《人体的社交网络——共生微生物》课外文献阅读：Commensal bacteria produce GPCR ligands that mimic human signaling molecules

HHS Public Access Author manuscript Nature. Author manuscript; available in PMC 2018 February 28 Published in final edited form as Nature.2017 September07,549(767048-53.doi:10.1038/ nature23874 Commensal bacteria produce GPCR ligands that mimic human signaling molecules Louis J. Cohen, 2, Daria Esterhazy, Seong-Hwan Kim, Christophe Lemetre', Rhiannon R. Aguilar', Emma A Gordon1, Amanda J. Pickard, Justin R Cross, Ana B. Emiliano4, Sun M. Han, John Chu, Xavier Vila-Farres', Jeremy Kaplitt, Aneta Rogoz, Paula Y. Calle Craig Hunter, J Kipchirchir Bitok', and Sean F. Brady Laboratory of Genetically Encoded Small Molecules, Rockefeller University Division of Gastroenterology, Department of Medicine, Icahn School of Medicine at Mount Sinai lAboratory of Mucosal Immunology, Rockefeller University 4Laboratory of Molecular Genetics, Rockefeller University cOmparative Biosciences Center, Rockefeller University 6Donald B. and Catherine C Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center Summary Statement Commensal bacteria are believed to play important roles in human health. The mechanisms by which they affect mammalian physiology are poorly understood; however, 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 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 al though future studies are needed to define their potential physiologic role in humans. This work suggests that chemical mimicry of eukaryotic signaling molecules may be common among commensal bacteria and download text and data.mine the content in such documents for the academic research, subjectalwaystothefullConditionsofusehttp://www.nature.com/authors/editorialpolicies/license.htmlterms Correspondence and requests for materials should be addressed to s.F. B. (sbrady(@rockefeller. edu). Contact: Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, 1230 York Avenue, New York, New York, 10065 Author Contributions: L.J.C. and S F B designed research, L.J.C. assisted with all experiments;, S.H. K. assisted with molecule characterization, E.A.G P.Y. C., J.K. B, and R.R.A. assisted with gene cloning:, D E, A B E, S M.H C H. and A.R. assisted with mouse experiments; J.C. X V-F, J K. assisted with molecule synthesis; A.J.P. and J. R C assisted with metabolite analysis in human/mouse samples; L.J.C. and C L analyzed data, LJ. C. and S.F.B. wrote the paper methods, figures and tables related to the structural determination of compound Competing Financial Interest Statement The authors of this study have no competing financial interests to declare

Commensal bacteria produce GPCR ligands that mimic human signaling molecules Louis J. Cohen1,2, Daria Esterhazy3, Seong-Hwan Kim1, Christophe Lemetre1, Rhiannon R. Aguilar1, Emma A. Gordon1, Amanda J. Pickard6, Justin R. Cross6, Ana B. Emiliano4, Sun M. Han1, John Chu1, Xavier Vila-Farres1, Jeremy Kaplitt1, Aneta Rogoz3, Paula Y. Calle1, Craig Hunter5, J. Kipchirchir Bitok1, and Sean F. Brady1 1Laboratory of Genetically Encoded Small Molecules, Rockefeller University 2Division of Gastroenterology, Department of Medicine, Icahn School of Medicine at Mount Sinai 3Laboratory of Mucosal Immunology, Rockefeller University 4Laboratory of Molecular Genetics, Rockefeller University 5Comparative Biosciences Center, Rockefeller University 6Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center Summary Statement Commensal bacteria are believed to play important roles in human health. The mechanisms by which they affect mammalian physiology are poorly understood; however, 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 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 physiologic role in humans. This work suggests that chemical mimicry of eukaryotic signaling molecules may be common among commensal bacteria Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence and requests for materials should be addressed to S.F.B. (sbrady@rockefeller.edu). Contact: Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, 1230 York Avenue, New York, New York, 10065. Author Contributions: L.J.C. and S.F.B. designed research; L.J.C. assisted with all experiments; S-H.K. assisted with molecule characterization; E.A.G., P.Y.C., J.K.B., and R.R.A. assisted with gene cloning; D.E., A.B.E., S.M.H., C.H. and A.R. assisted with mouse experiments; J.C., X.V-F., J.K. assisted with molecule synthesis; A.J.P. and J.R.C. assisted with metabolite analysis in human/mouse samples; L.J.C. and C.L. analyzed data; L.J.C. and S.F.B. wrote the paper Supplementary Information Extended data figures and tables are provided to accompany the main text and methods. Supplementary information contains the methods, figures and tables related to the structural determination of compounds. Competing Financial Interest Statement The authors of this study have no competing financial interests to declare. HHS Public Access Author manuscript Nature. Author manuscript; available in PMC 2018 February 28. Published in final edited form as: Nature. 