6 research outputs found

    ENZYMOLOGY AND MOLECULAR BIOLOGY OF BILE ACID 7alpha- AND 7beta- DEHYDROXYLATION BY THE INTESTINAL BACTERIA CLOSTRIDIUM SCINDENS AND CLOSTRIDIUM HYLEMONAE

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    The collective microbial genomes within our gut(microbiome) represent a powerful metabolic force, leading many authors to call our GI flora an organ within an organ , and the metagenomic sequencing of our microbiome, the second human genome project . Bile acids, endogenously produced by the host liver, represent both a strong selective pressure for potential colonizers, aswell as substrates for microbial metabolism. Indeed, microbes have evolved enzymes to deconjugate bile salts, epimerize bile acid hydroxyl groups, and 7alpha-dehydroxylateprimary bile acids. The products of microbial 7alpha-dehydroxylation, secondary bile acids, are suggested by numerous lines of evidence to be involved in promoting colon carcinogenesis. 7alpha-dehydroxylating activity is a multi-step pathway, genes of which have only been identified in a small number of organisms within the genusClostridium. The biochemistry of this pathway has been largely worked out. The third step in the pathway is introduction of a delta-4-double bond; however, the gene product(s) responsible have not been identified. The baiCD and baiH genes were cloned, expressed and shown to have NAD-dependent 3-oxo-delta-4-steroid oxidoreductase activity showing stereospecificity for 7alpha-hydroxy and 7beta-hydroxy bile acid, respectively.In addition, bai genes were isolated from C.hylemonae TN271 by bidirectional genome-walking by PCR. This represents the first report of bai genes from a low activity 7alpha-dehydroxylating bacterium. The gene organization and sequence of the baiBCDEFGHI operon was highly conserved between C. hylemonae TN271 and the high activity 7alpha-dehydroxylating bacterium C. scindens VPI12708. The baiA gene was located by PCR using degenerate oligonucleotides. Bi-directional genome-walking revealed what appears to be several novel genes involved in bile acid metabolism which were also located in C. scindens VPI 12708. Expression of a 62 kDa flavoprotein and reactionwith [24-14C] 3-oxo-DCA and NADP resulted in a product of greater hydrophilicity than deoxycholic acid. The identity of this product was not determined. A second gene appears to share a common evolutionary origin with the baiF gene. A hypothesis is offered regarding the function of these homologues as Type III CoA transferasesrecognizing 5alpha-bile acids, or 5beta-bile acids (allo-bile acids). A third gene encodes a putative short chain reductase, similar in size and predicted function to the baiA gene, which may be involved in the final reductive step in the pathway. These novel genes also contained a conserved upstream regulatory region with the baioxidative genes. Finally, two genes were identified which may serve as potential drug targets to inhibit bile acid 7alpha-dehydroxylation. The first is an ABC transporter which may be co-transcribed with the other novel bile acid metabolizing genes, and what appears to be a bile acid sensor/regulator similar to the Tryptophan-rich sensory protein (TspO)/mitochondrial peripheral benzodiazepinereceptor (MBR) family of proteins

    Characterization of enzymatic pathways involved in cortisol and bile acid metabolism by the gut microbiota

