Mycobacterium tuberculosis (M. tuberculosis) , the etiological agent of tuberculosis, is an intracellular bacterium which persists within macrophages. Successful control of tuberculosis depends on T-cell-mediated immunity. Immune protection involves the development of a Th1 response characterised by the secretion of cytokines such as IL-12, IFN-γ and TNF-α. The progression towards disease in humans and mice is often associated with a Th2 response characterised by the secretion of cytokines such as I L-4 and I L-10. Mycolic acids, the major cell wall lipid of M. tuberculosis, were previously shown to have a marginally protective effect on the development of disease in Balb/c mice when administered intravenously at an optimal dose of 25 µg one week before intravenous M. tuberculosis infection. Here it is shown that the protective effect is highly significant when infection is done intranasally. The protective effect of 25 µg mycolic acids against tuberculosis could not be explained by induction of a longer lasting Th1 response in Balb/c mice. This was determined by using semi-quantitative RT-PCR on the mRNA of cytokines characteristic of the different immune responses. It was observed that maximum sensitivity was obtained at the lowest possible PCR cycle and template concentrations for the samples. Mycolic acids were the first non-protein antigens shown to induce an immune response after presentation on CD1 membrane proteins. Balb/c mice predominantly generate a Th1 response during the first 3 - 4 weeks of M. tuberculosis infection, whereas they generate a Th2 response in the following weeks. Even though the protective effect of 25 µg mycolic acids could not be associated with a prolonged Th1 immune response in infected mice, it did induce IL-12 and IL-10 mRNA in uninfected mice. These cytokines are primarily. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2005. / Biochemistry / unrestricted

Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) and poses a global threat to human health. A third of the world’s population is infected with Mtb. Multi-drug resistant and extensively drug resistant Mtb strains are widespread and development of new drugs is urgently needed to treat drug resistant TB. This thesis focuses on the Mtb cytochrome P450 (P450) enzymes CYP126A1 and CYP143A1. P450s are heme-binding enzymes that catalyse activation of molecular oxygen and the oxidation of substrates bound close to the heme. CYP126A1 and CYP143A1 are “orphans” in terms of their functional characterization, but potential drug targets in view of ability of azole-based P450 inhibitors to inhibit growth and viability of Mtb. The CYP126A1 and CYP143A1 genes were cloned and expressed in Escherichia coli. Expression conditions and strains were optimised to maximise soluble protein production and methods were developed to purify the P450s using affinity, ion exchange and size exclusion chromatography. Both P450s were shown to bind heme b, and heme was shown to be axially coordinated by a cysteine thiolate and a water molecule in both cases using UV-visible and electron paramagnetic resonance (EPR) spectroscopy. Both P450s bound carbon monoxide (CO) in their reduced forms to produce heme Fe2+-CO complexes with absorption maxima at ~450 nm – characteristic of P450s. CYP126A1 and CYP143A1 bound avidly to a range of inhibitors, including several azole drugs. As examples, binding constant (Kd) values of 13.8 µM and 21.9 µM were determined for clotrimazole and econazole with CYP143A1; while ketoconazole bound CYP126A1 with a Kd of 0.20 µM. Each of these drugs is very effective in inhibiting Mtb growth. EPR confirmed inhibitory coordination of both P450s by azole drug nitrogen atoms; though indirect coordination via a retained axial water ligand may also occur in some cases. Extinction coefficients were determined as έ420 = 125 mM-1 cm-1 (CYP126A1) and έ415 = 105 mM-1 cm-1 (CYP143A1). CYP126A1’s heme iron redox potential was shown to be unusually positive (E°’ = -80 mV). Light scattering studies showed CYP126A1 to be a monodisperse, monomeric protein. CYP143A1 is also mainly a monomer, but with a small proportion of an oligomeric form. Despite its polydispersity, CYP143A1 was crystallized and its structure solved by X-ray diffraction to a resolution of 1.9 Å, using molecular replacement with the Mtb P450 CYP142A1. A limited compound screen of typical P450 substrates failed to provide “hits” to identify CYP143A1 substrate selectivity, but the presence of polyethylene glycol in the CYP143A1 active site in crystals suggests fatty acids as potential substrates. CYP126A1 was crystallized for studies to identify binding modes of small molecules (“fragments”) identified to interact with CYP126A1 by NMR. Crystal structures of CYP126A1 in complex with two such fragments (NMR401 and NMR343) were determined to ~2.0 Å resolution in ongoing research to build Mtb P450 isoform-specific inhibitors. Compounds identified as CYP126A1 substrates/inhibitors identified by high-throughput screening were validated by UV-visible titrations with the P450, and binding modes and affinity established. In conclusion, this thesis provides novel insights into the biochemical, biophysical and structural properties of two novel Mtb P450s that are potential targets for new anti-TB drugs.