Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-01T18:14:31Z No. of bitstreams: 1 Melissa Moura Abbehusen Estudo da imunogenicidade de vacinas de....pdf: 867164 bytes, checksum: 4ef801f12d349f64700eea900fccfc9b (MD5) / Made available in DSpace on 2012-08-01T18:14:31Z (GMT). No. of bitstreams: 1 Melissa Moura Abbehusen Estudo da imunogenicidade de vacinas de....pdf: 867164 bytes, checksum: 4ef801f12d349f64700eea900fccfc9b (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A Proteína ácida ribossomal de Leishmania infantum (L. infantum) - LiP0 é um componente estrutural da subunidade maior do ribossomo e já foi descrita como um antígeno imunodominante, que participa na síntese de outras proteínas e é capaz de induzir resposta imune humoral específica em soro de pacientes e cães infectados com Leishmania chagasi (L. chagasi). Mycobacterium smegmatis (M. smegmatis), é uma micobactéria oportunista, que apresenta crescimento rápido e uma poderosa capacidade adjuvante, já sendo utilizado como vetor de expressão para diversos antígenos. Neste trabalho avaliamos a capacidade imunoprotetora do Mycobacterium smegmatis recombinante (rM.smegmatis) expressando o gene que codifica a LiP0, bem como o plasmídeo e/ou proteína LiP0 utilizando estratégia homóloga (composta de plasmídeo de DNA) e heteróloga (composta de plasmídeo de DNA adicionado a proteína recombinante e CpG), contra a infecção causada por L. chagasi em hamsters. Hamsters foram imunizados e posteriormente infectados com L. chagasi por via endovenosa. Os animais imunizados com a micobactéria, apesar de apresentarem resposta imune humoral anti-M. smegmatis, não produziram anticorpo contra LiP0, que só foram detectados no soro dos animais que receberam a estratégia heteróloga de imunização. Na análise de citocinas, observou-se que os animais imunizados com rM.smegmatis LiP0 apresentaram maior concentração de IFN-γ e menor quantidade de TGF-ß e IL-10 quando comparado aos grupos controle, sugerindo uma resposta Th1. Em diferentes momentos após o desafio, o grau de proteção avaliado pela carga parasitária em órgãos alvo, foi estimado por ensaio de diluição limitante. Nenhuma diferença foi observada na carga parasitaria do baço, fígado e linfonodo em hamsters imunizados ou controles em todos os pontos da avaliação, sugerindo que a LiP0, não protegeu hamsters imunizados tanto com o Mycobacterium expressando o gene que codifica a proteína, nem como vacina de DNA utilizando a estratégia homóloga ou heteróloga contra infecção por L.chagasi. / The acid ribosomal protein of Leishmania infantum (L. infantum) - LiP0 is a structural component of the ribosomal subunit and it was described as an immunodominant antigen recognized either by serum of patients and dogs infected by Leishmania chagasi (L. chagasi) and cooperates in the synthesis of other proteins. Mycobacterium smegmatis (M. smegmatis) is a opportunistic bacteria, which presents rapid growth, is a potent adjuvant and has been used as carrier of antigens in several different experimental models of immunoprotection. In this work we evaluate the immunoprotective capacity of recombinant M. smegmatis (rM. smegmatis) carrying the gene of LiP0 and the DNA or protein of LiP0 using homologous strategy (composed of plasmid DNA) and heterologous (consisting of plasmid DNA and recombinant protein more CpG) to immunize hamsters against infection by L. chagasi. The immunized animals produced anti-M.smegmatis antibodies but they did not produce antibody against LiP0, detected only in animals who received the heterologous strategy of vaccination. In the analysis of cytokines, we observed that animals immunized with rM.smegmatis LiP0 had higher concentration of IFN-γ and lower amounts of TGF-ß and IL-10 compared to control groups, suggesting a Th1 response. At different times after challenge, the degree of protection, evaluated by parasite load in the target organs, was estimated by limiting dilution assay. No difference was observed in the parasite load in the spleen, liver and lymph node between immunized hamsters and controls at all points of evaluation. There was no protection in animals immunized with rM. smegmatis expressing the acidic ribosomal protein gene, suggesting that LiP0 did not protect hamsters immunized with either Mycobacterium expressing the gene encoding the protein or DNA as a vaccine strategy using homologous or heterologous against infection L.chagasi.

