The intestinal microbiota produces tens of thousands of metabolites. Here, we used host sensing of small molecules by G-protein coupled receptors (GPCRs) as a lens to illuminate bioactive microbial metabolites that impact host physiology. We screened 144 human gut bacteria against the non-olfactory GPCRome and identified dozens of bacteria that activated both well-characterized and orphan GPCRs, including strains that converted dietary histidine into histamine and shaped colonic motility; a prolific producer of the essential amino acid L-Phe, which we identified as an agonist for GPR56 and GPR97; and a species that converted L-Phe into the potent psychoactive trace amine phenethylamine, which crosses the blood-brain barrier and triggers lethal phenethylamine poisoning after monoamine oxidase inhibitor administration. These studies establish an orthogonal approach for parsing the microbiota metabolome and uncover multiple biologically relevant host-microbiota metabolome interactions.

Here, by building on recent developments in high-throughput screening of the complete GPCRome and activity-guided microbial metabolite identification approaches (), we developed a pipeline to screen human gut microbes for the ability to produce ligands that activate human GPCRs. In so doing, we established an orthogonal approach for elucidating biologically-relevant microbiota metabolite-host interactions and, in the process, uncovered multiple diet-microbe-host and microbe-microbe-host metabolic axes that shape both local and systemic host physiology.

G-protein coupled receptors (GPCRs) are the largest family of membrane proteins encoded in the human genome (including over 350 conventional non-olfactory GPCRs), are critical sensors of diverse small molecules, and regulate various aspects of host physiology, including vision, mood, pain, and immunity (). Specific GPCRs are also known to sense microbial metabolites, such as microbiota-derived short-chain fatty acids (SCFAs) (), and recent studies have continued to reveal novel microbiota-derived GPCR ligands that can shape host physiology (). Thus, the microbiota metabolome is a rich source of potential GPCR ligands.

The human gut microbiota produces thousands of unique small molecules that can potentially affect nearly all aspects of human physiology, from regulating immunity in the gut to shaping mood and behavior (). These metabolites can act locally in the intestine or can accumulate up to millimolar concentrations in the serum (). Recent studies employing state-of-the-art genomic and metabolomic approaches have begun to reveal the enormously complex intra- and inter-species microbial chemistries that potentially impinge on host physiology, as well as the impact of gut microbes on the processing of dietary small molecules and medical drugs (). In addition, they underscore the importance of continuing to develop new approaches to explore the bioactive microbiota metabolome ().

We next examined whether M. morganii would process B. theta C34-derived L-Phe into PEA in vitro and observed that B. theta C34-derived L-Phe was efficiently converted into PEA by M. morganii C135 ( Figure 7 D). To test whether metabolic exchange occurs in vivo, we colonized GF mice with either M. morganii C135 alone or with both B. theta C34 and M. morganii C135, fed these mice a simplified diet lacking L-Phe, and then treated them with phenelzine. Mice colonized with M. morganii C135 alone remained healthy and produced minimal PEA in the absence of dietary L-Phe ( Figure 7 E). In contrast, mice co-colonized with B. theta C34 and M. morganii C135 became lethargic by day 4 after MAOI treatment and exhibited significant accumulation of PEA ( Figure 7 E). This demonstrates that metabolic exchange between B. theta C34 and M. morganii C135 can contribute to the production of a bioactive trace amine that can have potent effects on systemic host physiology.

Our reductionist studies revealed that B. theta C34 produces large amounts of L-Phe while M. morganii C135 can process L-Phe into PEA. Thus, we wished to address whether these two bacteria might participate in an active metabolic exchange in vivo. Using a defined minimal bacterial medium that lacks L-Phe (standard amino acid complete medium or SACC; see STAR Methods ) (), we observed that B. theta C34 can directly synthesize large amounts of L-Phe in vitro ( Figures 7 A and 7B ). Furthermore, mice monocolonized with B. theta C34 and fed an L-Phe-deficient diet displayed significant intestinal accumulation of L-Phe as compared to GF mice ( Figure 7 C).

Data in all panels are representative of at least two independent experiments. Data are presented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test (B-C and E-F), ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

(E) B. theta C34 and M. morganii C135 can participate in active metabolic exchange to produce phenethylamine in vivo. Germ-free C57BL/6 mice were monocolonized with M. morganii C135 or co-colonized with B. theta C34 and M. morganii C135, fed a diet lacking L-Phe, and treated with the MAOI phenelzine. Activation of DRD2 by phenethylamine in cecal and colonic extracts was measured by DRD2-Tango. n = 4-6 mice per group.

(D) M. morganii C135 consumes B. theta C34-derived L-Phe to produce phenethylamine in vitro. B. theta C34 cultures were grown in SACC medium lacking L-Phe and then incubated with M. morganii C135. L-Phe and phenethylamine (PEA) concentrations were measured by QQQ-MS/MS.

(C) B. theta C34 produces L-Phe in vivo. Germ-free female C57BL/6 mice fed a conventional diet or a defined diet lacking L-Phe were colonized with or without B. theta C34. Fecal L-Phe concentrations were measured by QQQ-MS/MS one week after colonization. n = 4 mice per group.

(A and B) B. theta C34 can directly synthesize L-Phe. L-Phe concentrations in supernatants from C34 grown in a minimal medium (SACC) lacking L-Phe were evaluated by LC-MS (A) and quantitated by QQQ-MS/MS (B).

