2+–O2 + •NO  TrHbN-Fe3+ + NO3-) avec une constante bimoléculaire de 745 µM-1s-1 à 20°C. Cette haute efficacité, pratiquement limitée que par la vitesse de diffusion du substrat, a été associée en grande partie à la présence de deux tunnels hydrophobes visibles dans la structure de tridimensionnelle de TrHbN. L’objectif de cette thèse s’inscrit dans ce contexte, soit l’étude de la structure et de la dynamique de TrHbN à l’aide d’outils bio-informatiques, en particulier l’utilisation de simulations de dynamique moléculaire. Plusieurs simulations de dynamique moléculaire de TrHbN sous ses formes deoxy, oxygénée et cyanomet ont été menées. Ces simulations ont permis d’étudier la dynamique de la chaîne principale, du site actif et en particulier, celle des tunnels. Ces simulations ont révélé que les tunnels sont dynamiques, davantage complexes que le suggère la structure cristalline et que ceux-ci prennent place au cœur d’un repliement 2 sur 2 rigide. D’autres simulations incluant cette fois des molécules de •NO libres ont permis de mettre en évidence l’utilisation des tunnels de TrHbN par ceux-ci pour diffuser jusqu’au site actif. Ces simulations ont permis de proposer plusieurs hypothèses sur les routes utilisées et sur la diffusion des substrats du solvant vers le site actif. Pour valider ces hypothèses et pour pousser davantage nos connaissances, d’autres simulations couplées à diverses approches expérimentales ont été employées. D’abord, des simulations de TrHbN sous sa forme cyanomet couplées à une étude RMN approfondie ont permis de confirmer les résultats de DM quant à la rigidité du squelette de la protéine. De plus, ces derniers travaux ont révélé i) des mouvements lents (µs-ms) localisés le long des hélices B et G et ii) que la région pre-A n’est pas structurée contrairement à ce que suggère la structure cristalline. Enfin, d’autres simulations et des travaux de cinétiques enzymatiques ont été réalisés sur des mutants avec tunnel(s) obstrué(s). Ces travaux ont mené à des résultats démontrant que la matrice enzymatique de TrHbN est très plastique, permettant la diffusion du •NO malgré les mutations créées. Quoique notre compréhension sur les liens entre la structure et la fonction de TrHbN soit toujours incomplète, les travaux présentés dans cette thèse constituent un avancement considérable des connaissances. Plusieurs de nos découvertes mènent à une meilleure compréhension s’appliquant aux globines en général, aux protéines contenant un ou plusieurs tunnels et enfin, sur les mécanismes de diffusion des substrats gazeux à l’intérieur des enzymes. / The truncated hemoglobin N from Mycobacterium tuberculosis (TrHbN) protects aerobic respiration of Mycobacterium bovis BCG cells from the inhibitory effect of •NO. In addition, TrHbN catalyses the very rapid dioxygenation of •NO into the innocuous NO3- ions (NOD reaction: TrHbN-Fe2+–O2 + •NO  TrHbN-Fe3+ + NO3-) with a bimolecular rate constant of 745 µM-1s-1 at 20°C. This high efficiency was largely associated to the presence of two hydrophobic tunnels visible in the 3D-structure of TrHbN. In this context, the main goal of this thesis is to study TrHbN structure and dynamics with bioinformatics tools, especially molecular dynamics simulations. Several molecular dynamics simulations of TrHbN under its deoxy, oxygenated and cyanomet forms were conducted. These simulations allowed to study dynamics of TrHbN backbone, that of the active site and especially, that of the tunnels. As a main result, our simulations revealed that tunnels are highly dynamics, more complex than anticipated from the 3D-structure and that they are hosted in a very rigid two-on-two fold. Other simulations, this time including free •NO molecules, highlighted the use of these tunnels to reach the buried active site. These simulations allowed to propose many hypotheses regarding the preferred routes and to propose diffusions mechanisms from the solvent to the active site. In order to validate our hypotheses and to push further our knowledge on TrHbN, other simulations coupled with some experimental approaches were performed. First, simulations on TrHbN under its cyanomet form coupled with a detailed NMR confirmed that the backbone of the protein is ridig. In addition, this work revealed i) the presence of µs-ms motions localized along B and G helices and ii) that the pre-A region is not structured in contrast to the alpha helice seen in the crystal structure. Finally, other simulations along with kinetics characterizations of obstructed tunnel mutants were conducted. As a main result, the latter work revealed that TrHbN core is quite plastic, allowing substrate diffusion despite the presence blocking mutations. Our comprehension on TrHbN is still incomplete, however the work presented in this thesis constitutes a considerable progress. Moreover, the work presented herein contributes to other fields of research, especially on globins, to tunnel-containing proteins and finally, to gaseous substrates diffusion inside proteins.