One third of the world's population is infected with Mycobacterium tuberculosis, the bacterium that causes TB. During an infection, bacteria often survive host immune system attacks, which include oxidative stress conditions for bacteria growing inside macrophages. This makes treatment difficult and time-consuming. We hypothesize bacteria can adapt to environmental conditions by changing their mRNA maturation and degradation profiles. Using a model system, Mycobacteruim smegmatis, we focus on how mRNA expression is affected by oxidative stress. After construction and sequencing of RNA expression libraries, preliminary analysis showed that after three hours of H2O2 exposure most upregulated genes were related to DNA repair, while downregulated genes included transport proteins. After six hours of exposure, upregulated genes were similar to three hours and downregulated genes included tRNAs. 5' end mapping libraries were also constructed to access differential cleavage site abundance under oxidative stress conditions. We also investigated the roles RNase J may have in stress response and mRNA processing in Mycobacteria. RNase J and RNase E are thought to be the major RNases in bacteria. While most bacteria only have one of them, mycobacteria encode both in their genome, with RNase J being non-essential. We constructed a set of 4 strains (WT, RNase J overexpression, RNase J deletion, and complemented RNase J deletion) and tested their drug resistance and stress tolerance. Results suggests that RNase J deletion and overexpression alter drug sensitivity. Stress tolerance assays showed that WT is more tolerant to oxidative stress, followed by RNase J deletion strain and overexpression and complemented RNase J deletion strains, with the last two showing no growth when cultured with H2O2. Analysis of the expression profile of these strains was performed to help understand if gene expression differences are responsible for the phenotypes observed. For the complemented RNase J deletion, one operon had almost all its genes upregulated. This operon encodes a hydrogenase (Hyd3), suggesting that redox balance in the strain is perturbed.

Techn. Universiẗat, Diss., 1999--Berlin.

Tuberculosis is the leading cause of death by single infectious disease worldwide; novel antibiotics are needed to continue to treat this disease. To goal of this project is to provide proof-of-principle support for the idea that targeting protein-protein interactions (PPI) is an appropriate course for the discovery of new drugs. This study optimized the M-PFC assay, which allows detection of PPI in Mycobacteria, through the use of stronger promoters and inducible expression of a peptide blocker by riboswitch. To accomplish this, promoter induction studies were used to find stronger promoters for the M-PFC, optimization of the riboswitch as a method for inducible protein expression within this system, and the addition of both elements to the existing version of the M-PFC. This M-PFC targets DosR homodimerization; this process is known to be essential for survival within the host. This study optimizes a system that may be used to screen for drugs that are capable of interrupting this interaction.

The continuing emergence of multi-drug resistant Mycobacterium tuberculosis is threatening the ability to treat tuberculosis (TB) worldwide. The development of new anti-TB drugs requires new approaches and new drug targets. In this study, a mariner-based transposon, TnQuoVadis, was constructed to identify essential genes as potential drug targets. This transposon has an outward-facing anhydrotetracycline (ATc)-inducible promoter at each end. A mutant with TnQuoVadis inserted upstream of an essential gene may display normal growth in the presence of ATc, but exhibit no growth or severely diminished growth in the absence of ATc. TnQuoVadis was placed onto a vector with a temperature sensitive replication origin for more efficient mutagenesis of mycobacteria. In a preliminary genetic screen using the model organism Mycobacterium smegmatis, 13 mutants with ATc-dependent growth were identified. Identification of the insertion sites by cloning and sequencing indicated that there were nine genetic loci containing transposon insertions upstream of essential gene candidates in M. smegmatis. Further analysis of these genes indicated that many were previously known essential in both M. smegmatis and M. tuberculosis. These results demonstrate that TnQuoVadis and its delivery system can be utilized for the identification of essential genes in mycobacteria / Master of Science

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

