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A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master of Medicine (Internal Medicine) Johannesburg 2001 / Introduction and Aim: The waxy outer cell wall of mycobacteria consists mainly of mycolic acids (MA). The unique immuno-stimulatory properties of MA via the CD 1-restricted antigen presentation pathway have been demonstrated in humans. Purification and isolation of M.tuberculosis (MTB) MA has allowed them to be applied as an antigen in an ELISA-based sero-diagnostic assay to detect specific antibodies in the sera of humans. The aim of the study was to measure the levels of antibody to MA in the sera of patients with culture proven pulmonary tuberculosis (PTB), and in control subjects without evidence of tuberculosis. / IT2018

The immunological effects of mycolic acids (MA) from Mycobacterium tuberculosis on mouse peritoneal macrophages were studied. MA was solubilbized using various carriers. Phagosome uptake and maturation (into late stage phagolysosomes) were compared using fluorescent markers and the confocal microscope. During assessment on the effects of MA on mouse macrophages, changes in morphology and activation of the macrophages were found. This indicated that the MA was immune reactive towards macrophages. The phenotype of cell that develops after in vivo loading with MA was characterized by using cell surface markers: it was found that MA-Ioaded macrophages developed into foam cells. Cell survival, proliferation and macrophage cytokine production were examined to characterize the foam-like cells. The effect of MA-induced foam-like cells on living Mycobacterium tuberculosis was evaluated and increased bactericidal activity was found. The roles of reactive oxygen and nitrogen intermediates via myeloperoxidase were also examined and a theoretical mechanism for the formation of foam cells proposed. The possible role of myeloperoxidase in activation of macrophages, foam cell formation and killing of Mycobacterium tuberculosis is discussed. It is postulated that a possible relationship might exist between tuberculosis and atherosclerosis that is facilitated by mycolic acids. / Thesis (DPhil (Human Physiology))--University of Pretoria, 2005. / Physiology / unrestricted

Tuberculosis (TB) is the leading cause of death among HIV infected people. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of TB, features a distinctive lipid-rich cell wall with mycolic acids (MA) the major component in the outer layer. Mycolic acids are á-alkyl â-hydroxy long chain fatty acids, which exist in a number of chemical subclasses depending on the presence of functional oxygenated and non-oxygenated groups in the meromycolate chain. In numerous studies the different MA subclasses have been shown to play different roles in antibody recognition, virulence and the ability to attract cholesterol. It was previously suggested that the oxygenated MA might be important for these properties. Except for the mycolic acid motif, little is known about the stereochemistry of the other chiral centres. The importance of the different functional groups, their position and stereochemistry, for immunological properties, are not yet clarified. This study set out to resolve the structureactivity relationships of mycolic acids from M. tuberculosis in terms of their antigenicity and the ability to attract cholesterol. To determine fine specificity of interaction of MA with antibodies, the subclasses of MA from M. tuberculosis were separated and the antigenicity of two was determined. TB+ and TB- patient sera recognised natural MA, alpha-MA and methoxy-MA. It was confirmed that the carboxylic acid group played a fundamental role in its recognition. Interestingly, cord factor (trehalose-6,6’-dimycolate) was recognised specifically by TB+ sera. This implies multiple epitopes in the MA structure, some of which are very specific for TB patients. A stereocontrolled diastereomer of cis-cyclopropane methoxy-MA was synthesized and along with other synthetic methoxy-, keto- and hydroxy-MAs, were tested for antibody recognition. One diastereomer, SS-SR-methoxy-MA, was recognised stronger by TB+ serum than the other, it also is the one that closest approximates the signal strength of antibody binding to natural MA by TB+ patient sera. While the others are not specifically recognised, this SS-SR-methoxy-MA may well represent one of the antigenically active components that occurs in natural MA and that elicits specific antibodies in TB patients. This thesis reports a stereocontrolled chemical synthesis of biologically active mycolic acids and shows that a single component of the mycolic acid mixture can be sufficient to elicit an immunological response. / Thesis (PHD)--University of Pretoria, 2008. / Biochemistry / unrestricted