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    The gut microbiota consists of a complex network of distinct bacterial taxa that together affect the physiology of the human host. Of the many facets affected by gut microbiota, their impact on the endocrine system is both significant and understudied. The diversity observed in microbial isolates and operational taxonomic units from one human gut to another is due largely in part by the strain specific proteins expressed by the microbes that inhabit them. Some of these unique proteins have evolved to biotransform host sterols which can be reabsorbed by the body and potentially affect endocrine functions. Bacterial hydroxysteroid dehydrogenases (HSDHs) have the potential to significantly alter the physicochemical properties of bile acids with implications for increased/decreased toxicity for gut bacteria and the host. We located a gene-cluster in Eggerthella CAG:298 predicted to encode three HSDHs (CDD59473, CDD59474, CDD59475), synthesized the genes for heterologous expression in E. coli and then screened bile acid substrates against the purified recombinant enzymes, revealing novel 3-, 3-, and 12-HSDHs. We also developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase activity from recombinant enzymes encoded by the desA and desB genes from Clostridium scindens ATCC 35704. Steroid-17,20-desmolase is responsible for the side chain cleavage that biotransforms cortisol into 11β-hydroxyandrostenedione (11β-OHAD). The reaction performed by the steroid-17,20-desmolase appears to be regulated via pyridine nucleotide-dependent HSDHs, which are capable of converting cortisol into a form unable to be utilized by steroid-17,20-desmolase. A recently identified 20β-HSDH from Bifidobacterium adolescentis strain L2-32 has been biochemically characterized and crystallized. This 20β-HSDH has the potential to directly affect androgen formation by functioning as a metabolic rheostat controlling the steroid-17,20-desmolase activity of Clostridium scindens ATCC 35704

    A Gnotobiotic Mouse Model for Studying the Effect of Human Gut Community Ecology on a Pathobiont, Bacteroides fragilis

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    ABSTRACT OF THE DISSERTATION A Gnotobiotic Mouse Model for Studying the Effect of Human Gut Community Ecology on a Pathobiont, Bacteroides fragilis by Vitas Wagner Doctor of Philosophy in Biology and Biomedical Sciences Evolution, Ecology, and Population Biology Washington University in St. Louis, 2015 Professor Jeffrey I. Gordon, Chair Childhood undernutrition represents a pressing global health challenge. Epidemiologic studies have shown that undernutrition is not due to food insecurity alone, but rather represents a complex set of interactions between intra- and intergenerational factors. The gut microbiota has been implicated as one such factor. This thesis tested the hypothesis that enteropathogen burden affects the structure and expressed functions of the gut microbiota, and reciprocally, the gut microbiota affects susceptibility to the effects of enteropathogen invasion. To examine this hypothesis, groups of adult germ-free C57Bl/6 mice were colonized with fecal microbiota sampled from two 24 month-old members of a birth-cohort living in an urban slum in the Mirpur district of Dhaka, Bangladesh: one child had a healthy growth phenotype as judged by anthropometry, and the undernourished child was severely stunted and underweight and exhibited relative microbiota immaturity. The microbiota of both children contained Bacteroides fragilis, a pathobiont. Both groups of colonized mice were fed three diets that embodied the diets consumed by the population from which the microbiota donors were selected. Mice harboring the intact uncultured microbiota from the stunted donor exhibited severe weight-loss, while those receiving the healthy donor’s microbiota maintained weight on these diets. Clonally-arrayed, sequenced collections of cultured anaerobic bacteria strains, generated from the donors’ fecal microbiota, transmitted (i) the discordant weight phenotypes within and across generations of animals (in a diet-dependent fashion), as well as (ii) distinct host metabolic phenotypes (manifest by marked differences in tissue organic acid, amino acid and ceramide profiles as defined by mass spectrometry). The B. fragilis strain in the stunted donor’s culture collection was enterotoxigenic (ETBF), while the two B. fragilis strains in the healthy donor’s culture collection were non-toxigenic (NTBF). Through a series of experiments in which mice were colonized with either the stunted or healthy culture collection ± ETBF or ± NTBF, I demonstrated that ETBF was associated with weight loss as a member of the stunted donor’s community, but not the healthy donor’s community. Microbial RNA-Seq analysis revealed marked differences in ETBF gene expression in the two different community contexts, and as a function of the presence or absence of NTBF. Strikingly, ETBF induced expression of a large repertoire of virulence factor genes encoded in the genomes of the healthy culture collection members; these effects were mitigated when NTBF was present. The effects of ETBF on host metabolism were also community context-dependent. These results provide preclinical evidence that enteropathogen effects on host physiology and metabolism are greatly impacted by gut community ecology and illustrate the value of combining gnotobiotic mouse models, human diet embodiments, and ‘personal’ culture collections for dissecting microbial-microbial and microbial-host interactions. A parallel study using gnotobiotic mice and subsets of the culture collection from the healthy donor revealed how turmeric, a culturally relevant spice in the Bangladeshi diet, and microbial bile acid production/metabolism interact to impact gut motility

