Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist

Abstract

The trillions of bacteria that make up the human microbiome are believed to encode functions that are important to human health; however, little is known about the specific effectors that commensal bacteria use to interact with the human host. Functional metagenomics provides a systematic means of surveying commensal DNA for genes that encode effector functions. Here, we examine 3,000 Mb of metagenomic DNA cloned from three phenotypically distinct patients for effectors that activate NF-κB, a transcription factor known to play a central role in mediating responses to environmental stimuli. This screen led to the identification of 26 unique commensal bacteria effector genes (Cbegs) that are predicted to encode proteins with diverse catabolic, anabolic, and ligand-binding functions and most frequently interact with either glycans or lipids. Detailed analysis of one effector gene family (Cbeg12) recovered from all three patient libraries found that it encodes for the production of N-acyl-3-hydroxypalmitoyl-glycine (commendamide). This metabolite was also found in culture broth from the commensal bacterium Bacteroides vulgatus, which harbors a gene highly similar to Cbeg12. Commendamide resembles long-chain N-acyl-amides that function as mammalian signaling molecules through activation of G-protein-coupled receptors (GPCRs), which led us to the observation that commendamide activates the GPCR G2A/GPR132. G2A has been implicated in disease models of autoimmunity and atherosclerosis. This study shows the utility of functional metagenomics for identifying potential mechanisms used by commensal bacteria for host interactions and outlines a functional metagenomics-based pipeline for the systematic identification of diverse commensal bacteria effectors that impact host cellular functions.

Keywords: Cbeg; N-acyl amino acids; NF-κB; commendamide; functional metagenomics.

Fig. 1.

Overview of metagenomic methods. Step 1: The human reporter cell line and individual metagenomic bacterial clones are robotically arrayed in separate 384-well microplates. Step 2: Mature bacterial cultures are filter sterilized, and sterile spent culture broth is then transferred to plates containing the human reporter cell line. Human reporter cells that have been exposed to spent culture broth are imaged by fluorescent microscopy to identify metagenomic clones that activate the reporter. Step 3: Once an active metagenomic clone is confirmed, the specific effector genes are identified through sequencing and transposon mutagenesis, and the effector molecules (proteins or small molecules) are characterized from large-scale cultures of the active metagenomic clone.

Fig. 2.

Screen results. (A) Histogram of HEK293:NF-κB:GFP reporter activation Z scores for all 75,003 metagenomic clones screened in this study. The percentage of activated cells was determined on a per-well basis and normalized to negative control wells on the assay plate to give a Z score. The Z-score distribution from metagenomic clones demonstrates a positive skew toward increased GFP activation compared with negative control wells (normal curve, black line). (B) Images of a representative negative control well and a well from an active metagenomic clone. Nuclei appear blue (Hoechst 33442 stain). HEK293:NF-κB:GFP cells expressing GFP appear green. (C) Table of total metagenomic clones screened and hit rates for each library. Active clones have reproducible HEK293:NF-κB:GFP cell activation in a secondary assay. Effector genes were identified by transposon mutagenesis of unique cosmids recovered from the active hits. All hit rates are expressed relative to total metagenomic clones screened. *Library 1 was not robotically arrayed into a microplate but mechanically dispensed at a dilution of 0–3 metagenomic clones per well. Total number of clones screened in library 1 is therefore an estimate based on the number of wells screened and average number of clones per well.

Fig. 3.

Bacteroidetes species appear to be enriched in effector genes. (A) The percent identity for the top hit identified in a BlastN search of each effector gene against either the NCBI reference genome dataset or the Swissprot dataset is tabulated. (B) The phylogenetic origin of the sequence from NCBI reference genome dataset to which each effector gene is most closely related is tabulated [Bacteroidetes (blue), Firmicutes (orange)]. (C) A 16s gene analysis (V4 region) of each metagenomic library was carried out to assess phylogenetic diversity in these libraries. For each library, the percentage of effector genes predicted to arise form Bacteroidetes spp. (88%; B) is greater than the percentage of 16S genes predicted to arise from Bacteroidetes spp. (36–40%; C).

