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Tuberculosis (TB) claims the lives of millions of individuals each year, and is consequently the world’s second-most deadly infectious disease after acquired immune deficiency syndrome (AIDS), responsible for 1.4 million deaths in 2010 alone. Developing countries carry the heaviest burden, with the occurrence of multidrug-resistant (MDR) TB becoming more frequent, making more efficient vaccination and treatment strategies a necessity to combat this epidemic. The ESX-1 gene cluster (encoding the virulence-associated proteins ESAT-6 and CFP-10) and the Type Vll secretion system are thought to be responsible for the transport of extracellular proteins across the hydrophobic, and highly impermeable, cell wall of Mycobacterium, and consequently are thought to play a role in the virulence of this organism. To date, our understanding of tuberculosis pathophysiology and virulence has been described primarily using proteomic and genomic approaches. Subsequently, using the relatively new research approach called metabolomics, and interpreting the data using systems biology, we aimed to identify new metabolite markers that better characterise virulence and the proteins involved, more specifically related to the ESX-1 gene cluster. Using a GCxGC-TOFMS metabolomics research approach, we compared the varying metabolomes of M. smegmatis ESX-1 knock-out (ESX-1ms) to that of the wild-type parent strain and subsequently identified those metabolite markers differing between these strains. Multivariate and univariate statistical analyses of the analysed metabolome were used to identify those metabolites contributing most to the differences seen between the two sample groups. A general increase in various carbohydrates, amino acids and lipids, associated with cell wall structure and function, were detected in the ESX-1ms strain relative to the wild-type parent strain. Additionally, metabolites associated with the antioxidant system, virulence protein formation and energy production in these mycobacteria, were also seen to differ between the two groups. This metabolomics investigation is the first to identify the metabolite markers confirming the role of the ESX-1 gene cluster with virulence and the underlying metabolic pathways, as well as its associated role with increased metabolic activity, growth/replication rates, increased cell wall synthesis and an altered antioxidant mechanism, all of which are believed to contribute to this organism’s increased pathogenicity and survival ability. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Tuberculosis (TB) claims the lives of millions of individuals each year, and is consequently the world’s second-most deadly infectious disease after acquired immune deficiency syndrome (AIDS), responsible for 1.4 million deaths in 2010 alone. Developing countries carry the heaviest burden, with the occurrence of multidrug-resistant (MDR) TB becoming more frequent, making more efficient vaccination and treatment strategies a necessity to combat this epidemic. The ESX-1 gene cluster (encoding the virulence-associated proteins ESAT-6 and CFP-10) and the Type Vll secretion system are thought to be responsible for the transport of extracellular proteins across the hydrophobic, and highly impermeable, cell wall of Mycobacterium, and consequently are thought to play a role in the virulence of this organism. To date, our understanding of tuberculosis pathophysiology and virulence has been described primarily using proteomic and genomic approaches. Subsequently, using the relatively new research approach called metabolomics, and interpreting the data using systems biology, we aimed to identify new metabolite markers that better characterise virulence and the proteins involved, more specifically related to the ESX-1 gene cluster. Using a GCxGC-TOFMS metabolomics research approach, we compared the varying metabolomes of M. smegmatis ESX-1 knock-out (ESX-1ms) to that of the wild-type parent strain and subsequently identified those metabolite markers differing between these strains. Multivariate and univariate statistical analyses of the analysed metabolome were used to identify those metabolites contributing most to the differences seen between the two sample groups. A general increase in various carbohydrates, amino acids and