We next examined whether other orphan GPCRs might also respond to L-Phe by stimulating all adhesion and orphan GPCRs with pure L-Phe ( Figure S7 A). We found that GPR97/AGRG3 also responded to L-Phe and showed greater selectivity toward L-Phe than GPR56/AGRG1—L-Phe, but not L-Tyr, L-Trp, or L-His, activated GPR97/AGRG3 ( Figure S7 B). Like GPR56/AGRG1, the extracellular domain of GPR97/AGRG3 was required for its ability to respond to L-Phe ( Figure S7 C), and removal of L-Phe and L-Tyr from the medium increased the magnitude of activation of GPR97/AGRG3 by exogenous L-Phe ( Figures S7 B and S7D). Furthermore, L-Phe also activated G protein-dependent signaling downstream of GPR97/AGRG3 ( Figure S7 E). Notably, GPR56/AGRG1 and GPR97/AGRG3 are closely related evolutionarily ( Figure S7 F), which may explain their shared ability to detect L-Phe.

Data in all panels are representative of at least three independent experiments. Data are presented as mean ± SEM.

(F) GPR56/AGRG1 and GPR97/AGRG3 are evolutionarily related. A phylogenetic tree for a subset of GPCRs, including all adhesion GPCRs, was constructed and visualized with equal branch lengths using gpcrdb.org , PHYLIP, and jsPhyloSVG.

(E) L-Phe activates G protein-dependent signaling downstream of GPR97/AGRG3. Activation of G proteins downstream of GPR97/AGRG3 by L-Phe as measured by the CRE-SEAP assay. Gα s -Gα t and Gα s -Gα o chimeras were used to redirect GPR97/AGRG3 signaling to Gα s and enable use of the CRE-SEAP assay. n = 3 replicates per sample.

(D) L-Phe specifically activates GPR97/AGRG3. Activation of GPR97/AGRG3 by titrating doses of L-Phe, L-Tyr, L-Trp, and L-His was measured via GPR97 PRESTO-Tango in media lacking L-Phe and L-Tyr. n = 3 replicates per sample.

(C) The extracellular domain of GPR97/AGRG3 is indispensable for GPR97/AGRG3 activation by L-Phe. Activation of GPR97 or GPR97-ΔNT (a mutant lacking the extracellular domain) by titrating doses of L-Phe was measured via PRESTO-Tango. n = 3 replicates per sample.

(A) L-Phe activates GPR56/AGRG1 and GPR97/AGRG3. Activation of all orphan, adhesion and other potential amino acid-sensing GPCRs by L-Phe was evaluated via PRESTO-Tango. n = 3 replicates per sample.

Using the promiscuous Gα-Gαand Gα-Gαchimeras and the CRE-SEAP assay described above (), we found that L-Phe activated G protein-dependent signaling downstream of GPR56/AGRG1 ( Figure 6 H and Figure S6 F); however, since high concentrations of L-Phe (> 1mM) were required to activate GPR56/AGRG1, it remains unclear whether physiological concentrations of L-Phe will engage GPR56/AGRG1-mediated G protein signaling in vivo. GPR56/AGRG1 belongs to the adhesion GPCR family, whose members possess large extracellular domains that mediate interactions with a variety of protein ligands (). However, we found that the extracellular domain of GPR56/AGRG1 was also required for L-Phe-induced activation of GPR56/AGRG1 () ( Figure S6 E). Together, our data demonstrate that a unique strain of B. theta secretes high levels of L-Phe and that L-Phe is a novel agonist of the adhesion GPCR, GPR56/AGRG1.

Since there was no known endogenous small molecule ligand for GPR56/AGRG1 (), we next attempted to identify the specific metabolite produced by B. theta C34 that activated GPR56/AGRG1. B. theta C34 supernatants were extracted and subjected to fractionation by reversed-phase HPLC and all fractions were analyzed for activity via GPR56-Tango ( Figure 6 E). High resolution mass spectrometry, NMR and coinjection analyses of the active fraction (F11) revealed the essential amino acid phenylalanine (Phe) as the primary constituent of F11 ( Figures 6 E and S6 B) and structural characterization using advanced Marfey’s analysis confirmed that L-Phe is the likely bioactive ligand ( Figure S6 C) (). Accordingly, pure L-Phe and, to a lesser extent, L-Tyr stereoselectively activated GPR56/AGRG1, while L-Trp and L-His, D-Phe, D-Trp, D-His, and D-Tyr showed no activity ( Figures 6 F and S6 D). We hypothesized that L-Phe and L-Tyr in the medium used for the Tango assay might obscure the full extent of GPR56/AGRG1 activation by L-Phe. Indeed, removal of endogenous L-Phe and L-Tyr from the culture medium greatly increased the sensitivity and magnitude of GPR56 activation by L-Phe and L-Tyr as measured by GPR56-Tango ( Figures 6 G and S6 E); furthermore, GPR56 expression was essential for this response ( Figure S6 E). Finally, despite their differential secretion of L-Phe, the genomes of B. theta C34 and two strains of Bacteroides that failed to activate GPR56/AGRG1 all encoded the full suite of enzymes in the shikimate pathway that synthesize L-Phe ( Table S4 S5 ).

We observed that specific bacterial supernatants activated select orphan GPCRs ( Figure 6 A). To confirm these hits, we repeated our PRESTO-Tango screening procedure using a richer culture medium (Gifu) that supports more robust growth of many of the human gut microbes in our collection. This modified procedure significantly expanded the number of positive hits against orphan GPCRs: 17 orphan GPCRs showed greater than four-fold activation in response to at least one bacterial supernatant ( Figure 6 B). Metabolites from a strain assigned to the species B. theta (B. theta C34) activated GPR56/AGRG1 under both culture conditions ( Figure 6 C). In contrast, other strains of B. theta, as well as multiple related Bacteroides strains, failed to activate GPR56/AGRG1 ( Figure 6 D) despite similar bacterial growth ( Figure S6 A).

Data in all panels except B and C are representative of at least three independent experiments. Data are presented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test ∗ p < 0.05, ∗∗∗∗ p < 0.0001.

