Tissue-specific activities of an immune signaling module regulate physiological responses to pathogenic and nutritional bacteria in C. elegans

Abstract

Microbes represent both an essential source of nutrition and a potential source of lethal infection to the nematode Caenorhabditis elegans. Immunity in C. elegans requires a signaling module comprised of orthologs of the mammalian Toll-interleukin-1 receptor (TIR) domain protein SARM, the mitogen-activated protein kinase kinase kinase (MAPKKK) ASK1, and MAPKK MKK3, which activates p38 MAPK. We determined that the SARM-ASK1-MKK3 module has dual tissue-specific roles in the C. elegans response to pathogens--in the cell-autonomous regulation of innate immunity and the neuroendocrine regulation of serotonin-dependent aversive behavior. SARM-ASK1-MKK3 signaling in the sensory nervous system also regulates egg-laying behavior that is dependent on bacteria provided as a nutrient source. Our data demonstrate that these physiological responses to bacteria share a common mechanism of signaling through the SARM-ASK1-MKK3 module and suggest the co-option of ancestral immune signaling pathways in the evolution of physiological responses to microbial pathogens and nutrients.

Figure 1. SEK-1 activities in the intestine and the chemosensory neurons contribute to resistance to P. aeruginosa PA14

Shown are survival curves on P. aeruginosa PA14 of L4 stage WT (N2) and the sek-1(km4) mutant, along with different sek-1(km4) mutant strains carrying transgenes expressing the sek-1 cDNA fused at its C-terminus to GFP, under the control of tissue-specific promoters. Pathogenesis assays were carried out under standard lawn conditions (bacteria seeded in a small spot in the center of the plate). (A) Survival curves for WT N2, sek-1(km4), and three independent lines carrying the Pges-1::sek-1::GFP transgene in an extrachromosomal array, on the standard lawn. (B) Survival curves for WT N2, sek-1(km4), and three independent lines carrying the Punc-119::sek-1::GFP transgene in an extrachromosomal array, on the standard lawn. p < 0.001 for each transgenic line compared with sek-1(km4). (C) Survival curves for WT N2, sek-1(km4), and three independent lines carrying the Posm-5::sek-1::GFP transgene in an extrachromosomal array, on the standard lawn. p < 0.001 for transgenic lines compared with sek-1(km4).

Figure 2. Regulation of intestinal gene expression by the TIR-1-NSY-1-SEK-1-PMK-1 pathway

Shown are images of young adult animals carrying the integrated agIs219 transgene, a GFP reporter of T24B8.5 transcriptional activity. T24B8.5 encodes a ShK toxin-like peptide that is expressed in the intestine. Strains are (A) WT (N2), (B) tir-1(qd2) (C) nsy-1(qd6), (D) sek-1(qd39), (E) pmk-1(km25). (F) Quantitative RT-PCR analysis of endogenous T24B8.5 expression in WT N2, sek-1(km4), and sek-1(km4);qdEx3 (Pges-1::sek-1::GFP) and sek-1(km4);qdEx12 (Posm-5::sek-1::GFP) transgenic strains.

Figure 3. SEK-1 activity in the chemosensory neurons is required for behavioral avoidance to P. aeruginosa PA14

Shown are survival curves on P. aeruginosa PA14 of L4 stage WT (N2) and the sek-1(km4) mutant, along with different sek-1(km4) mutant strains carrying transgenes expressing the sek-1 cDNA fused at its C-terminus to GFP, under the control of tissue-specific promoters. Pathogenesis assays were carried out under big lawn (bacteria spread to cover the entire plate) conditions which, when compared to standard lawn assays (Figure 1), define contributions to survival caused by behavioral avoidance of the pathogenic lawn of bacteria. (A) Survival curves for WT N2, sek-1(km4), and three independent lines carrying the Pges-1::sek-1::GFP transgene in an extrachromosomal array, on the big lawn. (B) Survival curves for N2, sek-1(km4), and three independent lines carrying the Posm-5::sek-1::GFP transgene in an extrachromosomal array, on the big lawn.

Figure 4. Serotonin-mediated behavioral avoidance is required for resistance to pathogens

Survival curves of L4 stage WT (N2), tph-1(mg280) and tph-1(n4622) mutants on PA14, carried out under (A) standard pathogenesis assay conditions, and (B) big lawn pathogenesis assay conditions.

Figure 5. The SARM-ASK1-MKK3 pathway in C. elegans is required for pathogen induction of serotonin biosynthesis in the ADF chemosensory neuron pair

(A) Representation of Ptph-1:gfp expression in C. elegans WT worms under GFP channel, DIC and merged channels. The white arrowhead points to an ADF neuron. (B) Images show representative fluorescence microscopy images of one ADF neuron of L4 stage worms carrying an integrated Ptph-1:GFP transgene, nIs145, propagated on E. coli OP50 and after 6 h exposure to P. aeruginosa PA14 in the following strains: WT(N2), tir-1(qd4), nsy-1(ag3), sek-1(km4), and pmk-1(km25). (C) Quantitation of tph-1:GFP fluorescence in the ADF neurons of WT (N2), tir-1(qd4), nsy-1(ag3), sek-1(km4), pmk-1(km25) worms propagated on OP50, both without (yellow) and after 6 h of exposure to PA14 (blue). Error bars reflect standard error of the mean (SEM). Asterisks indicate results of Student’s t-test comparing values of fluorescence on OP50 vs. PA14 for indicated strains: *p < 0.01, **p < 0.05.

Figure 6. The SARM-ASK1-MKK3 pathway functions in the sensory nervous system of C. elegans to promote egg-laying behavior

(A) Percentage of eggs laid by synchronized adult animals for WT (N2), and tir-1(qd4), nsy-1(ag3), sek-1(km4), and pmk-1(km25) mutant animals at each of the following stages over a one hour period: 1–8 cell, 9–20 cell, 21+ cell, comma, 2-fold, 3-fold. The egg-laying defective phenotype is manifest by mutant worms laying eggs with more advanced developmental stage relative to wild-type worms. (B) Same assay carried out for WT (N2) and sek-1(km4) mutant animals along with sek-1(km4) strains carrying extrachromosomal arrays expressing the sek-1::GFP transgene under the control of tissue-specific promoters, Pges-1 (intestine) (qdEx4, qdEx5, qdEx6), Punc-119 (pan-neuronal) (qdEx7, qdEx8, qdEx10), and Posm-5 (ciliated sensory neurons) (qdEx11, qdEx12, qdEx13). Three independent transgenic lines for each tissue-specific promoter are presented.

Figure 7. Tissue-specific activities of the TIR-1-NSY-1-SEK-1 module in C. elegans responses to bacteria

In response to pathogen infection, the TIR-1-NSY-1-SEK-1 module is required in the intestinal and epidermal cells for the PMK-1 p38 MAPK-dependent, cell autonomous activation of innate immunity. In addition, the TIR-1-NSY-1-SEK-1 module is required in the sensory nervous system for serotonin-dependent neuroendocrine signaling from the ADF chemosensory neurons, which is required for behavioral avoidance to pathogens. Serotonin-dependent neuroendocrine pathways also mediate the behavioral reproductive egg laying response to nutritional bacteria through the TIR-1-NSY-1-SEK-1 signaling module.