Immune defenses are triggered by microbe-associated molecular patterns or as a result of damage to host cells. The elicitors of immune responses in the nematode Caenorhabditis elegans are unclear. Using a genome-wide RNA-mediated interference (RNAi) screen, we identified the G protein–coupled receptor (GPCR) DCAR-1 as being required for the response to fungal infection and wounding. DCAR-1 acted in the epidermis to regulate the expression of antimicrobial peptides via a conserved p38 mitogen-activated protein kinase pathway. Through targeted metabolomics analysis we identified the tyrosine derivative 4-hydroxyphenyllactic acid (HPLA) as an endogenous ligand. Our findings reveal DCAR-1 and its cognate ligand HPLA to be triggers of the epidermal innate immune response in C. elegans and highlight the ancient role of GPCRs in host defense.
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We thank A. Chisholm (University of California, San Diego) for vector pCZGY1434; S. Mitani (Tokyo Women's Medical University School of Medicine) for strain tir-1(tm3036); Y. Goshima (Yokohama City University) for strains dcar-1(nj66) and dcar-1(tm2484), dcar-1 cDNA, and plasmids sra-6p∷dcar-1∷Venus and sra-6p∷dcar-1; C. Bargmann, A. Chisholm, C. Couillault, P. Golstein and E. Vivier for critical reading of the manuscript; M. Metwaly, F. Montañana-Sanchis, S. Omi, J. Soulé and the staff at WormBase and ModulBio for technical support; and C. Melon for advice. Nematode strains pmk-1(km25) and gpa-12(pk322) were provided by the Caenorhabditis Genetics Center, which is funded by the Office of Research Infrastructure Programs of the US National Institutes of Health (P40 OD010440); dcar1(tm2484) was provided by the National Bioresource Project coordinated by S. Mitani. Supported by INSERM, CNRS, the PACA Regional Council, the Agence Nationale de Recherche (MIME-2007, ANR-12-BSV3-0001-01, ANR-11-LABX-0054 (Investissements d'Avenir–Labex INFORM) and ANR-11-IDEX-0001-02 (Investissements d'Avenir–A*MIDEX)), the US National Institutes of Health (GM088290 to F.C.S.) and the Fondation Association pour la Recherche sur le Cancer (B.S.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 The GPCR DCAR-1 controls AMP gene expression following fungal infection and wounding.
Normalized fluorescence ratio in wild type and dcar-1(tm2484) worms carrying the integrated array frIs7 (which contains the reporter transgenes nlp-29p::gfp and col-12p::DsRed) after infection by D. coniospora (left panel), wounding (middle left panel) and osmotic stress (middle right panel), and in wild type and dcar-1(tm2484) worms that in addition to frIs7 carry frIs30, an integrated transgene that encodes a constitutively active form of GPA-12 (GPA-12*; right panel). In all cases, the results are representative of at least three independent experiments with a minimum of 50 worms for each condition.
Supplementary Figure 2 DCAR-1 acts in an epidermis-specific and cell-autonomous manner to regulate the induction of nlp-29 AMP gene.
a, Normalized fluorescence ratio of wild type worms carrying frIs7 treated with RNAi against control (sta-1, gfp, sta-2) or candidate genes (osm-9 and ocr-2) and infected or not. b, Normalized fluorescence ratio of worms carrying frIs7 in rde-1(ne219);col-19p::rde-1 worms that are resistant to RNAi except in the adult epidermis treated with RNAi against control genes (sta-1, gfp, sta-2) or dcar-1 and infected or not. The results are representative of at least three independent experiments with a minimum of 50 worms for each condition.
Supplementary Figure 3 DCAR-1 acts in the epidermis to regulate nlp gene expression and resistance to infection with D. coniospora.
a, Survival of wild-type, dcar-1(tm2484), dcar-1;dcar-1p::dcar-1::gfp, dcar-1;col-12p::dcar-1::gfp and sra-6p::dcar-1::Venus worms after infection with D. coniospora. For the experiment shown here, n= 70, 65, 67, 67 and 70 respectively). The statistical significance of the differences between strains is shown below the graph (one-sided log rank test). b, c Quantitative RT-PCR analysis of the expression of genes in the nlp-29 cluster in wild-type, dcar-1(tm2484) and dcar-1;dcar-1p::dcar-1::gfp (b) or wild-type, dcar-1(tm2484), dcar-1;col-12p::dcar-1 and dcar-1;sra-6p::dcar-1 worms (c) after infection with D. coniospora. Data are from three independent experiments (average and SD). d, Survival of wild-type, dcar-1(tm2484), dcar-1;col-12p::dcar-1 and with sra-6p::dcar-1 worms after infection with D. coniospora. For the experiment shown here, n= 62, 71, 55 and 65 respectively). The statistical significance of the difference between strains is shown below the graph (one-sided log rank test). The difference in survival between the strains common to the experiments shown in a and d is linked to a variation in pathogenicity between different preparations of fungal spores used in experiments performed on different days.