(G) The extracellular domain of GPR56/AGRG1 is indispensable for GPR56/AGRG1 activation by L-Phe. Activation of GPR56 or GPR56-ΔNT (a mutant lacking the extracellular domain) by titrating doses of L-Phe was measured via PRESTO-Tango. n = 3 replicates per sample.

(F) L-Phe-induced activation of G protein-dependent signaling in HEK cells is GPR56-dependent. Activation of G proteins downstream of GPR56/AGRG1 by L-Phe as measured by the CRE-SEAP assay. Gα s -Gα t and Gα s -Gα o chimeras were used to redirect GPR56/AGRG1 signaling to Gα s and enable use of the CRE-SEAP assay. Cells transfected with DRD2-Tango and Gα s -Gα t and Gα s -Gα o chimeras failed to respond to L-Phe. n = 3 replicates per sample.

(E) L-Phe-induced Tango activation is GPR56/AGRG1-dependent. Luciferase expression (RLU) was measured after stimulation of cells transfected with GPR56-Tango or empty vector with titrating doses of L-Phe. n = 3 replicates per sample.

(D) L-Phe and L-Tyr stereoselectively activate the orphan receptor GPR56/AGRG1. Activation of GPR56/AGRG1 by titrating doses of pure L-Phe, L-Tyr, D-Phe, and D-Tyr (in L-Phe and L-Tyr-free medium) was measured via GPR56-Tango. n = 3 replicates per sample.

(C) Advanced Marfey’s analysis verified the stereochemistry of Phe in fraction 11 to be L-Phe. D-Phe in the active fraction was not detected. FDAA is 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey’s Reagent).

Effect of Different Bacterial and Culture Media on Bacterial Growth and GPR56/AGRG1 Activation, Structural Characterization of B. theta C34 Agonist L-Phe, and Role of N-Terminal Domain in GPR56/AGRG1 Activation by L-Phe, Related to Figure 6

Data in all panels except for A, B, and E are representative of at least three independent experiments. Data are presented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

(H) L-Phe activates G protein-dependent signaling downstream of GPR56/AGRG1 as measured by the CRE-SEAP assay. Gα s -Gα t and Gα s -Gα o chimeras were used to redirect GPR56/AGRG1 signaling to Gα s .

(F and G) L-Phe activates the orphan receptor GPR56/AGRG1. Activation of GPR56/AGRG1 by titrating doses of pure L-Phe, L-Tyr, L-Trp, and L-His was measured via GPR56-Tango using RPMI 1640 medium (F) or a custom medium lacking L-Phe and L-Tyr (G).

(E) B. theta C34-derived L-Phe activates GPR56/AGRG1. B. theta C34 supernatants were fractionated via reversed-phase HPLC and fractions were evaluated for activation of GPR56/AGRG1 via GPR56-Tango. The active fraction (F11) contained a primary constituent that was identified via LC-MS, HRMS-ESI-QTOF, NMR, and advanced Marfey’s analyses as L-Phe.

(D) B. theta strain C34 uniquely activates GPR56/AGRG1. Activation of GPR56/AGRG1 by supernatants from diverse species and strains from the genera Bacteroides and Parabacteroides cultured in GMM was measured via GPR56 PRESTO-Tango.

(C) A single isolate C34 assigned to the species Bacteroides thetaiotaomicron activates GPR56/AGRG1 when cultured in gut microbiota medium (GMM: top panel) or Gifu medium (bottom panel). Activation of GPR56/AGRG1 by supernatants from 144 human gut isolates as measured via GPR56-Tango.

(A and B) Activation of orphan GPCRs by metabolomes from a human gut microbiota culture (see Figure 1 ) grown in gut microbiota medium (A) or Gifu (B) as measured by PRESTO-Tango. Screening results are displayed on a phylogenetic tree of orphan GPCRs that was constructed and visualized with equal branch lengths using gpcrdb.org , PHYLIP, and jsPhyloSVG. Color intensities represent the magnitude of activation over media and radii of circles represent the number of bacteria that activated a given GPCR by more than two-fold.

Unlike histamine, we observed only low levels of colonic PEA in M. morganii monocolonized mice ( Figure 5 A). One potential explanation for this observation is that many biogenic amines, including PEA, are rapidly degraded in the intestine by host monoamine oxidases (MAOs) (). MAO inhibitors (MAOIs) were the first FDA-approved antidepressants (). Thus, to reveal the potential production of PEA in vivo, we treated germ-free mice or mice monocolonized with M. morganii C135 or a Bacteroides thetaiotaomicron strain (B. theta C34) that does not produce DRD agonists with the irreversible MAOI phenelzine. While colonic PEA remained undetectable in germ-free mice or mice colonized with B. theta even after phenelzine treatment, M. morganii-colonized mice treated with phenelzine exhibited high levels of colonic PEA ( Figure 5 A). M. morganii-colonized mice also became lethargic within days after MAOI treatment, and more than half of all M. morganii-colonized mice died by day seven post MAOI administration, while germ-free and B. theta C34-colonized mice remained healthy ( Figure 5 B). Morbidity and mortality after MAOI treatment correlated with elevated levels of PEA in the colon, serum, and brains of M. morganii-colonized mice ( Figures 5 C and S5 F, and S5G), and cecal and colonic contents, as well as serum and brain extracts from these mice activated DRD2 ( Figure 5 D). Finally, mice colonized with a mock community plus M. morganii also exhibited increased PEA in the colon and brain, as measured by DRD2 activation ( Figure 5 E). Together, these data show that M. morganii-derived phenethylamine can accumulate systemically, cross the blood-brain barrier, and trigger lethal phenethylamine poisoning in mice treated with MAOIs.

