Project description:The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many of these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs) in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising >90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism’s 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These results offer insight into how gut microbes adapt to dietary perturbations at both a community level and from the perspective of a well-adapted symbiont with exceptional saccharolytic capabilities, and illustrate the value of artificial communities. 611 samples total (221 from experiment 1, 390 from experiment 2). Evaluation of changes in an artificial gut community's structure over time as a result of dietary oscillation.
Project description:Sulfate-reducing bacteria (SRB) colonize the guts of ~50% of humans. We used genome-wide transposon mutagenesis and insertion-site sequencing (INSeq), RNA-Seq, plus mass spectrometry to characterize genetic and environmental factors that impact the niche of Desulfovibrio piger, the most common SRB in a surveyed cohort of healthy USA adults. Gnotobiotic mice were colonized with an assemblage of sequenced human gut bacterial species with or without D. piger and fed diets with different levels and types of carbohydrates and sulfur sources. Diet was a major determinant of functions expressed by this artificial 9-member community and of the genes that impact D. piger fitness; the latter includes high- and low-affinity systems for utilizing ammonia, a limiting resource for D. piger in mice consuming a polysaccharide-rich diet. While genes involved in hydrogen consumption and sulfate reduction are necessary for its colonization, varying dietary free sulfate levels did not significantly alter levels of D. piger, which can obtain sulfate from the host in part via cross-feeding mediated by Bacteroides-encoded sulfatases. Chondroitin sulfate, a common dietary supplement, increased D. piger and H2S levels without compromising gut barrier integrity. A chondroitin sulfate-supplemented diet together with D. piger impacted the assemblage’s substrate utilization preferences, allowing consumption of more reduced carbon sources, and increasing the abundance of the H2-producing Actinobacterium, Collinsella aerofaciens. Our findings provide genetic and metabolic details of how this H2-consuming SRB shapes the responses of a microbiota to diet ingredients, and a framework for examining how individuals lacking D. piger differ from those that harbor it. 8 samples total, 2 gropus of 4 mice: Proximal colon gene expression profiles of gnotobiotic mice colonized with an artificial gut community composed of 8 human gut species (group 1: NoDp) and from mice colonized with the same community plus D. piger (Dp). Mice were fed a HF/HS diet supplemented with 3% chondroitin sulfate. Animals were sacrificed 2 weeks after colonization
Project description:Determination of differentially expressed genes in the proximal colon and distal ileum tissue in MR1 and IL-17A deficiency at steady-state. Tissue from naïve mice was harvested, total RNA extracted and subjected to RNASeq analysis.
Project description:Gastrointestinal (GI) mucus is continuously secreted and lines the entire length of the GI tract. Essential for health, it keeps the noxious luminal content away from the epithelium and propels forward the digesta. The aim of our study was to characterize the composition and structures of mucus throughout the various GI segments in dog. Mucus from the stomach, small intestine (duodenum, jejunum, ileum), and large intestine (cecum, proximal and distal colon) was collected from 5 dogs. pH and water content of GI mucus and digesta were analyzed. Composition of all GI-tract segments from a domestic and a laboratory dog was determined by label-free global proteomics. A colonic-focussed composition analysis with TMT-labelled proteomics was used on jenunal and proximal and distal colonic mucus samples from 3 laboratory and 1 domestic dog. Finally, the composition of jejunal and colonic mucus samples of 3 laboratory and 1 domestic dog was evaluated with lipidomics and metabolomics. Structural properties were investigated using cryoSEM and rheology. The proteome was similar across the different GI segments. The highest abundant secreted gel-forming mucin in the gastric mucus was mucin 5AC, whether mucin 2 had highest abundance in the intestinal mucus. Lipid and metabolite abundance was generally higher in the jejunal mucus than the colonic mucus. In conclusion, the mucus is a highly viscous and hydrated material. The proteins, lipids and metabolites were similar throughout the GI tract, although abundances depended on location. These data provide an important baseline for future studies on human and canine intestinal diseases and the dog model in drug absorption.
Project description:Gut microbes elicit specific changes in gene expression in the colon of mice. We colonized germ-free mice with microbial communities from the guts of humans, zebrafish and termites, human skin and tongue, soil and estuarine microbial mats. We used microarrays to detail the differences in global gene expression in colon tissue that are caused by the different microbial communities 28 days after gavage into the germfree animal. Three biological replicates per group, male C57BL/6 mice (12-16 weeks old)
Project description:Opioid use is associated with worse outcome in HIV-infected patients. The exacerbated disease progression by opioids is mainly driven by increased epithelial damage and less regenerative response. The present study investigates how opioids potentiate HIV disease progression by impairing intestinal epithelial self-repair. Abnormal intestinal morphology and reduced epithelial proliferation were observed in HIV-infected humanized mice, which were exposed to opioids. For this study, we had 4 groups of BLT humanized mice- (A) Untreated, (B) Morphine treated, (C) HIV infected and (D) HIV infected + Morphine treated. HIV infection was done for 4 weeks and Morphine treatment was done for 7 days. Morphine treatment was done via subcutaneous implantation of slow-release morphine pellet in individual mouse in the appropriate groups. In other groups, a matching Placebo pellet was implanted.
Project description:The gastrointestinal mucus is a hydrogel that lines the luminal side of the gastrointestinal epithelium, offering barrier protection from pathogens and lubrication of the intraluminal contents. These barrier properties likewise affect nutrients and drugs that need to penetrate the mucus to reach the epithelium prior to absorption. In order to assess the potential impact of the mucus on drug absorption, we need information about the nature of the gastrointestinal mucus. Today, most of the relevant available literature is mainly derived from rodent studies. In this work, we used a larger animal species; the pig model to characterize the mucus throughout the length of the gastrointestinal tract. This is the first report of the physiological properties (physical appearance, pH and water content), composition and structural profiling of the porcine gastrointestinal mucus. Gastrointestinal mucus was collected from the stomach, small intestine (duodenum, jejunum, ileum) and large intestine (cecum, proximal and distal colon) from slaughtered pigs. The composition of the mucus was characterized both with labelfree and TMT proteomics, with lipidomics and metabolomics. The structural profiling was determined with rheological measurements and cryo-Scanning Electron Microscopy. These findings allow for direct comparisons between the characteristics of mucus from various segments and can be further utilized to improve our understanding of the role of the mucus on region dependent drug absorption. Additionally, the present work is expected to contribute to the assessment of the porcine model as a preclinical species in the drug development process.
Project description:Endogenous intestinal microbiota have wide-ranging and largely uncharacterized effects on host physiology. Here, we used reverse-phase liquid chromatography-coupled tandem mass spectrometry to define the mouse intestinal proteome in the stomach, jejunum, ileum, cecum, and proximal colon under three colonization states: germ-free, monocolonized with Bacteroides thetaiotaomicron, and conventionally raised. Our analysis revealed distinct proteomic abundance profiles along the gastrointestinal tract. Unsupervised clustering showed that host protein abundance primarily depended on gastrointestinal location rather than colonization state and specific proteins and functions that defined these locations were identified by random forest classifications. K-means clustering of protein abundance across locations revealed substantial differences in host protein production between conventionally raised mice relative to germ-free and monocolonized mice. Finally, comparison to fecal proteomic datasets suggested that the identities of stool proteins are not biased to any region of the gastrointestinal tract, but are substantially impacted by the microbiota in the distal colon.