The Glycolytic Versatility of Bacteroides uniformis CECT 7771 and Its Genome Response to Oligo and Polysaccharides.
ABSTRACT: Bacteroides spp. are dominant components of the phylum Bacteroidetes in the gut microbiota and prosper in glycan enriched environments. However, knowledge of the machinery of specific species isolated from humans (like Bacteroides uniformis) contributing to the utilization of dietary and endogenous sources of glycans and their byproducts is limited. We have used the cutting-edge nanopore-based technology to sequence the genome of B. uniformis CECT 7771, a human symbiont with a proven pre-clinical efficacy on metabolic and immune dysfunctions in obesity animal models. We have also used massive sequencing approaches to distinguish the genome expression patterns in response to carbon sources of different complexity during growth. At genome-wide level, our analyses globally demonstrate that B. uniformis strains exhibit an expanded glycolytic capability when compared with other Bacteroides species. Moreover, by studying the growth and whole-genome expression of B. uniformis CECT 7771 in response to different carbon sources, we detected a differential growth fitness and expression patterns across the genome depending on the carbon source of the culture media. The dietary fibers used exerted different effects on B. uniformis CECT 7771 activating different molecular pathways and, therefore, allowing the production of different metabolite types with potential impact on gut health. The genome and transcriptome analysis of B. uniformis CECT 7771, in response to different carbon sources, shows its high versatility to utilize both dietary and endogenous glycans along with the production of potentially beneficial end products for both the bacterium and the host, pointing to a mechanistic basis of a mutualistic relationship.
Project description:<h4>Background</h4>Associations have been made between obesity and reduced intestinal numbers of members of the phylum Bacteroidetes, but there is no direct evidence of the role these bacteria play in obesity. Herein, the effects of Bacteroides uniformis CECT 7771 on obesity-related metabolic and immune alterations have been evaluated.<h4>Methods and findings</h4>Adult (6-8 week) male wild-type C57BL-6 mice were fed a standard diet or a high-fat-diet HFD to induce obesity, supplemented or not with B. uniformis CECT 7771 for seven weeks. Animal weight was monitored and histologic, biochemical, immunocompetent cell functions, and features of the faecal microbiota were analysed after intervention. The oral administration of B. uniformis CECT 7771 reduced body weight gain, liver steatosis and liver cholesterol and triglyceride concentrations and increased small adipocyte numbers in HFD-fed mice. The strain also reduced serum cholesterol, triglyceride, glucose, insulin and leptin levels, and improved oral tolerance to glucose in HFD fed mice. The bacterial strain also reduced dietary fat absorption, as indicated by the reduced number of fat micelles detected in enterocytes. Moreover, B. uniformis CECT 7771 improved immune defence mechanisms, impaired in obesity. HFD-induced obesity led to a decrease in TNF-? production by peritoneal macrophages stimulated with LPS, conversely, the administration of B. uniformis CECT 7771 increased TNF-? production and phagocytosis. Administering this strain also increased TNF-? production by dendritic cells (DCs) in response to LPS stimulation, which was significantly reduced by HFD. B. uniformis CECT 7771 also restored the capacity of DCs to induce a T-cell proliferation response, which was impaired in obese mice. HFD induced marked changes in gut microbiota composition, which were partially restored by the intervention.<h4>Conclusions</h4>Altogether, the findings indicate that administration of B. uniformis CECT 7771 ameliorates HFD-induced metabolic and immune dysfunction associated with intestinal dysbiosis in obese mice.