    Etude des interactions bidirectionnelles entre le microbiote intestinal et les récepteurs aux xénobiotiques CAR et PXR

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    Le pregnane X receptor (PXR, NR1I2) et le récepteur constitutif aux androstanes (CAR, NR1I3) sont deux récepteurs nucléaires hépatiques et intestinaux qui régulent la transcription d'enzymes de détoxification des xénobiotiques. Des travaux antérieurs ont montré que l'expression des gènes cibles de CAR et PXR est significativement réduite dans le foie des souris axéniques. Dans ce projet de thèse, nous avions pour objectif de mieux comprendre les interactions bidirectionnelles entre le microbiote intestinal et ces xénosenseurs. Nous avons d'abord utilisé une approche pharmacologique chez les souris mâles WT vs Pxr-/- et comparé la signature transcriptomique des gènes régulés par PXR dans le foie lors de l'activation via le PCN. L'activation de PXR a augmenté l'accumulation de triglycérides hépatiques. Nous avons observé un chevauchement significatif entre les gènes régulés négativement lors de l'activation de PXR et une liste de gènes cibles de PPARδ induits par le jeûne. Parmi ceux-ci, nous avons identifié le facteur de croissance de fibroblastes 21 (Fgf21) comme un nouveau gène régulé par PXR. L'activation de PXR a aboli les taux plasmatiques de FGF21. Ces premiers résultats ont fourni une signature complète de l'activation de PXR dans le foie et ont identifié de nouveaux gènes cibles potentiellement impliqués dans les effets stéatogènes et pléiotropes de PXR. Ensuite, nous avons comparé la signature hépatique à la signature intestinale de l'activation pharmacologique de PXR, ce qui nous a permis d'identifier les gènes cibles communs de PXR dans ces 2 organes. Enfin, nous avons utilisé des souris Pxr+/+ et Pxr-/- littermate et supprimé le microbiote intestinal au moyen d'antibiotiques (ATB). En utilisant les gènes cibles de PXR identifiés précédemment, nous avons confirmé que les ATB réduisaient de manière significative l'activité de PXR dans le foie et l'iléon. Des analyses transcriptomiques hépatiques ont montré que les ATB diminuaient un nombre beaucoup plus élevé de gènes PXR-dépendants dans le foie des souris mâles que chez les femelles. Chez les mâles, l'axe microbiote intestinal-PXR contrôlait le métabolisme des xénobiotiques et le remodelage des lipides hépatiques. À l'inverse, le séquençage 16S et la métabolomique par RMN du contenu caecal ont révélé des différences subtiles mais significatives dans la composition du microbiote intestinal des souris Pxr-/- par rapport aux souris Pxr+/+, uniquement chez les mâles. Nos résultats démontrent donc que, dans le foie, PXR est un senseur majeur du microbiote intestinal qui contrôle les capacités de détoxication de l'hôte et le métabolisme des lipides de manière sexuellement dimorphique. Dans le dernier chapitre, nous avons étudié les interactions microbiote-CAR. Chez les souris Car+/+ et Car-/- littermates, la suppression du microbiote par les antibiotiques a diminué l'activité de CAR dans le foie et l'iléon des mâles, mais uniquement dans le foie des femelles. Dans le contenu caecal, le séquençage 16S et la metabolomique ont montré une différence significative dans la composition et l'activité métabolique du microbiote intestinal chez les souris Car+/+ vs Car-/- mâles, mais pas chez les femelles. Nous avons cherché les conséquences potentielles de cette dysbiose CAR dépendante et avons observé que la délétion de CAR augmentait l'accumulation de tissu adipeux chez les souris mâles à 37 semaines. Cependant, l'implication du microbiote CAR-dépendant dans ce phénotype reste à vérifier. Ainsi, nos résultats montrent pour la première fois que l'interaction CAR-microbiote est sexuellement dimorphique et pourrait contrôler le dépôt adipeux chez les souris mâles. Dans l'ensemble, nos résultats montrent que le dialogue entre le microbiote intestinal et les récepteurs aux xénobiotiques CAR et PXR est impliqué de façon sexuellement