Data in all panels are representative of at least two independent experiments. Data are presented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test (D-E), Kaplan-Meier and Log-rank analysis (B), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

(E) M. morganii-derived phenethylamine accumulates in the sera and brains of mice colonized with Mock Community A plus M. morganii. Germ-free female C57BL/6 mice were colonized with Mock Community A with or without M. morganii C135, or monocolonized with M. morganii C135. All mice were treated with the MAOI phenelzine in the drinking water for one week and phenethylamine accumulation was detected using DRD2-Tango as a proxy.

(C and D) M. morganii-colonized mice treated with phenelzine accumulated phenethylamine in the cecum, colon, serum, and brain. M. morganii C135 and B. theta C34 monocolonized female C57BL/6 mice were treated with or without the MAOI phenelzine in the drinking water. Phenethylamine was measured via QQQ-MS/MS (C) or DRD2 PRESTO-Tango (D). n = 4 mice per group.

(B) Mice colonized with M. morganii exhibit lethal phenethylamine poisoning after treatment with the MAOI phenelzine. Female germ-free C57BL/6 mice were monocolonized with M. morganii C135 or B. theta C34 for one week before treatment with phenelzine in the drinking water. Survival is depicted on a Kaplan-Meier curve. n = 4 mice per group.

(A) M. morganii produces phenethylamine in vivo. Female germ-free C57BL/6 mice were colonized with M. morganii C135 or B. theta C34 and treated with or without the MAOI phenelzine. Phenethylamine concentration in colonic extracts was examined using QQQ-MS/MS.

Together, these data demonstrate that M. morganii impacts intestinal motility through histamine secretion and activation of histamine receptors, that dietary histidine can enhance these effects, and that bacterial histidine decarboxylases (both generally and from M. morganii) are enriched in patients with CD.

To examine the potential importance of histamine production by M. morganii (or other microbes) in human physiology, we mined publicly available metagenomic and metabolomic data from the integrative Human Microbiome Project (see STAR Methods for details) to determine the relative abundance of histamine producing enzymes or histamine itself in microbiomes from patients with IBD versus healthy controls (). We found that CD patients exhibited an increased prevalence and abundance of histidine decarboxylase genes, including M. morganii-encoded histidine decarboxylase, as compared to healthy controls or UC patients ( Figure 4 G and Figure S5 E), and that histamine itself was increased in fecal samples from CD and UC patients as compared to controls ( Figure 4 H). This observation is in line with previous studies demonstrating increased intestinal histamine in patients with IBD, although this was largely attributed to host-derived histamine production ().

To test whether M. morganii can impact host physiology in the context of a more diverse microbial community, we colonized germ-free mice with a mock community of nine human gut microbes with or without M. morganii C135 and examined histamine accumulation in the gut and serum, as well as fecal output. Although the effects were less profound than those observed with monocolonizations, the addition of M. morganii to a mock community also led to an accumulation of histamine in the colon and serum ( Figures 4 E and S5 D) as well as increased fecal output ( Figure 4 E). Finally, we found that treatment with histamine receptor antagonists could largely block the effects of M. morganii on fecal output despite the accumulation of similar levels of histamine in the gut ( Figure 4 F).

Oral gavage with histamine increases colon motility in rodents (). We thus hypothesized that gut microbe-derived histamine might also increase intestinal motility. We monitored intestinal motility in gnotobiotic mice colonized with two mock communities (which do not produce histamine) or M. morganii C135 with or without administration of 1% L-His in the water and found that M. morganii induced a significant increase in fecal output, which was further increased upon supplementation with L-His ( Figure 4 D). Similarly, mice colonized with L. reuteri C93 exhibited increased fecal output as compared to mice colonized with L. reuteri C88 ( Figure 4 D).

To determine the location of M. morganii in vivo, we used modified Niven’s agar to enumerate M. morganii CFUs in gnotobiotic mice colonized with two mock communities of diverse human gut microbes plus M. morganii C135 (). We found that M. morganii constitutes approximately 5% of the microbiota in the context of a mock community, and primarily inhabits the cecum and colon ( Figure S5 C and Table S3 ). Notably, M. morganii also preferentially localizes in tissue- or mucus-associated niches in the colon in humans ().

Modification of Niven’s medium for the enumeration of histamine-forming bacteria and discussion of the parameters associated with its use.

All M. morganii strains and two L. reuteri strains in our collection generated histamine in vitro and supplementation with additional L-His significantly increased histamine production by these strains; in contrast, two distinct strains of L. reuteri failed to produce histamine regardless of supplementation with L-His ( Figure 4 A). To test whether M. morganii can also produce histamine in vivo, we colonized germ-free mice with two distinct mock communities containing 9 or 10 diverse human gut microbes or with M. morganii C135 with or without supplementation of 1% L-His in the drinking water to approximate an L-His-rich diet (e.g., a meat-heavy diet) ( Figure 4 B). In addition, we monocolonized mice with two L. reuteri strains with divergent histamine production capabilities: L. reuteri C93, which produced significant histamine in vitro, and L. reuteri C88, which failed to produce histamine in vitro. Mice colonized with M. morganii C135 or L. reuteri C93 exhibited high levels of intestinal histamine production, while mice colonized with the two mock communities or L. reuteri C88 showed nearly undetectable intestinal histamine ( Figure 4 C). In addition, supplementation with dietary L-His increased histamine production in M. morganii monocolonized mice ( Figure 4 C). Finally, we also detected increased histamine in the serum of mice colonized with M. morganii ( Figure S5 A).

(G) Accumulation of phenethylamine (PEA) in serum and brains of mice monocolonized with M. morganii C135 and treated with or without phenelzine (MAOI) as measured via QQQ-MS/MS. n = 4 mice per group.

(F) Quantification of phenethylamine (PEA) in the cecum, colon, serum, and brain from mice monocolonized with M. morganii C135 and treated with or without phenelzine (MAOI) via QQQ-MS/MS. n = 4 mice per group.