Le génome de l'agent étiologique de la tuberculose humaine, Mycobacterium tuberculosis, a été séquencé complètement et l' analyse a révélé l'existence de deux gènes, glbN et glbO, codant respectivement pour les hémoglobines 2/2 Mt-HbN et Mt-HbO. Chez M bovis BCG, l'homologue de Mt-HbO est exprimé de façon constitutive tandis que l'homologue de MtHbN est exprimé seulement en phase stationnaire, suggérant des fonctions différentes. MtHbN fixe l'O2 avec une grande affinité (8 nM) et possède une cavité optimisée pour la dioxygénation du NO en N03. En effet, les travaux présentés au chapitre 2 ont permis de démontrer, par inactivation génique, complémentation et spectrophotométrie ± stopped-flow ¿ que Mt-HbN possède une activité NO-dioxygénase très efficace et capable de protéger la respiration cellulaire contre les effets toxiques du NO. Mt-HbO possède, en plus d'une tyrosine en BIO, une tyrosine en CD 1 et un tryptophane en G8. Les chapitres suivants (3 - 5) décrivent mes travaux portant sur la caractérisation bio~himique et biophysique de Mt-HbO, et plus spécifiquement les réactions avec les ligands gazeux O2, CO et -NO et le H20 2. Par spectrophotométrie ± stopped-flow ¿, nous avons déterminé les constantes d'association et de dissociation de ligands gazeux chez la protéine sauvage ainsi que chez les variants Y(B1O)F, Y(CD1)F et W(G8)F. Les cinétiques d'association et de dissociation des ligands sont complexes et modulées par ces trois résidus distaux. Toutefois, le remplacement des résidus Y(CD1) et W(G8) montre davantage d'impact que celui de la Y(B10) avec, par exemple, des augmentations respectives de 25 à 115 fois pour la constante d'association de l'O2. De plus, des analyses de spectroscopie de résonance Raman (chapitre 4) ont démontré, en accord avec la structure cristalline, la participation des résidus Y(CD1) et W(G8) dans la formation d'un réseau complexe de ponts H stabilisant l'O2 lié au fer de l'hème. Le chapitre 5 décrit les travaux publiés portant sur la réaction entre la forme férrique de Mt-HbO et le H2O2. Cette étude a permis de constater que Mt-HbO possède une activité peroxydase significative, impliquant vraisemblablemnt des espèces spectrales différentes comparées à d'autres peroxydases très connues. Ces dernières observations ainsi que les fortes interactions avec 1'02 suggèrent que Mt-HbO et les autres trHbs du groupe II soient capables d'activer l'O2 et de catalyser des réactions d'oxydo-réduction de types peroxydase et oxygénase.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Drug resistance in Mycobacterium tuberculosis is an increasing global problem. Drug resistance is mostly caused by single nucleotide polymorphisms (SNPs) within the bacterial genome. This observed increase in global incidence of drug resistant tuberculosis (TB) has sparked the search for new anti-TB drugs and the repurposing of drugs that are currently used against other organisms or species of mycobacteria. One such repurposed drug, clofazimine (CFZ), is currently used for the treatment of leprosy, caused by Mycobacterium leprae. The mechanism of action of CFZ is not clear, but it is hypothesized that CFZ is reduced by a mycobacterial type II NADH oxidoreductase (NDH-2). The reduction of CFZ drives the production of reactive oxygen species (ROS) which is toxic to the pathogen. The aim of this study was to elucidate the mechanism of CFZ resistance. Towards this aim, spontaneous in vitro CFZ resistant mutants were selected, characterized and whole genome was used identify SNPs which may cause CFZ resistance. Mutations were identified in a transcriptional regulator encoded by Rv0678, fatty-acid-AMP ligase, or FadD28 (Rv2941) and glycerol kinase or GlpK (Rv3696c). Mutations in Rv0678 have previously been shown to play a role in both CFZ resistance and bedaquiline (BDQ) cross-resistance, while no link has been found between CFZ resistance and mutations in fadD28 and glpK. The novel, non-synonymous SNP identified in Rv0678 resulted in the replacement of an alanine residue with threonine at codon 84, which is located in the DNA binding domain. Virtual modelling of the mutated Rv0678 protein showed that the A84T mutation may influence DNA binding, possibly due to its proximity to the DNA binding domain. This mutation caused a change in hydrophobicity, which may influence binding to DNA. Previous studies showed that mutations in Rv0678 resulted in the upregulation of mmpL5, a putative efflux pump. However, the mechanism whereby CFZ resistance occurs via increased abundance of this efflux pump in the cell wall is not clear and needs further investigation. The cross-resistance between CFZ and BDQ, caused by mutations in Rv0678, is of concern and may influence the planning of anti-TB drug regimens for the future. The roles of the other two mutations identified in this study in CFZ resistance is also not clear and requires further investigation. Finally, the findings of this study support the role of Rv0678 in CFZ resistance thereby suggesting that this gene could be useful as a diagnostic marker to test for CFZ resistance in clinical isolates. / AFRIKAANSE OPSOMMING: Middelweerstandigheid in Mycobacterium tuberculosis is 'n wêreldwye toenemende probleem. Middelweerstandigheid word meestal veroorsaak deur enkel nukleotied polimorfismes (SNPs) in die bakteriële genoom. Hierdie toename in middelweerstandige tuberkulose (TB) het gelei tot die soektog na nuwe anti-TB-middels en die alternatiewe aanwending van middels wat tans teen ander organismes of spesies van mikobakterieë gebruik word. Een so 'n alternatiewe middel, clofazimine (CFZ), word tans gebruik vir