dimorphique dans le contrôle des capacités de détoxification de l'hôte, et joue un rôle dans l'homéostasie lipidique.The pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3) are two liver and intestine-enriched nuclear receptors that act as transcriptional regulators of enzymes critical for the detoxification of xenobiotics and endogenous metabolites. Previous works have shown that the expression of CAR and PXR target genes is significantly reduced in the liver of germ-free mice. In this PhD project, we aimed to gain insights into the bidirectional interactions between the gut microbiota and these xenosensors. We first used a pharmacological approach in WT vs Pxr-/- male mice and performed a transcriptomic comparison of the PXR-regulated genes in the liver upon activation via the rodent activator PCN. We confirmed that PXR activation increased liver triglycerideaccumulation and significantly regulated the expression of genes, mostly involved inxenobiotic metabolism. We also highlighted a significant overlap between the genes downregulated upon PXR activation and a list of fasting-induced PPARδ target genes. Among these, we identified the well-described PPARδ target fibroblast growth factor 21 (Fgf21) as a new PXR-regulated gene. PXR activation abolished plasmatic levels of FGF21. This first set of results provided a comprehensive signature of PXR activation in the liver and identified new PXR target genes that might be involved in the steatogenic and pleiotropic effects of PXR. Next, we compared the hepatic vs. intestinal signature of the pharmacological activation of PXR. This allowed us to unravel the strongest PXR target genes in both organs. Finally, we used Pxr+/+ and Pxr-/- littermate mice and suppressed the gut microbiota using antibiotics (ATB). Using the previously identified PXR targets, we confirmed that ATB significantly decreased Pxr activity in the liver and ileum. Liver transcriptomic analyses showed that ATB decreased a much higher number of PXR-dependent genes in the liver of male mice than in females. In males, this gut microbiota-PXR axis controlled xenobiotic metabolism and lipid remodelling. Conversely, 16S sequencing and 1H-NMR-based metabolic profiling of caecal content revealed subtle but significant differences in the gut microbiota composition of male Pxr-/- vs. Pxr+/+ mice, while no difference was observed in females. Our results therefore demonstrate that hepatic PXR is a major sensor of the gut microbiota that controls the host detoxifying capacities and lipid metabolism in a sexually dimorphic way. In the final chapter, we investigated the microbiota-CAR interactions. In Car+/+ and Car-/- littermate mice. Microbiota suppression by antibiotics decreased CAR activity in the liver and ileum of males but only in the liver of females. In caecal content, male-specific and CAR-dependant metabolites were also detected through 1H-NMR-based metabolomics. Furthermore, 16S sequencing confirmed a significant difference in gut microbiota composition of Car-/- vs Car+/+ male mice but not in females. We investigated the potential consequences of this sexually dimorphic CAR-dependent dysbiosis and observed that long-term Car deletion increased adipose tissue accumulation in male mice (at 37 weeks old). Whether the Car-dependent dysbiosis is responsible for this phenotype remains to be determined. In 37-week-old females, Car deletion induced a significant increase in spleen weight and a decrease in colon length, therefore suggesting a role for Car in systemic and intestinal inflammation. Thus, our result show for the first time that the CAR-gut microbiota interaction is sexually dimorphic and might control adipose deposition in male mice. Overall, our results shed new light into the crosstalk between the gut microbiota and the host's xenobiotic receptors CAR and PXR, demonstrating that this cross-talk might be involved in the control the host's hepatic lipid and xenobiotic metabolism
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