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Gut-inhabiting Clostridia build human GPCR ligands by conjugating neurotransmitters with diet- and human-derived fatty acids

Nature Microbiology volume 6, pages 792–805 (2021)Cite this article

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A Publisher Correction to this article was published on 22 April 2021

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Abstract

Human physiology is regulated by endogenous signalling compounds, including fatty acid amides (FAAs), chemical mimics of which are made by bacteria. The molecules produced by human-associated microbes are difficult to identify because they may only be made in a local niche or they require a substrate sourced from the host, diet or other microbes. We identified a set of uncharacterized gene clusters in metagenomics data from the human gut microbiome. These clusters were discovered to make FAAs by fusing exogenous fatty acids with amines. Using an in vitro assay, we tested their ability to incorporate 25 fatty acids and 53 amines known to be present in the human gut, from which the production of six FAAs was deduced (oleoyl dopamine, oleoyl tyramine, lauroyl tryptamine, oleoyl aminovaleric acid, α-linolenoyl phenylethylamine and caproyl tryptamine). These molecules were screened against panels of human G-protein-coupled receptors to deduce their putative human targets. Lauroyl tryptamine is found to be an antagonist to the immunomodulatory receptor EBI2 against its native oxysterol ligand (0.98 μM half-maximal inhibitory concentration), is produced in culture by Eubacterium rectale and is present in human faecal samples. FAAs produced by Clostridia may serve as a mechanism to modulate their host by mimicking human signalling molecules.

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Fig. 1: Mining Clostridia pathways from the human gut metagenome.
Fig. 2: The pathways from Clostridia produce FAAs.
Fig. 3: Enzyme activity against fatty acids and amines present in the human gut.
Fig. 4: Lauroyl tryptamine is an EBI2 antagonist that is present in human faecal material.

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Data availability

Genomic data used in this study are available in JGI (https://gold.jgi.doe.gov/) and NCBI (https://www.ncbi.nlm.nih.gov/) with identifiers listed in Supplementary Data 1. The FAA pathways found in this study are available in NCBI with identifiers listed in Supplementary Table 2. HMP faecal metagenomic data used in this study are available in NCBI with identifiers listed in Supplementary Table 7. HMP faecal metatranscriptomic data used in this study are available in NCBI with identifiers listed in Supplementary Table 8. BIO-ML OpenBiome Project untargeted metabolomics data used in this study are available in the Metabolomics Workbench (https://www.metabolomicsworkbench.org) under Project ID PR000804. Data supporting the findings of this study are available within the paper, supplementary materials and Source data are provided with this paper. Additional data are available from the corresponding author upon reasonable request. All strains and plasmids are available upon request.

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Acknowledgements

We thank T. Wyche and J. Clardy (Harvard Medical School) for help with nuclear magnetic resonance. We also thank S. Abubucker and H. Haiser (Novartis) for help with experimental design. The stool samples were graciously provided by E. Alm (MIT) and M. Poyet (MIT) of the OpenBiome. This work is funded by the US Defense Advanced Research Projects Agency (DARPA) Living Foundries 1KM award no. HR0011-15-C-0084. F.-Y.C., C.A.V. and D.B.G. are funded by a research award from the Novartis Institute for Biomedical Research (NIMBR).

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Authors and Affiliations

  1. Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Fang-Yuan Chang, Emerson Glassey, David Benjamin Gordon & Christopher A. Voigt

  2. The Foundry, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Fang-Yuan Chang, Thomas Williams, David Benjamin Gordon & Christopher A. Voigt

  3. Synthetic Biology Group, Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Cambridge, MA, USA

    Piro Siuti & Horst Hemmerle

  4. Disease Area X, Novartis Institutes for BioMedical Research, Basel, Switzerland

    Stephane Laurent & Andreas W. Sailer

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Contributions

F.-Y.C., P.S., D.B.G., H.H. and C.A.V. conceived the study and designed the experiments. F.-Y.C. performed the bioinformatics, experiments and data analysis. S.L. and A.W.S. performed and analysed the EBI2 assays. T.W., E.G. and D.B.G. analysed MS data and performed experiments with the faecal samples. F.-Y.C. and C.A.V. wrote the manuscript.

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Correspondence to Christopher A. Voigt.

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Peer review information Nature Microbiology thanks Vanessa Phelan for her contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Pathway boundary analysis.

a, Genetic diagrams for the eighteen Clostridia pathways, consisting of the eight HMP-derived pathways (top) and the ten NCBI nr database (bottom), with pathway boundaries predicted by antiSMASH. Pink = biosynthetic genes (1 = condensation domain protein, 2 = thiolation domain protein, 3 = adenylation domain protein). Yellow = accessory genes (4 = alpha/beta-fold hydrolase, 5 = PPTase, 6 = sterol transfer protein). Colorless = other genes (7 = TetR regulator, 8 = AraC regulator, 9 = Renal dipeptidase, 10 = HisJ phosphatase, 11 = MerR regulator, 12 = Cell wall glycosyltransferase, 13 = DMT transporter, 14 = CorA transporter, 15 = Citrate transporter. b, Annotation of the E. recale pathway including two additional genes outside of the antiSMASH-predicted boundary. Each gene was investigated for the presence of a homolog in other FAA pathways.

