Sonnenburg, Justin L.

Background Microbial conversion of dietary substrate to bioactive molecules represents a potential regulatory mechanism by which gut microbes can alter intestinal physiology. Tryptamine, a monoamine structurally similar to serotonin (5‐HT), can be generated via decarboxylation of tryptophan by the gut bacteria but its physiological effect is not known. Aim To determine the effect of bacterial metabolite tryptamine on colonic secretion. Methods We mounted proximal colon mucosa‐submucosa preparations from germ free (GF), humanized (HM; ex‐GF colonized with human gut bacteria for 4–5 weeks), conventionally raised WT and 5‐HT4 receptor knockout (5‐HT4R −/− ) male mice in the Ussing chamber. The change in short circuit current (ΔIsc) was determined in response to application of increasing concentrations of tryptamine (3μM – 3mM) on the submucosal (basolateral) side either alone or in the presence of 5‐HT3R or 5‐HT4R antagonist, ondansetron (100nM) and GR‐113808 (30nM) respectively, with and without TTX (500nM). Colonoids (3‐dimensional epithelial organoids derived from colon) from GF and HM were used for swelling assay and ELISA based cAMP measurement. Results Tryptamine application significantly increased Isc in both GF and HM mice (n=6, P < 0.05, two‐way ANOVA with Bonferroni post‐hoc). Tryptamine mediated ΔIsc response was not significantly affected by amiloride, an inhibitor of epithelial sodium channels and Na + /H + exchangers, which are primarily responsible for absorption but was significantly decreased by 5‐Nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB; anion blocker), a calcium‐sensitive chloride channel and sodium bicarbonate exchanger inhibitor responsible for anionic secretion in both GF and HM mice (n=4, P < 0.05, two‐way ANOVA with Bonferroni post‐hoc). Tryptamine evoked ΔIsc was not inhibited by 5‐HT3R (n=4, P > 0.05) but completely blocked by 5‐HT4R antagonist and was absent in 5HT4R knock out mice suggesting that the effect of tryptamine is mediated via 5HT4R. The increase in Isc in response to tryptamine and its inhibition by 5‐HT4R antagonist was also seen in presence of TTX (neuronal blocker; n=4, one‐way ANOVA , P < 0.05) suggesting that the epithelial 5‐HT4R alone can mediate the effect of tryptamine. Application of tryptamine (1mM) for 30 minutes led to a significant increase in the surface area of fluorescein labelled colonoids (n=5, one‐way ANOVA , P < 0.05), similar to forskolin, when compared to vehicle treated and 5HT4R antagonist pre‐treated GF and HM colonoids. Colonoids incubated with tryptamine (1mM) for 60 minutes had significantly higher cAMP levels (normalized to forskolin) compared to vehicle treated GF and HM colonoids, confirming that tryptamine activates 5‐HT4 GPCR (n=3, student's t‐test, P < 0.05). Conclusion Tryptamine, a tryptophan‐derived microbial metabolite, increases colonic secretion via 5HT4 GPCR activation. The identification of a bacterial metabolite activating a host GPCR to alter GI function represents an important advance in the field, and provides a distinct example of small molecule mediated host‐microbe interaction. Engineered probiotics producing tryptamine could be used as therapeutics in diseases associated with constipation. Support or Funding Information NIH DK111850, DK114007 (PCK) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract Microbiota-accessible carbohydrates (MACs) are powerful modulators of microbiota composition and function. These substrates are often derived from diet, such as complex polysaccharides from plants or human milk oligosaccharides (HMOs) during breastfeeding. Host-derived mucus glycans on gut-secreted mucin proteins serve as a continuous endogenous source of MACs for resident microbes; here we investigate the potential role of purified, orally administered mucus glycans in maintaining a healthy microbial community. In this study, we liberated and purified O-linked glycans from porcine gastric mucin and assessed their efficacy in shaping the recovery of a perturbed microbiota in a mouse model. We found that porcine mucin glycans (PMGs) and HMOs enrich for taxonomically similar resident microbes. We demonstrate that PMGs aid recovery of the microbiota after antibiotic treatment, suppress Clostridium difficile abundance, delay the onset of diet-induced obesity, and increase the relative abundance of resident Akkermansia muciniphila. In silico analysis revealed that genes associated with mucus utilization are abundant and diverse in prevalent gut commensals and rare in enteric pathogens, consistent with these glycan-degrading capabilities being selected for during host development and throughout the evolution of the host–microbe relationship. Importantly, we identify mucus glycans as a novel class of prebiotic compounds that can be used to mitigate perturbations to the microbiota and provide benefits to host physiology.

A reduction in intestinal barrier function is currently believed to play an important role in pathogenesis of many diseases, as it facilitates passage of injurious factors such as lipopolysaccharide, peptidoglycan, whole bacteria, and other toxins to traverse the barrier to damage the intestine or enter the portal circulation. Currently available evidence in animal models and in vitro systems has shown that certain dietary interventions can be used to reinforce the intestinal barrier to prevent the development of disease. The relevance of these studies to human health is unknown. Herein, we define the components of the intestinal barrier, review available modalities to assess its structure and function in humans, and review the available evidence in model systems or perturbations in humans that diet can be used to fortify intestinal barrier function. Acknowledging the technical challenges and the present gaps in knowledge, we provide a conceptual framework by which evidence could be developed to support the notion that diet can reinforce human intestinal barrier function to restore normal function and potentially reduce the risk for disease. Such evidence would provide information on the development of healthier diets and serve to provide a framework by which federal agencies such as the US Food and Drug Administration can evaluate evidence linking diet with normal human structure/function claims focused on reducing risk of disease in the general public.

