Lipid signalling couples translational surveillance to systemic detoxification in Caenorhabditis elegans

Abstract

Translation in eukaryotes is followed to detect toxins and virulence factors and coupled to the induction of defence pathways. Caenorhabditis elegans germline-specific mutations in translation components are detected by this system to induce detoxification and immune responses in distinct somatic cells. An RNA interference screen revealed gene inactivations that act at multiple steps in lipid biosynthetic and kinase pathways upstream of MAP kinase to mediate the systemic communication of translation defects to induce detoxification genes. Mammalian bile acids can rescue the defect in detoxification gene induction caused by C. elegans lipid biosynthetic gene inactivations. Extracts prepared from C. elegans with translation deficits but not from the wild type can also rescue detoxification gene induction in lipid-biosynthesis-defective strains. These eukaryotic antibacterial countermeasures are not ignored by bacteria: particular bacterial species suppress normal C. elegans detoxification responses to mutations in translation factors.

Figure 1. Translation inhibition using toxin or RNAi induces xenobiotic detoxification

A) The toxin G418 or inhibition of translation by iff-2(translation initiation factor) RNAi induces pgp-5::gfp expression in the intestine as assessed using a transcriptional promoter fusion. Scale bar, 50μm. B) RNAi of translation initiation factor (iff-2) or elongation factor (eft-4) induces pgp-5 mRNA as assessed by qRT-PCR. Fold change compared to control RNAi treated wildtype animals. **P<0.01. C) G418 induces pgp-5 and pgp-6 mRNA from the chromosomal locus but not mrp-1–multidrug resistance protein homolog mRNA as assessed by qRT-PCR. Fold change compared to non-toxin-treated wildtype animals. D) Hygromycin induces expression of particular xenobiotic efflux pump genes as assessed by qRT-PCR. Fold change compared to non-hygromycin-treated wildtype animals. E) Hygromycin induces expression of particular xenobiotic detoxification genes as assessed by qRT-PCR. Fold change compared to no hygromycin-treated wildtype animals. B, C, D and E: Unpaired t test; **P<0.01, *P<0.05. ns; no significant difference. ~300 worms per condition were washed off 1 plate for each experiment. Mean ± s.d from n= 3 independent experiments is shown.

Figure 2. Translation defects in the germline induce systemic xenobiotic detoxification response

A) Genetic defects in germline translation induce xenobiotic and innate immune response GFP fusion genes. iff-1, rpl-11.1(ribosomal protein L11) and eft-3 (an elongation factor 1-α ortholog) are expressed only in the germline and are required for translation in the germline only; they are not expressed in somatic cells or required for somatic translation. Scale bar, 50 μm. B) Quantification of pgp-5::gfp activation by germline translation defects. Fluorescence was measured using a COPAS Biosort. Unpaired t test; ***P<0.001. Mean ± s.d of n=413 and 1680 worms for pgp-5::gfp, and eft-3 (q145); pgp-5::gfp respectively. Data was consolidated from two independent experiments. C) Genetic defects in germline translation induce xenobiotic efflux pump expression, as measured using PCR-based quantitation of mRNA levels of endogenous genes. Fold change compared to wildtype animals. Unpaired t test; ***P<0.001. **P<0.01. *P<0.05. ~300 worms per condition were washed off 1 plate for each experiment. Mean ± s.d from n=3 independent experiments is shown. D) Genetic defects in germline translation induce xenobiotic and innate immune response genes, as measured using PCR-based quantitation of mRNA levels of endogenous genes. Fold change compared to wildtype animals. Statistical significance was determined using unpaired t test. **P<0.01. *P<0.05. ~300 worms per condition were washed off 1 plate for each experiment. Mean ± s.d from n=3 independent experiments is shown.

Figure 3. p38 MAPK signaling and zip-2–bZIP transcription factor are required for translation-inhibition-induced xenobiotic defense response

A) RNAi of the zip-2–bZIP transcription factor gene disrupts the induction of pgp-5::gfp in response to germline translation defects in eft-3(q145);pgp-5::gfp. Scale bar, 50μm. B) Kinase and transcription factor gene inactivations that disrupt pgp-5::gfp induction in response to germline translation defects in the eft-3(q145);pgp-5::gfp strain. n=60 worms. Mean ± s.d. n is consolidated from three independent experiments. C) p38–pmk-1 and zip-2 are required for G418 induction of pgp-5 mRNA as assessed by qRT-PCR. Fold change compared to no drug wildtype animals. One-way ANOVA; **P<0.01. ~300 worms per condition were washed off 1 plate for each experiment. Mean ± s.d from n=3 independent experiments is shown. D) zip-2 and skn-1 transcription factors are required for G418-induced pgp-5::gfp expression. Fluorescence was measured using a COPAS Biosort. Statistical significance was determined using one-way ANOVA. ***P<0.001. Mean ± s.e.m of n=400 worms for control RNAi and n=399, 563, and 99 worms, respectively for G418 in combination with dsRNA control, zip-2 RNAi and skn-1 RNAi treatments. Data was consolidated from two independent experiments. E) hpk-1,mig-15,and pak-1 are required for hygromycin-induced induction of pgp-5::gfp. One-way ANOVA; ***P<0.001. Mean ± s.d. of n=136 worms for control RNAi and n=397, 268,123, 143 worms respectively for hygromycin treatment in combination with control RNAi, hpk-1 RNAi, mig-15 RNAi and pak-1 RNAi. Data was consolidated from two independent experiments.

