Premièrement, nous avons analysé 14 génomes publiés de M. abscessus montrant quece taxon comprend au moins cinq taxons différents spécifiés par des caractéristiques microbiologiques d’intérêt médical. Au cours d'un deuxième travail, nous avons développé une technique d’identification et de génotypage de M. abscessus qui a permis de distinguer sans ambiguïté M. massiliense de M. bolletii et M. abscessus.Nous avons ensuite analysé le bactériophage de M. bolletii que nous avons nommé Araucaria. La résolution de sa structure 3D a montré une capside et un connecteur similaires à ceux de plusieurs bactériophages de bactéries à Gram négatif et positif; et une queue hélicoïdale décorée par des pointes radiales. La partie basale (baseplate) du phage Araucaria présente des caractéristiques observées dans les phages se liant à des récepteurs de protéines. Araucaria se lie à son hôte en deux temps, un premier par liaison de la queue aux saccharides de l'hôte puis un deuxième par liaison de la baseplate aux protéines de la paroi cellulaire.Nous avons analysé la présence de séquence de phages dans 48 génomes disponibles de M. abscessus. Notre analyse phylogénétique suggère que les espèces de M. abscessus ont été infectées par différents mycobactériophages et ont une histoire évolutive différente de celle des hôtes mycobactériens et contiennent aussi des protéines acquises par transfert horizontal.Enfin, nous avons séquencé et analysé deux mycobactéries non-tuberculeuses responsables d’infections opportunistes, Mycobacterium simiae et Mycobacterium septicum. / In a first step, we reviewed the published genomes of 14 M. abscessus strains showing that M. abscessus sensu lato comprises of five different taxons specified by particular characteristics of microbiological and medical interests. In a second step, based on sequencing of eight intergenic spaces, we developed a Multispacer Sequence Typing technique (MST) for M. abscessus group sub-species identification and strain genotyping. MST clearly differentiates formerly “M. massiliense” organisms from other M. abscessus subsp. bolletii organisms. We also analyzed a bacteriophage from M. bolletii that we named Araucaria. We resolved Araucaria 3D structure, its capsid and connector share close similarity with several phages from Gram- or Gram+ bacteria. The helical tail decorated by radial spikes, possibly host adhesion devices. Its host adsorption device, at the tail tip, assembles features observed in phages binding to protein receptors. All together, these results suggest that Araucaria may infect its mycobacterial host using a mechanism involving adhesion to cell wall saccharides and protein, a feature that remains to be further explored. We also analysed 48 M. abscessus sequenced genomes for encoding prophages. Our phylogenetic analyses suggested that M. abscessus species were infected by different mycobacteriophages and have a different evolutionary history than the bacterial hosts and some proteins that are acquired by horizontal gene transfer mostly mycobacteriophages’ proteins and hypothetical proteins. Finally, we sequenced and analyzed two non-tuberculosis mycobacterium causing human infections, Mycobacterium simiaie and Mycobacterium septicum.

Student Number : 0201444H - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Due to the paucity of sensitive mutation markers available for studying mycobacterial species it was decided to explore the suitability of mycobacteriophage L5 as an analogous mutation detection system to phage Lambda in E. coli. The system relies on the detection of an increased production of clear plaque mutants (CPM) arising from turbid plaques, in response to DNA damage. A number of L5 phage experimental tools were developed and optimized, including a lysogen-based CPM confirmation assay. The mutant induction system was applied to wild type M. smegmatis mc2155 and its recA mutant, dinP mutant as well as an M. smegmatis(L5) lysogen. The lysogen system proved to be insensitive with respect to mutant induction since elevated CPM frequencies could not be detected. Interestingly, the wild type M. smegmatis mc2155 system demonstrated slightly elevated CPM frequencies in response to transfection of untreated L5 on UV irradiated host cells. This result suggests that a host SOS mutagenic system is able to act on normal, undamaged DNA bases. The involvement of the SOS response in untargeted mutagenesis was confirmed by the abrogation of increased CPM frequency, in an M. smegmatis recA mutant. This data supports suggestions that RecA is responsible for the control of the SOS response. The M. smegmatis dinP mutant system showed a decrease in CPM frequency which supports evidence that this gene does have mutator polymerase activity, as is in seen E. coli dinP homologues.

