Mice cecum and faeces gut metagenome
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA686640 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13277300.fastq.gz | Fastqsanger.gz | |||
| SRR13277301.fastq.gz | Fastqsanger.gz | |||
| SRR13277302.fastq.gz | Fastqsanger.gz | |||
| SRR13277303.fastq.gz | Fastqsanger.gz | |||
| SRR13277304.fastq.gz | Fastqsanger.gz |
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Project description:A Metaproteomic Workflow for Sample Preparation and Data Analysis Applied to Mouse Faeces: 1 MTD project_description Many diseases have been associated with gut microbiome abnormalities. The root cause of such diseases is not only due to bacterial dysbiosis, but also to change in bacterial functions, which are best studied by proteomic approaches. Although bacterial proteomics is well established, metaproteomics is hindered by challenges associated with the sample physical structure, contaminating proteins, the simultaneous analysis of hundreds of species and the subsequent data analysis. Here, we present a systematic assessment of sample preparation and data analysis methodologies applied to LC-MS/MS metaproteomics experiment. We could show that low speed centrifugation (LSC) has a significant impact on both peptide identifications and reproducibility. LSC led to increase in peptide and proteins identifications compare to no LSC. Notably, the dominant bacterial phyla, i.e. Firmicutes and Bacteroidetes, showed divergent representation between LSC and no-LSC. In terms of data processing, protein sequence databases derived from mouse faeces metagenome provided at least four times more MS/MS identification compared to databases of concatenated single organisms. We also demonstrated that two-steps database search strategy comes at the expense of a dramatic rise in number of false positives compared to single-step strategy. Overall, we found a positive correlation between matching metaproteome and metagenome abundance, which could be linked to core microbial functions, such as glycolysis-gluconeogenesis, citrate cycle and carbon metabolism. We observed significant overlap and correlation at the phylum, class, order and family taxonomic levels between taxonomy-derived from metagenome and metaproteome. Notably, nearly all functional categories (e.g., membrane transport, translation, transcription) were differentially abundant in the metaproteome (activity) compared to what would be expected from the metagenome (potential). In conclusion, these results highlight the need to perform metaproteomics when studying complex microbiome samples.
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Project description:The large-scale application of genomic and metagenomic sequencing technologies has yielded a number of insights about the metabolic potential of symbiotic human gut microbes. Bacteria that colonize the mucosal layer that overlies the gut epithelium have access to highly-sulfated polysaccharides (i.e., mucin oligosaccharides and glycosaminoglycans), which they could potentially forage as nutrient sources. To be active, sulfatases must undergo a critical post-translational modification catalyzed in anaerobic bacteria by the AdoMet enzyme anSME (anaerobic Sulfatase-Maturating Enzyme). In the present study, we have tested the role of this pathway in the prominent gut symbiont Bacteroides thetaiotaomicron, which possesses more predicted sulfatases (28) than in the human genome and a single predicted anSME. In vitro studies revealed that deletion of its anSME (BT0238) results in loss of sulfatase activity and impaired ability to use sulfated polysaccharides as carbon sources. Co-colonization of germ-free animals with both isogenic strains, or invasion experiments involving the introduction of one then the other strain, established that anSME activity and the sulfatases that are activated via this pathway, are important fitness factors for B. thetaiotaomicron, especially when mice are fed a simple sugar diet that requires this saccharolytic bacterium to adaptively forage on host glycans as nutrients. Whole genome transcriptional profiling of wild-type and the anSME mutant in vivo revealed that loss of this enzyme alters expression of genes involved in mucin utilization and that this disrupted ability to access mucosal glycans likely underlies the observed dramatic colonization defect. Comparative genomic analysis reveals that 100% of 46 fully sequenced human gut Bacteroidetes contain homologs of BT0238 and genes encoding sulfatases, suggesting that this is an important and evolutionarily conserved feature. Three replicate samples from 4 different biological treatment groups: 1. Wild-type B. thetaiotaomicron from the cecum of gnotobiotic mice fed a simple-sugar diet; 2. chuR mutant B. thetaiotaomicron from the cecum of gnotobiotic mice fed a simple-sugar diet; 3. Wild-type B. thetaiotaomicron from the cecum of gnotobiotic mice fed a plant-rich diet; 4. chuR mutant B. thetaiotaomicron from the cecum of gnotobiotic mice fed a plant-rich diet.
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Project description:One week after completing the regulatory acclimation period, mice received daily oral gavages with PBS, EcN pCC29, EcN pCC31, and EcN pCC35 and Spermidine treatments for 14 days. The dose administered was 10^8 CFU per day for a probiotic group or 150uL of 0.1M Spermidine solution. In order to evaluate if differences in metabolite levels existed between the conditions we presented, we studied by LC-MS/MS the metabolic signature of stool or cecum of mice from the different groups. For this purpose, individual collection of faeces was performed. These samples were stored in ice before being resuspended in PBS following the following ratio: 10uL of PBS for 1mg of weighed material.The resuspended samples were then vortexed for 1min, before being incubated in ice for 5min. This last step was repeated 3 times.After resuspending and vortexing all the samples, each tube was centrifuged at 10,000g for 10min and the supernatant was collected and filtered with a 0.22um filter. These supernatants were then stored at -80C before finally being prepared for acquisition on the mass spectrometer.
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