Figure 4. Bile acid-like biosynthetic signaling is required for translation-defective-induced xenobiotic defense response

A) Germline translation defects in the eft-3(q145);pgp-5::gfp strain causes p38 MAPK phosphorylation and nuclear translocation of active p38 PMK-1 in the intestine. Arrows indicate the nucleus. Scale bar, 20μm. B) tir-1, kin-18, svh-2, mig-15,and prk-2 are required for germline translation-defect-induced activation of p38 in the intestine. One-way ANOVA; **P<0.01. ns, P>0.05. n=50 (dsRNA control),45 (tir-1 RNAi), 50 (kin-18 RNAi),28 (svh-2 RNAi), 40 (prk-2 RNAi), 50 (mig-15 RNAi) and 32 (zip-2 RNAi) nuclei, respectively. Data represent one out of two independent experiments. C) Inactivation of lipid and bile acid biosynthetic genes disrupts the induction of pgp-5::gfp in response to G418. Error bars represent SD. One-way ANOVA; **P<0.01. n=413 (dsRNA control non-G418 treated), n=2271(dsRNA control G418 treated), n=239 (daf-22 RNAi),n=514 (dhs-28 RNAi) worms. Data was consolidated from two independent experiments. D) Lipid and bile acid biosynthetic genes are required for hygromycin-induced pgp-5::gfp. Scale bar, 50μm. E) Inactivation of genes required for lipid–bile acid biosynthesis disrupts the induction of pgp-5::gfp in response to hygromycin. Error bars represent SD. One-way ANOVA; **P<0.01. n=10 worms for each condition. Data represent one out of two independent experiments. F) While about 40% of control RNAi treated wildtype animals treated with 0.2 mg/ml G418 grew to adulthood, RNAi of daf-22 and dhs-28 cause >70% of animals treated with 0.2 mg/ml G418 to arrest at L1-larval stage. n=20 worms for each condition. Data represent one out of two independent experiments. G) Mutations in genes required for lipid–bile acid biosynthesis cause hypersensitivity to G418. n=20 worms for each condition. Data represent one out of two independent experiments. H) Lipid and bile acid biosynthetic genes are required for germline translation-defective-induced p38 nuclear localization. One-way ANOVA; ***P<0.001. n=50 nuclei (control RNAi on pmk-1 (km25)) and n=42 (control RNAi),32 (dhs-28 RNAi), 18 (daf-22 RNAi), 35 (nlt-1RNAi), and 50 (K05B2.4 RNAi) intestinal nuclei, respectively for eft-3 (q145); pmk-1 (km25). Data represent one out of two independent experiments.

Figure 5. Bile acid signaling couples translation defects to the induction of xenobiotic defense genes

A) Mammalian bile acids rescue the defect in pgp-5::gfp induction caused by daf-22 or dhs-28 bile biosynthetic gene inactivations in the eft-3(q145);pgp-5::gfp strain with a germline translation elongation factor mutation. Scale bar, 50μm. B) Quantitation of the rescue by mammalian bile acids of the defect in pgp-5::gfp induction caused by RNAi inactivation of C. elegans lipid–bile acid biosynthetic genes. Error bars represent SD. One-way ANOVA; *P<0.01. ns, P>0.05. n=100 worms. Data represent one out of three independent experiments. C) Exogenous bile acids or lipid extracts from translationally-challenged C. elegans enhance the induction of pgp-5::gfp expression in response to mild translational inhibition by diluted eft-4 RNA. Data are from two independent experimental replicates. N=100 worms. Error bars represent SD. Statistical significance was calculated by comparing to solvent control treated animals on eft-4 dilution RNAi using one-way ANOVA. **P<0.01. ns, P>0.05. Data represent one out of three independent experiments.

Figure 6. Bacterial suppression of the induction of xenobiotic responses by a germline-translation mutation

A) C. elegans grown on lawns of Paenibacilli, Kocuria or Alcaligenes bacteria fail to induce pgp-5::gfp in response to the germline-translation-defects in eft-3(q145);pgp-5::gfp. Scale bar, 50 μm. B) C. elegans grown on various Kocuria species fail to induce pgp-5::gfp in response to the germline translation defects in eft-3(q145);pgp-5::gfp. Data represent one of two independent experiments. n denotes number of worms. C) C. elegans grown on K.rhizophila fail to induce pgp-5 and pgp-6 in response to germline translation defects in eft-3(q145). Error bars represent SD. Unpaired t test; **P<0.01. ~300 worms per condition were washed off 1 plate for each experiment. Mean ± s.d is from n=3 independent experiments. D) C. elegans grown on K.rhizophila fail to induce various chromosomal xenobiotic and innate immune response genes in response to germline translation defects in eft-3(q145). Error bars represent SD. Unpaired t test; **P<0.01. *P<0.05. ns, not significant. ~300 worms per condition were washed off 1 plate for each experiment. Mean ± s.d, n=3 independent experiments. E) Feeding on K.rhizophila disrupts both basal and constitutive p38 MAPK phosphorylation as assessed using an antibody that recognizes active phosphorylated PMK-1 protein. One-way ANOVA. ***P<0.001. For wildtype on E.coli, n=20, for eft-3(q145) n=32 (E.coli) and 40 (K. rhizophila) nuclei, respectively. Data represent one out of two independent experiments. F) Feeding on K.rhizophila disrupts the nuclear translocation of active phosphorylated p38 MAPK. Unpaired t test.. ***P<0.001. **P<0.01. For wildtype, n=50 (E.coli) 40 (K.rhizophila) nuclei, and for eft-3 (q145) n=55 (E.coli) and 48 (K. rhizophila) nuclei, respectively. Data represent one of two independent experiments.