No description available.

No description available.

No description available.

No description available.

Tuberculosis has been an important disease throughout human history, shaping countless past populations. The archaeological study of the causative agents of tuberculosis, members of the Mycobacterium tuberculosis Complex (MTBC), is hindered by the non-diagnostic nature of tuberculosis-associated skeletal changes. As such, ancient DNA (aDNA) or palaeogenetic analyses have become an important tool for identifying tuberculosis in past populations. However, due to the age and variable preservation of aDNA, there are often issues with sporadic results and false negatives. The overall aim of the work presented here was to use different methods, including traditional target-specific PCR, to identify and detect tuberculosis aDNA in archaeological remains. The main objectives within this overarching aim were to first test a method called whole genome amplification (WGA), used to non-specifically amplify all the DNA within a sample, and its potential to improve the yield of aDNA from skeletal remains (Chapters 3 and 4). To determine the extent of its impact, WGA was used in a comparative context, where each archaeological sample analysed was separately subjected to two methods of MTBC detection - the traditional targeted PCR method and the same method assisted by the initial application of WGA. The results show that applying WGA before the traditional targeted PCR methodology to detect the presence of MTBC pathogens in skeletal remains is only useful and viable in some cases, likely depending on the age and preservation of the sample. The second objective was to use next generation sequencing to obtain more information on the aDNA composition of certain archaeological samples and answer questions beyond the scope of traditional target-specific PCR techniques (Chapter 5). Although most of the sequencing runs were variably unsuccessful, the composition of two samples, both known to probably contain tuberculosis aDNA, could be analysed. The samples both contained similar amounts of mycobacterial aDNA and varying amounts of both human and even potentially human intestinal flora DNA. Finally, the third objective was to determine if MTBC aDNA could be detected in a rib sample from Private William Braine of the lost Franklin Expedition using standard target-specific PCR (Chapter 6). In this case study, no evidence of tuberculosis ancient DNA was found. The work done through-out highlights the difficulties of ancient DNA research and, in Chapter 4, shows the importance of using more than a single sample to evaluate methods for application in palaeogenetic contexts.

Publicación a texto completo no autorizada por el autor / Determina la bioactividad sobre Mycobacterium tuberculosis H37RV de los extractos, alcaloides y flavonoides de las especies Juglans neotropica Diels., Piper aduncum L., Croton lechleri Müll. Arg., Lantana camara L., Annona cherimola Mill, Annona muricata L. y Jatropha gossypifolia L., mediante el ensayo en microplacas con azul de alamar. Se recolectó hojas de las 7 especies vegetales seleccionadas según la quimiotaxonomía vigente: Juglans neotropica Diels., Piper Aduncum L., Croton lechleri Mull. Arg., Lantana camara L., Annona cherimola Mill, Annona muricata L. y Jatropha gossypifolia L. Se preparó extractos etanólicos y clorofórmicos. Se aisló alcaloides y flavonoides. Se determinó la bioactividad sobre Mycobacterium tuberculosis H37RV de los extractos, alcaloides y flavonoides mediante el screening antimicobacteriano basado en la reducción del azul de alamar a tres concentraciones (10, 100 y 1000 μg/ml); siguiendo el protocolo original de Collins y Franzblau 64 con algunas modificaciones. Se determinó la concentración mínima inhibitoria (CMI) in vitro de los extractos con bioactividad sobre Mycobacterium tuberculosis H37RV en el rango de concentraciones de 2000-15.63 μg/ml, mediante el ensayo en microplacas con azul de alamar; siguiendo el protocolo original de Collins y Franzblau 64. El screening antimicobacteriano basado en el azul de alamar determinó que los 14 extractos estudiados (100%) presentaron bioactividad sobre Mycobacterium tuberculosis H37RV a 1000 μg/ml. El ensayo en microplacas con azul de alamar determinó que el extracto etanólico de Piper Aduncum L presentó un CMI igual a 31.5 μg/ml. El Piper aduncum L. es una buena alternativa de extractos y metabolitos antituberculosos. / Tesis

This thesis consists of two related studies directed at aspects of M. tuberculosis biology. The first focuses on deciphering gene-regulatory circuits from ChIP-seq data, and the second focuses on alternative strategies for combatting drug-resistant infections. The first study describes Binding Resolution Amplifier and Cooperative Interaction Locator (BRACIL), a post-peak-caller computational method that predicts transcription-factor (TF) binding sites with high-resolution as well as cooperative TF interactions derived from ChIP-seq data. BRACIL integrates ChIP-seq coverage with motif discovery from a signal-processing perspective and uses a blind-deconvolution algorithm that predicts binding-site locations and magnitudes. BRACIL also explicitly considers a second-order signal, represented by DNA fragments with two sites bound simultaneously, and uses it to predict cooperative interaction. Cooperative interaction indicates that the binding to a first site influences the probability of binding to a second site. This method estimates the probability of a binding configuration from the ChIP-seq coverage and performs a likelihood ratio test to predict cooperative interaction. As a proof of principle, I validated this method using M. tuberculosis transcription factor DosR. The second study focuses on strategies to fight antibiotic resistance. In particular, recent reports have shown the existence of treatment conditions (called "antiR") that select against drug-resistant strains. I used a mathematical model of infection dynamics and immunity to simulate the growth of resistant and sensitive pathogens under different treatment conditions (no drugs, antibiotic present, and antiR), and could show how a precisely timed combination of treatments can defeat resistant strains. This analysis suggested that a time- scheduled, multi-treatment therapy could lead to complete elimination of both sensitive and resistant strains. Also, my results indicated that the time necessary to turn a resistant infection into a sensitive one ("tclear") depends on the experimentally measurable rates of pathogen division, growth and plasmid loss. Additionally, I estimated tclear for a specific case, using available empirical data, and found that resistance may be lost up to 15 times faster under antiR treatment as compared to a no-treatment regime. Finally, an extension of these findings to population models provides quantitative support for therapeutic plans to clear antibiotic-resistant infections, including novel drug-cycling strategies.