Oxidative stress is a universal phenomenon experienced by both aerobic and anaerobic organisms. Reactive oxygen species (ROS) are generated during the stress, which can damage most cellular components including proteins, lipids and DNA. Naturally, organisms have evolved defence mechanisms to prevent oxidative damage. In prokaryotic systems, Dps (DNA binding protein from stationary phase cells) forms an important component of the mechanisms. Dps is known to be produced maximally during the stationary phase of bacterial growth. They exhibit ferroxidase activity as well. Dps homologs have been identified in a variety of distantly related bacteria, thus implying that this protein has a crucial function. The crystal structures of these proteins from a few bacteria are available. The work reported here is concerned with structural studies on Dps molecules from Mycobacterium smegmatis. Well-established X-ray crystallographic techniques were used to study the structures reported here. Hanging drop vapour diffusion and microbatch methods were used for crystallization. X-ray intensity data were collected on MAR Research imaging plates mounted on Rigaku X-ray generators. The data were processed using the HKL program suite. All the structures were solved by the molecular replacement method using the programs AMoRe and PHASER. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using FRODO and COOT. PROCHECK, ALIGN, INSIGHT, NACCESS, HBPLUS, CONTACT and ESCET were used for validation and analysis of the refined structures. Figures were prepared using MOLSCRIPT, BOBSCRIPT, RASTER3D and PYMOL. The structure of the first Dps identified in M. smegmatis has been determined in three crystal forms and has been compared with those of similar proteins from other sources. The dodecameric molecule can be described as a distorted icosahedron. The interfaces among subunits are such that the dodecameric molecule appears to have been made up of stable trimers. The situation is similar in the proteins from Escherichia coli and Agrobacterium tumefaciens, which are closer to the M. smegmatis protein in sequence and structure than those from other sources, which appear to form a dimer first. Trimerisation is aided in the three proteins by the additional N-terminal stretches they possess. The M. smegmatis protein has an additional C-terminal stretch compared to other related proteins. The stretch, known to be involved in DNA binding, is situated on the surface of the molecule. A comparison of the available structures permits a delineation of the rigid and flexible regions in the molecule. The subunit interfaces around the molecular dyads, where the ferroxidation centres are located, are relatively rigid. Regions in the vicinity of the acidic holes centred around molecular threefold axes, are relatively flexible. So are the DNA binding regions. The crystal structures of the protein from M. smegmatis confirm that DNA molecules can occupy spaces within the crystal without disturbing the arrangement of the protein molecules. However, contrary to earlier suggestions, the spaces need not to be between layers of the protein molecules. The cubic form provides an arrangement in which grooves, which could hold DNA molecules, criss-cross the crystal. M. smegmatis Dps is characterised by a 26 residue C-terminal tail which has been shown to be involved in DNA binding. The protein spontaneously degrades into a species in which 16 C-terminal residues are cleaved away. This species does not bind DNA, but forms dodecamers. A second species in which all the 26 residues constituting the tail were deleted not only does not bind to DNA, but also fails to assemble into dodecamers, indicating a role in assembly also for the C terminal tail. Therefore, the crystal structure of the species without the entire C-terminal tail was carried out. The molecule of the C-terminal mutant has an unusual open decameric structure, resulting from the removal of two adjacent subunits from the original dodecameric structure of the native form. It has been earlier shown that a Dps dodecamer could assemble with a dimer or one of two trimers (Trimer-A and Trimer-B) as intermediate and that Trimer-A is the intermediate species in the M. smegmatis protein. Estimation of surface area buried on trimerisation indicates that association within Trimer-B is weak. It further weakens when the C-terminal tail is removed, leading to the disruption of the dodecameric structure. Thus, the C-terminal tail has a dual role, one in DNA binding and the other in the assembly of the dodecamer. M. smegmatis Dps also has a short N-terminal tail of 9 residues. A species with this tail deleted, forms trimers in solution, but not dodecamers unlike wild type M. smegmatis Dps, under the same conditions. The crystal structure of this N-terminal mutant was also determined. Unlike in solution, the N-terminal mutant forms dodecamers in the crystal. In native Dps, the N-terminal stretch of one subunit and the C-terminal stretch of a neighbouring subunit lock each other into ordered positions. The deletion of one stretch results in the disorder of the other. This disorder appears to result in the formation of a trimeric species of the N-terminal deletion mutant contrary to the indication provided by the native structure. The ferroxidation site is intact in the mutants. A second DNA binding protein from stationary phase cells of M. smegmatis (MsDps2) has been identified from the bacterial genome and its crystal structure determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps-DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. 50 bacteria contain 2 or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis In addition to the work on Dps molecules, the author was also involved in studies on the crystal structures of the adipic acid complexes of L- and DL-arginine and supramolecular association in arginine-dicarboxylic acid complexes. This investigation, carried out primarily to obtain a good grounding in crystallography, is presented in an appendix.