die behandeling van melaatsheid wat veroorsaak word deur Mycobacterium leprae. CFZ se meganisme van werking is nie duidelik nie, maar dit word vermoed dat CFZ gereduseer word deur 'n mikobakteriële tipe II NADH oksidoreduktase (NDH-2). Die reduksie van CFZ dryf die produksie van reaktiewe suurstof spesies wat giftig is vir die patogeen. Die doel van hierdie studie was om die meganisme van CFZ weerstandigheid te ondersoek. Om hierdie doel te bereik was spontane in vitro CFZ weerstandige mutante gekies, gekarakteriseer en heel genoom volgorde bepaling is gebruik om SNPs te identifiseer wat CFZ weerstandigheid veroorsaak. Mutasies in Rv0678, 'n transkripsie reguleerder, vetsuur-AMP ligase, of FadD28 (Rv2941) en gliserol kinase of GlpK (Rv3696c) geïdentifiseer. Dit is al voorheen gevind dat mutasies in Rv0678 ‘n rol speel in beide CFZ weerstandigheid en bedaquiline (BDQ) kruis-weerstandigheid, terwyl geen verband gevind is tussen CFZ weerstandigheid en mutasies in fadD28 en glpK nie. Die nuwe, nie-sinonieme SNP, geïdentifiseer in Rv0678 het gelei to die vervanging van 'n alanien aminosuur met treonien by kodon 84, wat geleë is in die DNS bindings domein. Virtuele modellering van die gemuteerde Rv0678 proteïen het getoon dat die A84T mutasie DNS binding moontlik kan beïnvloed, as gevolg van sy nabyheid aan die DNS bindings domein. Hierdie mutasie veroorsaak 'n verandering in die hidrofobiese natuur, wat DNS binding kan beïnvloed. Vorige studies het getoon dat mutasies in Rv0678 lei tot die opregulering van mmpL5, 'n waarskynlike uitvloei pomp. Die meganisme waardeur CFZ weerstandigheid veroorsaak, deur ‘n groot aantal van hierdie uitvloei pompe in die selwand, is nie duidelik nie en moet verder ondersoek word. Die kruis-weerstandigheid tussen CFZ en BDQ, wat veroorsaak word deur mutasies in Rv0678, is van belang en kan die beplanning van anti-TB middel behandeling vir die toekoms beïnvloed. Die rolle van die ander twee mutasies, wat in hierdie studie geïdentifiseer is, in CFZ weerstandigheid is ook nie duidelik nie en vereis verdere ondersoek. Ten slotte, die bevindinge van hierdie studie steun die rol van Rv0678 in CFZ weerstandigheid en dit dui daarop dat hierdie geen gebruik kan word as 'n diagnostiese merker om vir CFZ weerstandigheid te toets in kliniese isolate.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of the tuberculosis disease, is estimated to infect a third of the world’s population and is therefore, arguably, the most successful human pathogen in recorded history. Immense efforts to understand the genetic factors and biochemical processes underlying the complex interactions between M. tuberculosis and its host cells have delivered staggering insights into the profound proficiency by which this bacterium establishes and maintains an infection. It is now clear that M. tuberculosis can interfere with the immune responses initiated by host cells in such a manner as to subvert the various bactericidal conditions established by these cells and thus eliminate the tubercle bacilli that infect them. Specific characteristics of M. tuberculosis which provide it with this ability include a nearly impenetrable cell wall, secretion systems which secrete special factors which directly interact with host immune factors. This enables M. tuberculosis to modulate the activities of the host environment and unique metabolic adaptations of M. tuberculosis allows the organism to survive in the hypoxic, oxidative, nitrosative, acidic and nutrient poor environment of immune cell phagosomes and to persist for decades in a quiescent state in otherwise healthy people. New observations into the pathways which constitute energy, carbon and central nitrogen metabolism, among others, in M. tuberculosis, suggest that a carefully orchestrated homeostasis is maintained by the organism which may modulate the concentrations and ameliorate the effect of molecules that are important to defensive strategies employed by host cells. Here we discuss various recent studies as well as new information provided by this study, focusing on central metabolism and its regulation in M. tuberculosis. We aim to highlight the importance of nitrogen metabolism in the subversive response employed by M. tuberculosis to survive, colonise and persist in the host. We argue that the homeostatic regulation of nitrogen metabolism in M. tuberculosis presents a profound vulnerability in the pathogen which should be exploited with compounds that inhibit the activities of various effector proteins found in this pathway and that are unique to the organism. Such compounds may provide valuable novel chemotherapies to treat tuberculosis patients and may alleviate the burden of multiple drug resistance which plagues tuberculosis treatments. Specifically, in this study we investigate the role of M. bovis BCG glutamate dehydrogenase (GDH) and glutamate synthase (GltS) by subjecting knockout mutants of the aforementioned gene products to various cellular stress conditions. Furthermore, we investigated how the genomes of each M. bovis BCG strain was affected post deletion of the aforementioned protein products. The role of GDH was also tested in an murine macrophage model of infection to elucidate potential importance to colonisation and infection. This study provides novel results indicating an importance of GDH toward the resistance of nitrosative stress as well as a requirement for optimal persistence in RAW 264.7 macrophages. In addition, it was found that GltS is dispensable for resistance against nitrosative stress. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme wat die aansteeklike siekte tuberkulose veroorsaak, infekteer ongeveer ‘n dêrde van die wêreld populasie en is daarom, waarskynlik, een van die mees suksesvolle menslike patogene in geskiedenis. In die afgelope jare is daar noemenswaardige poging aangewend om genetiese faktore sowel as biochemiese prosesse te verstaan wat die komplekse interaksies tussen M. tuberculosis en sy gasheer selle verduidelik. Dit is nou voor die hand liggend dat M. tuberculosis kan inmeng met die reaksies van die immuun sisteem, om dus die bakteriosidiese omgewing wat geskep word deur die selle van hierde sisteem te vermy. Daar is spesifieke kenmerke van M. tuberculosis wat toelaat dat die bacilli so ‘n omgewing kan weerstaan. Hierdie kenmerke is, onder andere, ‘n byna ondeurdringbare selwand en uitskeiding sisteme wat spesiale faktore vrystel. Hierdie faktore het die vermoë om direk met die gasheer immuun sisteem ‘n interaksie te hê wat dus die immuun sisteem moduleer. Verder, is M. tuberculosis se metabolisme aan gepas om die organisme te help teen die lae suurstof, hoë oksidatiewe en stikstof stress, lae pH en lae voedingswaarde omgewing te oorleef. M. tuberculosis het ook die vermoë om vir ‘n onbeperkte tyd in ‘n statiese toestand te oorleef, in gashere wat toon as gesond. Nuwe waarnemings in die energie, koolstof en sentrale stikstof metaboliese paaie stel voor dat ‘n homeostase gehandhaaf word deur M. tuberculosis, wat die konsentrasies van verskeie molekules moduleer of die effek van molekules wat deur die gasheer vrygestel word as ‘n verdedigings meganisme versag. In hierdie dokument bespreek ons verskeie studies, asook nuwe inligting voortgebring deur hierdie studie, wat fokus op sentrale metabolisme en sy regulering in M .tuberculosis. Ons raak aan die vermoë van M. tuberculosis om intrasellulêr te oorleef, koloniseer en voort te bestaan in ‘n gasheer. Ons vemoed dat die homeostatiese regulering van stikstof metabolisme in M. tuberculosis n diepgaande kwesbaarheid in die patogeen skep wat die potentiaal het om uit gebuit te word. Molekules kan gesintiseer word wat die aktiwiteite van verskeie ensieme in hierdie padweg inhibeer en sodoende die organisme hinder. Sulke molekules mag dalk as waardevolle en oorspronklike medisynes ontwikkel word om tuberkulose patiënte meer suksesvol te behandel asook om die las van middelweerstandige bakterieë te verlig. Met betrokke tot hierdie spesifieke studie, het ons die rol van glutamaat dehidrogenase (GDH) en glutamaat sintase (GltS) van M. bovis BCG bestudeer deur om die uitslaan mutante van die genoemde geen produkte aan verskeie sellulêre stress toestande bloot te stel. Die effek van die verlore gdh en gltBD gene op die evolusie van die genome van elke M. bovis BCG uitslaan mutant ras ten opsigte van die wilde tipe was ook bestudeer. Die rol van GDH was getoets in ‘n muis makrofaag model van infeksie om te bepaal of GDH n funksie het in koloniseering en infeksie van M. bovis BCG. Hierdie studie het nuwe bevindinge voort gebring wat die belangrikheid van GDH in die weerstand teen stikstof oksied stress. Daar is verder bevind dat GDH n vereiste toon vir die suksessvolle oorlewing van M. bovis BCG in RAW 264.7 macrofage

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The role of bacterial small RNA (sRNA), i.e. RNA species between 50-500bp in size, in virulence, pathogenesis and drug resistance is gaining interest. In some bacterial species, it had been shown to play a crucial role in bacterial transcriptional and post-transcriptional regulation. sRNAs from various pathogenic bacteria were shown to modulate bacterial responses to the host and environment. In Mycobacterium tuberculosis, the causative agent of tuberculosis, more than 1000 sRNA species have been identified already; but the role of these sRNA in pathogenesis, virulence and stress responses is not well studied. Central dogma suggests that drug resistance in M. tuberculosis is associated with mutations in specific genes. However, a number of clinical drug resistant isolates do not harbour mutations in these genes, implicating other factors such as unknown mutations, as well as altered regulation of these resistance genes. Prediction of resistance, using molecular methods, can therefore be inaccurate in cases where known mutations are absent. In cases where known drug-resistance associated mutations are absent, mutations in other genes that regulate such resistance-associated genes might influence drug resistance. Growing evidence, in other bacteria and M. tuberculosis, hints at a role for mutations in intergenic regions and sRNAs species to play a role in bacterial growth and drug sensitivity. In light of this we hypothesised that mutations in sequences encoding sRNA or in sRNA target sequences influence the phenotype of M. tuberculosis clinical isolates. Using previously identified sRNA genes; we screened a genomic bank of clinical M. tuberculosis isolates for the presence of mutations in these sRNA encoding genes. A large number of isolates showed mutations in genes encoding for sRNAs. Furthermore, over-expression of sRNA using the plasmid pMV306 in Mycobacterium smegmatis showed differences in growth indicating that the presence of the extra copies of the three sRNA (mcr3, ASpks and mpr6) had a phenotypic effect on the bacterium. Overexpressed sRNAs did not affect the bacterial drug resistance phenotypes, although this requires further investigation before concluding the effect of sRNAs on drug resistance. We successfully modified a method to extract and purify sRNAs from Mycobacterium species, clean enough to perform Real Time Polymerase Chain Reaction even with small amounts. However challenges were faced in terms of quantification. Another challenge that still remains is obtaining reference genes specifically for sRNAs as we currently have none. / AFRIKAANSE OPSOMMING: Die rol van klein ribonukleïnsure ( m.a.w RNS spesies van ongeveer 50-250bp in grootte) in bakteriële virulensie, patogenese en antibiotika weerstandigheid word al hoe meer bevraagteken. 