Project description:We compared gene expression in the small intestine (ileum) of mice that were either (i) germ-free, (ii) colonized with a conventional mouse cecal microbiota, (iii) colonized with a conventional zebrafish gut microbiota, or (iv) colonized with Pseudomonas aeruginosa PAO1. Experiment Overall Design: Adult germ-free NMRI mice were colonized with either (i) a conventional mouse cecal microbiota harvested from adult Swiss-Webster mice (5 biological replicates), (ii) a conventional zebrafish intestinal microbiota harvested from adult C32 zebrafish (3 biological replicates), or (iii) a culture of Pseudomonas aeruginosa PAO1 (5 biological replicates). 14 days after colonization, total RNA was prepared from the ileum of each animal, with total RNA prepared from adult germ-free NMRI mouse ileum serving as negative controls (5 biological replicates). RNA was used as template to generate cRNA for hybridization to Affymetrix 430 v2 Mouse GeneChips.
Project description:Vertebrates are colonized at birth by complex microbial communities (microbiota) that influence diverse aspects of host biology. We have used a functional genomics approach to identify zebrafish genes that are differentially expressed in response to the microbiota. We assessed RNA expression profiles from zebrafish larvae at 6 days post-fertilization (dpf) that were either raised continuously in the absence of any microorganism (germ-free or GF), or raised GF through 3dpf then colonized with a normal zebrafish microbiota (conventionalized or CONVD). Total RNA was purified from pooled intact zebrafish larvae (28-80 larvae/pool, 3 biological replicate pools/condition) using Trizol reagent (Invitrogen) followed by DNase I digestion (DNA-Free, Ambion) according to manufacturers' protocols. Total RNA from each replicate pool (12ug RNA/replicate) was used as template for independent cDNA synthesis and in vitro transcription reactions (BioArray HighYield RNA Transcript Labeling Kit; Enzo Life Sciences) to generate biotinylated cRNA targets. cRNA targets (20ug/replicate) were fragmented using standard methods. Hybridization and scanning were performed using standard Affymetrix protocol. Raw expression values were normalized (Invariant set method) and modeled (PM-MM model), and present/absent calls were generated using dChip software (build date Dec.11, 2005).
Project description:Vertebrates are colonized at birth by complex microbial communities (microbiota) that influence diverse aspects of host biology. We have used a functional genomics approach to identify zebrafish genes that are differentially expressed in response to the microbiota. We assessed RNA expression profiles from zebrafish larvae at 6 days post-fertilization (dpf) that were either raised continuously in the absence of any microorganism (germ-free or GF), or raised GF through 3dpf then colonized with a normal zebrafish microbiota (conventionalized or CONVD).
Project description:Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. In a healthy physiological state of the gut (eubiosis), E. faecalis is a subdominant species in the intestinal microbiota. When the intestinal homeostasis is disrupted (dysbiosis), it becomes dominant species and can cause infections. The taurocholate bile acid (TCA) becomes prominent during dysbiosis. This experiment aimed to better understand how E. faecalis adapts to TCA. We showed that TCA reprograms genes involved in the pool management of amino acids and nucleotides.
Project description:To characterize the effect of microbiota on global gene expression in the distal small intestine during postnatal gut development we employed mouse models with experimental colonization by intestinal microbiota. Using microarray analysis to assess global gene expression in ileal mucosa at the critical stage of intestinal development /maturation associated with weaning, and asking how expression is affected by microbial colonization In the study presented here, preweaned and postweaned GF, SPF mouse small intestinal total RNAs were used. Also, 3-week-old gnotobiotic mouse as well as GF mouse small intestinal RNAs were used.
Project description:Olfactory systems are one of the most conserved and ancient sensory systems in vertebrates. The vertebrate olfactory epithelium is colonized by complex communities of commensal microorganisms, but their impact on olfactory epithelial development and function remains unknown. Using germ-free zebrafish model, we aim to understand the transcriptional responses that colonization with a microbiota induces in olfactory organs. This study was aimed to understand the changes in gene expression in the olfactory organ of Germ Free (GF) zebrafish compared to conventionalized (ConvD) zebrafish. This experiment is related to E-MTAB-5046 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5046)
Project description:Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Studies with germ-free or gnotobiotic animals represent the gold standard for research on bacterial-host interaction but they are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete murine intestinal microbiota and prove to have significant biologic validity. Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by approximately 400 fold while ensuring the animals’ health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer’s patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. We present a robust protocol for depleting mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion is phenotypic characteristics and epithelial gene expression profile similar to those of germ-free mice. Comparison of genome-wide gene expression of colon intestinal epithelial cells from mice subjected to microbiota depletion protocol against to control mice.
Project description:We performed RNA-seq from 6 days post fertilization hnf4a-/- and hnf4a+/+ zebrafish larval digestive tracts raised in the absence (Germ Free, GF) or presence (Conventionalized, CV) of microbiota. We found that zebrafish hnf4a activates almost half of the microbiota-suppressed genes, indicating that the microbiota supress Hnf4a trans-activity. We also provide evidence suggesting that microbial suppression of Hnf4a may contribute to IBD pathogenesis.
Project description:The human intestinal microbiota associated with rats produces in vivo a soluble(s) factor(s) that down-regulates the expression of genes encoding for the Shiga toxin II in E. coli O157:H7. The Shiga toxin II is one of the major virulence factors of E. coli enterohemorragic leading to the deadly hemolitic and uremic syndrome. Investigation of the effect of the human intestinal microbiota on the whole transcriptome of EHEC O157:H7 is of major importance to increase our understanding of the pathogen transcriptomic adaptation in response to the human microbiota. We analysed by microarray hybridization the gene expression pattern of EHEC O157:H7 grown in the caecal content of germ-free rats or rats associated with the human microbiota of a healthy human subject. By doing so, we increased our understanding of the regulatory activities of the human gut microbiota on E. coli O157:H7 A first group of twelve weeks old, male, germfree rats was colonized with the human fecal microbiota and a second group was kept germfree and condidered as a controle group. Rats were fed for two weeks with a sterile human type diet, and were sacrificed. E. coli O157:H7 was cultivated for 6 hours in the caecal content of germfree rats and rats associated with the human intestinal microbiota. RNAs were extracted and cDNAs were synthesized, fragmented and biotinylated before being hybridized on Affymetrix E. coli genome 2.0 arrays. The effect of the human intestinal microbiota was investigated by comparing the gene expression level in the caecal content of rats associated with the human microbiota with their expression level in the caecal content of the germfree rats.