Project description:Gnotobiotic NMRI mice were colonized at birth with B. thetaiotaomicron. Mice were sacrificed at P17 or P30, cecal contents were harvested and used for transcriptional profiling. Keywords: developmental timepoints, in vivo
Project description:Transcriptional profiling of Bacteroides thetaiotaomicron was performed on samples taken from (i) chemostat cultures (in vitro) and (ii) from the ceca of gnotobiotic monoassociated NMRI mice (in vivo). In vitro profiles were obtained from biological duplicate cultures taken at five timepoints from log to stationary phase in three types of media: (i) minimal media glucose (MM-G), (ii) minimal media maltotriose (MM-M), and (iii) rich media (TYG). In vivo profiling was performed on 12 mice, each colonized with B. thetaiotaomicron for ten days. The in vivo profiling was conducted in two separate experiments: (i) 6 male NMRI mice fed standard polysaccharide rich mouse chow and (ii) 3 male NMRI mice fed standard polysaccharide rich mouse chow and 3 male NMRI mice fed a simple sugar diet containing glucose and sucrose but deficient in polysaccharides. Keywords: other
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.
Project description:Rag1-/- C57BL/6 or Rag1-/- C57BL/6 injected with the IgA producing hybridoma producing 225.4 anti- Bacteroides thetaiotaomicron (Capsular polysaccharide 4 dependent carbohydrate epitope specific epitope) mice were colonized with B. thetaiotaomicron wildtype strain VPI-5482 for 10 days. Cecal bacteria were harvested and snap frozen and RNA isolated. Keywords: Single time point, experimental and control groups.
Project description:Genome wide transcriptional comparison of B. thetaiotaomicron or B. longum mono-association vs. B. thetaiotaomicron bi-association with B. longum. Cecal populations from 10d colonizations with NMRI gnotobiotic mice fed a standard-chow polysaccharide rich (PR) diet (n=5 samples/group) were profiled. Keywords: Mono-association vs. Bi-association, in vivo
Project description:Genome wide transcriptional comparison of B. thetaiotaomicron or B. longum mono-association vs. B. thetaiotaomicron bi-association with B. longum. Cecal populations from 10d colonizations with NMRI gnotobiotic mice fed a standard-chow polysaccharide rich (PR) diet (n=5 samples/group) were profiled. Total RNA was prepared from cecal contents of 10 day associated gnotobiotic mice and hybridized to B. thetaiotaomicron/B. longum GeneChips.
Project description:Comparative proteomics of Bacteroides thetaiotaomicron samples comparing the total membrane (TM) and outer membrane vesicles (OMV) of WT B. thetaiotaomicron and delta 4364
Project description:Purpose: Examining the transcriptome of Bacteroides thetaiotaomicron VPI-5482 challenged with Bacteroides phage to assess surface molecule expression changes Methods: Bacteroides thetaiotaomicron was grown in BPRM in vitro or Germ-Free mice were monocolonized with Bacteroides thetaiotaomicron and gavaged with ARB25 phage. Fold change was calculated as live phage versus heat-killed phage treated samples with n=3 biological replicates. Once cells reached an optical density corresponding to mid-log phase growth (absorbance between 0.4-0.5), RNA was isolated and rRNA depleted. Samples were multiplexed for sequencing on the Illumina HiSeq platform at the University of Michigan Sequencing Core. Data was analyzed using Arraystar software (DNASTAR, Inc.) using DEseq2 normalization with default parameters. Genes with significant up- or down-regulation were determined by the following criteria: genes with an average fold-change >5-fold and with at least 2/3 biological replicates with a normalized expression level >1% of the overall average, and a p-value < 0.05 (t test with Benjamini-Hochberg correction) Results: Specific capsule expression was increased in wild-type B. thetaiotaomicron during phage infection in vitro and in vivo. Many corresponding in vivo genes were upregulated as well as other surface layer proteins.