Project description:Faecalibacterium prausnitzii, a major commensal bacterium in the human gut, is well known for its anti-inflammatory effects, which improve host intestinal health. Although several studies have reported that inulin, a well-known prebiotic, increases the abundance of F. prausnitzii in the intestine, the mechanism underlying this effect remains unclear. In this study, we applied liquid chromatography tandem mass spectrometry (LC-MS/MS)-based multi-omics approaches to identify biological and enzymatic mechanisms of F. prausnitzii involved in the selective digestion of inulin. An LC-MS/MS-based intracellular proteomic and metabolic profiling was performed to determine the quantitative changes in specific proteins and metabolites of F. prausnitzii when grown on inulin. Interestingly, proteomic analysis revealed that the putative proteins involved in inulin-type fructan utilization by F. prausnitzii, particularly b-fructosidase and amylosucrase were upregulated in the presence of inulin. To investigate the function of these proteins, we overexpressed bfrA and ams, genes encoding beta-fructosidase and amylosucrase, respectively, in Escherichia coli, and observed their ability to degrade fructan. In addition, the enzyme activity assay demonstrated that intracellular fructan hydrolases degrade the inulin-type fructans taken up by fructan ATP-binding cassette transporters. Furthermore, we showed that the fructose uptake activity of F. prausnitzii was enhanced by the fructose phosphotransferase system transporter when inulin was used as a carbon source.
Project description:ObjectiveContrary to the long-standing prerequisite of inducing selective (i.e. bifidogenic) effects, recent findings suggest that prebiotic interventions lead to ecosystem-wide microbiota shifts. Yet, a comprehensive characterization of this process is still lacking. Here, we apply 16S rDNA microbiota profiling and matching (GC-MS) metabolomics to assess the consequences of inulin fermentation both on the composition of the colon bacterial ecosystem and fecal metabolites profiles.DesignFecal samples collected during a double blind, randomized, cross-over intervention study (NCT02548247) set up to assess the effect of inulin consumption on stool frequency in healthy adults with mild constipation were analyzed. Fecal microbiota composition and metabolite profiles were linked to the study’s clinical outcome as well as to quality-of-life measurements recorded.ResultsWhile fecal metabolite profiles were not significantly altered by inulin consumption, our analyses did detect a modest effect on global microbiota composition. At the same time, specific inulin-induced changes in relative abundances of Anaerostipes, Bilophila, and Bifidobacterium were identified. The observed decrease in Bilophila abundances following inulin consumption was associated with both softer stools and a favorable change in constipation-specific quality of life measures.ConclusionsEcosystem-wide analysis of the effect of a dietary intervention with prebiotic inulin-type fructans on the colon microbiota revealed that this effect is specifically associated to three genera, one of which (Bilophila) representing a promising novel target for mechanistic research.
Project description:It has been shown in vitro that only specific dietary-fibers contribute to immunity but studies in vivo are not conclusive. Here we investigated degree of polymerization (DP) dependent effects of β2→1-fructans on immunity via microbiota-dependent and -independent effects. To this end, conventional or germ-free mice received short- or long-chain β2→1-fructan for 5 days. Immune cell populations in the spleen, mesenteric lymph nodes (MLN), and Peyer's patches (PPs) were analyzed with flow cytometry, genome-wide gene expression in the ileum was measured with microarray, and gut microbiota composition was analyzed with 16S rRNA sequencing of fecal samples. We found that β2→1-fructans modulated immunity by both microbiota and microbiota-independent effects. Moreover, effects were dependent on the chain-length of the β2→1-fructans type polymer. Both short- and long-chain β2→1-fructans enhanced T-helper 1 cells in Peyer's patches, whereas only short-chain β2→1-fructans increased regulatory T cells and CD11b-CD103- DCs in the MLN. A common feature after short- and long-chain β2→1-fructan treatment was enhanced Fut2 expression and other IL-22-dependent genes in the ileum of conventional mice. These effects were not associated with shifts in gut microbiota composition, or altered production of short-chain fatty acids. Both short- and long-chain β2→1-fructans also induced immune effects in germ-free animals, demonstrating direct effect independent from the gut microbiota. Also, these effects were dependent on the chain-length of the β2→1-fructans. Short-chain β2→1-fructan induced lower CD80 expression by CD11b-CD103- DCs in PPs, whereas long-chain β2→1-fructan specifically modulated B cell responses in germ-free mice. In conclusion, support of immunity is determined by the chemical structure of β2→1-fructans and is partially microbiota-independent.
