Proteasome inhibition triggers tissue-specific immune responses against different pathogens in C. elegans

Abstract

Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF up-regulates proteostasis capacity after blockade of the proteasome and also promotes resistance against bacterial infection in the nematode Caenorhabditis elegans. SKN-1/NRF has 3 isoforms, and the SKN-1A/NRF1 isoform, in particular, regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants in skn-1a and its activating enzymes ddi-1 and png-1 show constitutive expression of immune response programs against natural eukaryotic pathogens of C. elegans. These programs are the oomycete recognition response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the intracellular pathogen response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently, skn-1a mutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programs are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner against natural eukaryotic pathogens of the C. elegans epidermis and intestine.

Fig 1. Proteasome impairment activates ORR in C. elegans.

(A) Schematic showing the proteasome surveillance pathway and the stepwise activation of SKN-1A for transcription of proteasomal subunits. (B) L4 stage C. elegans showing constitutive chil-27p::GFP expression in ddi-1(icb156), png-1(icb121), and skn-1a(mg570) mutant animals. Note that skn-1a and png-1 show a stronger, full body activation of the chil-27p::GFP marker in comparison to ddi-1 mutants, which might reflect the fact that png-1 and skn-1a are essential components of the proteasome surveillance pathway, while ddi-1 has been shown to be dispensable [19]. Scale bar is 100 μm. (C) Venn comparisons showing significant overlap between up-regulated genes in the transcriptome of ddi-1(icb156) and skn-1a(mg570) mutant, and BTZ-treated animals with ORR (RF 13.8, p < 6.171e-50 for comparison with ddi-1; RF 13.6, p < 5.518e-101 for comparison with BTZ and RF 36.0, p < 1.249e-59 value for comparison with skn-1a). (D) ddi-1(mg572), png-1(ok1654), and skn-1a(mg570) mutant animals exhibit reduced susceptibility to infection by M. humicola as compared to WT C. elegans (n = 60 per condition, performed in triplicates, p < 0.001 based on log-rank test, a representative graph for one of the 3 replicates is shown). The numerical data for all 3 replicates is available in Supporting information S1 Data. BTZ, bortezomib; ORR, oomycete recognition response.

Fig 2. Proteasome impairment in the epidermis is sufficient to activate the ORR.

(A) Epidermal (dpy-7p), neuronal (rab-3p), and intestinal (vha-6p) rescue of skn-1a function in skn-1a(mg570). Note loss of GFP puncta in the body (shown by arrowheads) corresponding to chil-27p::GFP expression specifically upon epidermal rescue (3 independent transgenic lines analyzed, representative image shown). In all cases, myo-2p::GFP has been used as a co-injection marker that labels the pharynx. (B) Tissue-specific proteasome dysfunction induced by epidermal-specific, intestine-specific, and neuronal-enhanced RNAi of rpt-5 (n > 50 per condition, performed in triplicates, representative image shown). Scale bars in A and B are 100 μm. (C) Survival analysis of skn-1a(mg570) mutants with skn-1a function rescued in neurons (rab-3p::skn-1a), epidermis (dpy-7p::skn-1a), and intestine (vha-6p::skn-1a) (n = 60 per condition, performed in triplicates, p < 0.001 based on log-rank test, a representative graph for one of the 3 replicates is shown). The numerical data for all 3 replicates is available in Supporting information S1 Data. ORR, oomycete recognition response.

Fig 3. Oomycete extract exposure does not cause broad proteasome dysfunction, and activation of the proteasome partially inhibits induction of ORR genes by oomycete extract.

(A) Heat map showing log2 fold change in the expression of proteasome components upon treatment with oomycete extract as opposed to BTZ treatment. None of these genes are induced as part of the ORR and are shown in white. The numerical data for the heat map is available in Supporting information S1 Data. (B) Induction of rpt-3p::GFP is observed upon BTZ treatment, but not upon extract treatment (n > 50, performed in triplicates, representative image shown). (C) Induction of sur-5p::UbV-GFP is observed upon BTZ treatment, but not upon extract treatment (n > 50, performed in triplicates, representative image shown). Scale bar in panels B and C is 100 μm. (D) RT-qPCR showing reduced induction of genes specific to ORR or genes in the overlap between ORR and IPR upon extract treatment in animals with constitutive expression of the activated form of SKN-1A [skn-1a(cut, 4ND) in skn-1a(mg570)] or constitutive activation of the proteasome [pas-3(α3ΔN)] (**p < 0.01, ****p < 0.0001 based on unpaired t test in comparison to extract-treated wild type). The numerical data for all 3 replicates is available in Supporting information S1 Data. BTZ, bortezomib; IPR, intracellular pathogen response; ORR, oomycete recognition response.

Fig 4. Proteasome impairment in the intestine leads to activation of the IPR.

(A) Venn diagram showing significant overlap of IPR with up-regulated genes in skn-1a mutants (RF 84.4, p < 1.790e-72) and IPR with BTZ-treated animals (RF 21.4, p < 8.653e-84). (B) Venn diagram showing that overlap between ORR and IPR involves genes induced upon BTZ treatment. (C) skn-1a(mg570) mutants display increased resistance to N. parisii at 30 hpi relative to WT animals when infected at young adult. The pals-22(jy1) mutant was used as a positive control for its known increased resistance to N. parisii. (D) Intestinal rescue of skn-1a, but not epidermal or neuronal rescue, restores the resistance of skn-1a(mg570) mutants to N. parisii infection to WT levels. Kruskal–Wallis test with Dunn’s multiple comparisons test was used for statistical analysis (**** p < 0.0001, *** p < 0.001. n = 150 animals per genotype). The numerical data for all 3 replicates of panels C and D is available in Supporting information S1 Data. BTZ, bortezomib; IPR, intracellular pathogen response; ORR, oomycete recognition response.

Fig 5. Common ORR and IPR genes can be induced in a tissue-specific manner.

Sections of straightened L2 stage animals showing mRNA distribution of some common ORR and IPR genes, namely, B0507.8 (A), pals-5 (B), cul-6 (C), and skr-3 (D) by smFISH upon 4 h extract treatment (ORR), prolonged heat stress at 30°C for 24 h (IPR) and 2 h of BTZ treatment (proteasome dysfunction). Epidermal nuclei are labeled in green with the dpy-7p::GFP-H2B marker. Co-localization of mRNA with green nuclei indicates epidermal expression as shown in zoomed-in panels (shown in magenta). Dashed yellow lines outline the intestine and orange arrows point to smFISH signal. Images are presented so that epidermal nuclei in the head region are in focus. The intensity of the GFP signal from out of focus epidermal nuclei in the posterior part of the body is reduced to highlight the signal in the intestine. Scale bar is 10 μm. See S6 Fig for quantification. BTZ, bortezomib; IPR, intracellular pathogen response; ORR, oomycete recognition response; smFISH, single molecule fluorescence in situ hybridization.

Fig 6. Model based on the findings of this study.

The model highlights the interplay of SKN-1A-mediated proteasomal gene expression and activation of ORR and IPR in the epidermis and the intestine, respectively. 1. Under normal conditions, proteasomal degradation of unknown positive regulators (denoted as X factor) keeps activation of immune responses in check. 2. When proteasome dysfunction happens either by BTZ treatment or loss-of-function of skn-1a, X-factor escapes degradation and activates ORR and IPR in the epidermis and the intestine. Simultaneously, BTZ-mediated inhibition increases proteasomal gene expression as a bounce-back response. Such X factors could be directly involved in the tissue-specific signaling pathway activated upon oomycete or microsporidia exposure, respectively, in the epidermis and the intestine. Alternatively, they could regulate ORR and IPR gene induction in parallel to the pathogen-induced signaling pathway. BTZ, bortezomib; IPR, intracellular pathogen response; ORR, oomycete recognition response.