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2015. / Made available in DSpace on 2016-05-24T17:52:39Z (GMT). No. of bitstreams: 1 334243.pdf: 5585985 bytes, checksum: 4f3d8bd503e7a8f2e8284a04905635ec (MD5) Previous issue date: 2015 / Proteínas tirosina fosfatases (PTP) têm um importante papel na transdução de sinal e no controle de diversas funções celulares. Bactérias patogênicas como as do gênero Yersinia e do complexo Mycobacterium tuberculosis (Mtb), utilizam suas PTP para subverter as células de defesa do hospedeiro. As bactérias patogênicas do gênero Yersinia possuem em comum a produção de uma PTP chamada YopH que modula a resposta inflamatória do hospedeiro à bactéria através de processos que envolvem a inibição da fagocitose, quebra de adesões focais e subversão da função dos linfócitos B e T prevenindo a resposta imune adaptativa. As doenças humanas causadas pelas espécies patogênicas de Yersinia variam desde síndromes gastrointestinais à peste bubônica, sendo esta última uma doença grave que ainda não foi erradicada e é associada a milhares de mortes no passado. O Mtb causa a tuberculose, doença que afeta principalmente o trato respiratório. Segundo dados da Organização Mundial da Saúde, a tuberculose foi responsável por 1,5 milhões de mortes somente no ano de 2013. Durantes a invasão das células de defesa, o Mtb produz duas PTP, a PtpA e a PtpB. Estas fosfatases, assim como a YopH, tem por função burlar o mecanismo de defesa do hospedeiro. A PtpA é responsável por modular a apoptose celular e impedir a acidificação do fagossomo e a fusão deste com o lisossomo. Já a PtpB, impede a produção de IL-6 e previne a morte do macrófago pela ativação da via de sinalização de Akt e bloqueio da atividade da caspase-3. A busca de inibidores da YopH de Yersinia spp. e das fosfatases do Mtb é de grande interesse para a produção de possíveis fármacos que poderiam ser utilizados no tratamento das doenças provocadas por estas bactérias. No presente trabalho, uma biblioteca de 398 compostos foi triada com o objetivo de identificar novos inibidores da YopH. Dentre os inibidores encontrados, 22 apresentaram valores de IC50 inferiores a 10 µM, sendo que 3 estão na faixa de nanomolar (o que os coloca entre os melhores inibidores descritos para esta fosfatase até o momento). Foram encontrados tanto inibidores competitivos (12 compostos) como não competitivos (6 compostos) da YopH e cinco compostos (delfinidina, AAA e os três complexos de vanádio) apresentaram valores de Ki na faixa de nanomolar. Avaliou-se também a citotoxicidade em células THP-1 e HepG2 além da atividade antitubercular de seis inibidores das fosfatases de Mtb (PtpA e PtpB) previamente relatados por nosso laboratório em 2012. Identificou-se dois compostos que não são citotóxicos (compostos 43 e 95) e dois compostos que apresentaram atividade em ensaios de infecção intracelular (compostos 95 e 96). De acordo com todos os resultados obtidos no presente trabalho, identificamos novas chalconas como eficientes inibidores da YopH além de 3 complexos de vanádio com uma marcante inibição em escala nanomolar. Quanto aos inibidores da PtpA e PtpB, o composto 95 mostrou-se não citotóxico e com atividade nos ensaios de infecção intracelular. Este composto poderia ser utilizado como molde na busca de outros compostos mais ativos ajudando assim no desenvolvimento de novas terapias contra o Mycobacterium tuberculosis.<br> / Abstract : Protein tyrosine phosphatases (PTP) have an important role in signal transduction and in the control of many cell functions. Pathogenic bacteria from Yersinia genus and Mycobacterium tuberculosis (Mtb) complex, utilize their PTP to subvert host?s defense cells. Pathogenic bacteria from Yersinia genus have in common the production of a PTP named YopH, this enzyme modules the host inflammatory response against the bacteria through processes that evolves the phagocytosis inhibition, focal adhesion disruption and impairing the function of B and T lymphocytes preventing the adaptive immune response. Diseases caused by pathogenic species of Yersinia range from gastrointestinal syndromes to bubonic plague. Bubonic plague is a not eradicated disease that is associated with thousands of deaths in the past. Mtb causes tuberculosis, a disease that affects mainly the respiratory