(E) Contribution of individual species to the relative abundance of histidine decarboxylase genes in the microbiomes of patients with IBD (CD and UC) as compared to controls (non-IBD). Metagenomic data from longitudinal stool samples from IBD patients (publicly available from the Human Microbiome Project 2; iHMP) were analyzed for the presence and relative abundance of histidine decarboxylase genes (see methods for details). Data shown are a compilation of all data across multiple collection time points.

(D) Groups of female germ-free C57BL/6 mice were colonized with a mock community of 9 phylogenetically diverse human gut bacteria (Mock Community A) with or without M. morganii C135. Mice were fed a conventional diet and administered 1% L-His ad libitum in the drinking water. Histamine concentrations in serum were measured via ELISA. n = 3-5 mice per group.

(B-C) M. morganii primarily inhabits the cecum and colon. Groups of female germ-free C57BL/6 mice were colonized with mock communities of 9 or 10 phylogenetically diverse gut microbes (Mock community A and B, respectively) with or without M. morganii C135. M. morganii CFUs can be distinguished from other bacteria based on their purple halos when plated on modified Niven’s agar. Gastric, small intestinal, cecal and colonic contents from mice colonized with Mock communities A or B and M. morganii were plated on Modified Niven’s agar to determine M. morganii colonization levels at various intestinal loci. Stacked barplot represents relative abundance of bacterial taxa in mice colonized with Mock community A plus M. morganii based on 16S rRNA gene sequencing (see also Table S3 ). n = 4 mice per group.

(A) Groups of female germ-free C57BL/6 mice were colonized with mock communities of 9 or 10 phylogenetically diverse human gut bacteria (Mock Community A or B) or monocolonized with M. morganii C135. Mice were fed a conventional diet with or without administration of 1% L-His ad libitum in the drinking water. Histamine concentrations in serum were measured via ELISA. n = 3-5 mice per group.

Data in all panels are representative of at least two independent experiments. Data are presented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test (C-F) or Kruskall-Wallis with Dunn’s multiple comparisons (G-H), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, NS not significant (p > 0.05).

(G and H) Relative abundances of genes encoding histidine decarboxylases (from all bacteria or M. morganii) are increased in the microbiomes of patients with Crohn’s disease as compared to healthy controls (G). Relative abundance of histamine is increased in IBD patients as compared to healthy controls as measured by metabolomics (H). Data are from longitudinal stool samples from IBD patients and are publicly available from the Human Microbiome Project 2 (iHMP). Total numbers of samples or subjects with detectable M. morganii are denoted below each plot; a subject was considered positive if M. morganii was detectable in one or more samples from that patient across the complete dataset.

(F) Histamine receptor inhibition partially reverses the impact of M. morganii on colon motility. Female germ-free C57BL/6 mice were colonized with Mock Community A or monocolonized with M. morganii C135 for two weeks. Mice were then treated with or without a cocktail of four histamine receptor inhibitors (targeting HRH1-4) in the drinking water for one week. Histamine concentrations in feces were measured via ELISA and fecal output was measured as in (D). n = 4-6 mice per group.

(E) M. morganii increases colon motility in the context of a mock gut microbial community. Female germ-free C57BL/6 mice were colonized Mock Community A with or without M. morganii C135 and administered 1% L-His ad libitum in the drinking water. Histamine concentrations in colonic extracts were measured via ELISA and fecal output was measured as in (D). n = 4-5 mice per group.

(D) M. morganii C135- and L. reuteri C93-derived histamine enhances colon motility. Fecal output for mice treated as described in B was measured by counting the number of fecal pellets produced by a single mouse in one hour. n = 3-5 mice per group.

(C) M. morganii- and L. reuteri-derived histamine accumulates in vivo in monocolonized mice. Female germ-free C57BL/6 mice were colonized with mock communities of 9 or 10 phylogenetically diverse human gut bacteria (Mock Community A or B) or monocolonized with M. morganii C135, L. reuteri C88 or C93. Mice were fed a conventional diet with or without administration of 1% L-His ad libitum in the drinking water. Histamine concentrations in cecal and colonic extracts and feces were measured via ELISA. n = 3-5 mice per group.

(A) Production of histamine by M. morganii and L. reuteri. L. reuteri and M. morganii strains were cultured in Gifu medium with or without supplemental L-His and histamine concentrations in the supernatants were measured by ELISA after 48 hours (background levels in controls containing supplemental histidine are due to slight cross-reactivity).

In mammals, PEA, dopamine, and tyramine are produced via decarboxylation of L-Phe, L-DOPA, and L-Tyr, respectively, by the aromatic L-amino acid decarboxylase (AADC; Figure S3 A) (). Thus, we tested whether M. morganii would similarly process these amino acids into their respective biogenic amines using a minimal medium (MM) lacking L-Phe, L-DOPA, L-Tyr, and L-His. Despite normal M. morganii growth in this medium, we could not detect any production of PEA, tyramine, dopamine, or histamine ( Figure 3 F). However, supplementation with L-Phe or L-His led to the production of high levels of PEA or histamine, and activation of DRD2-4 or HRH2-4 ( Figures 3 F and 3G). In contrast, supplementation with L-DOPA or L-Tyr failed to lead to the production of dopamine or tyramine, or activation of DRDs ( Figure S4 C). Thus, unlike mammalian AADC, M. morganii selectively converts L-Phe into PEA and cannot efficiently convert L-DOPA or L-Tyr into dopamine or tyramine. While it is currently unclear which genes are involved in the production of PEA by M. morganii, whole-genome sequencing revealed the presence of at least 17 decarboxylases that are shared between the two strains of M. morganii that we sequenced ( Table S4 S5 ).