Increased amino acids in the dysbiotic gut influences susceptibility to Clostridioides difficile infection in mice and humans.

Dietary modification has long been used empirically to modify symptoms in inflammatory bowel disease, irritable bowel syndrome, and a diverse group of diseases with gastrointestinal symptoms. There is both anecdotal and scientific evidence to suggest that individuals respond quite differently to similar dietary changes, and the highly individualized nature of the gut microbiota makes it a prime candidate for these differences. To overcome the typical confounding factors of human dietary interventions, here we employ ex-germfree mice colonized by microbiotas of three different humans to test how different microbiotas respond to a defined change in carbohydrate content of diet by measuring changes in microbiota composition and function using marker gene-based next-generation sequencing and metabolomics. Our findings suggest that the same diet has very different effects on each microbiota’s membership and function, which may in turn explain interindividual differences in response to a dietary ingredient.

AbstractThe production and handling of serotonin (5‐HT) is an important determinant of colonic motility and has been reported to be altered in gastrointestinal (GI) disorders such as irritable bowel syndrome (IBS). Recent studies suggest that the intestinal microbiota and sex of the host can influence expression of genes involved in 5‐HT biosynthesis and signaling. While expression of genes in serotonergic pathways has been shown to be variable, it remains unclear whether genes within this pathway are coregulated. As a first step in that direction, we investigated potential correlations in relative mRNA expression of serotonergic genes, in the proximal colon isolated from male and female mice in different states of microbial association: germ‐free (GF), humanized (ex‐germ‐free colonized with human gut microbiota, HM), and conventionally raised (CR) mice. Among the 10 pairwise comparisons conducted between five serotonergic transcripts, Tph1, Chga, Maoa, Slc6a4, and Htr4, we found a strong, positive correlation between colonic expression of Slc6a4 and Htr4 across different colonization states and sexes. We also identified a positive correlation between the expression of Tph1 and Chga; however, there were no correlations observed between any other tested pair of 5‐HT‐related transcripts. These data suggest that correlated expression of Slc6a4 and Htr4 likely involves coregulation of genes located on different chromosomes which modulate serotonergic activity in the gut. Further work will need to be done to understand the pathways and cell types responsible for this correlated expression, given the important role of 5‐HT in gastrointestinal physiology.

Gut microbiota alterations have been described in several diseases with altered gastrointestinal (GI) motility, and awareness is increasing regarding the role of the gut microbiome in modulating GI function. Serotonin [5‐hydroxytryptamine (5‐HT)] is a key regulator of GI motility and secretion. To determine the relationship among gut microbes, colonic contractility, and host serotonergic gene expression, we evaluated mice that were germ‐free (GF) or humanized (HM; ex‐GF colonized with human gut microbiota). 5‐HT reduced contractile duration in both GF and HM colons. Microbiota from HM and conventionally raised (CR) mice significantly increased colonic mRNAs Tph1 [(tryptophan hydroxylase) 1, rate limiting for mucosal 5‐HT synthesis; P < 0.01] and chromogranin A (neuroendocrine secretion; P < 0.01), with no effect on monoamine oxidase A (serotonin catabolism), serotonin receptor 5‐HT 4 , or mouse serotonin transporter. HM and CR mice also had increased colonic Tph1 protein ( P < 0.05) and 5‐HT concentrations (GF, 17 ± 3 ng/mg; HM, 25 ± 2 ng/mg; and CR, 35 ± 3 ng/mg; P < 0.05). Enterochromaffin (EC) cell numbers (cells producing 5‐HT) were unchanged. Short‐chain fatty acids (SCFAs) promoted TPH1 transcription in BON cells (human EC cell model). Thus, gut microbiota acting through SCFAs are important determinants of enteric 5‐HT production and homeostasis.—Reigstad, C. S., Salmonson, C. E., Rainey, III, J. F., Szurszewski, J. H., Linden, D. R., Sonnenburg, J. L., Farrugia, G., Kashyap, P. C. Gut microbes promote colonic serotonin production through an effect of short‐chain fatty acids on enterochromaffin cells. FASEB J. 29, 1395‐1403 (2015). www.fasebj.org

Significance Our data demonstrate that differences in host genotype that affect the carbohydrate landscape of the distal gut interact with diet to alter the composition and function of resident microbes in a diet-dependent manner.

Abstract Defining the functional status of host-associated microbial ecosystems has proven challenging owing to the vast number of predicted genes within the microbiome and relatively poor understanding of community dynamics and community–host interaction. Metabolomic approaches, in which a large number of small molecule metabolites can be defined in a biological sample, offer a promising avenue to ‘fingerprint’ microbiota functional status. Here, we examined the effects of the human gut microbiota on the fecal and urinary metabolome of a humanized (HUM) mouse using an optimized ultra performance liquid chromatography–mass spectrometry-based method. Differences between HUM and conventional mouse urine and fecal metabolomic profiles support host-specific aspects of the microbiota’s metabolomic contribution, consistent with distinct microbial compositions. Comparison of microbiota composition and metabolome of mice humanized with different human donors revealed that the vast majority of metabolomic features observed in donor samples are produced in the corresponding HUM mice, and individual-specific features suggest ‘personalized’ aspects of functionality can be reconstituted in mice. Feeding the mice a defined, custom diet resulted in modification of the metabolite signatures, illustrating that host diet provides an avenue for altering gut microbiota functionality, which in turn can be monitored via metabolomics. Using a defined model microbiota consisting of one or two species, we show that simplified communities can drive major changes in the host metabolomic profile. Our results demonstrate that metabolomics constitutes a powerful avenue for functional characterization of the intestinal microbiota and its interaction with the host.