The following work presents an evaluation of the antimycobacterial activity (against Mycobacterium smegmatis) of the extracts, fractions and some phenolic compounds present in the leaves of Ficus benjamina L. and Ficus luschnathiana (Miq.) Miq., along with an estimation of the total phenolic content and the quantification of some of these compounds by High Performance Liquid Chromatography (HPLC). The phenolic estimation (Folin-Ciocalteu method) revealed that the crude extracts and the high polarity fractions from these extracts (ethyl acetate and n-butanol) are rich in polyphenols, although, its amount are not directly related to the antimycobacterial activity. The evaluation of this biological activity was performed by broth microdilution method, furnishing the minimum inhibitory concentration (MIC) of the extracts, fractions and tested compounds. The better biological activity was verified with butanolic fraction from F. luschnathiana (MIC = 156,25 μg/mL) and ethyl acetate fraction from F. benjamina (MIC = 312,50 μg/mL). However, the correlation of these good results with specific compounds was not possible, since technical difficulties prevented the isolation of substances from these fractions; and concerning the screened polyphenols only quercetin was encountered in butanolic fraction. In addition, quercetin exerted weak antimycobacterial activity against M. smegmatis (MIC = 625,00 μg/mL), much weaker than that observed for the fraction. The other investigated standards that were encountered in the extracts and fractions (caffeic and chlorogenic acids, rutin and kaempferol), also exhibited weak inhibitory effect. Thus, the good antimycobacterial activity observed for the fractions above mentioned are probably not related to these phenolics, still it may be related to compounds of the same nature. Many polyphenols, such as flavonoids, have been reported as mycobacterial growth inhibitors, thus it is also possible to conclude that the structural characteristics of quercetin, rutin and kaempferol do not support the biological activity studied. / A seguinte dissertação apresenta uma avaliação da atividade antimicobacteriana (frente à Mycobacterium smegmatis) de extratos, frações e substâncias fenólicas presentes nas folhas de Ficus benjamina L. e Ficus luschnathiana (Miq.) Miq., juntamente com uma estimativa do teor de polifenóis totais e a quantificação de alguns destes compostos por Cromatografia Líquida de Alta Eficiência (CLAE). O doseamento de polifenóis (método de Folin-Ciocalteu) revelou que os extratos brutos e as frações mais polares destes extratos (acetato de etila e n-butanol) são bastante providos de substâncias fenólicas, ainda que este teor não esteja diretamente relacionado à atividade antimicobacteriana. A avaliação desta atividade biológica foi realizada por método de microdiluição em caldo, que fornece a concentração inibitória mínima (CIM) dos extratos, frações e substâncias testadas. Uma boa atividade inibitória foi constatada para a fração butanólica de F. luschnathiana (MIC = 156,25 μg/mL) e para a fração acetato de etila de F. benjamina (MIC = 312,50 μg/mL). Entretanto, não foi possível correlacionar estes bons resultados a compostos em específico, visto que por dificuldades técnicas não foi realizado o isolamento de substâncias destas frações; e dos polifenóis pesquisados, somente a quercetina foi encontrada na fração butanólica. Esta por sua vez apresentou fraca atividade inibitória sobre M. smegmatis (MIC = 625,00 μg/mL), inferior a própria fração. Outros padrões investigados e que foram encontrados nos extratos e frações (ácidos cafeico e clorogênico, rutina e canferol), também apresentaram fraca atividade antimicobacteriana. Desta forma, a boa atividade inibitória das frações citadas acima possivelmente não se deve a estes polifenóis, ainda que possa estar relacionada a substâncias de natureza semelhante. Diversos polifenóis, como os flavonóides, têm sido reportados como inibidores do crescimento de micobactérias, de forma que também se pode concluir que as características estruturais da quercetina, rutina e canferol não favoreçam esta atividade biológica aqui apresentada.