Previous reports have also suggested that M. morganii produces histamine (). We confirmed that M. morganii secreted significant amounts of histamine by ELISA and that our M. morganii strains encoded a previously described histidine decarboxylase; furthermore, 48 of 49 previously deposited M. morganii strains also encoded this histidine decarboxylase ( Figure 3 E and Table S4 S5 ). Two L. reuteri strains and two Enterobacteriaceae strains from our collections also secreted histamine ( Figure 3 E). Based on whole-genome sequencing, both the histamine-producing and non-histamine-producing strains of L. reuteri encoded an identical histidine decarboxylase proenzyme ( Table S4 S5 ). Together, these data reveal that M. morganii secretes high levels of PEA, which acts as a potent dopamine receptor agonist, and that M. morganii and select strains of L. reuteri secrete histamine.

We next used the cyclic AMP response element-secreted human placental alkaline phosphatase (CRE-SEAP) assay to examine whether PEA and related chemicals also activate G proteins (in addition to β-arrestin) downstream of DRDs (). To facilitate use of the CRE-SEAP assay for GPCRs that couple to G proteins other than Gα, such as DRD2-4, we used a Gα-Gαfusion protein (a kind gift of Stephen Liberles) to redirect DRD2-4 signaling to Gα). PEA activated G protein signaling downstream of all five dopamine receptors, which suggests that PEA is a full agonist for DRD2-4 and may be a biased agonist for DRD1 and 5 ( Figures S4 A and S4B).

(C) OD values for 24 hour cultures of M. morganii grown in minimal medium (MM) with or without L-Phe, L-Tyr, L-DOPA or L-His. n = 3 replicates per sample.

(B) Activation of G protein-dependent signaling downstream of DRD2-4 by titrating doses phenethylamine and related chemicals was measured by the CRE-SEAP assay. A Gα s -Gα o fusion was used to redirect DRD2-4 to Gα s and enable use of the CRE-SEAP assay. n = 3 replicates per sample.

(A) Activation of Gα s -dependent signaling downstream of DRD1, 5 and TAAR1 by titrating doses of phenethylamine and related chemicals was measured by the CRE-SEAP assay. n = 3 replicates per sample.

M. morganii was previously reported to produce various biogenic amines, including dopamine, tyramine, and phenethylamine (PEA) (). We noted that all M. morganii supernatants activated DRD2-4, but not DRD1 and 5 ( Figures 3 A, 3B, and S2 ). In contrast, dopamine itself activated all five dopamine receptors ( Figure S1 A). Therefore, we suspected that M. morganii might produce a metabolite that is structurally related to dopamine and can act as a selective ligand for DRD2-4 but not DRD1 or 5 ( Figure S3 A). We examined the ability of all possible upstream and downstream metabolites in the mammalian dopamine pathway to activate DRDs and found that PEA and tyramine showed identical activation patterns to M. morganii supernatant ( Figures S3 A–S3C). Accordingly, metabolomic analyses revealed that M. morganii produced only trace amounts of dopamine and no detectable tyramine, but instead secreted significant quantities of the potent trace amine PEA which, unlike dopamine and tyramine, can readily cross the blood-brain barrier ( Figures 3 D, S3 D, and S3E) ().

Besides the succinate receptor, the next most prevalent class of GPCRs activated by gut commensals was the aminergic receptors, which are expressed in diverse tissues and cell types and regulate a variety of core physiological processes ranging from neurotransmission to immunity ( Figure 2 ) (). More than a dozen commensal supernatants activated the dopamine (DRDs) or histamine (HRHs) receptors ( Figures 3 A and S2 ). For example, ten Proteobacteria strains activated both DRDs and HRHs, including all eight Morganella morganii strains in our collection ( Figures 3 B, 3C, and S2 ). In contrast, two Lactobacillus reuteri strains activated HRHs, while two distinct L.reuteri strains failed to activate HRHs despite displaying similar growth kinetics ( Figures 3 C and S2 ). Finally, one Streptococcus strain, but not two related isolates of Streptococcus, activated DRD2-4, and two unclassified Enterobacteriaceae strains activated HRH1-4 and DRD2 but failed to activate other DRDs ( Figures 3 C and S2 ).

Data for all panels other than A and B are representative of at least three independent experiments. Data are presented as mean ± SEM. n = 3 replicates per group (C-G).

(G) M. morganii-derived phenethylamine and histamine activate DRD2-4 and HRH2-4, respectively. M. morganii C135 were cultured as described in F and supernatants were screened for activity against DRDs and HRHs by PRESTO-Tango.

(F) Mass spectrometric traces of metabolite production by M. morganii C135. M. morganii was cultured in minimal medium (MM) with or without additional L-Phe, L-His, L-Tyr or L-DOPA for 48 hours. Metabolite production was analyzed by Liquid Chromatography-Mass Spectrometry (LC-MS).

(D) Quantification of dopamine, phenethylamine and tyramine production by M. morganii. Supernatants from 24-hour cultures of M. morganii C135 in gut microbiota medium were analyzed by Triple Quadrupole-Mass Spectrometry (QQQ-MS/MS). ND, not detected.

(B) Heatmap depicting the activation of aminergic GPCRs by metabolites from a human gut microbiota culture collection as measured by PRESTO-Tango. Fold induction over stimulation with bacterial media alone is depicted on a log2 scale.

(A) Activation of aminergic GPCRs by metabolomes from a 144 memeber human gut microbiota culture collection (see Figure 1 ). GPCR activation was measured by PRESTO-Tango. Screening results are displayed on a phylogenetic tree of aminergic GPCRs. Color intensity represents magnitude of activation over media alone and radii of the circles represent the number of bacteria that activated a given GPCR by more than two-fold over media alone.

