Commensal bacteria are believed to have important roles in human health. The mechanisms by which they affect mammalian physiology remain poorly understood, but bacterial metabolites are likely to be key components of host interactions. Here we use bioinformatics and synthetic biology to mine the human microbiota for N-acyl amides that interact with G-protein-coupled receptors (GPCRs). We found that N-acyl amide synthase genes are enriched in gastrointestinal bacteria and the lipids that they encode interact with GPCRs that regulate gastrointestinal tract physiology. Mouse and cell-based models demonstrate that commensal GPR119 agonists regulate metabolic hormones and glucose homeostasis as efficiently as human ligands, although future studies are needed to define their potential physiological role in humans. Our results suggest that chemical mimicry of eukaryotic signalling molecules may be common among commensal bacteria and that manipulation of microbiota genes encoding metabolites that elicit host cellular responses represents a possible small-molecule therapeutic modality (microbiome-biosynthetic gene therapy).
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(Wh)olistic (E)ndocannabinoidome-Microbiome-Axis Modulation through (N)utrition (WHEN) to Curb Obesity and Related Disorders
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We thank the High-Throughput and Spectroscopy Resource Center, Center for Clinical and Translational Science and the Comparative Bioscience Center at Rockefeller University for the use of their facilities; members of the Mangelsdorf laboratory at UT Southwestern and D. Drucker at Mount Sinai Hospital, Toronto for the use of the GLUTag cell line; A. Milshteyn, A. Estrela and J. Craig for their critical review of the manuscript. This work was supported in part by a grant from the Robertson Foundation, the Center for Basic and Translational Research on Disorders of the Digestive System, the Leona M. and Harry B. Helmsley Charitable Trust, Rainin Foundation, U01 GM110714-1A1 (S.F.B.), GM122559-01 (S.F.B.), the Crohn’s and Colitis Foundation Career Development Award (L.J.C.) and NIDDK K08 DK109287-01 (L.J.C.).
The authors declare no competing financial interests.
Reviewer Information Nature thanks D. J. Drucker and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
a, LC–MS analysis of crude extracts prepared from E. coli transformed with each hm-NAS gene expression construct (number 1–58, see Supplementary Table 1 for details about each clone number) compared to negative control extracts derived from E. coli containing an empty vector (Con). On the basis of metabolite retention time and observed mass hm-NAS genes could be grouped into 6 N-acyl amide families (1–6). The mass of the major metabolite (pictured) from each N-acyl amide family is shown in either the ESI+ or ESI− mass spectrometry detection mode for each hm-NAS extract, including the control extract. Functional differences in NAS enzymes follow the pattern of the NAS phylogenetic tree, with hm-NAS genes from the same clade or sub-clade mostly encoding the same metabolite family. Commendamide (Comm.) was previously isolated and is part of family 1 (ref. 3). b, Phylogenetic tree of PFAM13444, showing the location of each hm-NAS gene that we synthesized and examined by heterologous expression. c, Crude ethyl acetate extracts were prepared from cultures of bacterial species that contain the same or a highly related (>80% nucleotide identity) hm-NAS gene that was expressed by heterologous expression. The only exception was for the N-acyl alanine family, for which a representative cultured commensal bacterial species was not available. N-acyl glycines were previously analysed in the same manner3. The extracted-ion chromatogram for the hm-NAS gene family is shown for the E. coli clone compared to the crude extract from the commensal species. For family 6, hm-NAS clone 58 is pictured in c and not in a owing to formatting constraints.
Proposed two-step biosynthesis of N-acyl serinol using the two domains encoded by the hm-NAS N-acyl serinol synthase gene.
When structural analogues were independently screened in the GPCR panel (for example, N-oleoyl and palmitoyl serinol or N-3-hydroxypalmitoyl lysine and ornithine), they yielded the same GPCR profile and when N-acyl serinol was re-assayed across all GPCRs in the panel, it also yielded the same GPCR activity profile. a, b, N-3-hydroxypalmitoyl lysine and ornithine both interact with S1PR4. *Did not repeat. b, inset technical repeat of N-3-hydroxypalmitoyl ornithine dose–response curve. c, d, Technical repeats of N-palmitoyl serinol. c–e, N-palmitoyl and oleoyl serinol both interact with GPR119. Screening data were performed once, dose–response curves were performed in duplicate. Data are mean ± s.e.m.
Extended Data Figure 4 Combined analysis of protein and transcript expression of GPCRs in the gastrointestinal tract.