Project description:The formulation of next-generation probiotics requires competent preclinical studies to show their efficacy and safety status. This study aims to confirm the safety of the prolonged oral use of <i>Bacteroides uniformis</i> CECT 7771, a strain that protected against metabolic disorders and obesity in preclinical trials, in a sub-chronic 90 day trial in animals. The safety assessment was conducted in male and female Wistar rats (<i>n</i> = 50) administered increasing doses (10<sup>8</sup> CFU/day, 10<sup>9</sup> CFU/day, or 10<sup>10</sup> CFU/day) of <i>B. uniformis</i> CECT 7771, 10<sup>10</sup> CFU/day of <i>B. longum</i> ATCC 15707<sup>T</sup>, which complies with the qualifying presumption of safety (QPS) status of the EU, or vehicle (placebo), as the control. Pancreatic, liver, and kidney functions and cytokine concentrations were analyzed. Bacterial translocation to peripheral tissues was evaluated, and colon integrity was investigated histologically. No adverse metabolic or tissue integrity alterations were associated with treatments; however, alanine aminotransferase levels and the ratio of anti-inflammatory to pro-inflammatory cytokines in serum indicated a potentially beneficial role of <i>B. uniformis</i> CECT 7771 at specific doses. Additionally, the microbial community structure was modified by the interventions, and potentially beneficial gut bacteria were increased. The results indicated that the oral consumption of <i>B. uniformis</i> CECT 7771 during a sub-chronic 90 day study in rats did not raise safety concerns.
Project description:Bacteroides thetaiotaomicron was grown and transcriptionally profiled on a number of different host mucosal glycans and their component mono- and disaccharides. Overall design: Experiments are performed in biological duplicate (2 replicates per growth condition). All cultures were harvested at similar points in mid-log phase of growth in minimal medium plus 0.5% (total amount, w/v) of a defined carbon source. Cultures on individual carbon sources are designed to be referenced to a minimal medium glucose control (GSM302686 and 302791) grown in the same growth format. Note that the hybridization targets used for these two reference datasets are from the exact same prep used for similar targets on the GPL1821 platform (sets GSM301720 and 301721)
Project description:Gene expression profiles of Bacteroides thetaiotaomicron in vitro during growth on host mucosal polysaccharides as sole carbon sources. All substrates in this series are derived from porcine gastric mucin and include mucin O-glycans and glycosaminoglycans. Overall design: Two different culture formats used: 800ml batch-culture bioreactors and 5ml tube cultures (format is indicated within each sample title). Each set of growths was referenced to a minimal medium glucose reference corressponding to the same culture format. Unfractionated porcine mucosal glycan (PMG) growths were compared to previously published in vivo datasets, which were referenced to the 800ml minimal medium glucose reference dataset.
Project description:<i>?-</i>glucans are the dietary nutrients present in oats, barley, algae, and mushrooms. The macromolecules are well known for their immune-modulatory activity; however, how the human gut bacteria digest them is vaguely understood. In this study, <i>Bacteroides uniformis</i> JCM 13288 <sup>T</sup> was found to grow on laminarin, pustulan, and porphyran. We sequenced the genome of the strain, which was about 5.05 megabase pairs and contained 4868 protein-coding genes. On the basis of growth patterns of the bacterium, two putative polysaccharide utilization loci for <i>?-</i>glucans were identified from the genome, and associated four putative genes were cloned, expressed, purified, and characterized. Three glycoside hydrolases (GHs) that were endo-acting enzymes (<i>Bu</i>GH16, <i>Bu</i>GH30, and <i>Bu</i>GH158), and one which was an exo-acting (<i>Bu</i>GH3) enzyme. The <i>Bu</i>GH3, <i>Bu</i>GH16, and <i>Bu</i>GH158 can cleave linear exo/endo- <i>?-</i> 1-3 linkages while <i>Bu</i>GH30 can digest endo- <i>?-</i> 1-6 linkages. <i>Bu</i>GH30 and <i>Bu</i>GH158 were further explored for their roles in digesting <i>?-</i> glucans and generation of oligosaccharides, respectively. The <i>Bu</i>GH30 predominately found to cleave long chain <i>?-</i> 1-6 linked glucans, and obtained final product was gentiobiose. The <i>Bu</i>GH158 used for producing oligosaccharides varying from degree of polymerization 2 to 7 from soluble curdlan. We demonstrated that these oligosaccharides can be utilized by gut bacteria, which either did not grow or poorly grew on laminarin. Thus, <i>B. uniformis</i> JCM 13288 <sup>T</sup> is not only capable of utilizing <i>?-</i>glucans but also shares these glycans with human gut bacteria for potentially maintaining the gut microbial homeostasis.