PRESTO-Tango screening revealed that bacterial-derived metabolite mixtures activated both well-characterized GPCRs as well as orphan receptors, including GPCRs from nearly every class ( Figure 2 ). One specific pattern of activation tracked closely with gross phylogeny—Bacteroidetes and Proteobacteria potently activated the succinate receptor (Sucr1), while Firmicutes, Fusobacteria, and Actinobacteria largely failed to activate this receptor ( Figure 1 and Table S2 ). However, most activation patterns did not correlate with phylogeny, and multiple bacterial strains assigned to the same species exhibited unique GPCR agonist activities ( Table S2 ). GMM alone also activated select GPCRs when compared to PBS, and supernatants from specific microbes sometimes reversed these effects either due to bacterial consumption of GPCR ligands in the media or bacterial production of GPCR antagonists ( Table S3 ). Given the exquisite sensitivity of PRESTO-Tango, the veracity of all individual hits will need to be confirmed using alternative methods. Nonetheless, these data demonstrate that human gut microbes produce a remarkable array of GPCR ligands.

We previously assembled personalized gut microbiota culture collections from 11 inflammatory bowel disease patients through high-throughput anaerobic culture methods and next-generation sequencing (). This collection yielded 144 unique bacterial isolates from five phyla, nine classes, 11 orders, and 20 families, as well as multiple strains that were assigned to the same species ( Table S1 ). We cultured all members of our collection individually in a medium specialized for the cultivation of gut commensals (gut microbiota medium; GMM) () and screened their supernatants for activation or inhibition of nearly all conventional GPCRs (as compared to media alone) using PRESTO-Tango (see STAR Methods for details) ( Figures 1 and 2 ).

GPCR activation by metabolomes from a human gut microbiota culture collection consisting of 144 strains isolated from 11 IBD patients. Data are displayed on a hierarchical tree of GPCRs organized by class, ligand type, and receptor family. Color intensity represents the maximum magnitude of activation (log 2) over background (gut microbiota medium alone) across the complete dataset. Radii of the circles at each tip represent the number of strains that activated a given receptor or receptor family by more than two-fold over background. Graphics were generated in collaboration with visavisllc using d3.js.

We set out to establish a high-throughput screening system to identify specific human gut microbes that produce agonists or antagonists of conventional GPCRs. We developed a pipeline for parsing the microbiota metabolome based on the GPCR screening technology Parallel Receptor-ome Expression and Screening via Transcriptional Output-Tango (PRESTO-Tango) (). This technology leverages the Tango β-arrestin recruitment assay to simultaneously measure the activation of nearly all non-olfactory GPCRs ( Figures 1 S1 A and S1B) (). We thus proceeded to exploit this assay to perform a broad-ranging screen of bioactive metabolites produced by diverse members of the human gut microbiota.

We isolated 144 unique human gut bacteria spanning five phyla, nine classes, eleven orders, and twenty families from 11 inflammatory bowel disease patients via high-throughput anaerobic culturomics and massively barcoded 16S rRNA gene sequencing. Bacterial isolates were grown in monoculture in a medium specialized for the cultivation of human gut microbes (gut microbiota medium) and supernatants from individual bacterial monocultures were screened against the near-complete non-olfactory GPCRome (314 conventional GPCRs) using Parallel Receptor-ome Expression and Screening via Transcriptional Output-Tango (PRESTO-Tango).

Discussion

Donia and Fischbach, 2015 Donia M.S.

Fischbach M.A. HUMAN MICROBIOTA. Small molecules from the human microbiota. Fischbach, 2018 Fischbach M.A. Microbiome: Focus on Causation and Mechanism. The overwhelming complexity of the gut microbiota metabolome often obscures facile recognition of chemical communication between microbes and their hosts (). Here, we used host GPCR activation as a lens to detect bioactive metabolites produced by individual gut microbes. We found that dozens of phylogenetically diverse human gut bacteria produced small molecules that activated various GPCRs, including both well-characterized GPCRs and orphan GPCRs. We observed patterns of metabolite production that were largely predictable based on phylogeny, as well as strain-specific differences within a given species. Our approach thus revealed a plethora of novel microbiota metabolite-GPCR interactions of potential physiological importance. For example, we uncovered a diet-microbe-host axis that influences intestinal motility through microbial production of histamine and a tri-partite microbe-microbe-host relationship that results in the production of the potent trace amine phenethylamine. These chemicals exerted profound effects on both local and systemic host physiology. Overall, our results further support the notion that human-associated microbes represent a remarkably rich source of small molecules that impact human biology.

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Brady S.F. Accessing Bioactive Natural Products from the Human Microbiome. Prior studies have employed functional metagenomic screens as well as bioinformatics- and bioassay-guided natural product discovery approaches to uncover novel microbial-derived ligands for host GPCRs, including orphans (e.g., SCFA and GPR41 and 43, and commendamide and G2A and GPR119; (); however, these approaches also have notable limitations (). For example, while functional metagenomic screens enable identification of novel biosynthetic gene clusters and their products from unculturable microorganisms, they are restricted in scope to contiguous biosynthetic gene clusters that are active in heterologous hosts, require large-scale library construction, and necessitate extensive follow up to identify specific host receptors that recognize novel bioactive compounds. Similarly, while bioassay-guided natural product discovery efforts enable identification of compounds produced by native sources that engage a specific receptor or pathway, their utility is largely restricted to cultivatable microorganisms and they typically examine only a single receptor or activity at a time. In contrast, the high-throughput functional profiling approach that we employ here enables simultaneous interrogation of hundreds of receptors and thousands of chemicals and is unconstrained by prior annotations of biosynthetic gene clusters or metabolites (although still dependent on microbial cultivation). We thus anticipate that future expansions of our overall approach will continue to uncover microbial metabolites that impact host physiology and reveal novel natural ligands for orphan receptors.

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et al. Histamine-secreting microbes are increased in the gut of adult asthma patients. Smolinska et al., 2014 Smolinska S.

