DAF-16-dependent suppression of immunity during reproduction in Caenorhabditis elegans

Abstract

To further understand how the nematode Caenorhabditis elegans defends itself against pathogen attack, we analyzed enhanced pathogen resistance (epr) mutants obtained from a forward genetic screen. We also examined several well-characterized sterile mutants that exhibit an Epr phenotype. We found that sterility and pathogen resistance are highly correlated and that resistance in both epr and sterile mutants is dependent on DAF-16 activity. Our data indicate that a DAF-16-dependent signaling pathway distinct from previously described pathways is involved in the activation of genes that confer resistance to bacterial pathogens. The timing of DAF-16-dependent gene activation in sterile mutants coincides with the onset of embryonic development in wild-type animals, suggesting that signals from developing embryos normally downregulate the immune response.

Figure 1.—

C. elegans epr mutants exhibit enhanced resistance to both S. aureus and P. aeruginosa and various degrees of enhanced longevity on E. coli. L4 larval-stage epr mutants, ag12, ag15, ag17, and ag19, were transferred from E. coli OP50 plates onto plates containing S. aureus NCTC8325 (A), P. aeruginosa PA14 (B), and OP50 (C) and monitored for their survival over time at 25°. The time at which 50% of the worms had died (TD₅₀) for this assay for each epr mutant is shown in supplemental Table 1. daf-2(e1368) was included as positive controls (A and C). The experiment was repeated at least three times with similar results.

Figure 2.—

epr mutants require DAF-16 for pathogen resistance. epr mutants, ag12 and ag15 (A), ag17 and ag19 (B), were fed with either a vector control or daf-16 RNAi and then transferred to P. aeruginosa PA14 plates. The differences in survival kinetics between wild-type N2 and epr mutants (ag12, ag15, ag17, and ag19) treated with the control RNAi were significant (P < 0.0001). The differences in survival kinetics between the epr mutants treated with control RNAi and daf-16 RNAi were also significant (P < 0.0001). Experiments with ag12 and ag17 were repeated three times and experiments with ag15 and ag19 were repeated twice with similar results.

Figure 3.—

ag12 harbors a point mutation in the RBD of the PI(3)K AGE-1. Protein alignment of the RBDs of PI(3)Ks among different species including C. elegans, which is shown at bottom. The conserved leucine residue, which is mutated to phenylalanine in ag12, is indicated by an arrow.

Figure 4.—

epr mutants have a small brood size at 25°. N2, daf-2(e1368), age-1(ag12), ag13, ag14, inx-14(ag17), ag15, and ag19 were cultured at 15° and then transferred at the L4 larval stage to 25°. The total number of progeny that hatched from a single animal was counted. The average of more than eight animals for each genotype is shown. Error bars represent standard deviation.

Figure 5.—

inx-14(ag17) mutants and sterile mutants are more resistant than wild-type animals in the presence and absence of FUDR (A–D). N2 and inx-14(ag17) animals were transferred at the L4 larval stage onto plates preseeded with P. aeruginosa PA14 with and without FUDR (A). inx-14(ag17) exhibited enhanced pathogen resistance compared to wild type in the absence of FUDR (P < 0.0001) and in the presence of FUDR (P < 0.005). glp-1(e2141), fog-2(q71), and fem-3(q20) were grown at the nonpermissive temperature of 25° and then transferred onto plates preseeded with PA14 in the absence (B) or presence (C) of FUDR. Compared to wild-type N2, daf-2(e1368), glp-1(e2141), fog-2(q71), and fem-3(q20) exhibited significantly enhanced pathogen resistance in the absence (P < 0.0001) or presence of FUDR (P < 0.05). N2 and fer-15;fem-1 were pretreated or untreated with FUDR prior to infection with PA14 in the absence of FUDR (D). FUDR-pretreated fer-15;fem-1 exhibited enhanced pathogen resistance (P < 0.0001) compared to FUDR-pretreated wild-type N2. N2 and fer-15;fem-1 were mated with wild-type males prior to infection with PA14 (E). Mated fer-15;fem-1 animals no longer exhibited enhanced pathogen resistance (P = 0.2961) compared to mated N2. Four-day-old adults of N2 and fer-15;fem-1 were infected with PA14 (F). The difference in pathogen resistance phenotype between N2 and fer-15;fem-1 was much smaller in 4-day-old adults than in 1-day-old adults (E). Similar results were observed in two independent experiments for inx-14(ag17) and three independent experiments for the other sterile mutants.

