As enzimas da via do chiquimato são alvos atraentes para o desenvolvimento de agentes para tratar a tuberculose já que esta via é essencial para Mycobacterium tuberculosis e está ausente em humanos. A enzima chiquimato desidrogenase de M. tuberculosis (MtbSD), codificada pelo gene aroE, catalisa a quarta reação na via do chiquimato. Estudos de estrutura tridimensional, de perfis de pH e de mutagênese sítio-direcionada envolvendo muitas chiquimato desidrogenases sugerem a participação da Lisina-69 e do Aspartato-105 (numeração de acordo com a seqüência da MtbSD) na catálise e/ou na ligação ao substrato. A determinação das propriedades cinéticas de enzimas mutantes pode permitir importantes proposições acerca do papel destes resíduos para a enzima MtbSD. A mutagênese foi realizada usando uma técnica de amplificação por PCR para as seguintes proteínas mutantes: K69A, K69H, K69I, K69Q, D105A, D105N. Testes de diversas condições experimentais foram feitos para obter a expressão das proteínas MtbSD mutantes na fração solúvel. Além disso, empregamos um protocolo de purificação otimizado para obter as enzimas MtbSD não mutante e K69A aparentemente homogêneas. Realizamos ensaios cinéticos em estado estacionário e medidas espectrofluorimétricas destas duas enzimas, e os resultados indicam que o resíduo conservado Lisina-69 tem função catalítica e não está envolvido na ligação ao substrato para a MtbSD. Estudos estruturais, de mutagênese sítio-direcionada e de cinética enzimática podem trazer importantes contribuições para o desenho racional de novos e efetivos fármacos para tratar a tuberculose. / The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis but absent in humans. Mycobacterium tuberculosis aroE-encoded shikimate dehydrogenase (MtbSD) catalyzes the forth reaction in the shikimate pathway. Three-dimensional structure studies, pH-rate profiles and site-directed mutagenesis studies involving many shikimate dehydrogenases have suggested participation of Lysine-69 and Aspartate-105 (M. tuberculosis numbering) in catalysis and/or substrate binding. Importantly, investigation of the kinetic properties of mutant enzymes can bring important insights about the role of these residues for the MtbSD enzyme. Mutagenesis was performed using a PCR-amplification technique for the following mutant proteins: K69A, K69H, K69I, K69Q, D105A, D105N. Screening of experimental conditions has been performed to obtain the expression of the MtbSD mutant proteins in the soluble fraction. In addition, an improved purification protocol was used to obtain homogeneous wild-type MtbSD and K69A mutant enzymes. We have carried out steady-state kinetic assays and spectrofluorimetric measurements for the wild type and the K69A enzymes. Results indicate that the conserved Lysine-69 residue in MtbSD plays a catalytic role and is not involved in substrate binding. Enzyme kinetics, site-directed mutagenesis and structural studies provide a framework on which to base the rational design of new and effective agents to treat tuberculosis.

Para auxiliar no controle da tuberculose são necessários novos métodos de detecção de Mycobacterium tuberculosis que sejam rápidos, específicos e de aplicabilidade em laboratórios de saúde pública, principalmente em países em desenvolvimento. Vários métodos moleculares de detecção e identificação de micobactérias em amostras clínicas têm sido desenvolvidos. Com o objetivo de padronizar e aplicar essas metodologias em laboratório foi desenvolvido um protocolo baseado na amplificação de DNA por reação em cadeia da polimerase (PCR) e na detecção colorimétrica em microplacas. Uma região interna do elemento de inserção IS6110, específico do complexo M. tuberculosis, foi selecionada como alvo para amplificação por PCR com primers biotinilados. Uma sonda complementar a uma região interna do fragmento foi fixada em microplacas onde, posteriormente, foi hibridizado o produto amplificado. A detecção através de hibridização foi feita utilizando um conjugado estreptavidina peroxidase. A eficácia da técnica foi testada em amostras clínicas pulmonares de pacientes com suspeita de infecção e sem tratamento prévio para tuberculose. O padrão ouro no diagnóstico de infecção por M. tuberculosis foi a associação da baciloscopia com a cultura a partir da amostra clínica dos pacientes selecionados. Foram testadas 303 amostras de escarro induzido de 303 pacientes com suspeita de TB pulmonar, dos quais 69 apresentaram resultado positivo e 234 negativo para TB. A sensibilidade e especificidade obtidas foram 88% e 98% e os Valores Preditivos Positivo (VPP) e Negativo (VPN) foram 92% e 97%, respectivamente. Os resultados