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O’Mahony L. Histamine and gut mucosal immune regulation. We were particularly interested in examining the possibility that the microbiota-derived GPCR agonists we identified in vitro would also shape host physiology in vivo. We found that histamine production by M. morganii or L. reuteri increased colonic motility, that feeding with exogenous histidine enhanced this phenotype, and that histamine receptor inhibition reversed these effects. Since fecal output can be impacted by multiple factors (e.g., fluid secretion and modulation of the enteric nervous system), future studies will be necessary to determine the mechanistic basis of this phenotype. M. morganii monocolonized mice also exhibited elevated levels of serum histamine, indicating a potential systemic role for microbiota-derived histamine. Notably, a recent study found that M. morganii relative abundance was increased in asthmatics as compared to healthy controls (). Finally, we found that histamine decarboxylases (specifically from M. morganii) are enriched in patients with Crohn’s disease, which suggests that histamine production by the microbiota may directly impact IBD ().

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Burnett J.R. Phenylketonuria: an inborn error of phenylalanine metabolism. Our studies also uncovered a specific Bacteroides strain that produces high levels of the essential amino acid L-Phe and revealed that L-Phe activates the orphans GPR56/AGRG1 and GPR97/AGRG3. These findings raise multiple intriguing possibilities. GPR56/AGRG1 is highly expressed in the small intestine and human pancreatic islets (), and L-Phe concentrations in the jejunum can reach concentrations up to 2 mM after a meal (). Thus, GPR56/AGRG1 may act as a nutrient sensor to regulate digestion and satiety. In addition, although L-Phe concentrations in the serum are typically well below the levels necessary to activate GPR56/97, patients with phenylketonuria (PKU) can exhibit serum L-Phe concentrations greater than 1 mM (). Thus, it is also theoretically possible that GPR56/AGRG1 and/or GPR97/AGRG3 may be activated in extraintestinal tissues in PKU (e.g., GPR56/AGRG1 is highly expressed in the central nervous system).

The natural microbiota metabolome results from a complex web of interactions between diverse microbial species and strains, environmental inputs (e.g., diet), and host factors. Using a reductionist approach, we discovered two bacterial isolates that traffic in the same small molecule: a unique strain of B. theta that is a prolific producer of L-Phe and M. morganii, which converts L-Phe into PEA. These studies thus demonstrate that reductionist approaches can reveal metabolic exchanges that would be missed when examining endpoint microbiota metabolomes produced by complex mixtures of microorganisms. Understanding metabolic exchange networks will be essential to understand the effects of the microbiota metabolome on host biology under more physiological settings (i.e., in the context of complete gut microbial communities) and to eventually leverage microbial chemistries for therapeutic benefit. Toward these ends, we examined the effects of M. morganii on host physiology in the context of a mock gut microbial community consisting of nine phylogenetically diverse human gut microbes. We found that M. morganii continued to exhibit measurable (albeit more modest) metabolite-dependent impacts on the host in the context of this simplified community. However, there are almost certainly other gut microbial community contexts where competition for ecological space or metabolic precursors (e.g., L-His or L-Phe), or active degradation of M. morganii-derived metabolites, may reduce or eliminate the impact of M. morganii on the host (or, conversely, may enhance the effects of M. morganii).

Adibi and Mercer, 1973 Adibi S.A.

Mercer D.W. Protein digestion in human intestine as reflected in luminal, mucosal, and plasma amino acid concentrations after meals. Pezeshki et al., 2016 Pezeshki A.

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Chelikani P.K. Low protein diets produce divergent effects on energy balance. Our studies underscore the role of dietary amino-acids (e.g., L-His) in microbial production of biogenic amines. However, they also highlight the role of other members of the microbiota as sources of compounds that are often thought to be primarily derived from diet (e.g., essential amino acids). This leads to the question of when microbial-produced amino acids may potentially supplement or even replace dietary amino acids in microbial biotransformations. We modeled the possibility that microbe-derived L-Phe can be biotransformed by M. morganii using a simplified diet that lacks L-Phe. However, bacterial L-Phe may also be important under more physiological conditions. For example, dietary amino acids are largely absorbed in the small intestine and thus free amino acid concentrations in the colon are often limiting (); also, low-protein diets and fasting can dramatically reduce intestinal amino acid availability (). Thus, microbial production of amino acids may play a critical role in the production of bioactive microbiota metabolites.

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O’Mahony L. Histamine and gut mucosal immune regulation. While our reductionist approach revealed multiple potentially physiologically important host-microbiota metabolome interactions, it also suffers from notable limitations. For example, microbial metabolite production varies substantially depending on the media used for cultivation, and in vitro monoculture conditions fail to capture metabolites that result from interactions with the host organism, biotransformations of compounds absent from the cultivation medium, or interactions with other microbes. Furthermore, the metabolite concentrations produced during in vitro cultivation may not reflect in vivo metabolite production. Finally, in vitro screens fail to reveal the natural tissue distributions of gut microbiota metabolites. Understanding these distributions will be particularly important for metabolites that activate host receptors that are expressed in diverse cell types and tissues. For example, histamine and dopamine receptors are expressed on cells as diverse as immune cells, central and peripheral neurons, smooth muscle, epithelial and endothelial cells, and in essentially all tissues including the gut, lung, and brain ().

In conclusion, while the human gut microbiota metabolome is dauntingly complex and diverse, emerging approaches have begun to reveal key chemical interactions at the host-microbiota interface. We show here that high-throughput activity-based screening using potential host receptors as a lens can highlight physiologically relevant microbiota metabolites from complex metabolite mixtures. Such host-centric, functional profiling approaches can thus facilitate a mechanistic understanding of how we interact with and are affected by our microbial inhabitants, and have the potential to yield targeted therapeutic interventions aimed at the interface between indigenous microbes and their hosts.