tract. According to World Health Organization data, only in 2013 tuberculosis caused 1.5 million deaths. During the defense cell invasion, Mtb produces two PTP, PtpA and PtpB. These phosphatases act like YopH, they help the bacteria to evade the host defense mechanism. PtpA modulates the cellular aptotosis and it also impairs the phagosome acidification and its fusion with lysosome. PtpB prevents the production of IL-6 and macrophage death by activation Akt signaling pathway and by blocking caspase 3 activity. The search for inhibitors of YopH from Yersinia and Mtb phosphatases is of great interest for the production of drugs that could be used in the treatment of the diseases caused by these bacteria. In this work, an in-house library of 398 compounds was screened with the objective to search new YopH inhibitors. Out of the inhibitors found, 22 presented IC50 values below 10 µM, three of those in nanomolar range (this characteristic put these three compounds between the best described inhibitors for this phosphatase). We found competitive inhibitors (12 compounds) and non-competitive inhibitors (6 compounds) for YopH and five compounds (Delphinidin, AAA and three vanadium complexes) presented Ki values in nanomolar range. Cytotoxicity assays were made with two human cell lines THP-1 and HepG2 we also assayed the antitubercular activity of six inhibitors of Mtb PTP. The inhibitory activity against PtpA and PtpB of these six compounds was previously described in 2012 by our lab. The cytotoxicity and antitubercular assays resulted in two non-cytotoxic compounds (compound 43 and 95) and two compounds that are activity in intracell infection assays (compounds 95 and 96). In this present work we show new chalcones as eficient inhibitors of YopH, we also identified 3 vanadium complex with remarkable inhibition in nanomolar scale. According to the results obtained to PtpA and PtpB inhibitors, we identified the compound 95 which is no cytotoxic and has activity in intracell assays. This compoud could be used for the design of new compound with improved activity helping in the development of new therapies against Mycobacterium tuberculosis.

No description available.

Thiamine-phosphate kinase (or ATP:thiamine-phosphate phosphotransferase, ThiL) and homoserine (ThrB) kinases are essential to metabolism in Mycobacterium tuberculosis (Mtb). ThiL and ThrB respectively phosphorylate thiamine monophosphate (TMP) to thiamine diphosphate (TDP), the active form of vitamin B1, and L-homoserine to Ophosphohomoserine, critical to aspartate biosynthesis. In this study, ThiL and ThrB from Mtb were characterised structurally and functionally by producing the proteins recombinantly in E. coli. Proteins were purified by affinity, anion exchange and size exclusion chromatographies and purity checked by SDS-PAGE. ThiL and ThrB enzyme activities were confirmed and reaction products verified by high pressure liquid chromatography (HPLC). The crystal structure of ThiL was solved by molecular replacement using X-ray diffraction data. Functionally active ThiL, 36 kDa, produced hexagonal crystals belonging to space group P6122 with one monomer per asymmetric unit. Structurally it is related to ThiL from other organisms with minor structural deviations. Enzymatically active ThrB, 33 kDa, was crystallised. However, crystals failed to diffract Xrays to a suitable resolution. ThiL and ThrB could act as possible anti-TB drug targets against Mtb. / Dissertation (MSc)--University of Pretoria, 2017. / Biochemistry / MSc / Unrestricted

>Magister Scientiae - MSc / The generation of biomedical research data on the African continent is grow- ing, with numerous studies realizing the importance of African genetic diver- sity in discoveries of human origins and disease susceptibility. The decrease in costs to purchase and utilize such tools has enabled research groups to produce datasets of signi cant scienti c value. However, this success story has resulted in a new challenge for African Researchers and institutions. An increase in data scale and complexity has led to an imbalance of infrastructure and skills to manage, store and analyse this data. The lack of physical infrastructure has left genomic research on the continent lagging behind its counterparts abroad, drastically limiting the sharing of data and posing challenges for researchers wishing to explore secondary analysis, study veri cation and amalgamation. The scope of this project entailed the design and implementation of a proto- type genome archive to support the e ective use of data resources amongst researchers. The prototype consists of a web interface and storage backend for users to upload and browse projects, datasets and metadata stored in the archive. The server, middleware, database and server-side framework are