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The current anti-TB drugs have had success in decreasing the number of deaths caused by TB, however, this success is limited by the emergence of drug resistant TB strains. Therefore, a novel TB therapy that limits the development of resistance has become necessary in an attempt to effectively control TB. The anti-TB drugs directly target mycobacterial enzymes, and potentiate the development of this resistance, and have therefore provided the rationale for this study. The aim was therefore to identify host macrophage genes that affect M. tb intracellular survival. The proposed alternative anti-TB therapy potentially involves the application of RNA interference (RNAi) and RNA activation (RNAa) biological processes that will target host genes, thereby inducing an indirect bactericidal effect. We hypothesized that macrophage genes that are differentially expressed by pathogenic and non-pathogenic mycobacterial species may be important in the regulation of M. tb intracellular survival. The lipid-rich mycobacterial cell wall is implicated in the excessive clumping of the mycobacterial cells in liquid culture. In order to minimize this, Tween 80 detergent was supplemented (mycobacteriaT). However, due to substantial evidence emphasising the detrimental effects of Tween 80 on the mycobacterial cell wall, mycobacteria were also cultured without Tween 80 (mycobacteriaNT), in order to investigate if the perturbed mycobacterial cell wall induced by Tween 80 affects the transcriptional response of macrophages. We endeavoured to develop a new method to culture mycobacteria without Tween 80 that will still generate single cells. We further hypothesized that the macrophage gene expression profile induced by mycobateriaNT differs from the response induced by mycobacteriaT. Differentiated THP-1 (dTHP-1) cells were infected with pathogenic and non-pathogenic mycobacteria (for 3 h, 24 h and 48 h with M. tb and M. bovis BCG, and 3 h and 8 h with M. smegmatis) cultured in the presence or absence of Tween 80. The expression of 12 macrophage genes, selected based on their involvement in the phagocytic pathway and autophagy, as well as their general involvement in the immune response, was determined by qRT-PCR and further analysed on the REST programme. The expression of each target gene was normalised relative to the expression of the reference gene (Beta actin). We observed that out of the 12 genes, TLR7 and VAMP7 were consistently downregulated in dTHP-1 cells infected with M. tbNT and upregulated in dTHP-1 cells infected with M. smegmatisNT. Their response to M. bovis BCG was inconsistent and not significantly different, and therefore could not be interpreted. Furthermore, CCL1 was upregulated by all the mycobacterial species. However, its expression was more pronounced in response to mycobacteriaNT, when compared to mycobacteriaT. Differential gene expression of TLR7 and VAMP7 in response to pathogenic and non-pathogenic mycobacteriaNT suggests that these 2 genes may be potential targets for RNAa-based anti-TB therapy, even though we could not conclude whether their response was specific to macrophages. In addition, the observed difference in the expression of CCL1 induced by mycobacteriaNT, compared to mycobacteriaT suggests that the perturbation caused by Tween 80 on the mycobacterial cell wall most likely affected the response of macrophages to infection with mycobacteria. Furthermore, this study has demonstrated a feasible method by filtration to generate single cells from mycobacteriaNT, which should be considered for future mycobacterial infection studies. / AFRIKAANSE OPSOMMING: Die huidige anti-tuberkulose middels se sukses lê daarin dat dit die aantal sterftes verminder maar hierdie