Tuberculosis and malaria are among the most deadly infectious diseases in the world. The prevalence in regions without well-established public health causes economical and financial burdens for both society and patients. There is an urgent need to find effective treatments due to the emergence of drug-resistant strains. The aim of the studies reported here was to gain knowledge from the protein structures that can lead to the elimination of these pathogens. In these studies, protein crystallography is the main method used to solve protein structure. Based on the protein structure, we used different methods to characterize the protein function of three lipid-binding proteins (LprG, LprA, and gp232), and to identify potent inhibitors against Plasmodium falciparum enoyl-ACP reductase (PfENR), a drug target protein involved in central lipid metabolism. To characterize the function of two lipid-binding proteins (LprG and LprA), liquid chromatography-mass spectrometry (LC-MS) was used to analyze the ligand extract. In the study of tail fiber protein from mycobacteriophage, we used protein sequence alignment to identify gp232 as a major tail fiber protein, which potentially binds to lipids on the cellular surface of mycobacteria. A pull-down assay and imaging methods (fluorescence microscopy and electron microscopy) were conducted to confirm the function of gp232. In the structural study of PfENR, the structure-activity relationships method was used to find potent inhibitors against PfENR, which would show stronger inhibition than the known inhibitor triclosan. The triclosan-like analogs with modification at the 5-position revealed a new binding site in PfENR that has great potential for improving the potency of inhibition. We found that two inhibitors containing the core structure of piperidine and tetrahydroquinoline reached this new binding site and were 10-fold more potent than triclosan. The structural study of PfENR provides structural insights into the inhibitor-binding site that can lead to the discovery of new drugs. The comprehensive knowledge that we gained from the structural studies of these lipid-binding proteins provide new information that could lead to a greater understanding of pathogen physiology or guide the discovery of effective treatments to eliminate the pathogens.

Orientador: Fernando Rogério Pavan / Resumo: A Tuberculose é uma doença infecto contagiosa causada pelo agente etiológico Mycobacterium tuberculosis, sendo este a causa de 1,3 milhões de mortes ao redor do mundo no ano de 2018. O tratamento preconizado pela Organização Mundial da Saúde é eficaz para cepas sensíveis, porém os efeitos adversos dos fármacos levam muitos pacientes ao abandono da terapia e consequente surgimento de resistência micobacteriana. Dentro desse contexto e mediante o reduzido número de fármacos disponíveis para o tratamento se faz necessário o desenvolvimento de novas alternativas terapêuticas. Bacteriófagos, vírus que infectam bactérias, têm sido sugeridos como importantes agentes terapêuticos no combate a bactérias multirresistentes, os quais podem ser encapsulados em lipossoma objetivando a proteção contra a degradação pelo sistema imune. O objetivo deste trabalho foi avaliar o perfil do micobacteriófago D29 livre e encapsulado em lipossoma frente a cepa padrão de M. tuberculosis (H37Rv). Os lipossomas apresentaram tamanho, PDI e valores de potencial zeta pelo DLS (Dynamic Light Scaterring). De acordo com imagens obtidas por microscopia de transmissão, foram classificados com vesículas unilamelares gigantes com uma eficiência de encapsulação fágica de 9,4 % ± 0,023. Para garantir mais de 50 % em viabilidade celular, com a linhagem MRC-5 e J774A.1, respectivamente o volume máximo de 80% e 60% das amostras (tampão de fago, micobacteriófago D29, micobacteriófago D29 encapsulado em lipossoma e lipos... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

<p>Bacteriophages (phages) have broad applications in diverse areas including phage therapy, agriculture, food safety, and environmental protection. In order to fully realize the potential for phage applications, it is critical to understand phage-bacteria interactions and characterize bacterial responses/targets to phage infection. Previous studies have largely focused on other classes of phages other than mycobacteriophages. This research provides the first global proteome investigation of the dynamic relationship between a mycobacteriophage and a mycobacterial host. Mycobacteriophages are viruses that infect mycobacteria. They have been reported to have vital potential uses in various fields, especially as an alternative in the prevention and treatment of mycobacterial diseases such as tuberculosis. Despite their potential, not much is known about the molecular interaction with mycobacteria during a mycobacteriophage infection, especially at the translational level. To better understand this, a novel mycobacteriophage, Ochi17 was first isolated and characterized based on the genome and structure. I then applied label-free quantitative proteomics using the model host, <i>Mycobacteria smegmatis</i>,which<i> </i>was infected with Ochi17<i> </i>at different infection time points. Phage Ochi17 was found to be a temperate phage and classified as a Siphoviridae. The proteome changes occurring at the mid-lytic stage of Phage Ochi17 infection was first examined followed by a temporal study of the global changes. More than 2,000 <i>M. smegmatis</i> proteins and at least 50 Ochi17 proteins were identified across all time points. Homologous recombination and host macromolecular synthetic processes were significantly upregulated, while lipid metabolism was significantly downregulated. The results suggested that Ochi17 suppressed the growth of Mycobacterium smegmatis not just by utilizing the macromolecular synthesis of the host, but also by suppressing host transcription, and fatty acid biosynthesis, in addition to the degradation of fatty acids irrespective of infection time. The two-component system was a target at only 24 h post infection. I also showed that phage Ochi17 proteome expression is time-dependent and the proteins typically cluster based on functional relatedness. The results presented here may contribute in the development of mycobacteriophages as antimicrobial therapies that can overcome various defense strategies employed by host mycobacteria.</p>