components of the genome archive and form the software stack. The server component provides the shared resources such as network connectivity, le storage, security and metadata database. The database type implemented in storing the metadata relating to the sample les is a NoSQL database. This database is interfaced with the iRods middleware component which controls data being sent between the server, database and the Flask framework. The Flask framework which is based on the Python programming language, is the development platform of the archive web application. The Cognitive Walkthrough methodology was used to evaluate suitabil- ity of the software for its users. Results showed that the core conceptual model adopted by the prototype software is consistent and that actions available to the user are visible. Issues were raised pertaining to user feedback when per- forming tasks and metadata term meaning. The development of a continent wide genome archive for Africa is feasible by utilizing open source software and metadata standards to improve data discovery and reuse.

Mycobacterium tuberculosis (Mtb) remains a global challenge that has been worsened by the emergence of drug resistant strains of Mtb. We used publicly available Next Generation Sequencing (NGS) and drug susceptibility (DST) data to develop “Resistance sniffer”, an online software program for the rapid prediction of lineage and Mtb drug resistance. Based on the distribution of polymorphisms in the genomes of Mtb, we calculated the power of association between the polymorphisms in different clades of Mtb and resistance to 13 anti-TB drugs. Our data suggests that the development of drug resistance in Mtb is a stepwise process that involves the accumulation of polymorphisms in the Mtb genome. We carefully curated the polymorphisms based on their association powers to create a diagnostic key that captures patterns of these polymorphisms that can be used to predict lineage and drug resistance in Mtb. This diagnosis key was incorporated into the Resistance Sniffer tool, an online software program that we developed for the rapid diagnosis of drug resistance in Mtb. The tool was tested using sequence data from the South Africa Medical Research Council (SA-MRC). Our data suggests that the majority of the strains in SA may have been brought by the arrival of European settlers while the more resistant strains may have been introduced in the region by Asian travellers later on. We next sought to determine non-random associations between polymorphic sites in genomes of Mtb. Using the attributable risk (Ra) statistical methods, we distinguished between functional associations and associations that may have been due to genetic drift events for different Mtb clades. We then integrated the (Ra) data with drug susceptibility and annotation data to generate networks in Cytoscape 3.71. These networks were then used to infer evolutionary trajectories that drive the emergence and fixation of the drug resistant phenotype in different clades of Mtb. We demonstrate that strains from the Lineage 1.2 are associated with less complex functional associations compared to the strains from other clades such as the Asian and Euro-American clades. Our data also shows that the predisposition of strains from the Asian clades to develop multi-drug resistance may be attributed to a complex network of functional interactions of mutations in genes that are involved in several aspects of Mtb physiology such as cell wall modelling, lipid metabolism, stress response and DNA repair. / Dissertation (MSc)--University of Pretoria, 2019. / Biochemistry / MSc / Unrestricted

Philosophiae Doctor - PhD / Marine organisms including algae and bacteria are known to produce chemically diverse secondary metabolites for survival purposes in the marine environment. Scientists have identified some of these natural products as therapeutic agents including some antibiotics. Given the increase in the resistance of pathogenic microorganisms especially methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis to commonly prescribed antibiotics, researchers have turned towards exploiting marine natural products for new antibacterial compounds. Due to the proven success of finding bioactive compounds in the marine environment this study therefore aims to discover lead compounds against MRSA and Mycobacterium tuberculosis from two marine sources, the marine algae and the bacteria associated with marine invertebrates referred to as bacterial isolates. / 2024