Mycobacterium tuberculosis (M.tb) l'agent étiologique de la tuberculose infecte un tiers de la population mondiale avec 9 millions de nouveaux cas et 1.5 millions de décès chaque année. La capacité de la bactérie à persister dans son hôte ainsi que l'apparition croissante de souches multi-résistantes voire totalement résistantes expliquent ces statistiques dramatiques. La découverte de nouveaux traitements à travers une meilleure connaissance de la physiologie et des programmes génétiques adaptatifs du pathogène est donc une priorité mondiale. M.tb est un bacille à Gram+ avec une enveloppe particulière caractérisée par une membrane externe (la mycomembrane) essentielle à sa viabilité et sa virulence. Cette membrane est constituée majoritairement d'acides mycoliques (AMs), des acides gras à très longues chaînes modifiés par l'introduction de groupements fonctionnels. Bien qu'à ce jour la biosynthèse des AMs est relativement bien caractérisée d'un point de vue biochimique, certaines données nécessitent d'être confirmées in vivo, de même qu'il existe peu d'information sur la régulation et la contribution des gènes de biosynthèse à la capacité adaptative des mycobactéries. Une trentaine de gènes sont impliqués dans la biosynthèse des AMs dont hadA, hadB et hadC codant pour une réaction de déshydratation essentielle. Il a été démontré biochimiquement que HadB porte l'activité catalytique et que HadA et HadC apportent la spécificité de substrats. Au cours de ma thèse, par une approche génétique, nous avons montré que seule HadB était essentielle à la viabilité mais que HadA et HadC bien que non essentielles jouaient un rôle majeur dans la physiologie, la capacité adaptative et la virulence des mycobactéries, en relation avec leur rôle dans la structure des AMs. Ces résultats avaient non seulement confirmé les données biochimiques quant au rôle de HadC dans la biosynthèse des AMs, mais également souligné l'intérêt d'une stratégie de lutte basée sur l'affaiblissement du fitness de M.tb, rendant ainsi le pathogène plus sensible aux traitements déjà existants ainsi qu'aux défenses naturelles de l'hôte. Une analyse phylogénétique couplée à une analyse expérimentale de l'expression des gènes nous a permis de retracer et de rationaliser le scénario évolutif qui a façonné le locus hadABC. En accord avec l'organisation génétique, j'ai ainsi montré que la carence en nutriments, un stress rencontré par la bactérie lors de l'infection, conduisait à la co-répression des gènes hadABC ainsi que de la plupart des gènes de biosynthèse des AMs avec des gènes impliqués dans le processus de traduction. Le potentiel de traduction est connu pour être contrôlé par la disponibilité en nutriments, à travers notamment la réponse stringente, une réponse adaptative universellement conservée chez les bactéries. Suite à ces résultats, un modèle a été proposé selon lequel au cours de la réponse stringente, les intermédiaires de synthèse des AMs, seraient détournés au profit de la synthèse de lipides alternatifs dont des lipides de stockage. L'analyse phylogénétique a également suggéré une relation fonctionnelle étroite entre l'activité des enzymes HadABC et des enzymes responsables de la modification des AMs. Afin d'avoir une vision intégrée de la régulation de la synthèse de la mycomembrane, nous avons analysé le rôle biologique du gène rv0081 codant pour un facteur de transcription global. Différentes approches systémiques suggéraient que Rv0081 jouerait un rôle central dans la capacité adaptative de M.tb en régulant de nombreux gènes impliqués dans différentes fonctions cellulaires dont les gènes hadABC. J'ai pu montrer qu'un mutant rv0081 était hypervirulent et que l'absence d'une régulation naturelle du gène affectait la capacité de survie de la bactérie à l'intérieur des macrophages. / Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis infects one third of the world population with 9 million new cases and 1.5 million deaths each year. The capability of the bacteria to persist in its host and the emergence of multi- and totally-drug resistant strains explain these dramatic statistics. Therefore, the discovery of new drugs through a better understanding of the physiology and of the adaptive genetic programs of the pathogen is a priority. Mtb is a Gram + bacilli with an unusual cell envelope characterized by an outer membrane (the mycomembrane) essential to its viability and virulence. This membrane is mainly composed of mycolic acids (MAs), a class of very long chain fatty acids which are modified by the introduction of functional groups. To date the biosynthesis of MAs is biochemically well characterized, but some data need to be confirmed in vivo, likewise there is little information about the regulation and the contribution of biosynthesis genes in the adaptive capacity of mycobacteria. Around thirty genes are involved in the biosynthesis of MAs including hadA, hadB and hadC which are required