Comparative Study
doi: 10.1038/nature23874.
Epub 2017 Aug 30.
Commensal bacteria make GPCR ligands that mimic human signalling molecules
Affiliations
- PMID: 28854168
- PMCID: PMC5777231
- DOI: 10.1038/nature23874
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Comparative Study
Commensal bacteria make GPCR ligands that mimic human signalling molecules
Nature.
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Erratum in
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Corrigendum: Commensal bacteria make GPCR ligands that mimic human signalling molecules.Nature. 2018 Apr 4;556(7699):135. doi: 10.1038/nature25997. Nature. 2018. PMID: 29620727
Abstract
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).
Conflict of interest statement
The authors of this study have no competing financial interests to declare.
Figures
a, LCMS analysis of crude extracts prepared from E. coli transformed with each hm-NAS gene expression construct (number 1–43, see Supplementary Table 1 for details about each clone number) compared to negative control extracts derived from E. coli containing an empty vector (con). Based on 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(−) MS 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 largely encoding the same metabolite family. Commendamide was previously isolated and is part of family 1.
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 harbor the same or highly related (>80% nucleotide identity) hm-NAS gene that was expressed by heterologous expression. The only exception was for N-acyl alanines for which a representative cultured commensal bacterial species was not available. N-acyl glycines were previously analyzed in the same manner. The extracted ion 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 a due to formatting constraints.
Proposed two-step biosynthesis of N-acyl serinol using the two domains found in the enzyme predicted to be encoded by the hm-NAS N-acyl serinol synthase gene. Simple N-palmitoyl derivatives of all 20 natural amino acids did not activated GPR119 by more than 37% relative to OEA.
When structural analogs were independently screened in the GPCR panel (e.g., 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, d, e
N-palmitoyl and oleoyl serinol both interact with GPR119. Screening data performed in singlicate, dose response curves performed in duplicate. Error bars are mean +/− SEM.
Table links GPCR, N-acyl amide, bacterial genus and the site where these co-occur in the gastrointestinal tract (colored). Based on 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 known to encode metabolites that target these GPCR (Figure 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 value 2e-132) to those we found to encode GPR119 or S1PR4 ligands.,,
ACTOne HEK293 cells (control) and ACTOne HEK293 cells transfected with GPR119 were exposed to equimolar concentrations of the endogenous GPR119 ligand oleoylethanolamide or the bacterial ligand N-oleoyl serinol. Relative fluorescent intensity was recorded for each ligand concentration compared to background signal. All data points were performed in quadruplicate and error bars represent SD around the mean. An increase in cAMP concentration was observed in HEK293 cells expressing GPR119 but not in native 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 (Figure 5a).
a, LC-MS analysis of crude cecal 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 N-palmitoyl serinol parent ion and the product ion at m/z: 92.0706 shows presence of the serinol head group.
One week after inoculation with E. coli a single fecal 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. a) The number of colony forming units per 10−6 g of feces 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 there are other colonizing bacteria present. Stool samples produced threefold more colony forming units on unselected LB agar plates compared to LB agar plates with kanamycin. Error bars in both a, b are mean +/− SEM. In both cases when random colonies were picked from the LB/kanamycin plates they were all 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 (E94A). N-acyl serinol metabolites (e.g., N-palmitoyl serinol and N-oleoyl serinol) are absent from the point mutant culture broth (ESI(+) mode). This mutant was created to address the possibility that the observed mouse phenotype might be due to over-production of any protein by E. coli and not specifically from N-acyl serinol production.
High-resolution reversed-phase LC-MS analysis of human fecal extract pooled from 128 samples representing 21 individuals. Extracted ion chromatograms for individual N-acyl amides are shown within a 2 ppm tolerance of the exact mass (M+H). Compounds observed to be present in the human fecal extract were confirmed by alignment to authentic standards (top panel), 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.
a, Phylogenetic tree of N-acyl genes from PFAM13444. hm-NAS genes have a circle at the branch tip. Black dots were not synthesized, red dots were synthesized but no molecule was detected and large grey dots mark genes that produced N-acyl amides. Branches are colored by bacterial phylogeny. b, The major heterologously produced metabolite from each N-acyl family (1–6) is shown. c,
hm-NAS gene distribution and abundance [Reads per Kilobase of Gene Per Million Reads (RPKM)] based on molecule family (1–6). Body site and molecule designations in a are based on the analysis shown in c and b. Box plots are median from 1st to 3rd quartile. HMP patient samples analyzed include N = 133 tongue, 127 plaque, 148 stool, 122 buccal.
