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

According to the World Health Organization (WHO), M. tuberculosis, the causative agent of TB, accounts for approximately 1.7 million deaths annually. Further contributing causes to the worldwide TB incidence, is the widespread unavailability and ineffectiveness of TB vaccines, time consuming diagnostic methods and unsuccessful treatment approaches. Research for better characterising mycobacteria in general, or other Mycobacterium species, may help us to better understand M. tuberculosis and TB disease mechanisms, which will in turn lower the future TB disease prevalence, as this may lead to the development of better treatments, diagnostics and vaccines. Mycobacteria use various secretion pathways, including the ESX- or type VII secretion (T7S) system, to ensure transport across the complex cell wall. The genome of M. tuberculosis has five copies of a gene cluster known as the ESX gene cluster region, which is associated with virulence and viability of mycobacteria. The ESX-3 gene cluster is thought to be essential for growth of M. tuberculosis and proposed to be involved in iron / zinc homeostasis. Mycobacteria synthesise siderophores, which are proposed to be involved in the uptake of iron over their cell wall. M. tuberculosis are known to produce two types of siderophores, namely: carboxymycobactins and mycobactins. Loots and colleagues, however illustrated, that ESX-3 knockouts, show signs of iron overload, despite the absence of the mycobactins induced by knocking out the ESX-3 gene cluster. It was hypothesised, that this overload occurs due to an increase in exochelin synthesis, another iron uptake protein not associated with ESX-3, overcompensating for the perceived iron depletion in the knockout organism. A Metabolomics research approach was subsequently used in this study, to generate new information in order to better characterise the role of iron on the metabolism of these organisms, and additionally confirm the role of ESX-3 in iron uptake. In this study, we firstly determined the most optimal extraction conditions for this metabolomics investigation. Two extraction methods were subsequently investigated and compared, considering their repeatability and their respective capacities to extract those compounds which best differentiate the M. smegmatis ESX-3 knockouts and wild-type parent strains. Considering the results generated, the total metabolome method was chosen for further analyses, for the following reasons: 1) it is simpler, 2) faster, 3) showed better repeatability, 4) extracts those compounds best differentiating the compared groups and 5) has been previously described for metabolomics analyses characterising ESX-3 gene functionality, hence potentially allowing us to compare results to that previously generated and published data. Subsequently, we used the chosen extraction method, followed by GCxGC-TOFMS analysis of the separately cultured M. smegmatis wild-type sample extracts, cultured in normal, low and high iron conditions, to determine the influence of varying iron concentrations on the metabolome of this organism, by metabolomics comparisons of these groups. Following this, an identical research approach was used to compare the metabolome of a M. smegmatis ESX-3 knock-out strain, to that of a M. smegmatis wild type parent strain, both cultured in normal / standardised iron concentrations. Considering the results generated when comparing the metabolome of a M. smegmatis ESX-3 knock-out strain to that of a M. smegmatis wild type parent strain, the altered metabolome of the M. smegmatis ESX-3 knockouts correlated well to that of the M. smegmatis wild type cultured in elevated iron growth conditions. This suggests ESX-3 is involved in iron uptake, and that knocking out the ESX-3 gene cluster of M. smegmatis does in fact result in a metabolome profile suggesting iron overload, as was proposed by Loots et al (2012), most probably due the exochelins overcompensating for the absence of mycobactins, in M. smegmatis ESX-3 knockouts. / MSc (Biochemistry) North-West University, Potchefstroom Campus 2013.