for an essential dehydration reaction. It has been shown biochemically that HadB bears the catalytic activity and that HadA and HadC bring about the substrate specificity. In this study, using a genetic approach, we have shown that only HadB was essential to the viability but that the non-essential HadA and HadC proteins played a major role in the physiology, the adaptive capacity and the virulence of mycobacteria. These results have not only confirmed the biochemical data on the role of HadC in the biosynthesis of MAs, but have also underlined the relevance of a strategy based on weakening the fitness of Mtb, making the pathogen more susceptible to existing therapy as well as to the natural host defenses. Phylogenetic and experimental analyses of gene expression allowed us to rationalize the evolutionary scenario that has shaped the hadABC locus. In agreement with the genetic organization, I have shown that starvation, a stress experienced by the bacterium upon infection, resulted into the co-repression of the genes hadABC as well as most of the MAs biosynthetic genes with genes involved in the translation process. The translation potential is known to be controlled by nutrient avaibility, especially through the stringent response, an adaptive response widely conserved in bacteria. Following these results a model was proposed stating that during the stringent response, the MAs intermediate products would be redirected toward the synthesis of alternate lipids including storage lipids. Phylogenetic analysis also suggested a close functional relationship between the activity of HadABC proteins and the enzymes involved in the modification of MAs. In order to get an integrated picture of the regulation of the biosynthesis of the mycomembrane, we analyzed the biological role of rv0081, a gene encoding a transcription factor. Various comprehensive approaches suggested that Rv0081 plays a key role in M.tb adaptive capacity through the regulation of many genes involved in various cellular functions including the hadABC genes. In my PhD work I have shown that an rv0081 deleted strain was hypervirulent and that the inability of the bacteria to properly regulate the gene had prevented the bacteria to survive within macrophages.

Rhodococcus is an important industrial organism that possesses diverse metabolic capabilities, it also has a unique cell envelope, composed of an outer layer of mycolic acids and glycolipids (free or bound lipids generally linked to the sugar trehalose). Rhodococcus is able to transform nitriles to the corresponding amide by the enzyme Nitrile Hydratase (NHase), therefore rhodococcal cells can be utilized as biocatalysts in the detoxification of nitrile waste water or in the production of industrially important amides such as acrylamide. However, the NHase within the native cells must be stable with high activity. This research examined how NHase activity and stability can be increased in native cells by changing growth media composition, the impact on the rhodococcal cell envelope was also studied. Growth media composition was altered by supplementing different sugars such as fructose, maltose or maltodextrin to replace glucose in rich solid media containing cobalt and urea for induction of NHase. The supplementation of maltose or maltodextrin resulted in significantly higher NHase activities and greater NHase stability at 55„aC. The supplementation of these different sugars was shown to alter cellular and lipid bound trehalose levels, a sugar known to stabilize proteins and a component of the rhodococcal cell envelope. Cells that had higher levels of cellular trehalose had significantly greater NHase stability at 55„aC. The effect of the different sugar supplements and inducers of NHase, such as cobalt, on cell envelope components such as mycolic acids and glycolipids were examined by High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC). The results showed that changes in mycolic acids and glycolipids occurred when the cells were grown in the presence of different sugar supplements and when the cells were induced for NHase. Susceptibility of Rhodococcus sp DAP 96253 to different antibiotics was examined to indicate if changes were occurring in the cell envelope. Differences in antibiotic susceptibility were observed when the cells were grown on media with different sugar supplements and when the cells were induced for NHase. In the presence of cobalt Rhodococcus sp DAP 96253 showed a significant increase in sensitivity to antibiotics. Changes in growth media composition influences the cell envelope of Rhodococcus sp DAP 96253 and also affects NHase activity and stability. Therefore, achieving increased enzyme activity and stability is not entirely dependent on the actual enzyme, but is related to other aspects of the cell, such as the cell envelope and metabolites of the cell.

