The human gut is home to a significant number of microorganisms including the dominant symbiont Bacteroides thetaiotaomicron. This microbe is predicted to possess an array of glycoside hydrolases, majority of which are involved in starch utilization. Presented here is a comparative functional analysis of two alpha-glucosidases, Family 31 Glycoside Hydrolases from Bacteroides thetaiotaomicron. Enzymatic kinetics revealed these enzymes both preferentially cleave alpha-(1,6) linkage in comparison to the predicted alpha-(1,4) and favour maltose derived substrates of longer length.

The human gut is home to a significant number of microorganisms including the dominant symbiont Bacteroides thetaiotaomicron. This microbe is predicted to possess an array of glycoside hydrolases, majority of which are involved in starch utilization. Presented here is a comparative functional analysis of two alpha-glucosidases, Family 31 Glycoside Hydrolases from Bacteroides thetaiotaomicron. Enzymatic kinetics revealed these enzymes both preferentially cleave alpha-(1,6) linkage in comparison to the predicted alpha-(1,4) and favour maltose derived substrates of longer length.

The human gut is host to a vast consortium of microorganisms, collectively referred to as the microbiota or microflora, which play important roles in health and disease. Current applications focus only on a single type of bacteria, which are not the most dominant numerically, and without detailed knowledge of the specific functions of these bacteria. A good indicator of the function of a bacterial species involves detailed analysis of its enzymes. Bacteroides thetaiotaomicron is one of the predominant bacterial species with a great representation of the carbohydrate processing enzymes, glycoside hydrolases in its proteome. This thesis reports the production and purification of one such enzyme, BT3299, suitable for kinetic and structural studies. The enzyme displayed a broad substrate specificity with a slight preference for 1-->3 and 1-->6 glycosidic linkages and longer chain saccharides. Future work will focus on structural analysis as an aid to the understanding of the enzyme function.

The human gut is host to a vast consortium of microorganisms, collectively referred to as the microbiota or microflora, which play important roles in health and disease. Current applications focus only on a single type of bacteria, which are not the most dominant numerically, and without detailed knowledge of the specific functions of these bacteria. A good indicator of the function of a bacterial species involves detailed analysis of its enzymes. Bacteroides thetaiotaomicron is one of the predominant bacterial species with a great representation of the carbohydrate processing enzymes, glycoside hydrolases in its proteome. This thesis reports the production and purification of one such enzyme, BT3299, suitable for kinetic and structural studies. The enzyme displayed a broad substrate specificity with a slight preference for 1-->3 and 1-->6 glycosidic linkages and longer chain saccharides. Future work will focus on structural analysis as an aid to the understanding of the enzyme function.

Notre équipe vient de découvrir une méthode originale d’obtention d’extraits de Bacteroides thetaiotaomicron (E) qui préserve sa viabilité. Après culture anaérobie de ce commensal intestinal en milieu gélosé pauvre en facteurs de croissance, puis exposition à l’air, la bactérie semble posséder et générer dans E tout l’équipement de détoxication des espèces réactives de l’oxygène in vitro. Il laisse alors augurer d’un pouvoir thérapeutique à visée anti-inflammatoire.Objectifs et méthodes : Le but est d’abord de caractériser E, aux plans glucidique, lipidique et protéique. Dans ce dernier cas, il s’agit de séparer les protéines produites par la bactérie vivante et contenues dans E par électrophorèse bidimensionnelle et de les identifier par la technique des cartes peptidiques massiques. Les gels (n≥6) sont traités statistiquement (PDQuest®, Bio-Rad). Pour mieux localiser ces protéines dans la bactérie, elles sont comparées avec celles obtenues par destruction de B. thetaiotaomicron et identifiées dans la fraction cellulaire relative à la membrane bactérienne externe. Un travail de microscopie électronique est aussi entrepris pour visualiser les éventuels évènements intervenant pendant l’extraction.Le but est alors de vérifier, in vitro, l’effet antioxydant de l’extrait bactérien standardisé et d’en contrôler l’innocuité en modèles cellulaires utilisant le granulocyte neutrophile. L’effet thérapeutique anti-inflammatoire est ensuite recherché chez l’animal. L’action de E est d’abord évaluée en modèle murin d’inflammation cutanée auriculaire induite par dépôt de chlorure de benzalkonium, sous anesthésie générale. Des témoins positifs et négatifs de traitement et d’autres ne subissant pas d’irritation sont testés en parallèle. L’épaisseur des oreilles est mesurée toutes les heures pendant 5 h et des coupes histologiques d’oreilles, effectuées au bout de 2 h chez certains animaux. Deux colorations différentes permettent alors d’évaluer la quantité de mastocytes dégranulant localement.L’action de E, administré par voie intra-rectale (IR), est ensuite testée chez des souris subissant les premières phases d’un processus inflammatoire, en modèle de colite aiguë. Celle-ci est induite per os par du dextran sulfate sodium (DSS) ; elle évolue sur 8 jours. Sont considérés en parallèle des témoins positifs et négatifs de traitement et d’autres ne subissant pas de colite. Des scores cliniques et des scores histologiques de sévérité sont établis tous les jours de l’expérience. Des marqueurs de l’inflammation sont suivis dans les tissus murins après autopsie des animaux. [...] / Our team had discovered a new method to obtain extracts of Bacteroides thetaiotaomicron (E) which preserved its viability. This intestinal symbiont was anaerobically grown on an agar medium poorly supplemented in growth factors. After exposure to air, the bacterium seemed to possess and generate in E all the equipment able in vitro to detoxify reactive oxygen species. It let us expect a therapeutic power referred to anti-inflammatory properties.Objectives and methods: The aim was first to characterize E, in terms of carbohydrates, lipids and proteins. To achieve this last-mentioned goal, proteins contained in E coming from living bacteria were separated by two-dimensional electrophoresis and identified by the peptide mass fingerprinting technique. The gels (n ≥ 6) were statistically analyzed (PDQuest®, Bio-Rad). To find the origin of these proteins in bacteria, they were compared with those obtained by destruction of B. thetaiotaomicron (BT) and identified in the cell fraction containing the bacterial outer membrane proteins. Electron microscopy work was also undertaken to visualize any event occurring during extraction.The antioxidative effect of standardized E extracts was checked in vitro. E safety was also controlled in cell models using polymorphonuclear neutrophils. An E anti-inflammatory effect was then searched in animal models. E was first evaluated using a skin irritation mouse model. Inflammation was induced by benzalkonium chloride on ears of anesthetized mice. Positive and negative controls were treated in parallel. The ear thickness was measured every hour for 5 h and histological ear sections were performed after 2h for some animals. Two different staining methods enabled the enumeration of degranulating mast cells in ear sections.The effect of the bacterial extract was next tested locally by intrarectal (IR) instillations in mice undergoing the early stages of inflammation in a dextran sodium sulfate (DSS)-induced colitis. This acute model evolved over 8 days. In parallel, positive and negative animal controls underwent or not the colitis and were treated or not. Clinical and colonic histological severity scores were daily determined. Inflammation markers were measured in mouse colonic tissues after animal autopsy. [...]