'n Rol vir hierdie nukleinsure in transkripsie en post-transkripsie regulering was voorheen gewys in verskeie bakteriële studies, waar dit gedemonstreer was dat hierdie RNA spesies n rol speel vir die bakterieë om aan te pas in die gasheer se omgewing 1–3. Meer as 1000 klein RNS spesies is voorheen in Mycobacterium tuberculosis (die bakterie wat tuberkulosis veroorsaak) geïdentifiseer, maar die rol van hierdie RNA in patogenese, virulensie en stress reaksies is nie bekend nie. Antibiotika weerstandigheid in M. tuberculosis word tans geassosieer met mutasies in spesifieke gene. Daar is wel n aantal weerstandige isolate waar hierdie bekende mutasies heeltemal afwesig is, wat suggereer dat ander rolspelers aanleiding kan gee to middelweerstandigheid. Byvoorbeeld, veranderde regulering van transkripsie patrone van gene (wat n bekende rol in weerstandigheid het) mag ook aanleiding gee tot weerstandigheid, maar sulke alternatiewe meganismes is nog nie goed ondersoek in die bakterium nie. Dis belangrik om al die rolspelers te identifiseer, want bestaande molekulere diagnostiese tegnieke fokus slegs op bekende gene; dus sal weerstandigheid gemis word in isolate waar bekende mutasies afwesig is en slegs molekulere tegnieke gebruik word. Die potensiële assosiasie van klein RNS in tuberkulose antibiotika weerstandigheid is voorheen in n paar studies gemaak. In lig van hierdie studies, is dit voorspel dat mutasies in klein RNA kan aanleiding gee tot verandering in die sensitiwiteit teenoor antibiotika in M. tuberculosis. Vir hierdie studie het ons n genoom bank, wat bestaan uit individuele genome van kliniese M.tuberculosis isolate, geanaliseer vir die teenwoordigheid van mutasies in klein RNS. Daar was spesifiek gefokus op die klein RNS spesies wat in vorige studies met antibiotika weerstandigheid geassosieer was. Hierdie bio-informatiese analise het mutasies in klein RNS spesies in n groot aantal weerstandige stamme geïdentifiseer. Hierdie mutasies was nie in sensitiewe isolate gevind nie, Om die rol van spesieke RNS spesies te ondersoek, was rekombinante plasmiede geskep wat bestaan het uit spesifieke klein RNS spesies van M. tuberculosis en die plasmied pMV306. Hierdie rekombinante was getransformeer in Mycobacterium smegmatis. Die teenwoordigheid van hierdie M. tuberculosis klein RNS kopieë in M.smegmatis het n negatiewe impak gehad op groei, en dui aan dat hierdie RNA spesies, naamlik mcr3, ASpks and mpr6, n potensiele belangrike rol het in die fenotipe van mikobakterieë het. Die ekstra kopieë het nie veranderinge veroorsaak in sensitiwiteit van M.smegmatis teenoor die antibiotika moksifloksasien en kanamisien nie, hoewel meer studies gedoen moet word voordat definitiewe konklusies gemaak kan word. In die finale deel van die studie, is n metode ontwerp om klein RNS op n makliker,vinner manier te isoleer van mikobakterieë. Hierdie metode was suksesvol aangewend om DNA-vry, hoë kwaliteit RNS, beide groter RNA en klein RNS spesies te isoleer. Die klein RNS was goeie kwaliteit, DNA-vry en kon omskep word in DNA met retrotranskripsie. Laasgenoemde DNA kon ook gebruik word in verder polymerase kettingreaksies. Dit het dus potential vir kwantitatiewe studies om die regulering van klein RNS te studeer.

Thesis (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: This study exploits the Mycobacterium tuberculosis H37Rv genome sequence data in the context of M. tuberculosis clinical isolates, to elucidate genetic variation, and examine the phenotypic and molecular epidemiological implications thereof. The study was initiated by investigation of the insertion sequence IS6110, the primary DNA fingerprinting probe for the molecular epidemiology of tuberculosis. The transposable element is present in variable copy number and chromosomal location in clinical isolates of M. tuberculosis strains, giving rise to extensive genetic diversity. At the inception of this study, little was known about this element in terms of the genetic identity of its surrounding regions, its chromosomal distribution, and the mechanisms contributing to genetic diversity. These shortcomings were therefore addressed by a number of approaches. Firstly, to establish their genetic identity and chromosomal distribution, IS6110 insertion sites from clinical isolates of M. tuberculosis were cloned and sequenced. This data was examined in conjunction with available genome sequence data. The results demonstrated that the majority of insertions occurred within coding regions. Furthermore, the element was shown to have a non-random chromosomal distribution, and a number of preferential integration sites were identified. Secondly, the stability of chromosomal domains flanking IS611 0 elements was investigated by utilizing the insertion site clones as hybridization probes against clinical isolates. This allowed the identification of extensive genetic variation associated with these chromosomal domains, arising from IS6110 transpositions, deletions and point mutations. These events were expressed in terms of a phylogenetic tree which demonstrated ongoing genome evolution associated with IS6110. Thirdly, to investigate the hypothesis that IS6110-mediated deletions occur via homologous recombination between adjacent elements, deletion