According to the World Health Organization (WHO), M. tuberculosis, the causative agent of TB, accounts for approximately 1.7 million deaths annually. Further contributing causes to the worldwide TB incidence, is the widespread unavailability and ineffectiveness of TB vaccines, time consuming diagnostic methods and unsuccessful treatment approaches. Research for better characterising mycobacteria in general, or other Mycobacterium species, may help us to better understand M. tuberculosis and TB disease mechanisms, which will in turn lower the future TB disease prevalence, as this may lead to the development of better treatments, diagnostics and vaccines. Mycobacteria use various secretion pathways, including the ESX- or type VII secretion (T7S) system, to ensure transport across the complex cell wall. The genome of M. tuberculosis has five copies of a gene cluster known as the ESX gene cluster region, which is associated with virulence and viability of mycobacteria. The ESX-3 gene cluster is thought to be essential for growth of M. tuberculosis and proposed to be involved in iron / zinc homeostasis. Mycobacteria synthesise siderophores, which are proposed to be involved in the uptake of iron over their cell wall. M. tuberculosis are known to produce two types of siderophores, namely: carboxymycobactins and mycobactins. Loots and colleagues, however illustrated, that ESX-3 knockouts, show signs of iron overload, despite the absence of the mycobactins induced by knocking out the ESX-3 gene cluster. It was hypothesised, that this overload occurs due to an increase in exochelin synthesis, another iron uptake protein not associated with ESX-3, overcompensating for the perceived iron depletion in the knockout organism. A Metabolomics research approach was subsequently used in this study, to generate new information in order to better characterise the role of iron on the metabolism of these organisms, and additionally confirm the role of ESX-3 in iron uptake. In this study, we firstly determined the most optimal extraction conditions for this metabolomics investigation. Two extraction methods were subsequently investigated and compared, considering their repeatability and their respective capacities to extract those compounds which best differentiate the M. smegmatis ESX-3 knockouts and wild-type parent strains. Considering the results generated, the total metabolome method was chosen for further analyses, for the following reasons: 1) it is simpler, 2) faster, 3) showed better repeatability, 4) extracts those compounds best differentiating the compared groups and 5) has been previously described for metabolomics analyses characterising ESX-3 gene functionality, hence potentially allowing us to compare results to that previously generated and published data. Subsequently, we used the chosen extraction method, followed by GCxGC-TOFMS analysis of the separately cultured M. smegmatis wild-type sample extracts, cultured in normal, low and high iron conditions, to determine the influence of varying iron concentrations on the metabolome of this organism, by metabolomics comparisons of these groups. Following this, an identical research approach was used to compare the metabolome of a M. smegmatis ESX-3 knock-out strain, to that of a M. smegmatis wild type parent strain, both cultured in normal / standardised iron concentrations. Considering the results generated when comparing the metabolome of a M. smegmatis ESX-3 knock-out strain to that of a M. smegmatis wild type parent strain, the altered metabolome of the M. smegmatis ESX-3 knockouts correlated well to that of the M. smegmatis wild type cultured in elevated iron growth conditions. This suggests ESX-3 is involved in iron uptake, and that knocking out the ESX-3 gene cluster of M. smegmatis does in fact result in a metabolome profile suggesting iron overload, as was proposed by Loots et al (2012), most probably due the exochelins overcompensating for the absence of mycobactins, in M. smegmatis ESX-3 knockouts. / MSc (Biochemistry) North-West University, Potchefstroom Campus 2013.

Le système de sécrétion type VII (T7SS) présent chez les mycobactéries comporte cinq loci, nommés ESX-1 à ESX-5, chacun possédant leurs propres fonctions. Le système ESX-3, le plus conservé entre les espèces de mycobactéries, participe au transport du fer et à la sécrétion de protéines dont la protéine EsxH. EsxH interagit avec le système ESCRT dans le macrophage et contribue à la persistance de M. tuberculosis (Mtb) dans l’hôte. Afin de mieux comprendre le mécanisme du T7SS, nous avons surexprimé le locus ESX-3 de Mtb dans les organismes recombinants M. smegmatis et M. marinum. Nous avons purifié un coeur protéique partiel du complexe ESX-3 par chromatographie liquide de protéine rapide (FPLC) et déterminé différentes structures in vitro qui pourraient représenter son dynamisme par une analyse des particules individuelles. Nous avons localisé le complexe ESX-3 aux pôles chez M. marinum par microscopie de fluorescence (fLM) et microscopie super-résolution d’illumination de structure (SR-SIM). Finalement, nous avons reconstruit un modèle in vivo 3D de ce complexe en jumelant une technique de corrélation entre la microscopie par localisation photoactivée (PALM) et la tomographie électronique en conditions cryogéniques (cryo-ET). En jumelant nos structures in vitro et notre modèle in vivo, nous discutons d’un mécanisme possible du complexe ESX-3. Ces analyses pourront aussi supporter le criblage d’inhibiteurs potentiels pour traiter les infection mycobactériennes. / A novel, type VII secretion system (T7SS) was recently discovered in Mycobacteria. Five subsystems, called ESX-1 to ESX-5, have been identified, with the ESX-3 being the most conserved. The ESX-3 system is essential for growth and pathogenesis, and has been implicated in iron transport and secretion of effector proteins into the host. The secreted proteins are shown to prevent phagosome maturation by interacting with the ESCRT machinery of the macrophage. To characterize the structure and mechanism of secretion employed by the T7SS, we use the ESX-3 system from Mycobacterium tuberculosis (Mtb) and recombinantly express it in M. smegmatis and M. marinum cells. By combining Fast Protein Liquid Chromatography (FPLC) and Single Particle electron microscopy analysis, we have reconstructed several in vitro models that contain at least three of the ESX-3 cluster proteins and may represent the dynamic nature of the core complex. Using fluorescent light microscopy (fLM) and super-resolution structure illumination microscopy (SR-SIM), we localized the ESX-3 complex to the lateral pole in M. marinum cells. We also used super-resolution photoactivated localization microscopy (PALM) in correlation with cryo-electron tomography (cryo-ET) of whole M. marinum cells to localize and reconstruct an in vivo 3D model of the ESX-3 secretion system. By combining the single particle reconstructions and the cryotomography data, we discuss a possible mechanism of ESX-3 secretion. Such analysis may support future inhibitor screens to prevent mycobacterial infections.