obtidos demonstraram que a técnica desenvolvida no presente estudo pode ser uma importante ferramenta no diagnóstico de tuberculose e poderia ser utilizada como um teste complementar no diagnóstico da doença. / New methods to detect Mycobacterium tuberculosis rapidly, accurately and that are feasible to apply in laboratories of developing countries are necessaries. Several methods for direct detection and identification of mycobacteria in clinical samples have been developed. With the aim to standardize the application of such methods in laboratory, we developed a molecular method for DNA extraction and PCR detection using a microwell plate hybridization assay. An internal region of the repetitive element, specific of M. tuberculosis complex, called IS6110 was selected as a target for amplification using specific biotinylated primers. The detection of amplified fragments was performed using microwell plates. An aminated DNA fragment complementary to an internal region of the amplified fragment was used as a probe. The biotinylated amplified products were added to the plate and identified with the use of streptavidin conjugated to horseradish peroxidase. The efficacy of the method was tested to detect pulmonary tuberculosis in patients suspected of TB infection with no previous treatment. As gold standard, the bacteriological criteria was used. Three hundred and three induced sputum samples from 303 pulmonary TB suspects were evaluated, of which 69 showed positive result and 224 negative for TB. The sensitivity and specificity obtained were 88% and 98% and Positive and Negative Predictive Values were 92% and 98%, respectively. The obtained results demonstrated that the PCR detection using a microwell plate hybridization assay may be an important tool for the diagnosis of tuberculosis and suggest that it could be used as a complementary diagnostic method.

Made available in DSpace on 2014-06-12T16:32:45Z (GMT). No. of bitstreams: 2 arquivo6462_1.pdf: 937219 bytes, checksum: 4f6f89209360eea1e4631d3d2a07c29d (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / Lippia gracillis Shauer, Verbenaceae, é um arbusto com pequenas folhas aromáticas, encontrado em regiões semi-áridas do Nordeste do Brasil. É conhecida popularmente como alecrim de tabuleiro e usada no tratamento de infecções da boca, garganta e pele. Algumas espécies do gênero contêm em sua composição química o timol e carvacrol, compostos majoritários de reconhecida atividade antimicrobiana. Esta pesquisa objetivou o estudo da atividade tuberculostatica do óleo essencial de Lippia gracillis Shauer frente a cepas selvagens de Mycobacterium tuberculosis, e uma cepa padrão de referência H37Rv. Estas cepas foram isoladas e identificadas como Mycobacterium tuberculosis, a partir de escarro de pacientes com sinais e sintomas de tuberculose pulmonar, que demonstraram diferentes perfis de sensibilidade aos tuberculostáticos clássicos, pirazinamida, isoniazida, etambutol rifampicina, etionamida e estreptomicina. A extração do óleo essencial das folhas da planta foi realizada por hidrodestilação com aparelho de Clevenger adaptado. A sensibilidade do Mycobacterium tuberculosis aos tuberculostáticos e ao óleo essencial de Lippia gracillis foi realizado pelo método das proporções teste indireto e determinação da concentração inibitória mínima. Das quinze cepas estudadas, treze mostraram-se sensíveis ao óleo essencial de Lippia gracillis Shauer. Embora resistentes a isoniazida as cepas de Mycobacterium tuberculosis LC01, LC 02 e LC07 mostraram-se sensíveis ao óleo essencial de Lippia gracillis. Apenas duas cepas LC04 e LC12, que tiveram perfil de resistência a pelo menos três drogas tuberculostáticas, demonstraram também resistência ao óleo essencial. O óleo essencial de Lippia gracillis apresentou atividade tuberculostática frente a cepas sensíveis aos tuberculostáticos clássicos, frente a cepas com resistência isoniazida, isoniazida e rifampicina, não demonstrou atividade frente a cepas com três ou mais resistência a diferentes classes de tuberculostáticos