This first detailed description of the pathology of tuberculosis, caused by Mycobacterium tuberculosis in springbok is reported. The springbok were part of a semi free-ranging herd kept on the grounds of iThemba Laboratory for Accelerator Based Science (LABS) in the Kuils River district of the Western Cape Province, South Africa. Of the 33 animals sampled, two animals had tuberculosis lesions. Mycobacterium tuberculosis was isolated from these two animals, as well as an animal that did not show tuberculosis pathology. The index case was an adult ewe that was presented for necropsy in a severely weakened condition. The ewe showed advanced miliary tuberculosis with marked macroscopic lesions in the lungs, pleura and respiratory lymph nodes. Limited sampling was done but microscopic tuberculosis lesions were found in almost all the organs sampled, and acid-fast bacilli were generally numerous. Six healthy rams were culled nine months later and a pilot study indicated miliary tuberculosis lesions in one ram, which again were macroscopically most prominent in the lungs, pleura and respiratory lymph nodes. Macroscopic lesions were also noted in the sternal, iliac, prefemoral and retropharyngeal lymph nodes. Microscopy in this animal revealed lesions in the macroscopically affected organs as well as numerous other lymph nodes, and suspected lesions occurred in the testicle and colon. Acid-fast bacilli were scarce to moderate in affected organs. Because of the miliary nature of the lesions in both affected animals, the route of infection could not be established conclusively. The lesions in most affected organs of both animals resembled classical tuberculous granulomas, viz. central caseous necrosis, with various degrees of calcification, surrounded by various numbers macrophages, epithelioid cells, multinucleated giant cells and lymphoplasmacells, and mild to moderate fibrous encapsulation. Necrotic lesions in the spleen, liver and kidney of the ewe were more disseminate and coagulative. A main study conducted on healthy culled animals 19 months after the pilot study failed to find any animal with tuberculosis lesions in the group of 25 sampled. These animals were all negative for mycobacteria via mycobacterial culture. The Interferon-gamma (INFg) assay was performed on all the animals of the pilot and main study but failed to identify the culture-positive animals and showed one false-positive reaction. / Dissertation (MMedVet (Pathology))--University of Pretoria, 2007. / Paraclinical Sciences / unrestricted

A clone containing the terminal third of the Mycobacterium tuberculosis katG gene was previously shown to confer resistance to ethyl methane sulfonate on DNA repair-deficient Escherichia coli cells. The first aim of this study, therefore, was to examine the role played by the M tuberculosis katG gene in DNA repair. The strategy used was overexpression of different regions of the gene in DNA repair-deficient mutants of E. coli, and examination of the sensitivities of the transformants to DNA damaging agents. Overexpression of the gene resulted in an increase in the survival of recA mutants exposed to ultraviolet (UV) light irradiation (254 run) and hydrogen peroxide, and uvr mutants exposed to mitomycin C. Both the 5' and 3' regions of the M tuberculosis KatG protein conferred the above effects, and this was independent of the catalase or peroxidase activity of the enzyme. The results suggest that the M tuberculosis katG gene may encode a novel function related to the repair of DNA damage, and this may have implications for the survival of M tuberculosis in the presence of DNA damaging agents, for example, in the macrophage. UV sensitivity tests on M intracellulare and M tuberculosis strains mutant in katG revealed that the katG gene product does not play a demonstrable role in the survival of repair-competent mycobacterial cells after exposure to UV irradiation. The second aim of this study was to examine the regulation of expression of the M tuberculosis katG gene. An E. coli-mycobacterial shuttle vector, pJCluc, containing the luciferase reporter gene, was constructed and used to examine the katG promoter sequences. The region required for optimal expression in M. smegmatis was localized to a 559 hp fragment immediately upstream of the gene. Two transcription start sites were mapped and putative -10 and -35 promoter sequences identified. It was demonstrated that expression from the promoter peaks during late exponential phase, and declines during stationary phase, and that the promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. An upstream element that increased expression from the M. tuberculosis katG and the M. paratuberculosis PAN promoters was identified, and shown to bind to one or more M smegma/is proteins. Similar results were obtained in M bovis BCG. Understanding the regulation of gene expression in mycobacteria is essential for determining the processes that govern interaction with the host. This study provides information on both the mycobacterial transcription signals and gene regulatory mechanisms.