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:The intestine is a site of diverse functions including digestion, nutrient absorption, immune surveillance, and microbial symbiosis. As such, intestinal homeostasis is vital for overall wellbeing. Faecal microRNAs (miRNAs) offer valuable non-invasive insights into the transcriptional state of the intestine. However, typical faecal miRNA yields and profiles remain incompletely characterised. Here, we develop an optimised protocol for faecal miRNA detection, and describe a reproducible murine faecal miRNA profile across several studies by performing a meta-analysis. By examining faecal miRNA changes during chronic infection with the gastrointestinal helminth, Trichuris muris, we identify the altered expression of miRNAs associated with fibrosis, barrier integrity and wound healing. Fibrosis was confirmed in vivo, suggesting a role for these miRNAs in regulating wound healing during chronic infection where the production of classical wound healing Th2 cytokines are low. Further implementations of this technique can identify novel hypotheses and therapeutic strategies in diverse disease contexts.
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