The C. elegans CCAAT-Enhancer-Binding Protein Gamma Is Required for Surveillance Immunity

Abstract

Pathogens attack host cells by deploying toxins that perturb core host processes. Recent findings from the nematode C. elegans and other metazoans indicate that surveillance or "effector-triggered" pathways monitor functioning of these core processes and mount protective responses when they are perturbed. Despite a growing number of examples of surveillance immunity, the signaling components remain poorly defined. Here, we show that CEBP-2, the C. elegans ortholog of mammalian CCAAT-enhancer-binding protein gamma, is a key player in surveillance immunity. We show that CEBP-2 acts together with the bZIP transcription factor ZIP-2 in the protective response to translational block by P. aeruginosa Exotoxin A as well as perturbations of other processes. CEBP-2 serves to limit pathogen burden, promote survival upon P. aeruginosa infection, and also promote survival upon Exotoxin A exposure. These findings may have broad implications for the mechanisms by which animals sense pathogenic attack and mount protective responses.

Figure 1. cebp-2 is required for infection response gene induction upon P. aeruginosa PA14 infection

(A-C) irg-1p::GFP animals treated with either (A) L4440 RNAi control, (B) zip-2 RNAi, or (C) cebp-2 RNAi and infected with PA14. (D-F) irg-1p::GFP expression in (D) wild-type, (E) zip-2(tm4248), or (F) cebp-2(tm5421) animals infected with PA14. In (A)-(F), green is irg-1p::GFP, red is myo-2::mCherry expression in the pharynx as a marker for presence of the transgene. Images are overlays of green, red and Nomarski channels and were taken with the same camera exposure for all. Scale bar, 200 μm. (G) qRT-PCR comparison of PA14-induced gene expression in control RNAi (L4440), zip-2 RNAi, and cebp-2 RNAi treated animals. (H) qRT-PCR comparison of PA14-induced gene expression in wild-type, zip-2(tm4248), and cebp-2(tm5421) animals. For (G) and (H), results shown are the average of two independent biological replicates, error bars are SD. ** p < 0.01, * p < 0.05 with a two-tailed t test.

Figure 2. cebp-2 and zip-2 control pathogen burden and promote survival upon P. aeruginosa infection

(A) Survival of RNAi control (L4440), zip-2 RNAi, and cebp-2 RNAi treated animals on P. aeruginosa PA14. zip-2 RNAi and cebp-2 RNAi treated animals were more susceptible to killing by PA14 than control (p < 0.0001 for each). (B) Survival of wild-type, zip-2(tm4248), cebp-2(tm5421), and cebp-2(tm5421);zip-2(tm4248) animals on PA14. zip-2(tm4248), cebp-2(tm5421), and cebp-2(tm5421);zip-2(tm4248) animals were more susceptible to killing by PA14 than wild-type (p < 0.001 for each); there was not a significant difference between cebp-2(tm5421), and cebp-2(tm5421);zip-2(tm4248), p=0.7. For (A) and (B), graph shows a representative assay of three independent replicates. (C-E) Images of (C) wild-type, (D) zip-2(tm4248) and (E) cebp-2(tm5421) animals after 16 hours of exposure to dsRed-expressing PA14. In each panel, the left image shows an overlay of Nomarski with red fluorescence and the right image shows red fluorescence alone. Scale bar, 200 μm. (F) Quantification of dsRed fluorescence levels in the intestine of wild-type, zip-2(tm4248), cebp-2(tm5421), and cebp-2(tm5421); zip-2(tm4248) animals after 16 hours of infection with dsRed-expressing PA14. Fluorescence was measured with a COPAS Biosort machine. Results shown are a representative assay of two independent replicates, with at least 500 animals measured for each sample. Error bars are SEM. ***, p < 0.001 with a two-tailed t test; n.s., not significant.

Figure 3. CEBP-2::GFP is expressed in intestinal nuclei independently of infection and independently of ZIP-2

(A) CEBP-2::GFP transgenic animals express GFP throughout the body with strong nuclear localization. (B) CEBP-2::GFP transgenic animals show nuclear GFP in the intestine when infected with P. aeruginosa. (C-D) CEBP-2::GFP transgenic animals have GFP in intestinal nuclei after treatment with (C) control RNAi or (D) zip-2 RNAi. (A-D) Images are overlays of green and Nomarski channels, with left panels imaged at 10× (scale bar, 200 μm) and right panels imaged at 40× (scale bar, 20 μm). White arrows indicate intestinal nuclei. (E) qRT-PCR comparison of gene expression in control RNAi (L4440) and zip-2 RNAi treated animals, shown as the fold change relative to L4440. (F) qRT-PCR measurement of gene expression shows that the CEBP-2::GFP transgene rescues the irg-1 induction defect of cebp-2(tm5421) mutants. For (E) and (F), results shown are the average of two independent biological replicates, error bars are SD. ** p < 0.01 with a two-tailed t test; n.s., not significant.

Figure 4. cebp-2 is required for induction of gene expression and survival upon translational inhibition by ToxA

(A-B) RNAi control (L4440) treated irg-1p::GFP animals after exposure to E. coli expressing either (A) the empty vector control or (B) ToxA. (C) zip-2 RNAi and (D) cebp-2 RNAi treated irg-1p::GFP animals after exposure to E. coli expressing ToxA. (E-F) Wild-type irg-1p::GFP animals after exposure to E. coli expressing either (E) the empty vector control or (F) ToxA. (G-H) irg-1p::GFP expression in (G) zip-2(tm4248) and (H) cebp-2(tm5421) animals after exposure to E. coli expressing ToxA. (A-H) Images are overlays of green, red, and Nomarski channels and were taken with the same camera exposure for all. Green is irg-1p::GFP, red is myo-2::mCherry expression in the pharynx as a marker for presence of the transgene. Scale bar, 200 μm. (I) qRT-PCR comparison of ToxA-induced gene expression in control RNAi (L4440), zip-2 RNAi, and cebp-2 RNAi treated animals. (J) qRT-PCR comparison of ToxA-induced gene expression in wild-type, zip-2(tm4248), and cebp-2(tm5421) animals. For (I) and (J), results shown are the average of two independent biological replicates, error bars are SD. *** p < 0.001, ** p < 0.01, * p < 0.05 with a two-tailed t test. n.s., not significant. (K) Survival of wild-type N2, zip-2(tm4248), cebp-2(tm5421), and cebp-2(tm5421); zip-2(tm4248) animals on E. coli expressing either ToxA or an empty expression vector starting at the L4 stage. Graph shows a representative assay of three independent replicates. N2 worms had no difference in life span when fed E. coli expressing either ToxA or the vector control (p = 0.7), while zip-2(tm4248), cebp-2(tm5421), and cebp-2(tm5421); zip-2(tm4248) animals had significantly shorter life spans on E. coli expressing ToxA as compared to the vector control (p < 0.001 for each). (L) Model for ZIP-2/CEBP-2 activation of gene expression after P. aeruginosa infection.