Ph.D. / Background: AIDS is characterized by a number of opportunistic infections and the immune depletion caused by HIV infection is the strongest risk factor for both reactivation of tuberculosis (TB) and progression of Mycobacterium tuberculosis (Mtb) infection to disease. Numerous studies have shown that concurrent infection of the same host cell by HIV,and M.tb stimulates replication of both pathogens. The interaction between the two is lethal. A synergistic relationship exists between Mtb and HIV. While HIV spurs the spread of TB, mycobacterial infection results in acceleration of HIV disease progression. The requirement for iron as a crucial factor for cellular processes has long been demonstrated. Excess iron leads to infections with harmful consequences such as cell death and function impairment. During infection, iron is required by both the host cell and the pathogens. Iron chelation is believed to modulate some of these effects. Objectives: Mtb, HIV and Fe-overload are common in sub-Saharan Africa and iron plays a major role in determining the outcome of several infections. In view of this, we wanted to (1) investigate the effect of excess iron on host cell defences during co-infection with the mentioned microorganisms, (2) evaluate the differences in both host and pathogen responses during acute and chronic infection in the presence of iron overload and (3) Determine the efficacy of iron chelation (with DFO) as a means of counteracting conditions associated with iron overload. Hypotheses: The combination of Fe-overload and co-infection of host cells with HIV and Mtb in an in vitro model should stimulate replication of the pathogens, which would ultimately result in host cell stress manifesting as lower viability or cell death and impaired immune defence functions. Also the detrimental effects of excess iron on host cell viability could be counteracted through the use of iron chelators. Methods: We analyzed the in vitro effect of Mtb in bothchronically and acutely HI V-infected cells (PBMC's and monocytes), exposed to 500 uM FeSO 4 and/or DFO for 4 days. Host cell viability, survival and death were assessed through viability assays (MIT and Alamar Blue) and flow cytometric analyses of apoptosis/necrosis (using Annexin V and propidium iodide). Secretion of IL- 6 and TNF-a and production of total nitrate were monitored as host immune/defence responses using specialized ELISAs. HIV replication was investigated by looking at core protein (p24) contents and reverse transcriptase (RT) activity. Mtb replication and growth was monitored using the microplate Alamar Blue assay (MABA) and quantitative culturing.Results: Co-infection caused a reduction of host cell viability (± 20% and 45% inhibition during chronic and acute infection respectively;, as measured by MTT), increases in the numbers of viral particles (2.3 times and 20% increases for chronic and acute infections respectively) and stimulation of both bacterial viability (36%) and host defence responses (30% increase in TNF-ct secretion). Excess iron further decreased viability with a marked increase in necrosis of cells and was found to enhance pathogen replication and growth (26% for HIV and 47% for Mtb). Chelation of iron with DFO abrogated the enhanced replication of the pathogens with a marginal restoration of host viability. Conclusion: The results obtained demonstrate the deleterious effect of excess iron during concurrent infection with both pathogens as well as its stimulating/enhancing properties on pathogens. On the other hand, DFO inhibited pathogen replication and host viability.

Masters of Science / Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). TB mainly affects lungs of patients but other parts of the body can also be affected. It kills approximately 2 million people annually. HIV/AIDS and drug resistance make TB difficult to control. Mtb CarD protein forms a physiological complex with Ribonucleic Acid Polymerase (RNAP). This complex causes Mtb to undergo dormancy rendering it difficult to control using current antibiotics. CarD and a size-reduced subunit β1 (denoted β1m for “minimized”) of Thermus thermophilus RNAP, in which the central domain has been replaced by a Gly-Gly linker, were produced and purified using affinity nickel nitrilotriaceticacid and glutathione-Stransferase (GST) affinity chromatography techniques respectively. CarD N terminal domain (CarDN) was generated from CarD by inserting a stop codon by site directed mutagenesis. CarD was stabilised by adding 5 % (v/v) glycerol to PBS pH 7.4 ensuring protein stability of up to 67 days rather than 2 days without glycerol. CarDN was stable in PBS pH 7.4 without addition of glycerol. This suggests that the CarD C terminal domain may be responsible for CarD instability. To further purify the proteins both anion exchange and gel permeation chromatography techniques were used. CarD and CarDN degrade immediately after anion exchange potentially because of the high ion concentration which partially unfolds the protein making it prone to proteolytic cleavage. GST-pull down assays were used to demonstrate complex formation between RNAP β1m and both CarD and CarDN confirming that complex formation is dependent on the N-terminal domain of CarD.