Project description:Bacteroides thetaiotaomicron was grown and transcriptionally profiled on a number of different host mucosal glycans and their component mono- and disaccharides. Experiments are performed in biological duplicate (2 replicates per growth condition). All cultures were harvested at similar points in mid-log phase of growth in minimal medium plus 0.5% (total amount, w/v) of a defined carbon source. Cultures on individual carbon sources are designed to be referenced to a minimal medium glucose control (GSM302686 and 302791) grown in the same growth format. Note that the hybridization targets used for these two reference datasets are from the exact same prep used for similar targets on the GPL1821 platform (sets GSM301720 and 301721)
Project description:Gene expression profiles of Bacteroides thetaiotaomicron in vitro during growth on host mucosal polysaccharides as sole carbon sources. All substrates in this series are derived from porcine gastric mucin and include mucin O-glycans and glycosaminoglycans. Two different culture formats used: 800ml batch-culture bioreactors and 5ml tube cultures (format is indicated within each sample title). Each set of growths was referenced to a minimal medium glucose reference corressponding to the same culture format. Unfractionated porcine mucosal glycan (PMG) growths were compared to previously published in vivo datasets, which were referenced to the 800ml minimal medium glucose reference dataset.
Project description:Analysis of colonic epithelial cell gene expression in germ-free, Bacteroides uniformis-colonized, and Clostridia-colonized gnotobiotic mice. Bacteria were isolated from our SPF mouse facility and were used to selectively colonize germ-free mice. Germ free mice were left germ free or were colonized with Bacteroides uniformis or a consortium of Clostridia. Total RNA was extraced from colonic epithelial cells.
Project description:Despite accepted health benefits of dietary fiber, little is known about the mechanisms by which fiber deprivation impacts the gut microbiota and alters disease risk. Using a gnotobiotic model, in which mice were colonized with a synthetic human gut microbiota, we elucidated the functional interactions between dietary fiber, the gut microbiota and the colonic mucus barrier, which serves as a primary defence against pathogens. We show that during chronic or intermittent dietary fiber deficiency, the gut microbiota resorts to host-secreted mucus glycoproteins as a nutrient source, leading to erosion of the colonic mucus barrier. Dietary fiber deprivation promoted greater epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium, but only in the presence of a fiber-deprived microbiota that is pushed to degrade the mucus layer. Our work reveals intricate pathways linking diet, gut microbiome and intestinal barrier dysfunction, which could be exploited to improve health using dietary therapeutics. Germ-free mice (Swiss Webster) were colonized with synthetic human gut microbiota comprising of 14 species belonging to five different phyla (names of bacterial species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides caccae, Bacteroides uniformis, Barnesiella intestinihominis, Eubacterium rectale, Marvinbryantia formatexigens, Collinsella aerofaciens, Escherichia coli HS, Clostridium symbiosum, Desulfovibrio piger, Akkermansia muciniphila, Faecalibacterium prausnitzii and Roseburia intestinalis). These mice were fed either a fiber-rich diet or a fiber-free diet for about 6 weeks. The mice were then sacrificed and their cecal tissues were immediately flash frozen for RNA extraction. The extracted RNA was subjected to microarray analysis based on Mouse Gene ST 2.1 strips using the Affy Plus kit. Expression values for each gene were calculated using robust multi-array average (RMA) method. Overall design: Fiber-rich diet group contained 4 replicate mice (2 independent experiments) and Fiber-free diet group contained 3 replicate mice (2 independent experiments)