Accurate diagnosis of active tuberculosis is required to improve treatment, reduce transmission of the disease and control the emergence of drug resistance. A rapid and reliable test would make a considerable contribution to the management of the TB epidemic, especially in HIV-burdened and resource-poor countries where access to diagnostic laboratories are limited. Surrogate marker antibody detection to mycobacterial lipid cell wall antigens gave promising results, in particular with cord factor. The specific advantage of using mycolic acids as lipid antigens in comparison to protein antigens is that mycolic acid is a CD1 restricted antigen with the ability to induce proliferation of CD4/CD8 double negative T-cells, which may explain the sustained antibody production in AIDS patients. Traditional end-point assays to detect anti-MA antibodies showed an unacceptable number of false positive and negative test results. Here a much improved biosensor method (the MARTI-assay, i.e. Mycolic acid Antibody Real-Time Inhibition assay) was developed to detect antibodies to mycolic acid in patient sera as surrogate markers of active tuberculosis. The test was assessed on an IAsys optical biosensor and gave an accuracy of 82%. The technology was transferred to an SPR (ESPRIT) biosensor to economise and simplify the assay. Mycolic acid containing liposomes were immobilized on the SPR gold surface pre-coated with octadecanethiol. The following parameters were optimized on the ESPRIT biosensor to enable reliable TB diagnosis: effect of degassed buffer, saponin blocking, first exposure to serum at low concentration and second exposure to antigen inhibited serum at high concentration. The IAsys biosensor system has a weakness in the double channel cuvette system, in which the channels often do not give matching results, while being ten times more expensive than the gold discs provided for the ESPRIT biosensor. The ESPRIT biosensor is provided with an adjustable laser setting to compensate for differences in the channel readings as well as an automated fluidic system that reduces variance from one sample to the next. First indications are that the test can also be used for prognosis of TB during treatment. It is hoped that the ESPRIT biosensor will improve the accuracy of the test to more than 90%. If the MARTI-assay technology could be made amenable for high throughput screening, it may provide the solution to the serodiagnosis of tuberculosis and monitoring of progress during TB treatment both in adult and children, thereby reducing the spread of TB within the communities. / Thesis (PhD)--University of Pretoria, 2009. / Biochemistry / unrestricted

Tuberculosis (TB) is a disease caused by the infection of Mycobacterium tuberculosis, which is progressively becoming multi-drug resistant (MDR). Understanding the mechanism by which the organism interacts with host lipids, infect macrophages and how components redistribute within the host could open the investigation of new ways of inhibiting and eradicating the infection suffered by patients world wide. Flow fluorometry of liposomes containing mycolic acids, which are â-hydroxy fatty acids with a long á-alkyl side chain of mycobacteria, may be useful to determine the dynamics of interaction of these lipids with the host membrane lipids and with cholesterol. This will increase the understanding about the structure-function relationship of mycolic acids in M.tb. It was shown in this thesis that natural mycolic acids had a unique property, it could exchange rapidly between liposomes in the presence and absence of cholesterol even at low temperatures. Rapid exchange of mycolic acids within the host could be the mechanism by which trafficking of mycobacterial lipids comes about, ultimately leading to immune response modulation beyond the infected cell. It also provides direction for future investigation to bring about new serodiagnostic tests based on lipid antigens. Although flow fluorometry as a modern technique was unable to resolve the exchange of mycolic acids in relation with other lipids, a unique property of mycolic acids was demonstrated for the first time, that of rapid exchange. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Biochemistry / unrestricted