Masters of Science / The three-dimensional structures of proteins are important in understanding their function and interaction with ligands and other proteins. In this work, the structures of two proteins, ImuA’ from mycobacterium tuberculosis and GH9 C1 cellulase from a metagenomic library, were analysed using structural biological and modelling techniques. The gene encoding ImuA’ was amplified by two-step PCR, cloned, and expressed in E. coli. The recombinant ImuA’ produced was found to be largely insoluble. The insoluble protein was successfully solubilized in 8M urea but refolding the protein to its native structure was unsuccessful. By homology modelling, a 3D model of ImuA’ was obtained from a partly homologous protein RecA. In comparison to RecA, ImuA’ appears to lack some loop amino acids critical for DNA binding. Hence ImuA’ is postulated to not bind DNA. The second protein, GH9 C1 cellulase, was produced in E. coli. The protein was purified by chromatographic techniques and crystallized in a precipitant to protein ratio of 1:2 by hanging and sitting drop crystallization methods. The reservoir solution was made up of 15-30% (w/v) PEG 3350, 200 mM salt and 100 mM Tris-HCL pH 7.5-8.5. The protein crystals only diffracted x-rays to 4 å resolution which could not be used to obtain a crystal structure of the protein. The diffraction data, however, showed the crystal to be monoclinic with space group P2. Homology modelling revealed GH9 C1 cellulase to be a two domain protein with a smaller N-terminal Ig-like domain and a larger catalytic domain.The catalytic domain retains two ca2+ binding sites, which potentially stabilize the active site conformation and increase thermostability of the protein. Overall GH9 C1 cellulase is structurally similar to other GH9 cellulases, suggesting that its catalytic mechanism may be conserved.

The serious threat of tuberculosis, especially XDR-TB, is a reality in Southern Africa particularly in individuals with HIV/AIDS. Therefore the importance of development of new or improved anti-TB treatment must now be emphasized more than ever. In this study, a model was created to target isoniazid (toxophore) specifically to a cholesterol rich environment where mycobacteria reside in macrophages, by making use of a sterol binding drug, Amphotericin B (haptophore). Isoniazid was covalently linked to Amphotericin B via a Schiff base to a linker molecule, terephthalaldehyde. Although this molecule showed a loss of biological activity, a discovery was made by serendipity that could have great impact in understanding how Mycobacterium tuberculosis enters and survives in the host macrophage. During the testing of the compound, it was discovered that Amphotericin B bound to mycolic acids at least as well as it binds to cholesterol, its natural ligand. This could provide proof of the structural similarity between mycolic acids and cholesterol but many more controls need to be investigated. As cholesterol was previously shown in literature to be critical for entry and survival of Mycobacterium tuberculosis in macrophages, the indication of a structural mimicry between the cell wall mycolic acids and cholesterol and the attraction of these two chemical entities to one another seems to be highly relevant. This characteristic can now be further explored to improve the understanding of the process of entry and survival of Mycobacterium tuberculosis in the macrophage host. Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Benadie, Y 2006, Amphotericin B as a mycolic acid specific targeting agent in tuberculosis, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-04212008-151642 / > / Dissertation (MSc (Biochemistry))--University of Pretoria, 2008. / Biochemistry / unrestricted

Literature highlights the effects of mycolic acid (MA) fine-structure on biological activity, pathogenicity, virulence and cell wall structure and permeability. Knowledge on MA-structure and how their conformations are dependent on their precise molecular composition becomes essential in exploiting these properties in drug-design and in advancing our understanding of the disease. In our group evidence for a structural or functional relationship between cholesterol and MAs have been discovered. The aim of the experimental part of this work was to study this relationship further by attempting to quantify the interaction between cholesterol and MAs in liposomes on an evanescent field biosensor. The binding profiles that were obtained could not be evaluated with kinetic software and the interaction between cholesterol and MAs was not linearly dependant on the concentration of cholesterol. However, novel insight into the interaction was gained when it was observed that cholesterol only accumulated on MA liposomes when cholesterol liposomes containing concentrations of cholesterol resulting in a suspected liquid ordered phase, were used. This is significant since it implies that cholesterol in membrane rafts of the host cell that exist in a liquid ordered phase would be able to interact with MAs under physiological conditions. The theoretical part of this work represents the first molecular modeling study in which MAs are allowed to fold with no conformational restrictions. It is proposed that MAs fold as a function of their functional groups, stereochemistry, and various chain lengths. It was also investigated whether methylation of the acid group changes conformational preferences. The effect of