Project description:The intestinal microbiota plays a key role in shaping host homeostasis by regulating metabolism, immune responses and behaviour. Its dysregulation has been associated with metabolic, immune and neuropsychiatric disorders and is accompanied by changes in bacterial metabolic regulation. Although proteomic is well suited for analysis of individual microbes, metaproteomic of faecal samples is challenging due to the physical structure of the sample, presence of contaminating host proteins and coexistence of hundreds of species. Furthermore, there is a lack of consensus regarding preparation of faecal samples, as well as downstream bioinformatic analyses following metaproteomic data acquisition. Here we assess sample preparation and data analysis strategies applied to mouse faeces in a typical LC-MS/MS metaproteomic experiment. We show that low speed centrifugation (LSC) of faecal samples leads to high protein identification rates but possibly enriched for a subset of taxa. During database search, two-step search strategies led to dramatic and underestimated accumulation of false positive protein identifications. Regarding taxonomic annotation, the MS-identified peptides of unknown origin were annotated with highest sensitivity and specificity using the Unipept software. Comparison of matching metaproteome and metagenome data revealed a positive correlation between protein and gene abundances. Notably, nearly all functional categories of detected protein groups were differentially abundant in the metaproteome compared to what would be expected from the metagenome, highlighting the need to perform metaproteomic when studying complex microbiome samples.
Project description:RNAseq and LC/MS metabolomics analysis of C. difficile strain 630 grown in BHIS media with 50% (vol/vol) faecal water added, compared with control BHIS containing only the additional PBS used for prep of Faecal water. Cells grown in biological triplicates to late log phase (T=6h) prior to harvest. Goal was to determine changes in gene expression caused by exposure to Faecal water, and changes in the metabolite profile of faecal water containing medium when incubated with actively growing C. difficile cells
Project description:In this randomised placebo-controlled trial, irritable bowel syndrome (IBS) patients were treated with faecal material from a healthy donor (n=8, allogenic FMT) or with their own faecal microbiota (n=8, autologous FMT). The faecal transplant was administered by whole colonoscopy into the caecum (30 g of stool in 150 ml sterile saline). Two weeks before the FMT (baseline) as well as two and eight weeks after the FMT, the participants underwent a sigmoidoscopy, and biopsies were collected at a standardised location (20-25 cm from the anal verge at the crossing with the arteria iliaca communis) from an uncleansed sigmoid. In patients treated with allogenic FMT, predominantly immune response-related genes sets were induced, with the strongest response two weeks after FMT. In patients treated with autologous FMT, predominantly metabolism-related gene sets were affected.
Project description:Comparison of faecal flora of three healthy individuals and a patient suffering from Ulcerative Colitis during disease and remission states. Faecal samples were taken and frozen at -80 within one hour.