a, Gene expression analysis for an N-acyl glycine hm-NAS gene in a stool metatranscriptome dataset and an N-acyloxyacyl lysine hm-NAS gene in a supragingival plaque metatranscriptome dataset. Gene expression is normalized to the expression of all genes from a bacterial genome containing the hm-NAS gene that was heterologously expressed – Bacteroides dorei in stool, Capnocytophaga ochracea in plaque (1 highly expressed, 0 not expressed). b, Comparison of hm-NAS gene abundance based on RNA or DNA derived reads obtained from individual patient stool samples. Abundance is measured in RPKM. N = 24 patient stool samples analyzed – 8 patients with 3 samples per patient. N = 38 patient plaque samples analyzed.
Screen of N-acyl amides (color coded) for agonist activity against 168 GPCRs with known ligands (a) as well as 72 orphan GPCRs (b). Dot plots display data for all N-acyl amides assayed against all GPCRs. Screen performed in singlicate. Bar graphs show the strongest N-acyl GPCR agonist interactions compared to all GPCRs. Insets show dose response curves and EC50 data (each dose performed in duplicate). c, Screen of N-acyl amides as antagonists in the presence of endogenous ligands. Screen performed in singlicate. i, PTGIR is specifically inhibited by N-acyloxyacyl glutamine. ii, PTGER4 is inhibited by structurally diverse hm-N-acyl amides. Error bars are mean +/− SEM.
Comparison of microbiota encoded and human GPCR ligands suggests a structural and functional complementarity.
a,
β-arrestin GPR119 activation assay using microbiota (green) and human (blue) ligands (each dose performed in duplicate). b,
β-arrestin assay comparing microbiota ligands and 20 synthesized N-palmitoyl amino acids (screen performed in singlicate). c, Release of GLP-1 by GLUTag cells (ANOVA, p < 0.05, data combined from 2 independent experiments, N = 4 for DMSO and 2-oleoyl glycerol, N = 6 for OEA and N-oleoyl serinol). d, Oral glucose tolerance test (OGTT) in gnotobiotic mice. Treatment mice (n = 6 mice, data combined from 2 independent experiments) were colonized with E. coli producing N-acyl serinols and control mice (n = 8 mice, data combined from 2 independent experiments) were colonized with E. coli containing an empty vector (two way ANOVA, bonferroni post-hoc) e, OGTT after withholding IPTG to stop N-acyl gene expression (no difference, two way ANOVA, N is the same as in d). f, OGTT in an antibiotic treated mouse cohort (n = 9 mice in both groups, data combined from 2 independent experiments, two way ANOVA, bonferroni post-hoc). g, Insulin (n = 6 mice in both groups, one experiment, technical triplicates) and h, GLP-1 (n = 9 control mice, n = 10 treatment mice, data combined from 2 independent experiments, technical replicates) measured at 15 min after glucose gavage in the antibiotic treated cohort (unpaired T test, two tailed). Key Bacterial (green) and human (blue) GPR119 ligands reference figure colors. Error bars (mean +/− SEM) * p < 0.05, ** p < 0.01 *** p < 0.001.
Comment in
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Gut microbiota: Microbial metabolites as mimickers of human molecules.Nat Rev Gastroenterol Hepatol. 2017 Nov;14(11):630-631. doi: 10.1038/nrgastro.2017.131. Epub 2017 Sep 13. Nat Rev Gastroenterol Hepatol. 2017. PMID: 28900240 No abstract available.
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Host-microbe interactions: Aiming at GPCRs.Nat Chem Biol. 2017 Oct 18;13(11):1139. doi: 10.1038/nchembio.2507. Nat Chem Biol. 2017. PMID: 29045380 No abstract available.
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G protein-coupled receptors: Gut feeling on bacterial GPCR agonists.Nat Rev Drug Discov. 2017 Oct 30;16(11):754. doi: 10.1038/nrd.2017.205. Nat Rev Drug Discov. 2017. PMID: 29081522 No abstract available.
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Gut microbiota in 2017: Contribution of gut microbiota-host cooperation to drug efficacy.Nat Rev Gastroenterol Hepatol. 2018 Feb;15(2):69-70. doi: 10.1038/nrgastro.2017.170. Epub 2017 Dec 20. Nat Rev Gastroenterol Hepatol. 2018. PMID: 29259330 No abstract available.
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The Gut Feeling: GPCRs Enlighten the Way.Cell Host Microbe. 2019 Aug 14;26(2):160-162. doi: 10.1016/j.chom.2019.07.018. Cell Host Microbe. 2019. PMID: 31415748 Free PMC article.