Links between GPCR, N-acyl amide, bacterial genus and the site where these co-occur in the gastrointestinal tract (coloured) are shown. On the basis of protein expression data (Human Protein Atlas) GPR119 is most highly expressed in the pancreas and duodenum, S1PR4 in the spleen and lymph node, G2A in the lymph node and appendix, PTGIR in the lung and appendix and PTGER4 in the bone marrow and small intestine. From gene expression data in the colon (GTEx dataset, n = 88 patient samples from small intestine, 345 patient samples from colon) GPR132, PTGER4 and PTGIR are all expressed alongside the N-acyl synthase genes that are known to encode metabolites that target these GPCRs (Fig. 1). In the gastrointestinal tract, GPR119 and S1PR4 are most highly expressed in the small intestine where 16S studies have identified bacteria from the genera Gemella and Neisseria. All known reference genomes (NCBI) from these genera contain N-acyl synthase genes that are highly similar (BLASTN, E = 2 ×10 −132) to those we found to encode GPR119 or S1PR4 ligands37,43,44.
ACTOne HEK293 cells (control) and ACTOne HEK293 cells transfected with GPR119 were exposed to equimolar concentrations of the endogenous GPR119 ligand OEA or the bacterial ligand N-oleoyl serinol. Relative fluorescent intensity was recorded for each ligand concentration compared to the background signal. All data points were performed in quadruplicate and data are mean ± s.d. An increase in cAMP concentration was observed in HEK293 cells expressing GPR119, but not in control HEK293 cells. The DCEA (5-(N-ethylcarboxamido)adenosine) control is presented to confirm cAMP response of the parental cell line. The EC50 for N-oleoyl serinol (bacterial) was 1.6 μM and for oleoylethanolamide was 5.1 μM, which are consistent with data from the β-arrestin assay (Fig. 5a).
a, LC–MS analysis of crude caecal extracts. Extracted-ion chromatograms for palmitoyl serinol ([M + H]+ m/z: 330.3003) are shown. A peak with the same exact mass and chromatographic retention time as the N-palmitoyl serinol standard was present in treatment mice but not control mice. Treatment mice were colonized with E. coli containing the N-acyl serinol synthase gene. Control mice were colonized with E. coli containing the empty pET28c vector. b, Identification of N-palmitoyl serinol by MS/MS fragmentation of the m/z 330.3003 ion. In the MS2 spectrum the diamond indicates the N-palmitoyl serinol parent ion and the product ion at m/z: 92.0706 shows the presence of the serinol head group.
One week after inoculation with E. coli, a single faecal pellet from a colonized mouse was collected, resuspended in 400 μl PBS and plated at a 1:100 dilution onto LB agar plates with or without kanamycin 50 μg ml−1. a, The number of colony-forming units per 10−6 g of faeces observed on LB agar plates with kanamycin was similar for the treatment group (E. coli with hm-NAS gene, n = 6 mouse stool samples) and the control group (E. coli with empty vector, n = 8 mouse stool samples). b, In the antibiotic-treated mouse cohort, other colonizing bacteria are present. Stool samples produced threefold more colony-forming units on unselected LB agar plates compared to LB agar plates with kanamycin. Data are mean ± s.e.m. In both cases, when random colonies were picked from the LB with kanamycin plates, all colonies were found to contain the cloning vector. indicating these were in fact E. coli colonizing bacteria.
LC–MS analysis of crude extracts prepared from cultures of E. coli expressing either the N-acyl serinol synthase gene or the N-acyl serinol synthase gene with an active site point mutation (E91A). N-acyl serinol metabolites (for example, N-palmitoyl serinol and N-oleoyl serinol) are absent from the culture broth with the point mutant (ESI+ mode). This mutant was created to address the possibility that the observed mouse phenotype might be due to overproduction of any protein by E. coli and not specifically from N-acyl serinol production.
High-resolution reversed-phase LC–MS analysis of human faecal extract pooled from 128 samples representing 21 individuals. Extracted-ion chromatograms for individual N-acyl amides are shown within a 2 p.p.m. tolerance of the exact mass (M + H). Compounds observed to be present in the human faecal extract were confirmed by alignment to authentic standards (top) and by spiked addition of the pure compound (data not shown). No zwitterionic N-acyl amides (N-acyl or N-acyloxyacyl ornithine/lysines) were detected. Patient demographics are provided in the table. Human stool samples were collected with informed consent at MSKCC under institutional review board numbers 09-167, 09-141, and 06-107.
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Cohen, L., Esterhazy, D., Kim, SH. et al. Commensal bacteria make GPCR ligands that mimic human signalling molecules. Nature 549, 48–53 (2017). https://doi.org/10.1038/nature23874
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