Fig. 4.

Summary of commensal bacterial effector genes (Cbegs). Each effector gene was queried by BLASTn against the NCBI nr dataset to determine the reference genome from which the most closely related sequence arises (column 1) and to predict the domain architecture and makeup of each Cbeg protein (columns 2 and 3, respectively). The metagenomic library from which each Cbeg was recovered is shown in column 1. In column 4, we have grouped Cbegs based on their general predicted functions. *Cbeg2 and Cbeg3 are found on the same unique cosmid. ^†Cbeg6 and Cbeg7 are found on the same unique cosmid. ^Cbeg12 is found at 98% identity in three unique cosmids, each in a different patient library (Cbeg12-1, Cbeg12-2, Cbeg12-3).

Fig. 5.

General schematic of predicted Cbeg effector functions. Cbegs are predicted to encode proteins that result in activation of the HEK293:NFκB:GFP reporter through different mechanisms. This includes proteins that likely induce major transcriptional changes in the bacterial host as well proteins with diverse catabolic, anabolic, and ligand-binding functions. See the main text for a detailed discussion of each Cbeg.

Fig. 6.

Characterization of commendamide. (A) Electrospray ionization (ESI)–mass spectroscopy (MS) traces of culture broth extracts from E. coli transformed with an empty pJWC1 cosmid vector (i), cosmid Cbeg12-1 (ii), cosmid Cbeg12-2 (iii), cosmid Cbeg12-3 (iv), cosmid Cbeg12-1 with a transposon insertion in the Cbeg12-1 gene (v), Cbeg12-1 subcloned into pJWC1 (vi), and synthetic commendamide (vii). (B) Key NMR correlations used to define the structure of commendamide (1), the major clone-specific peak found in cultures of E. coli transformed with Cbeg12-1, are shown. (C) The structures for three minor clone-specific metabolites related to commendamide (compounds 2–4) were also determined using NMR and MS data. (D) Purified commendamide activates the HEK293:NFκB GFP reporter assay. (E) Endogenous long-chain N-acyl-amides that are structurally related to commendamide are reported to function as agonists for numerous receptors, including many GPCRs. Such signaling systems have been therapeutically targeted for the treatment of pain, inflammation, depression, obesity, and diabetes.

Fig. 7.

Phylogeny of the commendamide N-acyl synthase. A BLASTn search of the three Cbeg12 genes against the NCBI reference genome dataset. Genes similar to Cbeg12 at >70% identity are represented in a phylogenetic tree annotated by reference genome. All genes are from commensal bacteria except Desulfosporosinus acidophilus SJ4, which was isolated from an acid mine. B. vulgatus ATCC 8482 is 100% identical to Cbeg12-1, and Bacteroides dorei HS1 is 100% identical to Cbeg12-2 and Cbeg12-3. B. vulgatus ATCC 8482 was purchased from ATCC and grown for 14 d in LY-BHI medium under anaerobic conditions. The B. vulgatus culture was extracted 1:1 with ethyl acetate, and crude extracts were fractionated by flash column chromatography (water:methanol, 0.1% TFA). MS [electrospray ionization (ESI)] analysis of extract fractions identified a metabolite with the same mass and retention time as commendamide in the B. vulgatus culture broth.

Fig. 8.

Commendamide activates the human GPCR G2A. (A) Using the β-arrestin Pathunter assay (DiscoveRx), commendamide was screened (10 μM) for agonist activity against a panel of 242 GPCRs including 73 orphan GPCRs. For each GPCR, percent activity is expressed relative to baseline activity of the receptor. (B) Dose–response of natural and synthetic long-chain N-acyl-amides in the Pathunter assay using the receptor G2A. Synthetic (green) and natural product (blue) commendamide are equipotent. Two of the minor metabolites (compound 2: 3-OH-16:1; and compound 4: 3-OH-14:0) activate G2A but are slightly less potent than commendamide. Dramatic changes in either the amino acid head group (glycine to tyrosine) or fatty acid tail (3-OH-16:0 to 3-OH-10:0) result in analogs that do not appreciably activate the G2A receptor.