junctions were mapped and sequenced in clinical isolates representing predecessor and descendant strains. While these results support the involvement of IS6110 as a mediator of genetic deletion, they suggest either alternative mechanisms or the existence of unidentified intermediates. The investigation of IS6110 flanking regions identified the disruption of a number of members of the PPE gene family, leading to the second main area of investigation. The PPE gene family was newly identified as a result of the M. tuberculosis genome sequencing project, and its products are speculated to be of antigenic importance. However, at the commencement of this study very little data was available regarding the biological role of PPE proteins. Therefore, to explore the phenotypic implications of PPE gene disruption, various aspects of the gene family were investigated. Firstly, phylogenetic relationships between members of the PPE family were elucidated, which suggested an evolutionary progression, and highlighted the possibility that there may be functional subdivisions within the gene family. Secondly, the extent and mechanisms of PPE gene variation were analyzed by a combination of hybridization, peR and sequence analysis. This approach revealed extensive variation associated the gene family, although different members of the family exhibit different levels of variation. Of special interest was the discovery that long tandem repeat regions (~69 bp) found within 3 members of the gene family demonstrate variation in the numbers of these tandem repeats. A third avenue of investigation focused on in vitro and in vivo PPE gene expression profiles. RT- , peR was utilized to demonstrate in vitro expression of PPE genes, while RNA:RNA in situ hybridization demonstrated the expression of PPE genes in human tissue samples. Intriguingly, in situ hybridization suggests that there is variable PPE gene expression within the human granuloma. The final approach reported here focused on the subcellular localization of one member of the PPE family, Rv1917c. A combination of cell fractionation and whole-cell antibody binding experiments suggest that the Rv 1917c protein is a cell wallassociated, surface exposed molecule. In summary, the results obtained have potential implications for the interpretation of molecular epidemiological data, support the role of IS6110 as an agent of genome evolution, and emphasize the potential for IS6110 to impact on strain phenotype. Investigation of the PPE family demonstrated that this gene family contributes to genetic variation, is expressed in vitro and in vivo and that at least one protein encoded by the gene family is cell wall associated. Together, the results obtained support the hypothesis that selected members of the PPE gene family may encode products involved in antigenic variation. / AFRIKAANSE OPSOMMING: Dié studie maak gebruik van die Mycobacterium tuberculosis H37Rv genoom volgorde data in die konteks van M. tuberculosis kliniese isolate, om genetiese variasie toe te lig en die fenotipiese en molekulêre epidemiologiese implikasies daarvan te ondersoek. Die studie het 'n aanvang geneem deur die ondersoek van die inset-volgorde /S6110, wat die primêre DNS vingerafdruk pylfragment vir die molekulêre epidemiologie van tuberkulose is. Hierdie transponerende element is in wisselende kopiegetal en chromosomale posisies teenwoordig in kliniese isolate van M. tuberculosis stamme, en gee so oorsprong aan omvangryke genetiese afwisseling. Met die aanvang van hierdie studie was min bekend omtrent hierdie element betreffende die genetiese identiteit van die areas wat die insetsels omring, die chromosomale distribusie van insetsels, asook die meganismes wat bydra tot genetiese afwisseling. Hierdie gebreke is dus deur 'n aantal benaderings aangespreek. Eerstens is IS6110 insettingsetels van kliniese M. tuberculosis isolate gekloneer en hul nukleotiedvolgorde bepaal om sodoende hul genetiese identiteit en chromosomale verspreiding vas te stel. Hierdie data is in oorleg met beskikbare genomiese volgorde data geanaliseer. Die resultate het gewys dat die meerderheid van insetsels binne koderende gebiede plaasgevind het. Verder is gewys dat hierdie element nie na willekeur deur die chromosoom versprei is nie, en 'n aantal gebiede waar insetting by voorkeur plaasvind, is geïdentifiseer. Tweedens is die stabiliteit van die chromosomale gebiede wat IS6110 elemente flankeer ondersoek deur die insettingsetel klone as pylfragmente te gebruik in hibridisasie van kliniese isolate. Dit het die identifisering van omvangryke genetiese afwisseling binne hierdie chromosomale gebiede, wat ontstaan deur IS611 0 transposisies, delesies en puntmutasies, tot gevolg gehad. Hierdie afwisselings is uitgedruk as 'n filogenetiese boom waarin die voortdurende genomiese evolusie wat geassosieer word met IS6110 gewys word. Derdens, om die teorie dat IS6110-gedrewe delesies deur middel van homoloë rekombinasie tussen naasliggende elemente plaasvind te ondersoek, is die grense van delesies gekarteer en die nukleotiedvolgorde daarvan bepaal in kliniese isolate wat voorganger- en afstammelingstamme verteenwoordig. Alhoewel die resultate die betrokkenheid van IS6110 as 'n bemiddelaar van genetiese delesie ondersteun, stel dit ook die bestaan van of alternatiewe meganismes of van onbekende intermediêre vorme voor. Ondersoek van die IS6110-flankerende gebiede het gelei tot die ontdekking van ontwrigting van 'n aantal gene wat behoort tot die PPE geenfamilie, en het so gelei tot die tweede hoof ondersoek tema. Die PPE geenfamilie is ontdek as gevolg van die M. tuberculosis