lipids, associated with cell wall structure and function, were detected in the ESX-1ms strain relative to the wild-type parent strain. Additionally, metabolites associated with the antioxidant system, virulence protein formation and energy production in these mycobacteria, were also seen to differ between the two groups. This metabolomics investigation is the first to identify the metabolite markers confirming the role of the ESX-1 gene cluster with virulence and the underlying metabolic pathways, as well as its associated role with increased metabolic activity, growth/replication rates, increased cell wall synthesis and an altered antioxidant mechanism, all of which are believed to contribute to this organism’s increased pathogenicity and survival ability. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Thesis (MScMedSc (Biomedical Sciences. Human Biology and Human Genetics))--University of Stellenbosch, 2010. / Thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Medical Biochemistry at the Faculty of Health Sciences, University of Stellenbosch. / ENGLISH ABSTRACT: Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease which causes approximately 2 million deaths each year. Despite extensive research on tuberculosis and M. tuberculosis, little is understood of the mechanisms of pathogenicity of the organism. The genome of M. tuberculosis contains five ESAT-6 gene cluster regions, each of which contains genes encoding proteins involved in the formation of a dedicated protein secretion system. Included in these regions are genes encoding exported T-cell antigens, serine proteases, ATP-binding proteins and other membrane-associated proteins. Although it is known that some of these secretion systems are involved in virulence and phagosomal escape of M. tuberculosis, and that deletion thereof causes attenuation of the organism, the structure, substrates and functions of the systems are largely unknown. Understanding the structure of the ESX secretion systems will advance our understanding of the mechanisms of mycobacterial pathogenicity and provide clues to ways in which to interfere with these virulence mechanisms. The ESAT-6 gene cluster region 3, encoding the ESX-3 secretion machinery, is the only ESAT-6 gene cluster region which is essential for the in vitro growth of M. tuberculosis. It is however not required for the growth of the saprophytic mycobacterium M. smegmatis. In this study we have identified proteinprotein interactions within the ESX-3 secretion system, using the Mycobacterial – Protein Fragment Complementation (M-PFC) mycobacterial two-hybrid system, and created a model of the M. tuberculosis ESX-3 secretion system. According to this model, the EsxG-EsxH and PE5-PPE4 substrate protein complexes bind to the same components of the ESX-3 secretion machinery and are secreted via the same mechanism. A knock-out of the ESX-3 secretion system in M. smegmatis was generated by homologous recombination to allow further research into the functions and properties of this secretion system. This knock-out was used, together with wild-type M. smegmatis, to investigate the secretion of the M. tuberculosis EsxH protein by the M. smegmatis ESX-3 secretion system. The ESX-3 secretion system interactome may serve as a model for the ESX secretion systems and assist in our understanding of this secretion machinery which is key to the virulence and survival of M. tuberculosis and other pathogenic mycobacteria. Improved understanding of these mechanisms and their role in pathogenicity and survival may provide means of interfering with the secretion machinery, potentially leading to developments in the prevention and treatment of tuberculosis disease. / AFRIKAANSE OPSOMMING: Tuberkulose, wat veroorsaak word deur Mycobacterium tuberculosis, eis jaarliks ongeveer 2 miljoen lewens. Ten spyte van uitgebreide navorsing oor tuberkulose en M. tuberculosis is min bekend oor die meganismes van patogenisiteit van díe organisme. Die genoom van M. tuberculosis bevat vyf ESAT-6 geen groep gebiede wat elk proteïene kodeer wat ‘n toegewyde sekresie sisteem vorm. Ingesluit in elk van díe geen groep gebiede is gene wat T-sel antigene, serien proteases, ATP-bindingsproteïene en ander membraan-geassosieërde proteïene kodeer. Alhoewel dit bekend is dat sekere van hierdie sekresie sisteme betrokke is by