Research ArticleCELLULAR NEUROSCIENCE

Neuronal GPCR NPR-8 regulates C. elegans defense against pathogen infection

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Science Advances  20 Nov 2019:
Vol. 5, no. 11, eaaw4717
DOI: 10.1126/sciadv.aaw4717
  • Fig. 1 Functional loss of NPR-8 enhances C. elegans survival against pathogen infection and increases pathogen clearance from the intestine.

    (A) Wild-type (WT) and npr-8(ok1439) animals were exposed to P. aeruginosa and scored for survival over time. The graphs are the combined results of three independent experiments. Each experiment included n = 60 adult animals per strain. Error bars represent the SEM. P value represents the significance level of the mutants relative to the WT, P < 0.0001. (B) WT and npr-8(ok1439) animals were exposed to heat-killed P. aeruginosa and scored for survival over time. The graphs are the combined results of two independent experiments. Each experiment included n = 60 adult animals per strain. Error bars represent SEM. P value represents the significance level of the mutants relative to the WT, P = 0.50. (C) WT and npr-8(ok1439) animals were subjected to lawn occupancy assays in which animals were placed on a small spot of P. aeruginosa in a 3.5-cm plate and monitored over time for their presence on the lawn. The graphs are the combined results of three independent experiments. Each experiment included n = 60 adult animals per strain. Error bars represent SEM. P values represent the significance level of the mutants relative to the WT, P > 0.05, for all five time points. (D) WT and npr-8(ok1439) animals were exposed to P. aeruginosa in full lawn plates for 24 hours. Pharyngeal pumping rates of animals were counted as the number of pumps per 30 s. Counting was conducted in triplicate and averaged to give a pumping rate. The graphs are the combined results of three independent experiments. Each experiment included n = 60 adult animals per strain. Error bars represent SEM. P value represents the significance level of the mutants relative to WT, P = 0.3934. (E) WT and npr-8(ok1439) animals were exposed to GFP-expressing P. aeruginosa in full lawn plates for 24 hours, and the CFU of live bacterial cells recovered from the intestine were quantified. The graph represents the combined results of three independent experiments. n = 10 animals per strain were used for each experiment. Error bars represent SEM. Asterisk (*) denotes a significant difference (P < 0.05) between WT and npr-8(ok1439) animals. (F) WT and npr-8(ok1439) animals were exposed to P. aeruginosa in full lawn plates for 24 hours. Defecation rates of animals were measured as the average time of 10 intervals between two defecation cycles. n = 10 animals per strain were used for the measurements. P value represents the significance level of the mutants relative to WT, P = 0.0003. (G) WT and npr-8(ok1439) animals grown on double-stranded RNA (dsRNA) for empty vector (EV) control or dsRNA for aex-4 were exposed to P. aeruginosa and scored for survival over time. The graphs are the combined results of two independent experiments. Each experiment included n = 60 adult animals per strain. Error bars represent the SEM. P value represents the significance level: WT + EV versus npr-8(ok1439) + EV, P < 0.0001; WT + aex-4 RNAi versus npr-8(ok1439) + aex-4 RNAi, P < 0.0001. (H) WT and npr-8(ok1439) animals grown on dsRNA for EV control or dsRNA for nhr-2 were exposed to P. aeruginosa and scored for survival over time. The graphs are the combined results of two independent experiments. Each experiment included n = 60 adult animals per strain. Error bars represent the SEM. P value represents the significance level: WT + EV versus npr-8(ok1439) + EV, P < 0.0001; WT + nhr-2 RNAi versus npr-8(ok1439) + nhr-2 RNAi, P < 0.0001.

  • Fig. 2 NPR-8-regulated collagen genes are required for C. elegans defense against P. aeruginosa infection.