Mycobacterium abscessus est une mycobactérie à croissance rapide qui a récemment émergé en tant que pathogène opportuniste, en particulier chez les patients atteints de mucoviscidose. Elle est naturellement résistante aux antibiotiques les plus couramment disponibles, limitant sérieusement les options thérapeutiques chez les patients infectés. Il apparaît donc urgent d’identifier et de caractériser de nouvelles cibles d’intérêt thérapeutique dans la lutte contre ce pathogène. Contrairement au variant lisse (S) de M. abscessus, caractérisé par une forte production de glycopeptidolipides (GPL), le variant rugueux (R) associé à une faible production de GPL est responsable des manifestations cliniques les plus sévères. Chez l’embryon de poisson zèbre, le variant R s’accompagne d’une virulence accrue avec formation de cordes mycobactériennes extracellulaires et d’abcès, engendrant une mortalité rapide des larves infectées. Les mécanismes moléculaires de la pathogénicité de M. abscessus demeurent toutefois très peu connus.Dans cette étude, nous avons identifié le gène MAB_4780, codant une déshydratase distincte du complexe HadABC impliqué dans la biosynthèse des acides mycoliques. Le gène MAB_4780, tout comme son homologue chez M. smegmatis, MSMEG_6754, ont été identifiés comme responsables de la résistance innée de ces deux espèces au thiacetazone (TAC), un agent antituberculeux de seconde intention. Nous avons inactivé le gène MAB_4780 dans le variant R de M. abscessus, ce qui a entraîné une modification de la composition en acides mycoliques, un défaut de formation des cordes mycobactériennes ainsi qu'un phénotype extrêmement atténué chez les embryons de poisson zèbre. L'atténuation in vivo du mutant MAB_4780 qui résulte très vraisemblablement de l’incapacité i) à former des cordes et ii) à inhiber la fusion phagolysosomale, s’accompagne d’une croissance intracellulaire diminuée. En outre, le mutant MAB_4780, tout comme le mutant de M. smegmatis dépourvu de MSMEG_6754, présente une croissance altérée dans l’amibe, qui représente un réservoir pour les mycobactéries environnementales. Ces résultats reflètent le rôle crucial de cette nouvelle déshydratase dans la survie de M. abscessus dans son environnement et dans l'établissement d'infections aiguës et létales. Par conséquent, cibler MAB_4780 pourrait représenter une stratégie particulièrement prometteuse pour contrôler les infections à M. abscessus. De futures études seront consacrées à l'identification d’inhibiteurs spécifiques de MAB_4780, grâce au développement d’un test d’activité et d’une structure cristalline de la protéine obtenue à très haute résolution.Nous avons également criblé contre M. abscessus une chimiothèque d’analogues structuraux du TAC, préalablement validés pour leur activité inhibitrice de la déshydratase HadABC de Mycobacterium tuberculosis. Trois d’entre eux ont montré une efficacité accrue d’un facteur 50 par rapport à la molécule parentale. La surexpression d’EthA, l’activateur du TAC, augmente la susceptibilité de M. abscessus à ces trois analogues. Ces résultats suggèrent que leur mode d'activation est similaire à celle de la TAC et indiquent que l'optimisation d’analogues du TAC pourrait conduire à une nouvelle génération de composés plus efficaces pour le traitement de M. abscessus. Des études de relations structure/activité sont envisagées afin d’améliorer l’efficacité et les propriétés pharmacologiques des analogues du TAC. / Mycobacterium abscessus, a rapidly-growing mycobacterium (RGM), has emerged in recent years as an important opportunistic pathogen especially in cystic fibrosis (CF) patients. M. abscessus is naturally resistant to most commonly available antibiotics, which seriously limits the treatment options, emphasizing the urgent need for more efficient drugs and innovative therapeutic strategies to combat M. abscessus infections. The M. abscessus rough (R) low-glycopeptidolipids (GPL) producer is responsible for more severe clinical infections than the smooth (S) high-GPL producer, and is associated with increased virulence in zebrafish, including the formation of massive serpentine cords, abscesses, and rapid larval death. However, the molecular mechanisms responsible for the pathogenicity of the R strains remain elusive.Herein, we identified a novel gene, MAB_4780, encoding a dehydratase distinct from the HadABC complex, known to participate in mycolic acid biosynthesis. Both MAB_4780 and its homologue in