virulensie en fagosoom-ontsnapping, en dat delesie daarvan die organisme attenueer, is die struktuur, substrate en funksies van die sisteme grootliks onbekend. Kennis van die struktuur van die ESX sekresie sisteme sal ons verstaan van die meganismes van mikobakteriele patogenisiteit verbeter en leidrade verskaf na maniere om in te meng by díe meganismes van virulensie. Die ESAT-6 geen groep gebied 3, wat die ESX-3 sekresie sisteem kodeer, is die enigste ESAT-6 geen groep gebied wat noodsaaklik is vir die in vitro groei van M. tuberculosis. Dit is egter nie nodig vir die groei van die saprofitiese mikobakterium M. smegmatis nie. In hierdie studie het ons proteïenproteïen interaksies van die ESX-3 sekresie sisteem geïdentifiseer, deur middel van die Mikobakteriële - Proteïen Fragment Komplementasie (M-PFC) mikobacteriële twee-hibriede stelsel. Die interaksies is gebruik om ‘n model van die M. tuberculosis ESX-3 sekresie sisteem te skep. Volgens díe model bind die EsxG-EsxH en PE5-PPE4 substraat proteïen komplekse aan dieselfde komponente van die ESX-3 sekresie apparaat en word deur dieselfde meganisme uitgevoer. ‘n uitklopmutant van die ESX-3 sekresie sisteem word deur homoloë rekombinasie in M. smegmatis gegenereer om verdere ondersoeke na die funksies en eienskappe van hierdie sekresie sisteem in staat te stel. Hierdie uitklopmutant is tesame met die wilde-tipe M. smegmatis gebruik om die sekresie van die M. tuberculosis EsxH proteïen deur die M. smegmatis ESX-3 sekresie sisteem te ondersoek. Die ESX-3 sekresie sisteem interaktoom kan dien as ‘n model vir die ESX sekresie sisteme om te help om ons kennis van hierdie sekresie apparaat, wat belangrik is vir die virulensie en oorlewing van M. tuberculosis en ander patogeniese mikobakterieë, te verbeter. Kennis van hierdie meganismes en hul rol in patogenisiteit en oorlewing mag maniere verskaf om by díe sekresie sisteme in te meng, wat moontlik kan lei tot ontwikkelings in die voorkoming en behandeling van tuberkulose.

Tuberculosis is an enormous global health problem. Despite decades of research, the mechanism(s) by which Mycobacterium tuberculosis (Mtb) mediates virulence remains incompletely understood. The ESX-1 secretion system is critical for Mtb to survive and cause disease in vivo, but its primary function and mechanism of action are unclear. The many inherent challenges of working with this slow-growing pathogen often limit the experimental approaches that can be used to address these questions. Thus, we have developed a model system in the nonpathogenic bacterium Bacillus subtilis to study ESX-type secretion systems. Here, we demonstrate that the B. subtilis yuk operon encodes an ESX-type secretion system responsible for the secretion of YukE. Additionally, we demonstrate that the yuk system is active in B. subtilis during conditions of nutrient deprivation and is required for normal biofilm formation. Interestingly, this is similar to our findings that the Mtb ESX-1 system plays dual roles in protein secretion and modulating cell wall integrity. One defining feature of all ESX loci is the presence of an FtsK/SpoIIIE family ATPase. Interestingly, these ATPases have a domain structure unique to ESX-associated ATPases, where each protein contains multiple (2-3) enzymatic domains. We used our B. subtilis system to dissect the mechanism of action of this unique class of motor proteins. We find that the yuk-encoded ATPase YukBA dimerizes to form a hexamer of enzymatic subunits that are differentially required for secretion. Strikingly, we find a unique requirement for rotational symmetry in the nucleotide binding activity of the subunits. Finally, we compared the energy requirements of the Mtb ESX-1 system and the B. subtilis yuk system. We find that these systems have some overlapping ATPase requirements for protein secretion and cell wall integrity/biofilm formation, suggesting that there is a conservation of function among ESX-type systems. We also find that some ATPase domains are differentially required for function between these two systems, which we postulate is due to the split protein architecture of the ESX-1-encoded ATPases. Together, these findings highlight the power of using a B. subtilis model system to understand the function and mechanism of action of ESX-type secretion systems.

Stuttgart, Univ., Fachstudie, 2006.