    (A) npr-8(ok1439) and (B) WT animals grown on dsRNA for collagen genes or EV control were exposed to P. aeruginosa and scored for survival over time. The graphs are the combined results of three independent experiments. Each experiment included n = 60 adult animals per strain. P values in (A) are relative to npr-8 + EV: npr-8 + col-80 RNAi, P < 0.0001; npr-8 + col-93 RNAi, P = 0.0030; npr-8 + col-98 RNAi, P < 0.0001; npr-8 + col-101 RNAi, P < 0.0001; npr-8 + col-103 RNAi, P = 0.0601; npr-8 + col-160 RNAi, P = 0.0333; npr-8 + col-179 RNAi, P < 0.0001. P values in (B) are relative to WT + EV: WT + col-80 RNAi, P = 0.2555; WT + col-93 RNAi, P = 0.5575; WT + col-98 RNAi, P = 0.5250; WT + col-101 RNAi, P < 0.0001; WT + col-103 RNAi, P = 0.6950; WT + col-160 RNAi, P = 0.0494; WT + col-179 RNAi, P = 0.0004. (C) WT, npr-8(ok1439), col-8(sun1), and npr-8;col-80 animals were exposed to P. aeruginosa and scored for survival over time. P values are as follows: WT versus npr-8(ok1439), P < 0.0001; WT versus col-80(sun1), P = 0.8761; npr-8(ok139) versus npr-8;col-80, P < 0.0001. (D) WT, npr-8(ok1439), col-98(sun2), and npr-8;col-98 animals were exposed to P. aeruginosa and scored for survival over time. P values are as follows: WT versus npr-8(ok1439), P < 0.0001; WT versus col-98(sun2), P = 0.7044; npr-8(ok139) versus npr-8;col-98, P < 0.0001. (E) WT, npr-8(ok1439), col-179(ok3010), and npr-8;col-179 animals were exposed to P. aeruginosa and scored for survival over time. P values are as follows: WT versus npr-8(ok1439), P < 0.0001; WT versus col-179(ok3010), P < 0.0001; npr-8(ok139) versus npr-8;col-179, P < 0.0001.

  • Fig. 3 NPR-8 regulates the dynamics of cuticle structure in response to pathogen infection.

    WT and npr-8(ok1439) animals were exposed to E. coli OP50 or P. aeruginosa PA14 for 24 hours and imaged using FEI Quanta 600 FEG SEM.

  • Fig. 4 NPR-8 is expressed in amphid sensory neurons.

    (A) The transcriptional reporter strain npr-8p::gfp expresses GFP under the control of the npr-8 native promoter. (B) npr-8p::gfp expression and DiI dye–filled neurons overlapped in AWB and ASJ neurons (dorsal view). (C) npr-8p::gfp expression and DiI dye–filled neurons overlapped in AWB and ASJ neurons (ventral view). (D) npr-8p::gfp expression and str-2p::mcherry expression overlapped in AWC neurons (dorsal view). DIC, differential interference contrast microscopy; DiI, DiI staining of amphid neurons; mCherry, mCherry fluorescence microscopy; Merge, overlay of GFP and DiI or mCherry images.

  • Fig. 5 NPR-8 functions in neuronal cells not in the gut to defend C. elegans against P. aeruginosa infection.

    (A) WT, npr-8(ok1439), and rescue animals were exposed to P. aeruginosa and scored for survival over time. JRS17, npr-8 rescue by its own promoter; JRS18, npr-8 rescue in AWB neurons; JRS19, npr-8 rescue in AWC neurons; JRS20, npr-8 rescue in ASJ neurons; JRS21, npr-8 pan-neuronal rescue. The graphs are the combined results of three independent experiments. Each experiment included n = 60 adult animals per strain. P values are relative to npr-8(ok1439) animals: WT, P < 0.0001; JRS17, P < 0.0001; JRS18, P < 0.0001; JRS19, P < 0.0001; JRS20, P < 0.0001; JRS21, P < 0.0001. (B) qRT-PCR analysis was performed to measure the expression of collagen genes in WT, npr-8(ok1439), JRS18, JRS19, and JRS20 animals exposed to P. aeruginosa. In all assays, pan-actin served as internal controls. Bars represent mean ± SEM, and values are the average of two independent experiments. Asterisks (*) denote a significant difference (P < 0.0001) in expression of collagen genes between npr-8(ok1439) animals and the rescue strains. (C) WT and VP303 (for intestine-specific RNAi) animals grown on dsRNA for npr-8 or EV control were exposed to P. aeruginosa and scored for survival over time. The graphs are the combined results of three independent experiments. Each experiment included n = 60 adult animals per strain. P values are relative to WT + EV: WT + npr-8 RNAi, P = 0.0062; VP303 + vector, P = 0.1141; VP303 + npr-8 RNAi, P = 0.2153. (D) WT, npr-8(ok1439), and JRS24 animals were exposed to P. aeruginosa and scored for survival over time. JRS24, npr-8 rescue in the intestine. The graphs are the combined results of two independent experiments. Each experiment included n = 60 adult animals per strain. WT versus npr-8(ok1439), P < 0.0001; WT versus JRS24, P < 0.0001; npr-8(ok1439) versus JRS24, P = 0.6979.

  • Table 1 Cuticular collagen genes were up-regulated in npr-8(ok1439) animals relative to wild-type animals exposed to P. aeruginosa.