chain length on cyclopropane structure and the viability of systematic conformational searching in MAs were shown using quantum mechanics. Replicate molecular dynamics simulations were done for 4 ns in vacuo on alpha-; methoxy-; methoxy methyl ester- and keto-MAs. MAs had an open starting conformation without conformational restrictions. Results were analysed using eight distances characteristic of the conformational fold. Using these distances, W-, U- and Z-shaped folds were identified. Principal component analysis (PCA) and self-organising maps (SOMs) were used to evaluate differences and trends in MA-conformations. Quantum chemical results showed that chain length did not affect cyclopropane structure and that the systematic plotting of potential energy surfaces is an effective tool to analyse effects of changes in geometry on the energy of the molecule and to predict favoured conformations. Remarkably, single MAs assumed W-, U- and Z-folds in vacuo during molecular dynamics simulations that have previously been observed in monolayers. PCA and SOM plots showed that keto-MA folded faster than other MAs. Alpha-MA showed the highest frequency of W-, U- and Z-folds. Methoxy-MA did not readily fold at its cis-cyclopropane group. Methylation of the acid group of methoxy-MA did not show remarkable differences in the conformations assumed, but almost doubled the frequency of WUZ-structures obtained as compared to non-esterified methoxy-MA. The inherent structural differences between MA-subclasses clearly affect the trends in structural folds that they assume. Molecular modeling of MAs proved to be a versatile tool for resolving structure-function relationships at the molecular level. Copyright 2008, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Prinsloo, W 2008, Computational models for conformations of cell wall mycolates from Mycobacterium tuberculosis, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-06122009-114802 / > E1400/gm / Dissertation (MSc)--University of Pretoria, 2009. / Biochemistry / unrestricted

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 a collective name for the bacterial infection, which is caused by members of the Mycobacterium tuberculosis (M. tb) complex and can infect the lungs (pulmonary) as well as the kidneys, lymph nodes, bones and joints (extra-pulmonary). The re-emergence of drug-resistant strains and the HIV epidemic are among the main reasons for the resurgence of TB and there is a need for new drugs and diagnostic assays which are rapid and sensitive. Serodiagnostic assays have the potential of being rapid, inexpensive and relatively non-invasive. The most abundant antigen in the cell wall of M. tb, which has been analysed with ELISA and resonant mirror biosensor assays for use in serodiagnosis, is mycolic acid (MA). The sensitivity previously obtained in the ELISA assay was however inadequate for serodiagnostic purposes. It was believed that MA mimicked the structure of cholesterol, thereby causing anti-cholesterol human antibodies from TB negative sera to bind to MA and result in a large number of false positives. Within this work the apparent molecular mimicry between MA and cholesterol was investigated using a competitive enzyme linked inhibition assay (CELIA) assay. The results suggested that MA in liposomes resembled the liquid ordered arrangement of cholesterol in liposomes, rather than a direct mimicry of individual molecules. The nature of the antibody from TB negative patient sera binding to MA coated onto ELISA plates was also investigated. The results obtained from this study have not disproved the hypothesis of a cross-reactive anti-cholesterol antibody, but it would appear that the MA signal from TB negative serum was partially due to the binding of anti-MA antibodies. The presence of anti-MA antibodies in TB negative serum could have been the result of prior BCG vaccination, latent infection or due to constant immune stimulation from saprophytic mycobacteria. This creates the potential of using antibodies to MA to distinguish between latent TB infection and active disease. Furthermore, in order to overcome the low sensitivity of the ELISA assay due to high background signals from TB negative serum, members of our group previously developed a resonant mirror biosensor inhibition assay based on MA contained in liposomes. The biosensor measured mass accumulation and the identity of the binding molecules were unknown. It was shown here that one of the serum components binding to the immobilised MA liposomes in the biosensor inhibition assay was immunoglobulin G antibodies. The specificity of both the ELISA and biosensor assays previously analysed using a natural mixture of MA however, remained poor, and in the search for a more specific antigen, this study investigated the potential of MA subclasses for TB serodiagnosis using ELISA. It was observed that the antibody binding signal to the MA subclasses depended on the polarity of the coating solution, for which hexane was the preferred solvent. Both the alpha- and keto-MA subclasses could better distinguish between a range of TB positive patient and TB negative sera compared with the natural mixture of MA. These results suggested that a particular subclass applied in the biosensor inhibition assay could enhance the test to reach the required sensitivity and specificity required for the serodiagnosis TB. / Dissertation (MSc)--University of Pretoria, 2011. / Biochemistry / unrestricted