2017 September 07; 549(7670): 48–53. doi:10.1038/nature23874. Author Manuscript Author Manuscript Author Manuscript Author Manuscript

and that manipulation of microbiota genes encoding metabolites that elicit host cellular responses represents a new small molecule therapeutic modality (microbiome-biosynthetic-gene-therapy) Keywords GPCR; microbiome; metagenome; signaling: N-acyl amide Although the human microbiome is believed to play an important role in human physiology Bacteria rely heavily on small molecules to interact with their environment. While it is likely that the human microbiota similarly relies on small molecules to interact with its human host, the identity and functions of microbiota-encoded effector molecules are largely unknown. The study of small molecules produced by the human microbiota and the identification of the host receptors they interact with should help to define the relationship between bacteria and human physiology and provide a resource for the discovery of small We recently reported on the discovery of commendamide, a human microbiota encoded, G protein-coupled receptor(GPCR) active, long-chain N-acyl amide that suggests a structural onvergence between human signaling molecules and microbiota encoded metabolites. N- acyl amides, like the endocannabinoids, are able to regulate diverse cellular functions due part, to their ability to interact with GPCRs. GPCRs are the largest family of membrane receptors in eukaryotes and are likely to be key mediators of host-microbial interactions the human microbiome. The importance of GPCRs to human physiology is reflected by the fact that they are the most common targets of therapeutically approved small molecule drugs. The GPCRs with which human N-acyl amides interact are implicated in diseases including diabetes, obesity, cancer, and inflammatory bowel disease among others. 4, With numerous possible combinations of amine head groups and acyl tails, long-chain N-acyl amides represent a potentially large and functionally diverse class of microbiota-encoded GPCR-active signaling molecules Here, we combined bioinformatic analysis of human microbiome sequencing data with targeted gene synthesis, heterologous expression and high-throughput GPCR activity screening to identify GPCR-active N-acyl amides encoded by the human microbiota. The human microbiome and suggest these GPCR-active small molecules and their associate bacterial effectors we identified provide mechanistic insights into potential functions of the microbial biosynthetic genes have the potential to regulate human physiology Isolation of commensal N-acyl amides To identify N-acyl synthase(NAS)genes within human microbial genomes, the Human Microbiome Project(HMP)sequence data was searched with BLASTN using 689 NAS genes associated with the N-acyl synthase protein family PFAM134443 The 143 unique human microbial N-acyl synthase genes(hm-NASs)we identified fall into four major clades Nature. Author manuscript; available in PMC 2018 February 28

and that manipulation of microbiota genes encoding metabolites that elicit host cellular responses represents a new small molecule therapeutic modality (microbiome-biosynthetic-gene-therapy). Keywords GPCR; microbiome; metagenome; signaling; N-acyl amide Introduction Although the human microbiome is believed to play an important role in human physiology the mechanisms by which bacteria affect mammalian physiology remain poorly defined.1 Bacteria rely heavily on small molecules to interact with their environment.2 While it is likely that the human microbiota similarly relies on small molecules to interact with its human host, the identity and functions of microbiota-encoded effector molecules are largely unknown. The study of small molecules produced by the human microbiota and the identification of the host receptors they interact with should help to define the relationship between bacteria