No description available.

The gut microbiota play a significant role in human health and nutrition, although the mechanisms these organisms use to survive in this densely populated environment are not well understood. Bacteroides thetaiotaomicron is a dominant member of the gut bacterial community whose genome sequence reveals large expansions in protein families involved in the sensing, acquisition and utilisation of complex carbohydrates, pointing to the ability to access a wide range of glycans as playing a significant role in becoming a successful resident of the human gut. Here we have characterised components of the B. thetaiotaomicron polysaccharide utilisation apparatus at molecular level, focusing mainly on fructan sensing, binding and degradation systems. Microarray data from our collaborators revealed that growth of B. thetaiotaomicron on inulin (β-2,1-linked fructan) specifically upregulated a locus of nine genes BT1757-BT1765 and an orphan gene BT3082, encoding a glycoside hydrolase from family 32 (GH32). The locus contains three other GH32s, two predicted polysaccharide binding outer membrane proteins (SusC and SusD homologues), a fructokinase, and an inner membrane monosaccharide transporter. Together these components form a polysaccharide utilisation locus (PUL). The nearest regulatory gene to the PUL is BT1754, a hybrid two component system. Here we show that the periplasmic sensor domain of BT1754 (BT1754peri) binds specifically to fructose, with a Kd of ~2 μM and a stoichiometry of 1:1, but not fructooligosaccharides or other monosaccharides. The crystal structure of BT1754peri revealed a two domain periplasmic binding protein (PBP)-fold with the ligand fructose sandwiched between the two domains. BT1754 is the first periplasmic sensor histidine kinase domain to display a non-PAS fold. The structure in combination with biophysical and site-directed mutagenesis studies also shows how the protein displays such specificity in ligand recognition and provides insights into the mechanism of signal transduction across the inner membrane. The four glycoside hydrolase family 32 members regulated by BT1754 were also biochemically and structurally characterised in this thesis. Three of four GH32 enzymes BT1759, BT1765 and BT3082, digest both β-2,1-linked (inulin) and β-2,6-linked (levan) fructans, indicating that levan is also utilised by the same locus. BT1759 and BT3082 are exo-acting enzymes releasing fructose from both long-chain and short-chain inulins and levans, while BT1765 is also exo-acting and produces fructose, but preferred short-chain sugars. BT1760 is unusual for GH32 as it is specific for levan and has an endo-like activity, releasing a range of different size oligosaccharides from the polysaccharide. The crystal structures of wild type BT3082 and a nucleophile mutant in complex with substrate (kestose) were solved to 2.2 Å and revealed a typical GH32 β-propeller fold as the catalytic domain. Like all other GH32s solved to date, the enzyme has a C-terminal β-sheet domain of unknown function that was shown to be necessary for correct folding of the enzyme. BT3082 also has a unique N-terminal β-sheet domain that was shown to be essential for enzyme activity but not correct folding. Extensive site-directed mutagenesis was carried out to provide insight into the relative importance of different residues in substrate binding and catalysis in BT3082. The crystal structure of wild type BT1760 was solved to 2.6 Å, revealing a surprisingly similar structure to that of the exo-acting enzymes. A rationale for the endo-like activity of this enzyme and the role each of the four GH32s in fructan utilisation by B. thetaiotaomicron is discussed. The outer membrane SusD homologue BT1762 from the fructan locus was shown to bind preferentially to long chain levans, with no recognition of inulin. The crystal structure of BT1762 was solved to 1.9 Å and was shown to share the same novel α-helical fold as SusD. Site-directed mutagenesis of a number of residues in the same region as the SusD binding site showed that while the location of the ligand binding sites are conserved between these two proteins, the identity of the residues involved in polysaccharide recognition are not the same. A model is proposed for levan recognition in BT1762 and also its role in polysaccharide utilisation. B. thetaiotaomicron also has at least 12 ECF sigma/anti-sigma factor gene pairs likely involved in polysaccharide utilisation. Here we show that these systems form a trans-envelope signalling apparatus with their cognate SusC-transducer homologue, but that there is no cross talk between different systems. The significance of this finding in relation to survival of this important gut bacterium is discussed.

No description available.