Quantitative RT-PCR analysis of the expression of genes in the nlp-29 cluster in wild-type and pmk-1 worms treated with 5 mM DHCA for 2 h (a) or of the intestinal defence genes F49F1.6 and F57F4.4 (that require PMK-1 for their expression) and pgp-5, irg-1, irg-3 (that do not require PMK-1 for their expression)1,2 in wild-type and dcar-1(tm2484) worms after exposure to 5 mM DHCA for 2 h (b) or 5 h (c). Data are from three independent experiments (average and SD). The qualitative effect on nlp gene expression in the pmk-1 mutant mirrors that seen upon infection3, also with an apparent partial pmk-1-independent component for the induction of nlp-31 and nlp-34. Although exposure of worms to DHCA was sufficient to increase epidermal AMP gene expression, pre-treatment of worms with it did not provide any measurable protection against infection (results not shown).1 - Estes, K. A., Dunbar, T. L., Powell, J. R., Ausubel, F. M. & Troemel, E. R. bZIP transcription factor zip-2 mediates an early response to Pseudomonas aeruginosa infection in Caenorhabditis elegans. Proc Natl Acad Sci U S A 107, 2153-2158 (2010).2 - Dierking, K. et al. Unusual regulation of a STAT protein by an SLC6 family transporter in C. elegans epidermal innate immunity. Cell Host Microbe 9, 425-435 (2011).3 - Pujol, N. et al. Distinct innate immune responses to infection and wounding in the C. elegans epidermis. Curr Biol 18, 481-489 (2008).
Supplementary Figure 5 Molecules that are structurally related to DHCA but do not trigger expression of the nlp-29p::gfp reporter gene.
Chemical structures of 3,4-hydroxy phenyl propionic acid (HPA), 4-hydroxyphenylpyruvic acid (HPPA), 3-4-dihydroxyphenyl lactic acid (DPLA), tyrosine, L-3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydrobenzaldehyde (DHB), 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihyroxybenzoic acid (DHBA), and 3,4 dihydroxymandelic acid (DHMA), together with normalized fluorescence ratio of young adult wild type worms carrying frIs7 treated for 2 h at 25°C with increasing concentrations of the indicated chemical. Data are representative of at least three independent experiments with a minimum of 50 worms for each condition. Compounds previously found to act as DCAR-1 ligands in a Xenopus oocyte system, and their relative potency are indicated (*** medium; ** low; * weak). Tyrosine and DOPA were also tested and had no effect4. As the epidermis of C. elegans is surrounded by a cuticle that is relatively impermeable to many compounds5–6, we cannot formally exclude the possibility that lack of reporter gene expression in our assay results from a failure of a given compound to reach DCAR-1 in vivo. Nor can we exclude the possibility that different compounds have different pharmacokinetic properties.4 - Aoki, R. et al. A seven-transmembrane receptor that mediates avoidance response to dihydrocaffeic acid, a water-soluble repellent in Caenorhabditis elegans. J Neurosci 31, 16603-16610 (2011).5 - Partridge, F. A., Tearle, A. W., Gravato-Nobre, M. J., Schafer, W. R. & Hodgkin, J. The C. elegans glycosyltransferase BUS-8 has two distinct and essential roles in epidermal morphogenesis. Dev Biol 317, 549-559 (2008).6 - Zheng, S. Q., Ding, A. J., Li, G. P., Wu, G. S. & Luo, H. R. Drug absorption efficiency in Caenorhbditis elegans delivered by different methods. PLoS One 8, e56877 (2013).
Supplementary Figure 6 Knocking down individual enzymes potentially involved in HPLA metabolism has no effect on nlp-29p::gfp expression.
a, Part of the tyrosine metabolic pathway, showing the names and structures of intermediates and the names of selected candidate enzymes which potentially catalyze each step. To the best of our knowledge, how the keto of HPPA is converted into the hydroxy function of HPLA has not been elucidated in any eukaryotic species. b, Normalized fluorescence ratio of young adult IG1502 rde-1(ne219) V; Is[wrt-2p::rde-1; myo-2p::rfp]; frIs7 IV worms that are resistant to RNAi except in the epidermis (infected or not, left panel) or dpy-10 worms (right panel) carrying frIs7 treated with RNAi against control (sta-1, gfp and dcar-1) or candidate genes. Data are representative of at least two independent experiments with a minimum of 50 worms for each condition.
Supplementary Figure 7 Pathways leading to expression of nlp-29, highlighting the newly discovered roles of HPLA and DCAR-1.
Infection with D. coniospora and physical injury trigger up-regulation of the AMP nlp genes in the epidermis via the Gα protein GPA-12 that acts upstream of the PKCδ TPA-1, the TIR-domain adapter protein TIR-1 and a p38 MAPK cascade involving NSY-1, SEK-1 and PMK-1, as well as the STAT transcription factor-like protein STA-2. We found that the level of HPLA, which is potentially derived from tyrosine, increases upon infection. The precise site and mode of production of HPLA awaits elucidation; tyrosine is placed here at the level of the cuticle for the purpose of illustration. HPLA triggers nlp-29 gene expression via its cognate receptor DCAR-1, which is expressed at the apical surface of the epidermal syncytium hyp7. dcar-1 acts upstream of GPA-12, the p38 MAPK cassette and STA-2.
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Zugasti, O., Bose, N., Squiban, B. et al. Activation of a G protein–coupled receptor by its endogenous ligand triggers the innate immune response of Caenorhabditis elegans. Nat Immunol 15, 833–838 (2014). https://doi.org/10.1038/ni.2957
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