Extended Data Fig. 2 In vitro substrate screening for pathways producing same major compounds.

Six Clostridia-derived pathways encoding for major FAA compounds that are equivalent to the pathways from Fig. 3d. Top: Major compound based on fatty acid and amine substrate with highest activity level in the panel assay. Bottom: Bar graph representing adenylation and condensation activity from the substrate panel assay. The amines are color-coded based on the FAA classes as in Fig. 3c. The data represent means of three experiments performed on different days. The data values are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Fatty acid specificity profile.

a, Overall average of product peak area of the eighteen Clostridia A domain proteins on the following fatty acid substrates (from left to right data point): stearic acid (F12), palmitic acid (F11), arachidonic acid (F22), docosahexaenoic acid (F25), linoleic acid (F18), α-linolenic acid (F20), and oleic acid (F16). This is correlated with the dissociation constant of human brain fatty acid binding protein measured by isothermal titration calorimetry (ITC), as reported in previous study74. b, Substrate specificity profile of the Clostridia adenylation protein by the length of fatty acid substrate structure. The reported value of product peak is the overall average from the eighteen Clostridia A domain proteins. The structure length is calculated by importing the structure from PubChem onto Chem3D version 15 (in the U-bent configuration for unsaturated fatty acids) and measuring the distance from the carboxylic acid carbon to the most distal carbon.

Extended Data Fig. 4 GPCR activity assay for oleoyl dopamine.

Cell-based β-arrestin reporter assay (DiscoverX) with a panel of 168 GPCRs with known ligands in agonist mode and antagonist mode, and also 73 orphan GPCRs in agonist mode at 10 µM. Agonist mode measures % activity of target GPCR by the compound, relative to the baseline value (0% activity) and maximum value with a known ligand, or two-fold increase over baseline for orphan GPCR (100% activation). Antagonist mode measures % inhibition of target GPCR by the compound in the presence of a known ligand, relative to the value at the EC80 (0% inhibition) and basal value (100% inhibition). GPCR targets with activity/inhibition higher than the empirical threshold provided by DiscoverX (30%, 35%, or 50% for GPCR agonist, GPCR antagonist, or orphan GPCR agonist, respectively; plotted as dotted line) are highlighted in red.

Extended Data Fig. 5 GPCR activity assay for oleoyl tyramine.

Cell-based β-arrestin reporter assay (DiscoverX) with a panel of 168 GPCRs with known ligands in agonist mode and antagonist mode, and also 73 orphan GPCRs in agonist mode at 10 µM. Agonist mode measures % activity of target GPCR by the compound, relative to the baseline value (0% activity) and maximum value with a known ligand, or two-fold increase over baseline for orphan GPCR (100% activation). Antagonist mode measures % inhibition of target GPCR by the compound in the presence of a known ligand, relative to the value at the EC80 (0% inhibition) and basal value (100% inhibition). GPCR targets with activity/inhibition higher than the empirical threshold provided by DiscoverX (30%, 35%, or 50% for GPCR agonist, GPCR antagonist, or orphan GPCR agonist, respectively; plotted as dotted line) are highlighted in red.

Extended Data Fig. 6 GPCR activity assay for oleoyl aminovaleric acid.

Cell-based β-arrestin reporter assay (DiscoverX) with a panel of 168 GPCRs with known ligands in agonist mode and antagonist mode, and also 73 orphan GPCRs in agonist mode at 10 µM. Agonist mode measures % activity of target GPCR by the compound, relative to the baseline value (0% activity) and maximum value with a known ligand, or two-fold increase over baseline for orphan GPCR (100% activation). Antagonist mode measures % inhibition of target GPCR by the compound in the presence of a known ligand, relative to the value at the EC80 (0% inhibition) and basal value (100% inhibition). GPCR targets with activity/inhibition higher than the empirical threshold provided by DiscoverX (30%, 35%, or 50% for GPCR agonist, GPCR antagonist, or orphan GPCR agonist, respectively; plotted as dotted line) are highlighted in red.

Extended Data Fig. 7 Activity of FAAs.