Thesis (PhD)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative organism of tuberculosis (TB), is a major cause for mortality and morbidity world-wide with a death toll only second to HIV among infectious diseases. Drug resistance is widespread and cases of multiple drug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB) have emerged in several countries. Drug treatment is problematic and new drugs are not developed rapidly enough to offset the rapid drug resistance mutation rate of M. tuberculosis. Simple and effective diagnostics are required to contain the spread of the disease as current routine diagnostics are not fulfilling this role. Additionally, current rapid TB diagnostics are out of reach to resource poor settings due to infrastructure, cost and skill requirements. Novel TB diagnostics are thus required that meet these requirements. Mycobacteriophages are phages that infect mycobacteria and could offer a viable and cost effective alternative rapid TB diagnostics. In this study, an affinity-tagged fluorescent reporter mycobacteriophage is described, which was engineered to act as a TB diagnostic. Its performance proved favourable and superior to current existing mycobacteriophage-based TB diagnostics. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme verantwoordelik vir tuberkulose (TB), is `n groot bron van mortaliteit en morbiditeit wêreldwyd en slegs HIV is verantwoordelik vir groter getalle sterftes as gevolg van n aansteeklike siekte. Middelweerstandigheid is algemeen en gevalle van meervoudigemiddelweerstandige tuberkulose (MDR-TB) en uiters weerstandige tuberkulose (XDR-TB) kom in verskeie lande voor. Antibiotika behandeling is problematies en nuwe anti-TB middels word nie vinnig genoeg ontwikkel om die antibiotika weerstandigheid mutasie spoed van M. tuberculosis te bekamp nie. Doeltreffende diagnostiese toetse word benodig om die verspreiding van die siekte te beheer en bestaande roetine diagnostiese toetse voldoen tans nie aan hierdie vereiste nie. Behalwe hiervoor, is huidige vinnige TB diagnostiese toetse buite bereik van arm instansies weens vereistes aan infrastruktuur, meegaande kostes en werknemervaardigheid. Nuwe TB diagnostiese toetse is dus nodig om aan hierdie vereistes te voldoen. Mikobacteriofaage is fage wat mikobacteria infekteer en kan moontlik 'n lewensvatbare en koste-effektiewe alternatief bied vir vinnige TB diagnostiese toetse. In hierdie studie word 'n affiniteitgekoppelde fluoreserende rapporteringsmikobakteriofaag beskryf wat ontwerp is om op te tree as `n nuwe vinnige TB diagnostiese toets. Die werking hiervan vertoon gunstige en beter resultate as die huidige, mikobacteriofaaggebaseerde TB-diagnostiese toetse.

<p>Bacteriophages are viruses that infect and kill bacteria. They are the most abundant organism on the planet and the largest source of untapped genetic information. Every year, more bacteriophages are isolated from the environment, purified, and sequenced. Once sequenced, their genomes are annotated to determine the location and putative function of each gene expressed by the phage. Phages have been used in the past for genetic engineering and new research is being done into how they can be used for the treatment of disease, water safety, agriculture, and food safety. </p> <p>Despite the influx of sequenced bacteriophages, a majority of the genes annotated are hypothetical proteins, also known as No Known Function (NKF) proteins. They are expressed by the phages, but research has not identified a possible function. Wet lab research into the functions of the hundreds of NKF phages genes would be costly and could take years. Bioinformatics methods could be used to determine putative functions and functional categories for these hypothetical proteins. A new bioinformatics method using algorithms such as Domain Assignments, Hidden Markov Models, Structure Prediction, Sub-Cellular Localization, and iterative algorithms is proposed here. This new method was tested on the bacteriophage genome PotatoSplit and dropped the number of NKF genes from 57 to 40. A total of 17 new functions were found. The functional class was identified for an additional six proteins, though no specific functions were named. Structure Prediction and Simulations were tested with a focus on two NKF proteins within lytic phages and both returned possible functional categories with high confidence.</p> <p>Additionally, this research focuses on the possibility of phage therapy and FDA regulation. A database of phage proteins was built and tested using R Statistical Analysis to determine proteins significant to phage infecting <i>M. tuberculosis</i> and to the lytic cycle of phages. The statistical methods were also tested on both pharmaceutical products recalled by the FDA between 2012 and 2018 to determine ingredients/manufacturing steps that could affect product quality and on the FDA Adverse Event Reporting System (FAERS) data to determine if AERs could be used to judge the quality of a product. Many significant excipients/manufacturing steps were identified and used to score products on their quality. The AERs were evaluated on two case studies with mixed results. </p>