La réduction du taux de cholestérol (CH) sanguin est un point clé dans la lutte contre les maladies cardiovasculaires. L’efficacité contrastée des médicaments disponibles actuellement ainsi que l’intérêt porté autour du microbiote intestinal dans la régulation de la physiologie de l’hôte nous amènent à envisager cette voie comme alternative thérapeutique. La production de coprostanol (CO), dérivé très peu absorbé du CH, par des bactéries de ce microbiote a été corrélée positivement à une faible cholestérolémie. Les objectifs de cette thèse sont i) d’isoler et d’identifier de nouvelles souches bactériennes ayant cette activité, ii) d’identifier les gènes bactériens responsables de cette transformation et iii) de détereminer l’impact de ce métabolisme sur la physiologie de l’hôte. Nous avons isolé 22 nouvelles souches productrices de CO à partir des selles d’un individu en produisant beaucoup. Nous avons choisi les souches Bacteroides sp. D8 et Bacteroides sp. BV pour la construction de deux banques génomiques et huit autres pour des essais d’implantation in vivo dans le tractus gastro-intestinal (TGI) de souris axéniques. Nous avons identifié 55 clones potentiellement positifs par le criblage fonctionnel des banques génomiques. Leurs analyses supplémentaires devraient nous apporter des informations sur les gènes impliqués dans cette activité. Toutes les bactéries sélectionnées sont capables de coloniser le TGI de la souris axénique. La souche Parabacteroides distasonis est la meilleure souche productrice de CO in vivo. Nous avons testé son effet sur la cholestéolémie chez des souris axéniques soumises à un régime riche en CH sur 11 semaines en comparaison avec une souche non productrice in vitro, B. dorei, et avec des souris conventionnalisées comme contrôle. La souche B. dorei produit du CO in vivo, soulignant l’importance de l’environnement dans l’activité de production de CO déjà supposée d’après la littérature et nos résultats in vitro. Des gènes impliqués dans l’excrétion du CH de l’organisme vers les selles sont surexprimés chez ces souris et celles colonisées avec P. distasonis. Cependant seules ces dernières présentent une cholestérolémie plus faible que les souris conventionnalisées. Le mécanisme impliqué semble indépendant de la production de CO et de l’excrétion de CH car les mêmes quantités de ces composés sont retrouvées dans les selles indépendamment du statut bactérien. Les concentrations en acides biliaires totaux dans la bile et dans les selles sont supérieures pour les souris monocolonisées comparées au conventionnalisées. Les selles des souris colonisées avec P. distasonis présentent plus d’acides urso- et chénodésoxycholiques que les souris conventionnalisées et plus d’acide cholique que les souris colonisées avec B. dorei. En conclusion, nous avons isolé de nouvelles souches et identifier des clones potentiellement positifs. Les études in vivo tendent à montrer que l’activité de production de coprostanol n’a pas d’effet sur la cholestérolémie. En revanche, la souche P. distasonis semble diminuer la cholestérolémie par un mécanisme encore inconnu. / Cholesterol (CH) level management is a keystone to limit cardiovascular diseases. The contrasted efficiency of the drugs currently available as well as the interest around the intestinal microbiota in regulating the host physiology lead us to consider this pathway as a therapeutic alternative. The production of coprostanol (CO), a very poorly absorbed CH derivative, by bacteria of this microbiota has been positively correlated with low CH plasma level. The aims of this thesis are (i) isolate and identify new bacterial strains possessing this activity, (ii) identify the bacterial genes responsible for this transformation and (iii) determine the impact of this metabolism on host physiology. We isolated 22 new strains producing CO from the stools of a high-coprostanol producing individual. We chose Bacteroides sp. D8 and Bacteroides sp. BV for the construction of two genomic libraries and eight others for in vivo implantation tests in the gastrointestinal tract (GIT) of germ-free mice. We identified 55 potentially positive clones by functional screening of these genomic libraries. Their additional analyzes should provide us with information about the genes involved in this activity. All selected bacteria are capable of colonizing the GIT of germ-free mice. Parabacteroides distasonis is the best strain producing CO in vivo. We tested its effect on blood cholesterol level in germ-free mice subjected to an 11-week CH-rich diet compared to an in vitro non-producing strain, B. dorei, and with conventionalized mice as control. The B. dorei strain produces CO in vivo, emphasizing the importance of the environment in the CO production activity already assumed from the literature and our results in vitro. Genes involved in the excretion of CH from body to feces are overexpressed in these mice and those colonized with P. distasonis. However, only the latter have lower cholesterolemia than conventional mice. The mechanism involved appears to be independent of CO production and CH excretion because the same amounts of these compounds are found in feces independently of bacterial status. Total biliary acids concentrations in bile and feces are higher for monocolonized mice compared to conventionalized mice. The feces of mice colonized with P. distasonis exhibited more urso- and chenodeoxycholic acids than conventionalized mice and more cholic acid than mice colonized with B. dorei. In conclusion, we have isolated new strains and identified potentially positive clones. In vivo studies tend to show that coprostanol production activity has no effect on plasma cholesterol. In contrast, P. distasonis seems to decrease plasma cholesterol by a still unknown mechanism.