Mycobacterium abscessus est une mycobactérie à croissance rapide, et un pathogène opportuniste responsable d’infections pulmonaires notamment chez les patients atteints de la mucoviscidose, et d’infections cutanéomuqueuses. La source de contamination pourrait être environnementale mais des contaminations interhumaines ne sont pas exclues. Les amibes environnementales pourraient jouer un rôle de réservoir. M. abscessus est capable de résister aux mécanismes de défense bactéricides des phagocytes environnementaux et humains. Le génome complet de M. abscessus a été séquencé mettant en évidence de nombreux facteurs de virulence non mycobactériens. Certains sont des facteurs de virulence connus dans le monde bactérien, comme la phospholipase C ou le facteur de captation du magnésium MgtC. Ces facteurs ont été montré induits en présence d’amibes, mais ne peuvent à eux seuls expliquer la survie intracellulaire et la virulence de M. abscessus. Nous avons donc, au cours de ce projet, criblé une banque de mutants générée par transposition chez M. abscessus, à la recherche de mutants dénués de croissance intracellulaire en amibes et macrophages. Cette approche a permis d’identifier, de façon majeure, 5 gènes du locus ESX-4 de M. abscessus codant un système de sécrétion de type VII avec tous ces composants cœur conservés. Pour mieux comprendre la contribution d’ESX-4 dans la survie intracellulaire de M. abscessus, un mutant obtenu par double recombinaison au sein du gène eccB4 dans la souche type de M. abscessus (ΔeccB4) a été construit. EccB4 est un élément structurel central du système de sécrétion codé par ESX-4. ΔeccB4 présente un défaut de survie au sein des cellules, lié à déficit de blocage de l’acidification phagosomale ainsi qu’un défaut de dégradation de la membrane phagosomale, empêchant un contact phagosome-cytosol. Ce travail a permis de révéler pour la première fois dans le monde mycobactérien le rôle d’un locus ancestral de sécrétion ESX-4 au sein d’une mycobactérie. L’étude des protéines secrétées par ce locus est actuellement en cours au laboratoire, afin d’envisager des approches thérapeutiques et vaccinales pour contrer cette mycobactérie multirésistante aux antibiotiques. / Mycobacterium abscessus is a fast growing mycobacterium, and an opportunistic pathogen responsible for lung infections particularly in patients with cystic fibrosis, and for mucocutaneous infections. The source of contamination could be environmental but human-to-human contaminations are not excluded. Environmental amoeba could play a role as a reservoir. M. abscessus is able to resist to the bactericidal defense mechanisms of environmental and human phagocytes. The complete genome of M. abscessus has been sequenced and presents numerous non-mycobacterial virulence factors. Some are known virulence factors in the bacterial world, such as phospholipase C or the magnesium uptake factor MgtC. These factors have been shown to be induced in the presence of amoeba, but cannot alone explain the intracellular survival and virulence of M. abscessus. We thus, in the course of this project, screened a library of mutants generated by transposition in M. abscessus, in search of mutants lacking intracellular growth in amoeba and macrophages. This approach made it possible to identify, in a major way, 5 genes of the ESX-4 locus of M. abscessus encoding a type VII secretion system with all these conserved core components. In order to better understand the contribution of ESX-4 to the intracellular survival of M. abscessus, a mutant obtained by double recombination within the eccB4 gene in the M. abscessus type strain (ΔeccB4) was constructed. EccB4 is a central structural element of the secretion system encoded by ESX-4. ΔeccB4 has a defect of survival within the cells, linked to deficiency of blockage of the phagosomal acidification as well as a defect of degradation of the phagosomal membrane, preventing phagosome-cytosol contact. This work made it possible to reveal for the first time in the mycobacterial world the role of an ancestral locus ESX-4 secretion within a mycobacterium. The study of the proteins secreted by this locus is currently underway in the laboratory, in order to consider therapeutic and vaccine approaches to counter this multiresistant antibiotic mycobacterium.

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.