Figure 6.—

daf-16 is required for the pathogen resistance of sterile mutants. The survival kinetics of N2 were compared to those of daf-16 and daf-2 upon P. aeruginosa PA14 infection (A). Similarly, the survival kinetics of the following sterile mutants were compared to their respective daf-16 and daf-2 mutants; glp-1 to daf-16;glp-1 and daf-2 glp-1 (B), fog-2 to daf-16;fog-2 and daf-2;fog-2 (C), fer-15;fem-1 with control RNAi to fer-15;fem-1 with daf-16 RNAi and with daf-2 RNAi (D), and fem-3 to daf-16;fem-3 and daf-2;fem-3 (E). In wild-type N2, a daf-16(mgDf47) mutation caused no significant change in pathogen resistance (P = 0.6281), whereas a daf-2(e1368) mutation enhanced the pathogen resistance significantly (P < 0.0001). In glp-1(e2141), fog-2(q71), fer-15;fem-1, and fem-3(q20) animals, either the daf-16(mgDf47) mutation or daf-16 RNAi suppressed the pathogen resistance phenotype of the respective sterile mutants (P < 0.0001). daf-2(e1368) or daf-2 RNAi significantly increased the pathogen resistance phenotype of the respective sterile mutants (P < 0.05). Similar results were observed in three independent experiments with glp-1(e2141), fer-15;fem-1, and fem-3(q20) and two independent experiments with fog-2(q71). N2 and glp-1(e2141) animals were fed either with vector control RNAi, daf-16 RNAi, or kri-1 RNAi and then tested for PA14 resistance (F). The differences between glp-1(e2141) treated with control RNAi and with daf-16 RNAi were significant (P < 0.0001). The differences between glp-1(e2141) treated with control RNAi and with kri-1 RNAi were not significant (P = 0.3990). kri-1 RNAi in glp-1 animals appeared to be effective because 100% of glp-1 animals (n = 94) treated with kri-1 RNAi appeared paler and smaller compared to the glp-1 animals treated with the control RNAi, as previously reported (Berman and Kenyon 2006). This experiment was repeated three times with similar results.

Figure 7.—

DAF-16∷GFP is localized to the nucleus in animals treated with RNAi clones that cause sterility. Shown are representative images of DAF-16∷GFP protein in intestinal cells of TJ356 animals fed with various RNAi clones including L4440 vector control (A and B), glp-1 (C and D), inx-14 (E and F), or fog-2 (G and H). Differential interference contrast images are in A, C, E, and G and GFP images are in B, D, F, and H. Only fully sterile animals were analyzed for DAF-16∷GFP.

Figure 8.—

DAF-16-dependent genes are upregulated in adult sterile mutants. The expression of four DAF-16-dependent genes, mtl-1, sod-3, lys-7, and dod-6, in gravid adult animals (A) or young adult animals without embryos (B) was determined by qRT–PCR and compared to that of wild-type N2. Mutants analyzed were daf-2(e1368), daf-16(mgDf47), glp-1(e2141), fer-15;fem-1, fem-3(q20), and inx-14(ag17). DAF-16-dependent gene expression in 4-day-old fer-15;fem-1 adults was compared to 4-day-old wild-type N2 (C). Fold changes represent the average of at least three different biological samples. Error bars represent standard error of the mean.