a, Dose response curves (sigmoidal) of the inhibitory activity of lauroyl tryptamine, tryptamine, and lauric acid on P2RY4 in the presence of the native agonist UTP at 2.79 µM (EC80). IC50 > 100 µM for all three compounds. The points were measured in duplicate. b, Dose response curves (sigmoidal) of the stimulatory activity of lauroyl tryptamine, tryptamine, and lauric acid on GPR132. EC50: lauroyl tryptamine = 1.45 µM; lauric acid = 25.2 µM; tryptamine > 100 µM. The points were measured in duplicate. c, Summary table of EC50s and IC50s calculated using data from the DiscoverX cell-based assay. d, Dose response curves (sigmoidal) of the calcium release-based stimulatory activity on EBI2. The error bars are the standard deviations of the three mean quadruplicate values. EC50: lauroyl tryptamine > 20 µM; 7α,25-OHC = 0.002 µM. e, Dose response curves (sigmoidal) of the radioligand binding activity of different compounds on EBI2. The error bars are the standard deviations of the three mean quadruplicate values. f, Dose response curves (sigmoidal) of the calcium release-based inhibitory activity of different compounds on EBI2 in the presence of the native agonist 7α,25-OHC at 3.7 nM (EC80). The error bars are the standard deviations of the three mean quadruplicate values. g, Dose response curves (sigmoidal) of the calcium release-based stimulatory activity of different compounds on EBI2. The error bars are the standard deviations of the three mean quadruplicate values. h, Summary table of EC50 and IC50 from radioligand binding and calcium release assays. NIBR51 and NIBR189 are known EBI2 antagonists. 7α,25-Dihydroxycholesterol (7α,25-OHC), 7β,25-Dihydroxycholesterol (7β,25-OHC), 25-Hydroxycholesterol (25-OHC), cholesterol, and 7β-Hydroxycholesterol (7-OHC) are known EBI2 agonists.

Extended Data Fig. 8 Lauroyl tryptamine produced by Eubacterium rectale.

a, Lauroyl tryptamine production by E. rectale in culture. Extracts were evaluated without substrate feeding in RCM media. MS chromatogram: (EIC ESI+ [M + H]+ m/z of lauroyl tryptamine, theoretical = 343.2744, experimental = 343.2369). The samples are injected alongside a chemically-synthesized and structurally-verified standard. The data shown are representative of three experiments performed on different days, all showing similar results. b, MS-MS chromatogram (lauroyl tryptamine quantifier ion 343.3 m/z -> 144.1) is shown for the product obtained from an E. rectale culture alongside a structurally-verified standard. c, UV spectrum for lauroyl tryptamine in E. rectale culture alongside a structurally-verified standard. d, Standard curve of 280 nm absorbance (0.5 nm tolerance) peak area by injection of the chemically synthesized lauroyl tryptamine standard. Standard curve was used to calculate the FAA concentrations in E. rectale culture, including Fig. 4e. The standards were originally measured in ng/mL in solution, as injected into the LC-MS. This was converted to mg/L, where L is bacterial culture volume, by multiplying by a factor of 0.002 (Methods). The FAA concentration was measured on three different days. A new standard curve was constructed for each day, one curve of which is shown.

Extended Data Fig. 9 Lauroyl tryptamine in human fecal samples.

a, Total count of MS peaks with the matching exact mass (within 0.002 ppm), or isomers, of the primary FAAs (bold) and secondary FAAs that are present in BIO-ML collection of human fecal samples89. The number indicates the isomers that have the same mass as the compound indicated, but one could not be proven to be that compound without additional experimentation. b, Standard curve of LCMS-MS peak area by injection of structurally-verified standard for quantification of lauroyl tryptamine titer in fecal samples. The standard curve was used to calculate the concentrations in c (Methods). The standards were originally measured in pg/mL in solution, as injected into the LC-MS. This was converted to ng/g, where g is “fresh fecal” wet weight, by multiplying by a factor of 0.04 (Methods). c, Summary FAA titers in select BIO-ML fecal samples (ng/g fresh fecal weight). ‘**’ indicates no compound detected.

Extended Data Fig. 10 Enzyme activity assay time course.

Fatty acid (left) and amine (right) substrate panel assay for C. eutactus pathway proteins. a, Unnormalized product level data are shown for 8 representative fatty acid (left) or amine (right) substrates. Product level data with adenylation protein (black lines) are normalized with respect to data without adenylation protein (red lines) for enzyme activity data with fatty acid substrates (left). Product level data with condensation protein (black lines) are normalized with respect to data without condensation protein (red lines) for enzyme activity data with amine substrates (right). b, For all 25 fatty acid (left) or 53 amine (right) substrates, normalized activity data are shown. The time point with the largest difference between the highest point and the remainder of the curves is marked with an arrow. The means were calculated based on three triplicates performed in different days and the error bars are the standard deviations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–12, Tables 1–8 and references.

Reporting Summary

Supplementary Data 1

Human gut-associated bacteria genomic datasets.

Supplementary Data 2

Data values for Supplementary Fig. 10.

Source data

Source Data Fig. 3

Data values for Fig. 3d.

Source Data Extended Data Fig. 2

Data values for Extended Data Fig. 2.

Source Data Fig. 4

Full scan of Fig. 4h. Lanes for Ladder and Stool (ere) are shown in Fig. 4h. Different BIO-ML stool DNA was subject to PCR detection for each lane. BIO-ML stool ID for each lane is as follows: lane 1, af-0003; 2, am-0015; 3, am-0111; 4, aq-0015; 5, bu-0080; 6, cj-0004; and Stool (ere), dc-0028.

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Chang, FY., Siuti, P., Laurent, S. et al. Gut-inhabiting Clostridia build human GPCR ligands by conjugating neurotransmitters with diet- and human-derived fatty acids. Nat Microbiol 6, 792–805 (2021). https://doi.org/10.1038/s41564-021-00887-y

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