and human physiology and provide a resource for the discovery of small molecule therapeutics. We recently reported on the discovery of commendamide, a human microbiota encoded, G protein-coupled receptor (GPCR) active, long-chain N-acyl amide that suggests a structural convergence between human signaling molecules and microbiota encoded metabolites.3 N￾acyl amides, like the endocannabinoids, are able to regulate diverse cellular functions due, in part, to their ability to interact with GPCRs. GPCRs are the largest family of membrane receptors in eukaryotes and are likely to be key mediators of host-microbial interactions in the human microbiome. The importance of GPCRs to human physiology is reflected by the fact that they are the most common targets of therapeutically approved small molecule drugs. The GPCRs with which human N-acyl amides interact are implicated in diseases including diabetes, obesity, cancer, and inflammatory bowel disease among others.4,5 With numerous possible combinations of amine head groups and acyl tails, long-chain N-acyl amides represent a potentially large and functionally diverse class of microbiota-encoded GPCR-active signaling molecules. Here, we combined bioinformatic analysis of human microbiome sequencing data with targeted gene synthesis, heterologous expression and high-throughput GPCR activity screening to identify GPCR-active N-acyl amides encoded by the human microbiota. The bacterial effectors we identified provide mechanistic insights into potential functions of the human microbiome and suggest these GPCR-active small molecules and their associated microbial biosynthetic genes have the potential to regulate human physiology. Isolation of commensal N-acyl amides To identify N-acyl synthase (NAS) genes within human microbial genomes, the Human Microbiome Project (HMP) sequence data was searched with BLASTN using 689 NAS genes associated with the N-acyl synthase protein family PFAM13444.3 The 143 unique human microbial N-acyl synthase genes (hm-NASs) we identified fall into four major clades Cohen et al. Page 2 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Cohen et al (clades A-D, Fig. la) that are divided into a number of distinct sub-clades( Fig. la). Forty four phylogenetically diverse hm-NAS genes were selected for synthesis and heterologous expression. This set included all hm-NAS genes from clades sparsely populated with hm- NAS sequences and representative examples from clades heavily populated with hm-NAS sequences(Fig. la) Liquid chromatography-mass spectrometry(LCMS)analysis of ethyl acetate extracts derived from E. coli cultures transformed with each construct revealed clone specific peaks in 31 cultures. hm-NAS gene functions could be clustered into 6 groups based on the retention time and mass of the heterologously produced metabolites(Extended Data Fig. I and Supplementary Table 1) Molecule isolation and structural elucidation studies were carried out on one representative culture from each group(Supplementary Information) This analysis identified six N-acyl amide families that differ by amine head group and fatty acid tail(Fig. Ib, families 1-6): 1)N-acyl glycine, 2)N-acyloxyacyl lysine, 3)N acyloxyacyl glutamine, 4)N-acyl lysine/ornithine, 5)N-acyl alanine, 6) N-acyl serinol. Each family was isolated as a collection of metabolites with different acyl substituents. The most common analog within each family is shown in Figure 1b. Long-chain N-acyl ornithine, lysines and glutamines have been reported as natural products produced by soil bacteria and some human pathogens.6, 7, 8 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(Fig. la). with the exception of one nAs that is predicted to use lysine and ornithine as substrates, hm-NASs appear to be selective for a single amine-containing substrate. The most common acyl chains incorporated by hm-NASs are from 14-18 carbons in length These can be modified by B-hydroxy lation or a single unsaturation. Three hm-NAS enzymes ontain two domains. The second domain is either an aminotransferase that is predicted to alyze the formation of serinol from glycerol (Fig. Ib, family 6, Extended Data Fig. 2)or an additional acy transferase that is predicted to catalyze the transfer of a second acyl grot Fig. Ib, families 2, 3). To explore NAS gene synteny we looked for gene occurrence patterns around NAS genes in the human microbiome. The only repeating pattern that we saw was that some nAs genes appear adjacent to genes predicted