genoomprojek, en dit word gespekuleer dat die produkte van hierdie gene van antigeniese belang mag wees. Daar was egter met die aanvang van hierdie studie baie min data beskikbaar omtrent die biologiese rol van die PPE proteïene. Om die fenotipiese implikasies van ontwrigting van PPE gene te ondersoek is daar dus ondersoek ingestel na verskeie aspekte van hierdie geenfamilie. Eerstens is filogenetiese verwantskappe tussen lede van die PPE familie bepaal, wat gedui het op 'n evolusionêre progressie en wat ook aangedui het dat daar moontlik funksionele onderverdelings binne hierdie geenfamilie mag bestaan. Tweedens is die omvang en meganismes van PPE geenvariasie geanaliseer deur 'n kombinasie van hibridisasie, PKR en nukleotiedvolgorde analise. Hierdie benadering het omvangryke afwisseling binne hierdie geenfamilie getoon, alhoewel verskillende lede van die familie verskillende vlakke van afwisseling demonstreer. Wat veral interessant was, was die ontdekking dat lang tandem herhalingsvolgordes (~69 bp) wat in 3 lede van hierdie geenfamilie voorkom, variasie toon in die getalle van hierdie tandem herhalingsvolgordes. 'n Derde been van ondersoek het gefokus op in vitro en in vivo PPE geen uitdrukkingsprofiele. RT-PKR is gebruik om te toon dat PPE gene in vitro uitgedruk word, terwyl RNA:RNA in situ hibridisasie getoon het dat PPE gene ook in menslike weefsel uitgedruk word. Interessant genoeg dui in situ hibridisasie daarop dat daar wisselende PPE geen uitdrukking binne die menslike granuloom voorkom. Die laaste benadering wat hier gerapporteer word fokus op die sub-sellulêre lokalisering van een lid van die PPE familie, Rv1917c. 'n Kombinasie van selfraksionering en heel-sel antiliggaam-bindingseksperimente dui daarop dat Rv1917c 'n selwand-geassosieerde molekuul is wat aan die oppervlak blootgestel word. Ter opsomming het die resultate wat bereik IS potensiële implikasies vir die interpretasie van molekulêr-epidemiologiese data, dit ondersteun die rol van IS6110 as 'n bemiddelaar van genoom evolusie en beklemtoon die potensiaal vir IS6110 om 'n invloed te hê op die fenotipe van die stam. Ondersoek van die PPE familie het getoon dat hierdie geenfamilie bydra tot genetiese afwisseling, dat dit uitgedruk word beide in vitro en in vivo en dat ten minste een lid van hierdie geenfamilie geassossieer word met die selwand. Tesame ondersteun hierdie resultate die teorie dat geselekteerde lede van die PPE geenfamilie wel produkte enkodeer wat betrokke is by antigeniese variasie.

Thesis (PhD)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The evaluation of the immune responses in peripheral blood and at the site of disease of people with differential outcomes following M.tb exposure will lead to the discovery of host biomarkers that will increase our understanding of the protective and non-protective immune responses against the bacterium. The main study consisted of a number of pilot studies and the objectives of the studies were: (1) To determine the background and stimulated whole blood cytokine profiles of children and adults of the community; (2) to establish biomarker profiles in whole blood of children with different M.tb infection phenotypes; (3) to investigate the anti-mycobacterial whole blood immune responses in HIV infected and uninfected children; (4) and to investigate the role of the innate immune system during human tuberculosis disease. The study designs were as follow: (1) Adults and children were enrolled in order to determine cytokine profiles in the community. Whole blood was stimulated with BCG and ESAT-6 or left unstimulated. Eighteen cytokines were measured in supernatants of each condition. Progression to active tuberculosis in the years after study participation was assessed by searching for patient entries in the tuberculosis register. (2) Children with known tuberculosis exposure in their households and with M.tb infection as assessed through interferon-ã release assays, children with exposure but no infection and a control group with no exposure and no infection were investigated. Whole blood was stimulated in QuantiFeron tubes overnight and 21 cytokines were measured in antigen stimulated and unstimulated supernatants by multiplex cytokine arrays. (3) HIV infected and uninfected children were enrolled in a hospital based study. Whole blood interferon-ã responses against specific mycobacterial antigens were investigated in a diluted 7 day whole blood assay and compared to QuantiFeron supernatants from the same participants. (4) Tuberculosis diseased adults were enrolled before the onset of treatment and innate and adaptive cell populations were investigated upon start of treatment and at treatment end. In addition, pleural effusion fluid was collected from tuberculosis and cancer patients and innate cell populations further investigated. The studies were performed in Cape Town, South Africa and included Tygerberg Academic Hospital and the surrounding neighbourhoods of Ravensmead, Uitsig and Elsies River. The main findings of the studies included: (1) We showed age related cytokine differences in our study community. Tuberculosis progressors had significantly higher levels of IL-10 in the unstimulated sample several years before the onset of tuberculosis disease. (2) Cytokines that distinguished best between children with tuberculosis infection and no infections were all cytokines that correlated with interferon-ã (interferon-ã was used to make the classification of M.tb infected and uninfected). Higher IL-1â and lower IL-17 levels in children with tuberculosis exposure without subsequent M.tb infection compared to children with