Project description:Abdominal and pelvic radiotherapy (RT) reduces the renewal capacity of the epithelium. Rectal biopsies obtained from patients receiving pelvic RT have revealed atrophy of surface epithelium, acute cryptitis, crypt abscesses, crypt distortion and atrophy, and stromal inflammation. Modifications in intestinal microbiota, such as an increase in the number of pathogens, may contribute to intestinal injury. The prebiotic effect of a carbohydrate is assessed by its capacity to stimulate the proliferation of healthy bacteria (Bifidobacterium, Lactobacillus) rather than pathogenic bacteria (Clostridium, E. coli).
The hypothesis of the study is that a mixture of inulin and fructooligosaccharide could modulate Lactobacillus and Bifidobacterium and reduce the intestinal injury in patients affected of gynaecological cancer and treated with abdominal radiotherapy.
Project description:Our study in mice investigated the potential effects of the prebiotics GOS and inulin administrated during gestation on the development of DSS-induced chronic colitis in the offspring. Mothers were given or not during gestation a diet enriched in the prebiotics GOS and inulin. Eight to ten weeks old male offspring were treated or not with 3 cycles of DSS in drinking water, one cycle consisting of 2 days with DSS and 5 days without DSS.
Project description:MicroRNA (miRNA) profiles vary with the nutritional and pathological conditions of cattle. In this study, we aimed to investigate the effects of inulin supplement on miRNA profiles derived from serum extracellular vesicles (EVs). Our goal was to determine the differences in miRNA expressions and analyse the pathways in which they are involved. Based on the results of California mastitis test and milk somatic cell counts, ten lactating cows with subclinical mastitis were randomly divided into two groups: an inulin group and a control group (n = 5 in each group). The inulin group received a daily supplement of 300 g of inulin while the control group did not receive any supplementation. After a 5-week treatment period, serum-derived EV-miRNAs from each cow were isolated. High-throughput sequencing was conducted to identify differentially expressed miRNAs. GO and KEGG bioinformatics analysis was performed to examine the target genes of these differentially expressed miRNAs. The EV-RNA concentration and small RNA content were not affected by the inulin treatment. A total of 162 known miRNAs and 180 novel miRNAs were identified from 10 samples in the two groups. Among the known miRNAs, 23 miRNAs were found to be differentially expressed between the two groups, with 18 upregulated and five downregulated in the inulin group compared to the control group. Pathway analysis revealed the involvement of these differentially expressed miRNAs in the regulation of cell structure and function, lipid oxidation and metabolism, immunity and inflammation, as well as digestion and absorption of nutrients. Overall, our study provides a molecular-level explanation for the reported beneficial health effects of inulin supplementation in cows with subclinical mastitis.
Project description:Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide (FOS)/inulin utilisation genes induced during growth on inulin included one encoding a b-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, while a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis demonstrated that the b-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked PTS II transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the utilization of inulin. The R. inulinivorans B-fructofuranosidase was over-expressed in E. coli and shown to hydrolyse fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosacharides. Genes induced on starch included the major extra-cellular a-amylase and two distinct a-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles.
Project description:Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide (FOS)/inulin utilisation genes induced during growth on inulin included one encoding a b-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, while a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis demonstrated that the b-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked PTS II transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the utilization of inulin. The R. inulinivorans B-fructofuranosidase was over-expressed in E. coli and shown to hydrolyse fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosacharides. Genes induced on starch included the major extra-cellular a-amylase and two distinct a-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles. RNA was purified from mid-exponential phase (OD650 = 0.4) cultures of R. inulinivorans grown on basal YCFA supplemented with a single substrate of either starch or inulin using the RNeasy RNA purification kit (Qiagen), and the mRNA component enriched using the MICROBExpress system (Ambion). The purified RNA (1 ug) was labelled by reverse transcription (Amersham), employing random nonamer extension incorporating either dCTP-Cy3 or dCTP-Cy5 dyes. In order to ensure reproducibility, and to obtain statistically significant results, the dye labelling was swapped for a second hybridisation. RNA purified from a separate biological replicate was labelled and hybridised twice in the same way.