to encode acyltransferases This is reminiscent of the two domain NASs that we found produce di-acyl lipids(families 2 and 3). There were rare instances where NASs potentially occur in gene clusters, but none of these were used in this study To look for native N-acyl amide production by commensal bacteria, organic extracts from ultures of species containing the hm-NAS genes we examined were screened by LCms Based on retention time and mass we detected the production of the expected N-acyl amides by commensal species predicted to produce N-acyl glycines, N-acyloxyacyl lysines, N-acyl lysine/ornithine and N-acyl serinols. The only case where we did not detect the expected N- acyl amide was for N-acyloxyacyl glutamines(Extended Data Fig. 1) Nature. Author manuscript; available in PMC 2018 February 28

(clades A–D, Fig. 1a) that are divided into a number of distinct sub-clades (Fig. 1a). Forty￾four phylogenetically diverse hm-NAS genes were selected for synthesis and heterologous expression. This set included all hm-NAS genes from clades sparsely populated with hm￾NAS sequences and representative examples from clades heavily populated with hm-NAS sequences (Fig. 1a). Liquid chromatography-mass spectrometry (LCMS) analysis of ethyl acetate extracts derived from E. coli cultures transformed with each construct revealed clone specific peaks in 31 cultures. hm-NAS gene functions could be clustered into 6 groups based on the retention time and mass of the heterologously produced metabolites (Extended Data Fig. 1 and Supplementary Table 1). Molecule isolation and structural elucidation studies were carried out on one representative culture from each group (Supplementary Information). This analysis identified six N-acyl amide families that differ by amine head group and fatty acid tail (Fig. 1b, families 1–6): 1) N-acyl glycine, 2) N-acyloxyacyl lysine, 3) N￾acyloxyacyl glutamine, 4) N-acyl lysine/ornithine, 5) N-acyl alanine, 6) N-acyl serinol. Each family was isolated as a collection of metabolites with different acyl substituents. The most common analog within each family is shown in Figure 1b. Long-chain N-acyl ornithines, lysines and glutamines have been reported as natural products produced by soil bacteria and some human pathogens.6,7,8 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 (Fig. 1a). With the exception of one NAS that is predicted to use lysine and ornithine as substrates, hm-NASs appear to be selective for a single amine-containing substrate. The most common acyl chains incorporated by hm-NASs are from 14–18 carbons in length. These can be modified by β-hydroxylation or a single unsaturation. Three hm-NAS enzymes contain two domains. The second domain is either an aminotransferase that is predicted to catalyze the formation of serinol from glycerol (Fig. 1b, family 6, Extended Data Fig. 2) or an additional acyltransferase that is predicted to catalyze the transfer of a second acyl group (Fig. 1b, families 2, 3). To explore NAS gene synteny we looked for gene occurrence patterns around NAS genes in the human microbiome. The only repeating pattern that we saw was that some NAS genes appear adjacent to genes predicted to encode acyltransferases. This is reminiscent of the two domain NASs that we found produce di-acyl lipids (families 2 and 3). There were rare instances where NASs potentially occur in gene clusters, but none of these were used in this study. To look for native N-acyl amide production by commensal bacteria, organic extracts from cultures of species containing the hm-NAS genes we examined were screened by LCMS. Based on retention time and mass we detected the production of the expected N-acyl amides by commensal species predicted to produce N-acyl glycines, N-acyloxyacyl lysines, N-acyl lysine/ornithines and N-acyl serinols. The only case where we did not detect the expected N￾acyl amide was for N-acyloxyacyl glutamines (Extended Data Fig. 1). Cohen et al. Page 3 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Cohen et al hm-NAs genes are enriched in gl bacteria A BLASTN search of NAs genes against human microbial reference genomes and metagenomic sequence data from the HMP revealed that NAS genes are enriched gastrointestinal(Gi) bacteria relative to bacteria found at other body sites( Fischers exact test p<0.05, gastrointestinal versus non gastrointestinal sites, Supplementary Table 2, Figure 1). Within gastrointestinal sites that were frequently sampled in the context of the HMP(e.g, stool, buccal