Mycobacterium smegmatis, MSMEG_6754, are responsible for the innate resistance in these species to thiacetazone (TAC), a second-line antitubercular drug. The successful deletion of MABS_4780 in the R variant of M. abscessus resulted in an altered mycolic acid composition, a pronounced defect in cording, and an extremely attenuated phenotype in zebrafish embryos. The in vivo attenuation of the MAB_4780 mutant results from both the deficiency in cord formation and the impaired intracellular growth, presumably due to limited inhibition of the phagolysosomal fusion events. In addition, similarly to the MSMEG_6754 deletion mutant, the MAB_4780 mutant showed impaired growth in amoeba, which represents a possible reservoir for environmental mycobacteria. These results reflect the critical role of this new dehydratase in the survival of M. abscessus in its environmental hosts and its importance in establishing acute and lethal infections. Therefore, targeting MAB_4780 may represent a promising strategy to control M. abscessus infections. Future work will focus on identifying specific inhibitors targeting MAB_4780, which could greatly benefit from the combination of our dehydratase assay and our high-resolution crystal structure of the protein.We have also screened against M. abscessus a library of TAC analogues, previously validated for their inhibitory effects against the Mycobacterium tuberculosis HadABC dehydratase. Among these compounds, three exhibited a 50-fold increased potency as compared to TAC. Overexpression of EthA, known as the activator of TAC, increased the susceptibility of M. abscesuss to the three analogues, suggesting that that their mode of activation is similar to that of TAC. Overall, these data indicate that optimizing the TAC scaffold may lead to more efficient compounds against M. abscessus. Additional structure/activity relationship studies are required to further improve the efficacy and pharmacological properties of TAC analogues.

Mycolic acids are long chain lipids from the cell walls of Mycobacterium tuberculosis. The Nkuku phage display library was previously used to obtain monoclonal antibody binders to mycolic acids. In total 11 binders were obtained of which one was selected (MAC10) for further investigation by genetic engineering as presented in this dissertation. The antibodies of the Nkuku phage display library are in the format of single chain variable fragments (scFv). ScFv’s constitute only the epitope binding domains of an antibody consisting of the VH and VL domains fused into a single chain by a flexible linker protein. The selected anti-mycolic acid scFv is referred to as mycolic acid clone 10 (MAC10). Genes encoding the scFv’s of the Nkuku phage display library were cloned into the plasmid pHEN-1, a phage display vector. This vector is not commercially available or ideally suited for expression of scFv proteins. Therefore two vectors were investigated as possible targets for subcloning. The plasmids pGE20 and pAK400 were previously used for the expression of scFv antibody proteins. Subcloning into plasmid pAK400 proved to be the more efficient of the two investigated for subcloning. This subcloning yielded the recombinant plasmid pAKJS. Following the subcloning scFv protein expression was attempted using the plasmids pMAC10 (derived from pHEN-1) and pAKJS (derived from pAK400). Expression of MAC10 using plasmid pMAC10 in both Escherichia coli TG-1 and HB2151 was constitutive. This demonstrates that plasmid pHEN-1 is a non ideal vector as expression should not occur unless induced. Expression of MAC10 did not occur when pAKJS and Escherichia coli HB2151 were used. This was due to both the vector and expression host producing inhibitor protein for the Lac Z promoter controlling expression of the scFv. The MAC10 gene was subsequently randomized using the directed evolution method, error prone PCR. Sequence analysis of the five selected mutants indicated an average mutation rate of 8.6 mutations per 1000 base pairs. From the combined total of all five mutants, transversions made up the majority of substitutions. The majority of transversion mutations occurred at A-T base pairs. Transition substation mutations that made up the minority of total mutations occurred mostly at G-C base pairs. / Dissertation (MSc)--University of Pretoria, 2012. / Biochemistry / unrestricted