Den mest välkända arten i släktet Mycobacterium som kan orsaka sjukdom hos människan är Mycobacterium tuberculosis (MTB). Infektioner av andra mykobakterier ökar världen över. Dessa benämns icke-tuberkulösa mykobakterier (NTM) och orsakar ofta liknande symtom som de vid MTB-infektion. Vanligtvis krävs dock olika behandlingar beroende på om infektionen är orsakad av MTB eller av NTM. Nuvarande diagnostik bygger på odling och mikroskopi, men analyser som innefattar molekylärbiologiska metoder för undersökning av mykobakteriers DNA blir allt vanligare. I denna studie jämfördes olika PCR-metoder för identifiering av NTM, där metoderna baserades på två tidigare publicerade artiklar. Tester gjordes på DNA från sex NTM-stammar med MTB-DNA som referens. För påvisning av hela släktet Mycobacterium användes primers riktade mot hsp65 och 16S, och för att kunna urskilja MTB från NTM användes primers riktade mot IS6110, IS1081 och ITS-MTC. Undersökningarna visade lägre Ct-värden och högre PCR-effektivitet för hsp65 än för 16S men tester med avseende på MTB-specifika primers visade samtliga amplifiering även för NTM. / The most well-known species of the genus Mycobacterium that can cause human disease is Mycobacterium tuberculosis (MTB). Infections caused by other mycobacteria is an increasing problem worldwide. These mycobacteria are known as non-tuberculous mycobacteria (NTM) and they often cause similar symptoms as those in MTB-caused infections. Usually different treatments are required depending on if the infection is caused by NTM or MTB. Current diagnostic methods are based on culture and microscopy, though molecular methods are becoming more common. In this study different PCR-methods for identification of NTM’s were compared. The different methods were based on two earlier published articles. Experiments were made with DNA from six NTM-species and with DNA from MTB as reference. To detect all mycobacteria primers targeting hsp65 and 16S were used, and primers targeting IS6110, IS1081 and ITS-MTC were used to separate MTB from NTM. This study showed lower Ct-values and higher PCR-efficiency for hsp65 than for 16S, but comparative tests regarding MTB-specific primers showed, with all three primer pairs, amplification of NTM as well.

