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 28hm-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