mucosa, supragingival plaque, tongue)hm-NAS gene families show distinct distribution patterns(Fig. Ic, two way ANOVA p<2e-16). Despite tremendous person-to-person variation in microbiota species composition, most N-acyl amide synthase gene families we studied can be found in over 90% of patient samples. N-acyoxyacyl glutamine(12%)and N-acyl alanine(not detected) synthase genes are the only exceptions Taken together, these data suggest that NAs genes are highly prevalent in the human microbiome and unique sites within the gastrointestinal tract are likely exposed to different sets of N-acyl amide structures When we searched existing metatranscriptome sequence data from stool and supragingival plaque microbiomes to look for evidence of hm-NAS gene expression in the gastrointestinal tract we observed site-specific hm-NAS gene expression that matches the predicted body si localization patterns for hm-NAS genes in metagenomic data. Across patient samples hm NAS genes are transcribed to varying degrees relative to the average level of transcription for each gene in the bacterial genome(Fig. 2a). In the stool metatranscriptome dataset both RNA and DNa sequencing datasets were available allowing for a more direct sample-to- sample comparison of hm-NAS gene expression levels. When metatranscriptome data were normalized using the number of hm-NAS gene specific DNA sequence reads detected in each sample, we observed what appears to be differential expression of hm-NAS genes in different patient samples(Fig. 2b ). Datasets whereby bacterial genes, transcripts and metabolites can be tracked in a single sample will be necessary to explore how hm-NAS gene transcription variation impacts metabolite production. hm-N-acyl-amides interact with gl GPCRs The major N-acyl amide isolated from each family was assayed for agonist and antagonist activity against 240 human GPCRs(Fig 3 and Extended Data Fig 3). The strongest agonist interactions were: activation of GPR119 by N-palmitoyl serinol(EC509 HM), activation of phingosine-l-phosphate receptor 4(SIPR4) by N-3-hydroxypalmitoyl ornithine(EC50 32 uM) and activation of G2a by N-myristoyl alanineEC50 3 uM). Interactions between bacterial N-acyl amides and GPCRs were also specific(Fig 3a and b). In each survey experiment, no other GPCRs reproducibly showed greater than 35% activation relative to the endogenous ligands. The strongest antagonist activities were observed for N-acyloxyacyl glutamine against two prostaglandin receptors, PTGIR and PTGER4(Fig 3c, PTGIR IC50 15 AM, PIGER4 IC50 43 uM). PTGiR was specifically antagonized by N-acyloxyacyl glutamine, while PTGER4 was antagonized by N-acyloxyacyl glutamine as well as other N- acyl amides [Fig 3c(i)and 3c(ii]. Alternative GPCR screening methods could identify interactions in addition to those uncovered here Nature. Author manuscript; available in PMC 2018 February 28

hm-NAS genes are enriched in GI bacteria A BLASTN search of NAS genes against human microbial reference genomes and metagenomic sequence data from the HMP revealed that NAS genes are enriched in gastrointestinal (GI) bacteria relative to bacteria found at other body sites (Fischer’s exact test p < 0.05, gastrointestinal versus non gastrointestinal sites, Supplementary Table 2, Figure 1). Within gastrointestinal sites that were frequently sampled in the context of the HMP (e.g., stool, buccal mucosa, supragingival plaque, tongue) hm-NAS gene families show distinct distribution patterns (Fig. 1c, two way ANOVA p < 2e-16). Despite tremendous person-to-person variation in microbiota species composition, most N-acyl amide synthase gene families we studied can be found in over 90% of patient samples. N-acyoxyacyl glutamine (12%) and N-acyl alanine (not detected) synthase genes are the only exceptions. Taken together, these data suggest that NAS genes are highly prevalent in the human microbiome and unique sites within the gastrointestinal tract are likely exposed to different sets of N-acyl amide structures. When we searched existing metatranscriptome sequence data from stool and supragingival plaque microbiomes to look for evidence of hm-NAS gene expression in the gastrointestinal tract we observed site-specific hm-NAS gene expression that matches the predicted body site localization patterns for hm-NAS genes in metagenomic data. Across patient samples hm￾NAS genes are transcribed to varying degrees relative to the average level of transcription for each gene in the bacterial genome (Fig. 2a). In the stool metatranscriptome dataset both RNA and DNA sequencing datasets were available allowing for a more direct sample-to￾sample comparison of hm-NAS gene expression levels. When metatranscriptome data were normalized using the number of hm-NAS gene specific DNA sequence reads detected in each sample, we observed what appears to be differential expression of hm-NAS genes in different patient samples (Fig. 2b). Datasets whereby bacterial genes, transcripts and metabolites can be tracked in a single sample will be necessary to explore how hm-NAS gene transcription variation impacts metabolite production. hm-N-acyl-amides interact with GI GPCRs The major N-acyl amide isolated from each family was assayed for agonist and antagonist activity against 240 human GPCRs (Fig. 3 and Extended Data Fig. 3). The strongest agonist interactions were: activation of GPR119 by N-palmitoyl serinol (EC50 9 µM), activation of sphingosine-1-phosphate receptor 4 (S1PR4) by N-3-hydroxypalmitoyl ornithine (EC50 32 µM) and activation of G2A by N-myristoyl alanine (EC50 3 µM). Interactions between bacterial N-acyl amides and GPCRs were also specific (Fig. 3a and b). In each survey experiment, no other GPCRs reproducibly showed greater than 35% activation relative to the endogenous ligands. The strongest antagonist activities were observed for N-acyloxyacyl glutamine against two prostaglandin receptors, PTGIR and PTGER4 (Fig. 3c, PTGIR IC50 15 µM, PTGER4 IC50 43 µM). PTGIR was specifically antagonized by N-acyloxyacyl glutamine, while PTGER4 was antagonized by N-acyloxyacyl glutamine as well as other N￾acyl amides [Fig. 3c(i) and 3c(ii)]. Alternative GPCR screening methods could identify interactions in addition to those uncovered here. Cohen et al. Page 4 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Cohen et al Based on data from the Human Protein Atlas(HPA) GPCRs targeted by human microbial acyl amides are localized to the gastrointestinal tract and its associated immune cells. In mouse models, this collection of gastrointestinal tract localized GPCRs have been reported to affect diverse mucosal functions including metabolism(GPRI19), immune cell differentiation (SIPR4, PTGIR, PTGER4), immune cell trafficking(SIPR4, G2A)and tissue repair(Ptgir) It is not possible at this time to look for co-localization of GPCR and hm-NAS gene expression in specific gastrointestinal niches, as neither the HMP nor the HPa are sufficiently comprehensive in their survey of human body sites. Nonetheless, 16S and metagenomic deep sequencing studies link bacteria containing hm-NAS genes or hm- NAS genes themselves to specific locations in the gastrointestinal tract where GPCRs of nterest are expressed (Extended Data Fig 4) Bacterial and human ligands share structure and function Human microbiota-encoded N-acyl amides bear structural similarity to endogenous GPCr- active ligands(Fig. 4). The clearest overlap in structure and function between bacterial and human GPCR-active ligands is for the endocannabinoid receptor GPR119(Fig. 4 and 5) Endogenous GPRI19 ligands include oleoylethanolamide(OEA)and the dietary lipid derivative 2-oleoyl glycerol(2-0G) 15, 16 In our heterologous expression experiment we isolated both the palmitoyl and oleoyl analogs of N-acyl serinol. The latter only differs from 2-OG by the presence of an amide instead of an ester and from Oea by the presence of an dditional ethanol substituent. N-oleoyl serinol is a similarly potent GPR119 agonist compared to the endogenous ligand OEa(EC50 12 AM vS. 7 uM) but elicits almost a 2-fold greater maximum GPRI19 activation(Fig 5a). N-palmitoyl derivatives of all 20 natural amino acids were synthesized and none activated GPri19 by more than 37% relative to OEA (Fig. 5b). The generation of a potent and specific long-chain N-acyl-based GPRI ligand therefore necessitates a more complex biosynthesis than the simple N-acylation of an amino acid as is commonly seen for characterized NAs enzymes. In this case, the biosynthesis of N-acyl serinols is achieved through the coupling of an NAs domain with an aminotransferase that is predicted to generate serinol from glycerol(Extended Data Fig. 2 The endogenous agonist for SIPR4, sphingosine-l-phosphate(SIP)and the M-3- hydroxypalmitoyl ornithine/lysine family of bacterial agonists share similar head group charges. SIP is a significantly more potent agonist(EC500.09 HM vS EC50 32 uM) however, the bacterial agonists are more specific for SIPR4. The bacterial