sukses word weer beperk met die ontstaan van middel-weerstandige M.tb stamme. Daarom is nuwe middels nodig wat die ontwikkeling van middel-weerstandigheid beperk in ʼn poging om effektiewe TB behandeling te bewerkstellig. Anti-tuberkulose middels teiken hoofsaaklik mycobakteriële ensiemsisteme en ontlok sodoende weerstandigheid in M.tb stamme en dit vorm die rasionale vir hierdie studie. Die doel was om gasheer makrofaag gene te identifiseer wat M.tb oorlewing intrasellulêr bewerkstellig. Die voorgestelde alternatiewe anti-TB behandeling sal dan behels die toepassing van RNA intervensie (RNAi) en RNA aktivering (RNAa) tegnologie wat gasheer selgene teiken (inaktiveer) en sodoende ʼn bakterisidiese respons induseer. Die kanse is skraal dat mycobakterieë weerstandigheid sal kan ontwikkel onder hierdie omstandighede. Ons hipotetiseer dus dat makrofaag gene wat differensieel uitgedruk word deur patogeniese en nie-patologiese mycobakteriële spesies belangrik mag wees vir die oorlewing van M.tb intrasellulêr. Die lipiedryke selwand van mycobakterieë word geïmpliseer in die oormatige sameklomping van die bakterieë in vloeistofkulture. Om hierdie effek te minimaliseer word Tween 80 normaalweg tot die medium gevoeg (mycobakterieëT). Maar weens genoegsame bewyse dat Tween-80 die selwand van bakterieë nadelig beïnvloed, is mycobakterieë ook in die afwesigheid van Tween 80 gekultureer (mycobakterieëNT) om te bepaal of die nadelige effek van Tween 80 op die selwand die transkripsionele respons in makrofage beïnvloed post-infeksie. Dit was daarom ook ons doelstelling om ʼn nuwe tegniek te ontwikkel om mycobakterieë te kultureer in die afwesigheid van Tween 80 wat ook enkelselle sal genereer vir beter gekontroleerde makrofaag infeksie. Ons hipotetiseer ook verder dat makrofaag geenuitdrukking-profiele verskil afhangende of infeksie gedoen is met mycobakterieë wat in die afwesigheid of teenwoordigheid van Tween 80 gekultureer is. Gedifferensieerde THP-1 (dTHP-1) was geïnfekteer met patogeniese en nie-patogeniese mycobakterieë (vir 3 h, 24 h en 48 h met M.tb en M.bovis BCG, en 3 h en 8 h met M.smegmatis) gekultureer in die teenwoordigheid en afwesigheid van Tween 80. Die uitdrukking van 12 makrofaag gene, geselekteer op grond van hul betrokkenheid in die fagositose meganisme en in outofagie asook hul betrokkenheid in die immuunrespons, is gekwantifiseer met qRT-PCR en daaropvolgens geanaliseer met die REST-program. Die uitdrukking van elke geen is genormaliseer relatief tot die uitdrukking van die verwysingsgeen (Beta actin). Daar is bevind dat van die 12 gene, TLR7 en VAMP7 deurlopend afgereguleer was in dTHP-1 selle geïnfekteer met M.tbNT en opgereguleer was in dTHP selle geïnfekteer met M.smegmatisNT. Selrespons met M.bovis BCG was onbeduidend en derhalwe kon geen gevolgtrekking hier gemaak word nie. Ook, CCL1 was opgereguleer met infeksie deur enige van die mycobakteriële spesies, maar CCL1 se uitdrukking was groter in respons tot mycobakterieëNT wanneer vergelyk word met respons tot mycobakterieëT. Differensiële geenuitdrukking van TLR7 en VAMP7 in respons tot patogeniese en nie-patogeniese mycobakterieëNT impliseer dat hierdie twee gene potensiële teikens kan wees vir RNAa-gebaseerde anti-TB behandeling, alhoewel ons nie kon beslis of hierdie respons spesifiek vir makrofage was nie. Ook, die verskille waargeneem in die uitdrukking van CCL1 geïnduseer deur mycobakterieëNT, vergeleke met mycobakterieëT, impliseer dat die steuring in die selwand veroorsaak deur Tween 80, heelwaarskynlik die respons van die makrofaag beïnvloed het. Hierdie studie beskryf ook ʼn filtrasiemetode om enkele mycobakteriële selle te genereer wat oorweeg moet word by toekomstige mycobakteriële infeksiestudies.

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