La capacidad de Mycobacterium tuberculosis para sobrevivir dentro del macrófago contribuye grandemente a su patogenicidad, latencia y persistencia durante la infección. Este bacilo induce alteraciones en el ambiente intrafagosomal e inhibe la maduración del fagosoma, favoreciendo su supervivencia intracelular. M. tuberculosis PknG secuestra al macrófago precisamente al evitar la fusión fagosoma-lisosoma. En este sentido, PknG representa una familia de dianas novedosas para enfrentar la necesidad de nuevos antimicrobianos para la tuberculosis latente. Aquí, apuntamos a: (i) elucidar la base estructural-molecular del ATP y Mg2+ como cofactores de PknG; (ii) caracterizar los parámetros cinéticos que gobiernan la formación del complejo PknG:ATP; e, (iii) identificar péptidos capaces de unirse a PknG para investigar experimentalmente su interactoma usando enfoques combinatorios como “Phage Display”. Nuestros resultados confirman que PknG se une exclusivamente al ATP con una constante de disociación (KD) de 108.8  22.9 µM. El Mg2+ estabiliza térmicamente a PknG de forma ATP-dependiente. Análisis de estado pre-estacionario muestran que la unión y disociación del ATP es rápida en el complejo PknG:ATP. Usando PknGN-Ext, TPR resolvimos la estructura cristalina en el estado unido al ADP mientras que demostramos que el ATP imposibilita la cristalización. Los análisis bioinformáticos de las librerías enriquecidas por Phage Display identificaron 57 potenciales peptidos que interactuarían con PknG. Una comparación cercana con el proteoma de M. tuberculosis proporcionó un subconjunto de 20 proteínas que podrían interactuar con PknG. Nuestros resultados confirmaron cinco proteínas asociadas a PknG previamente reportadas: PknG, DnaK chaperona, transportador ABC Rv1747, Proteína Ribosomal L23 y Factor de Elongación Tu, resaltando la validez de nuestra plataforma para descubrir el interactoma de PknG. Así, nuestros resultados revelan interacciones proteína-proteína putativas que podrían participar en la supervivencia micobacteriana, mientras que también proporcionan bases sólidas para desarrollar drogas antituberculosas al interrumpir estas interacciones o explotar estos peptidos tipo compuesto líder. / The ability of Mycobacterium tuberculosis to survive inside the macrophage greatly contributes to its pathogenicity, latency and persistence during infection. This bacillus induces alterations in the intraphagosomal environment and inhibits phagosome maturation, thus promoting mycobacterial survival. M. tuberculosis PknG hijacks the macrophage precisely by avoiding phagosome-lysosome fusion. In this sense, PknG represents a family of novel targets to cope with the need for new antimicrobials for latent tuberculosis. Here, we aimed to: (i) elucidate the structural-molecular basis of ATP and Mg2+ as PknG cofactors; (ii) characterize the kinetic parameters governing PknG:ATP complex formation; and, (iii) identify PknG-binding peptides to experimentally query PknG’s interactome using combinatorial approach such as Phage Display. Our results confirm that PknG exclusively binds to ATP with a dissociation constant (KD) of 108.8  22.9 µM. Mg2+ thermally stabilizes PknG in an ATP-dependent manner. Pre-steady-state analyses show that ATP binding and dissociation are rapid in the PknG:ATP complex. Using PknGN-Ext, TPR we solved the ADP-state crystal structure while showing that ATP precludes crystallization. Phage Display and bioinformatic analyses identified 57 potential PknG binders. A close comparison to the M. tuberculosis proteome provided a subset of 20 proteins that may interact with PknG. Our results confirmed five previously reported PknG-associated proteins: PknG, DnaK chaperone, ABC transporter Rv1747, Ribosomal Protein L23 and Elongation Factor Tu, highlighting our platform’s validity to uncover the PknG interactome. Altogether, our results reveal putative protein-protein interactions that may play a role in mycobacterial survival, while also providing solid bases for the development of anti-tuberculosis drugs by disrupting these interactions or exploiting these lead-like peptide molecules. / Tesis

Thesis (MSc. (Medical Sciences)) -- University of Limpopo, 2015 / Introduction: Multidrug-resistant tuberculosis (MDR-TB) caused by resistance to at least rifampicin (RIF) and isoniazid (INH) drugs is a growing public health concern in South Africa. The detection of MDR-TB still relies on culture despite advancement in molecular diagnostic technology. Currently MTBDRplus and GeneXpert are the only available assays used in rapid diagnosis of MDR-TB using chromosomal mutations in drug target regions. Some strains are missed by these assays due to their limitation in mutational detection profile. Novel Seegene Anyplex assays simultaneously detect TB and resistance to RIF and INH using fifteen and six mutational probes, respectively within 3 hours. Limpopo Province has limited information on the circulating strains of TB. Aim: To determine drug-resistant Mycobacterium tuberculosis (M. tuberculosis) mutations using Anyplex™ MTB/NTM/MDR-TB real time assay and characterise the drug-resistant strains. Methods: We prospectively collected 204 clinical samples at Modimolle MDR-TB unit and retrospectively used 104 culture isolates from MRC laboratory in Pretoria. The MTBDRplus assay was used to screen for M. tuberculosis and drug resistant mutations to RIF and INH drugs. Anyplex™ MTB/NTM/MDR-TB assay was used for rapid detection of M. tuberculosis and drug resistance to RIF and INH within 3 hours. The discordance between phenotypic and genotypic assays was resolved by sequencing and the Anyplex™ resistant profiles were spoligotyped. Diagnostic data was collected from NHLS and MRC databases and analysed using the Microsoft excel and Epi Info version 3.5. Descriptive statistics (percentages and frequencies) were used to explain proportions. Results: The Anyplex™ MTB/NTM assay detected M. tuberculosis in 69/111(62%) and 100/104 (96%) of clinical and culture samples respectively. The sensitivities, specificity, PPV and NPV obtained for both RIF and INH resistance by Anyplex™ MDR-TB assay were 67%, 59%, 67%, 55% and 15%, 100%, 100% and 17%, respectively. Anyplex™ MTB/NTM/MDR-TB resolved 23/45 (51%) of discordant vi samples. Sequencing of remaining discordant isolates revealed L511P, L533P and D516Y mutations within rpoB gene. A novel R385W mutation within katG was also detected. Spoligotyping of Anyplex™ MDR-TB resistant clinical isolates revealed Euro American clade with 20% followed by 15% Manu2, 5% East African Indian, 5% H37Rv, 5% atypical and 50% were orphans. Conclusion: The novel Anyplex™ MTB/NTM/MDR-TB assay is a rapid and valid technique for detecting M. tuberculosis and most common mutations conferring resistance to RIF and INH. However further investigations are required, as the assay has a lower sensitivity as compared to already endorsed techniques. / National Research Foundation (NRF) and University of Limpopo TB Grant