    A. Enrichment of molecular functions revealed by GO analysis of up-regulated genes in npr-8(ok1439) animals relative to wild-type animals exposed
    to P. aeruginosa.
    GO termDescriptionP*FDR qEnrichment (N, B, n, b)
    GO:0042302Structural constituent of cuticle3.08 × 10−86.87 × 10−57.96 (10,359, 121, 129, 12)
    B. Enrichment of cuticular collagen genes in npr-8(ok1439) animals relative to wild-type animals with or without exposure to P. aeruginosa.
    Enriched collagen
    genes
    Infected with P. aeruginosaUninfected
    npr-8(ok1439) animals relative to wild-type animalsnpr-8(ok1439) animals relative to wild-type animals
    Fold changeAdjusted P§Significantly
    different?
    Fold changeAdjusted P§Significantly
    different?
    col-934.63.75 × 10−82Yes1.32.82 × 10−2Yes
    col-803.52.03 × 10−38Yes1.32.17 × 10−2Yes
    col-1792.51.31 × 10−22Yes1.21.09 × 10−1No
    col-982.46.85 × 10−22Yes1.34.35 × 10−2Yes
    col-1602.49.91 × 10−33Yes1.41.37 × 10−3Yes
    col-1242.42.97 × 10−14Yes1.29.33 × 10−2No
    col-1032.21.35 × 10−17Yes1.33.74 × 10−2Yes
    col-1012.18.11 × 10−14Yes1.21.78 × 10−4Yes
    col-1462.09.72 × 10−8Yes3.39.24 × 10−23Yes
    col-1592.01.20 × 10−6Yes1.83.05 × 10−6Yes
    col-202.02.13 × 10−16Yes1.34.75 × 10−2Yes
    Y69H2.142.01.13 × 10−14Yes1.36.05 × 10−2No

    *P value is the enrichment P value computed according to the mHG model (Eden et al. 2007 PLoS Comp Bio 3 (3):e39).

    †FDR q value is the correction of the above P value for multiple testing using the Benjamini and Hochberg method (Benjamini and Hochberg 1995 J R Statist Soc B 57 (1):289–300).

    ‡Enrichment (N, B, n, b) is defined as follows: N, total number of genes; B, total number of genes associated with a specific GO term; n, number of genes in the target set; b, number of genes in the intersection; Enrichment = (b/n)/(B/N).

    §Adjusted P value is the correction of the P value for multiple testing using the Benjamini and Hochberg method (Benjamini and Hochberg 1995 J R Statist Soc B 57 (1):289–300).

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaaw4717/DC1

      Fig. S1. Functional loss of NPR-8 enhances C. elegans survival against pathogen infection and increases pathogen clearance from the intestine.

      Fig. S2. NPR-8 functions in AVL and DVB neurons to regulate defecation but not survival against infection.

      Fig. S3. NPR-8 does not play a role in conserved innate immune pathways.

      Fig. S4. NPR-8–regulated collagen genes are involved in C. elegans defense and defecation.

      Fig. S5. NPR-8 regulates collagen expression in the cuticle and hypodermis and controls the dynamics of cuticle structure in response to infection.

      Fig. S6. NPR-8 is expressed in amphid sensory neurons and throughout developmental stages.

      Fig. S7. NPR-8 is not involved in bacteria sensing, as determined by food choice assays.

      Table S1. Differential expression of genes in conserved innate immune pathways in npr-8(ok1439) animals relative to wild-type animals exposed to P. aeruginosa.

      Table S2. Ontology analyses of up-regulated genes in P. aeruginosa–infected wild-type animals relative to uninfected controls or in uninfected npr-8(ok1439) animals relative to wild-type animals.

      Table S3. List of transgenic C. elegans strains generated in this study.

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. Functional loss of NPR-8 enhances C. elegans survival against pathogen infection and increases pathogen clearance from the intestine.
      • Fig. S2. NPR-8 functions in AVL and DVB neurons to regulate defecation but not survival against infection.
      • Fig. S3. NPR-8 does not play a role in conserved innate immune pathways.
      • Fig. S4. NPR-8–regulated collagen genes are involved in C. elegans defense and defecation.
      • Fig. S5. NPR-8 regulates collagen expression in the cuticle and hypodermis and controls the dynamics of cuticle structure in response to infection.
      • Fig. S6. NPR-8 is expressed in amphid sensory neurons and throughout developmental stages.
      • Fig. S7. NPR-8 is not involved in bacteria sensing, as determined by food choice assays.
      • Table S1. Differential expression of genes in conserved innate immune pathways in npr-8(ok1439) animals relative to wild-type animals exposed to P. aeruginosa.
      • Table S2. Ontology analyses of up-regulated genes in P. aeruginosa–infected wild-type animals relative to uninfected controls or in uninfected npr-8(ok1439) animals relative to wild-type animals.
      • Table S3. List of transgenic C. elegans strains generated in this study.

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