Project description:M cells are the main site of bacterial translocation in the intestine. We used the in vitro M cell model to study the effect of the commensal bacteria; Lactobacillus salivarius, Eschericha coli and Bacteroides fragilis, on M cell gene expression. Bacterial translocation across the gut mucosa has traditionally been based on the detection of commensals in the mesenteric lymph node. Differential rates of commensal translocation have been reported in vivo, however fewer studies have examined translocation of commensals at the level of the gut epithelial M cell. In this study we employed an in vitro M cell model to quantify translocation of various bacteria. C2BBe1 cells were differentiated into M cells and the gene expression profile and transport kinetics of different bacterial strains, namely Lactobacillus salivarius, Escherichia coli, and Bacteroides fragilis, was assessed. For comparison with M cell uptake, the THP-1 monocytic cell line was used to analyze bacterial internalization and resulting cytokine production. The commensal bacterial strains were translocated across M cells with different efficiencies; E. coli and B. fragilis translocated with equal efficiency while L. salivarius translocated with less efficiency. In contrast, L. salivarius was internalized by THP-1 cells to a higher degree than B. fragilis or E. coli and was associated with a different cytokine profile. Microarray analysis showed both common and differential gene expression amongst the bacteria and control polystyrene beads. In the presence of bacteria, but not beads, upregulated genes were mainly involved in transcription regulation and dephosphorylation, e.g. EGR1, JUN; whereas proinflammatory and stress response genes were primarily upregulated by E. coli and B. fragilis, but not L. salivarius nor beads, e.g. IL8, TNFAIP3. These results demonstrate that M cells have the ability to discriminate between different commensal bacteria and modify subsequent immune responses. C2bbe1 cells were converted to M cells (C2M) following 21 days of culture on Transwells in the presence of Raji B cells. C2M cells were co-cultured alone, Lactobacillus salivarius, Eschericha coli, Bacteroides fragilis and control beads. Total RNA was extracted and processed for Affymetrix array hybridisation

Project description:M cells are the main site of bacterial translocation in the intestine. We used the in vitro M cell model to study the effect of the commensal bacteria; Lactobacillus salivarius, Eschericha coli and Bacteroides fragilis, on M cell gene expression. Bacterial translocation across the gut mucosa has traditionally been based on the detection of commensals in the mesenteric lymph node. Differential rates of commensal translocation have been reported in vivo, however fewer studies have examined translocation of commensals at the level of the gut epithelial M cell. In this study we employed an in vitro M cell model to quantify translocation of various bacteria. C2BBe1 cells were differentiated into M cells and the gene expression profile and transport kinetics of different bacterial strains, namely Lactobacillus salivarius, Escherichia coli, and Bacteroides fragilis, was assessed. For comparison with M cell uptake, the THP-1 monocytic cell line was used to analyze bacterial internalization and resulting cytokine production. The commensal bacterial strains were translocated across M cells with different efficiencies; E. coli and B. fragilis translocated with equal efficiency while L. salivarius translocated with less efficiency. In contrast, L. salivarius was internalized by THP-1 cells to a higher degree than B. fragilis or E. coli and was associated with a different cytokine profile. Microarray analysis showed both common and differential gene expression amongst the bacteria and control polystyrene beads. In the presence of bacteria, but not beads, upregulated genes were mainly involved in transcription regulation and dephosphorylation, e.g. EGR1, JUN; whereas proinflammatory and stress response genes were primarily upregulated by E. coli and B. fragilis, but not L. salivarius nor beads, e.g. IL8, TNFAIP3. These results demonstrate that M cells have the ability to discriminate between different commensal bacteria and modify subsequent immune responses.

Project description:Genome scale metabolic model of Drosophila gut microbe Acetobacter fabarum Abstract - An important goal for many nutrition-based microbiome studies is to identify the metabolic function of microbes in complex microbial communities and their impact on host physiology. This research can be confounded by poorly understood effects of community composition and host diet on the metabolic traits of individual taxa. Here, we investigated these multiway interactions by constructing and analyzing metabolic models comprising every combination of five bacterial members of the Drosophila gut microbiome (from single taxa to the five-member community of Acetobacter and Lactobacillus species) under three nutrient regimes. We show that the metabolic function of Drosophila gut bacteria is dynamic, influenced by community composition, and responsive to dietary modulation. Furthermore, we show that ecological interactions such as competition and mutualism identified from the growth patterns of gut bacteria are underlain by a diversity of metabolic interactions, and show that the bacteria tend to compete for amino acids and B vitamins more frequently than for carbon sources. Our results reveal that, in addition to fermentation products such as acetate, intermediates of the tricarboxylic acid (TCA) cycle, including 2-oxoglutarate and succinate, are produced at high flux and cross-fed between bacterial taxa, suggesting important roles for TCA cycle intermediates in modulating Drosophila gut microbe interactions and the potential to influence host traits. These metabolic models provide specific predictions of the patterns of ecological and metabolic interactions among gut bacteria under different nutrient regimes, with potentially important consequences for overall community metabolic function and nutritional interactions with the host.IMPORTANCE Drosophila is an important model for microbiome research partly because of the low complexity of its mostly culturable gut microbiota. Our current understanding of how Drosophila interacts with its gut microbes and how these interactions influence host traits derives almost entirely from empirical studies that focus on individual microbial taxa or classes of metabolites. These studies have failed to capture fully the complexity of metabolic interactions that occur between host and microbe. To overcome this limitation, we reconstructed and analyzed 31 metabolic models for every combination of the five principal bacterial taxa in the gut microbiome of Drosophila This revealed that metabolic interactions between Drosophila gut bacterial taxa are highly dynamic and influenced by cooccurring bacteria and nutrient availability. Our results generate testable hypotheses about among-microbe ecological interactions in the Drosophila gut and the diversity of metabolites available to influence host traits.

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Colonic goblet cell-associated antigen passages mediate physiologic and beneficial translocation of live gut bacteria in preweaning mice

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