N-3- hydroxypalmitoyl ornithine did not activate SIPRl, 2, or 3 in our GPCR screen, whereas SIP activates all four members of the SIP receptor family tested No direct comparison could be made between the microbiota-derived and endogenous ligands for PTGiR or PtgeR4 as there are no known endogenous antagonists for these receptors. Many human GPCRs remain orphan receptors lacking known endogenous ligands. Ligands for at least some of these receptors will undoubtedly be found among the small molecules produced by the human microbiota. G2A is an orphan receptor and therefore does not have a well-defined endogenous agonist, although it has been reported to respond to lysophosphatidylcholine. 7, 18 We found that the bacterial metabolites N-3- hydroxypalmitoyl glycine(commendamide) and N-palmitoyl alanine, both activate g2A Nature. Author manuscript; available in PMC 2018 February 28

Based on data from the Human Protein Atlas (HPA) GPCRs targeted by human microbial N￾acyl amides are localized to the gastrointestinal tract and its associated immune cells. In mouse models, this collection of gastrointestinal tract localized GPCRs have been reported to affect diverse mucosal functions including metabolism (GPR119), immune cell differentiation (S1PR4, PTGIR, PTGER4), immune cell trafficking (S1PR4, G2A) and tissue repair (PTGIR).9–14 It is not possible at this time to look for co-localization of GPCR and hm-NAS gene expression in specific gastrointestinal niches, as neither the HMP nor the HPA are sufficiently comprehensive in their survey of human body sites. Nonetheless, 16S and metagenomic deep sequencing studies link bacteria containing hm-NAS genes or hm￾NAS genes themselves to specific locations in the gastrointestinal tract where GPCRs of interest are expressed (Extended Data Fig. 4). Bacterial and human ligands share structure and function Human microbiota-encoded N-acyl amides bear structural similarity to endogenous GPCR￾active ligands (Fig. 4). The clearest overlap in structure and function between bacterial and human GPCR-active ligands is for the endocannabinoid receptor GPR119 (Fig. 4 and 5). Endogenous GPR119 ligands include oleoylethanolamide (OEA) and the dietary lipid derivative 2-oleoyl glycerol (2-OG).15,16 In our heterologous expression experiment we isolated both the palmitoyl and oleoyl analogs of N-acyl serinol. The latter only differs from 2-OG by the presence of an amide instead of an ester and from OEA by the presence of an additional ethanol substituent. N-oleoyl serinol is a similarly potent GPR119 agonist compared to the endogenous ligand OEA (EC50 12 µM vs. 7 µM) but elicits almost a 2-fold greater maximum GPR119 activation (Fig. 5a). N-palmitoyl derivatives of all 20 natural amino acids were synthesized and none activated GPR119 by more than 37% relative to OEA (Fig. 5b). The generation of a potent and specific long-chain N-acyl-based GPR119 ligand therefore necessitates a more complex biosynthesis than the simple N-acylation of an amino acid as is commonly seen for characterized NAS enzymes. In this case, the biosynthesis of N-acyl serinols is achieved through the coupling of an NAS domain with an aminotransferase that is predicted to generate serinol from glycerol (Extended Data Fig. 2). The endogenous agonist for S1PR4, sphingosine-1-phosphate (S1P) and the N-3- hydroxypalmitoyl ornithine/lysine family of bacterial agonists share similar head group charges. S1P is a significantly more potent agonist (EC50 0.09 µM vs. EC50 32 µM); however, the bacterial agonists are more specific for S1PR4. The bacterial N-3- hydroxypalmitoyl ornithine did not activate S1PR1, 2, or 3 in our GPCR screen, whereas S1P activates all four members of the S1P receptor family tested. No direct comparison could be made between the microbiota-derived and endogenous ligands for PTGIR or PTGER4, as there are no known endogenous antagonists for these receptors. Many human GPCRs remain orphan receptors lacking known endogenous ligands. Ligands for at least some of these receptors will undoubtedly be found among the small molecules produced by the human microbiota. G2A is an orphan receptor and therefore does not have a well-defined endogenous agonist, although it has been reported to respond to lysophosphatidylcholine.17,18 We found that the bacterial metabolites N-3- hydroxypalmitoyl glycine (commendamide) and N-palmitoyl alanine, both activate G2A. Cohen et al. Page 5 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript