Inactivation of conserved C. elegans genes engages pathogen- and xenobiotic-associated defenses

Abstract

The nematode C. elegans is attracted to nutritious bacteria and is repelled by pathogens and toxins. Here we show that RNAi and toxin-mediated disruption of core cellular activities, including translation, respiration, and protein turnover, stimulate behavioral avoidance of normally attractive bacteria. RNAi of these and other essential processes induces expression of detoxification and innate immune effectors, even in the absence of toxins or pathogens. Disruption of core processes in non-neuronal tissues was sufficient to stimulate aversion behavior, revealing a neuroendocrine axis of control that additionally required serotonergic and Jnk kinase signaling pathways. We propose that surveillance pathways overseeing core cellular activities allow animals to detect invading pathogens that deploy toxins and virulence factors to undermine vital host functions. Variation in cellular surveillance and endocrine pathways controlling behavior, detoxification, and immunity selected by past toxin or microbial interactions could underlie aberrant responses to foods, medicines, and microbes.

Figure 1. Inactivation of essential cellular pathways stimulates microbial avoidance behavior

(A) Schematic of RNAi microbial aversion assay. (B) Example of control and aversion phenotypes after 48hr of growth on elt-2 or RNAi control bacteria. elt-2 encodes a transcription factor necessary for gut development and homeostasis. (C) Developmental stage- and rank-ordered aversion levels +SEM for 379 gene inactivations (of 4,062 screened) exhibiting aversion ≥10% off bacteria at 48-58hr of growth on RNAi bacteria. (D) DAVID bioinformatic analysis of aversion genes showing enrichment for specific functional categories. *enriched gene classes that were not statistically significant due to small N. ^high intra-class homology that could produce an elevated false positive rate due to off-target RNAi effects. (See also Figure S1, Table S1, Movies S1–3.)

Figure 2. Toxins stimulate the microbial aversion behavior

(A) Toxin-induced microbial aversion phenotypes. Drug concentrations are provided in Supplemental Materials. (B) Aversion levels +/−SEM for each drug at peak time points: Zeocin (16 hr); Paraquat (8 hr); Antimycin A (6 hr); Tunicamycin (24 hr); Geneticin (3 hr); Bortezomib (6 hr); Concanamycin A (8 hr). *p<0.01; *p<0.001; ***p<0.0001 by t-test.

Figure 3. Activation of pathogen-associated and detoxification responses

(A) Innate immunity-associated reporter induction in response to growth on RNAi lawns inactivating the C. elegans eIF2γ homolog Y39G10AR.8, emb-5 and sca-1 genes. (B) RNAi of eIF-2γ and emb-5 stimulates cyp-35B1::GFP, a reporter for cytochrome P450 enzymes involved in phase I drug detoxification. (C) RNAi of eat-6 and rpt-3 stimulates a gst-4::GFP reporter for induction of glutathione S-transferase, a phase II detoxification enzyme. (See also Figure S2, Table S2.)

Figure 4. Neuroendocrine control of aversion behavior

(A) Aversion is stimulated by inactivation of the threonyl tRNA synthetase tars-1 in the hypodermis but not the intestine. (B-D) Aversion +/−S.D. is induced by RNAi of (B) mitochondrial ATP synthase subunits and (C) vacuolar ATPase subunits in the hypodermis or intestine, and (D) proteasome subunits in the hypodermis only. Representative experiments +/−S.D. shown. *p<0.01; **p<0.001; ***p<0.0001; n.s. not significant. (See also Figures S3–S4, Table S3.)

Figure 5. The Jnk MAP kinase pathway is required for aversion

(A) Four candidate pathogen and stress response pathways in C. elegans. (B) Aversion phenotypes for Jnk pathway mutants following inactivation of vha-6 (vacuolar ATPase subunit). (C-E) The Jnk pathway is required for aversion induced by RNAi of (C) translation components, (D) mitochondrial ETC components and (E) proteasome subunits. Representative experiment +/− SD shown. * p<0.01, ** p<0.001 and *** p<0.0001. n.s. not significant. (See also Figure S5.)

Figure 6. Learned avoidance and serotonergic signaling mediate aversion behavior

(A) Schematic outline of learning experiment. (B) Aversion time courses for animals grown on RNAi bacteria inactivating the kars-1 tRNA synthetase, hsp-60 mitochondrial chaperone, nhx-2 Na⁺/K⁺ potassium pump, or Y71H2AL.1 calcineurin B. Control experiments: Top left, animals grown on HT115 RNAi control and tested on HT115, HB101 or OP50 E. coli. Top middle, sid-1 RNAi defective animals were grown and tested on the same bacterial type (e.g. grown and tested on nhx-2 RNAi). For graphical clarity, only +S.D. bars shown. (C) Serotonin deficient tph-1(mg280) animals exhibited a partial aversion defect (~35% of wild type) in response to gene inactivations representing major functional classes.*p<0.01; **p<0.001; ***p<0.0001, ****p<10⁻¹⁹. (See also Figures S6–S7.)

Figure 7. Model of aversion behavior

Distress signals arising in internal tissues are integrated with sensory inputs to control the microbial aversion response. *Although Jnk pathway components are depicted as acting in the intestine, the site(s) of action have not yet been determined.