no exposure were shown. (3) HIV infected children showed better responses after 7 day whole blood antigen stimulation compared to the overnight stimulation in QuantiFeron tubes. TB10.4 stimulation in HIV infected TST positive children gave higher interferon-ã responses than ESAT-6 and CFP-10. (4) The presence of myeloid derived suppressor cells is shown during tuberculosis disease circulating in peripheral blood. Upon treatment a decrease in the population is observed. No differences were seen in the myeloid derived suppressor cell frequencies between tuberculosis and cancer patients, however significantly lower frequencies were seen in healthy controls. The immune response against M.tb is complex and interactions between the different cell types are essential to control and fight infection and disease. In this thesis we presented new biomarkers that play important roles during different stages of M.tb pathogenesis from exposure to infection and even during disease. These may shed light on mechanisms of protection against M.tb, relevant to development of tuberculosis diagnostics and vaccine strategies. Combinations of multiple biomarkers including cytokines and chemokines and cell subsets are required to characterize biosignatures relevant to the diagnosis of tuberculosis infection and disease. / AFRIKAANSE OPSOMMING: Deur die immuunreaksie te ondersoek, in heelbloed en in die setel van infeksie, in mense met verskillende uitkomste van M.tb blootstelling sal lei tot die ontdekking van biologiese merkers en sal bydra tot ons begrip van die beskermde en nie-beskermde immuunreaksies teen die bakterium. Die hoofstudie het bestaan uit ‘n aantal loodsstudies en die doel van die studies was: (1) Om die sitokienprofiele in gestimuleerde heelbloed, asook agtergrond waardes, van kinders en volwassenes te bepaal, in die gemeenskap; (2) om die profiele van biologiese merkers in heelbloed van kinders met verskillende M.tb infeksie fenotipes te bepaal; (3) om die anti-mykobakteriële immuunreaksies in heelbloed by MIV geïnfekteerde en nie-geïnfekteerde kinders te bepaal; (4) om ondersoek in te stel na die doel van die aangebore immuunsisteem tydens tuberkulose siekte. Die studie ontwerpe was soos volg: (1) Volwassenes en kinders het deelgeneem aan die ondersoek van sitokienprofiele in die gemeenskap. Heelbloed is gestimuleer met BCG en ESAT-6 of is ongestimuleerd gelaat. Agtien sitokiene is gemeet in die bo-stand verkry van elke kondisie. Mense wat aktiewe tuberkulose siekte in die jare na die studie ontwikkel het, is geïdentifiseer deur die pastiëntinligting in die tuberkulose-register. (2) Kinders met gedokumenteerde huishoudelike tuberkulose blootstelling en met M.tb infeksie, soos bepaal deur vrygelate interferon-ã toetse, kinders met blootstelling maar geen infeksie en ‘n kontrole groep met geen blootstelling en geen infeksie, is ondersoek. Heelbloed is gestimuleer in die QuantiFeron buise oornag en 21 sitokiene is gemeet in die antigeen gestimuleerde en ongestimuleerde bostande deur die multiplex sitokienpaneel. (3) MIV geïnfekteerde en nie-geïnfekteerde kinders het deelgeneem aan ‘n hospitaal baseerde studie. Heelbloed interferon-ã reaksies teen spesifieke mykobakteriële antigene is bestudeer in ‘n verdunde 7 dag heelbloed toets en vergelyk met die QuantiFeron bostande van dieselfde deelnemers. (4) Siek tuberkulose volwassenes wat nie op behandeling is nie, het deelgeneem. Die aangebore en verworwe selpopulasies is bepaal aan die begin van behandeling asook voor die einde van behandeling. Verder is pluralevog van tuberkulose en kanker pastiënte bestudeer vir aangebore selpopulasies. Die studies is uitgevoer in Kaapstad, Suid-Afrika en sluit in Tygerberg Akademiese Hospitaal en die gemeenskappe van Ravensmead, Uitsig en Elsiesrivier. Die hoofbevindinge van die studies sluit in: (1) Ons het gewys dat daar ouderdomsverwante sitokien verskille in die studie gemeenskap is. Mense wat tuberkulose siekte ontwikkel het, het beduidende hoër vlakke van IL-10 in die ongestimuleerde monsters getoon ‘n paar jaar voor die begin van die siekte. (2) Sitokiene wat die beste onderskeiding gewys het tussen infeksie en geen infeksie was sitokiene wat ook korrelasie getoon het met interferon-ã (interferon-ã is gebruik om die klassifikasie te maak van M.tb infeksie of geen infeksie). Hoër IL-1â en laer IL-17 vlakke in kinders met tuberkulose blootstelling en sonder M.tb infeksie, is gewys wanneer dit vergelyk is met kinders sonder blootsteling. (3) MIV geïfekteerde kinders het beter reaksies getoon na 7 dag heelbloed antigeen stimulasie as met die oornag stimulasie in QuantiFeron buise. TB10.4 stimulasie in MIV geïnfekteerde TST positiewe kinders het hoër interferon-ã reaksies getoon as na stimulasie met ESAT-6 en CFP-10. (4) Die teenwoordigheid van miloïed afgeleide onderdrukkende selle in heelbloed, is getoon tydens tuberkulose siekte. Na behandeling is ‘n afname in die populasie gesien. Geen verskille is gesien in die aantal miloïed afgeleide onderdrukkende selle tussen tuberkulose en kanker pastiënte nie, alhoewel beduidende laer getalle is waargeneem in gesonde kontrole deelnemers. Die immuunreaksie teen M.tb is kompleks en interaksies tussen die verskillende seltipes is belangrik om infeksie en siekte te kontroleer en te beveg. In die tesis het ons nuwe biologiese merkers geïdentifiseer wat belangrike funksies het, tydens die verskillende stadiums van M.tb patogenesiteit, van blootstelling tot infeksie asook tydens siekte. Dit kan gebruik word as biologiese merkers betrokke by die immuunreaksie teen M.tb en sal bydra tot die diagnose van tuberkulose infeksie en siekte.