TRPM channels mediate learned pathogen avoidance following intestinal distention

Abstract

Upon exposure to harmful microorganisms, hosts engage in protective molecular and behavioral immune responses, both of which are ultimately regulated by the nervous system. Using the nematode Caenorhabditis elegans, we show that ingestion of E. faecalis leads to a fast pathogen avoidance behavior that results in aversive learning. We have identified multiple sensory mechanisms involved in the regulation of avoidance of E. faecalis, including the GPCR NPR-1-dependent oxygen-sensing pathway, an ASE neuron-dependent pathway, and an AWB and AWC neuron-dependent pathway. Colonization of the anterior part of the intestine by E. faecalis leads to AWB and AWC mediated olfactory aversive learning. Finally, two transient receptor potential melastatin (TRPM) channels, GON-2 and GTL-2, mediate this newly described rapid pathogen avoidance. These results suggest a mechanism by which TRPM channels may sense the intestinal distension caused by bacterial colonization to elicit pathogen avoidance and aversive learning by detecting changes in host physiology.

Dissection of a sensorimotor circuit that regulates aversion to odors and pathogenic bacteria inC. elegansby whole-brain simulation

“…To investigate the neural circuitry governing the translation of pathogen chemosensory cues into the motor neuron activity necessary for avoiding said cues, we directly tested avoidance locomotion by making use of an assay that would allow us to quickly assess individual neuron requirements for reflexive aversion both before and after exposure to a pathogen (Figure 1A). The pathogenic bacteria P. aeruginosa and E. faecalis are initially attractive to C. elegans and only induce an avoidance response after many hours of exposure (4, 9, 10, 13). This learning process involves the association of infection and subsequent physiological responses, including intestinal distention, engagement of RNAi pathways, and immune activation, with bacterial cues, resulting in avoidance of the bacteria (10, 12, 25).…”

“…The pathogenic bacteria P. aeruginosa and E. faecalis are initially attractive to C. elegans and only induce an avoidance response after many hours of exposure (4, 9, 10, 13). This learning process involves the association of infection and subsequent physiological responses, including intestinal distention, engagement of RNAi pathways, and immune activation, with bacterial cues, resulting in avoidance of the bacteria (10, 12, 25). Using the reflexive aversion assay, we found that naïve animals do not respond to drops of P. aeruginosa (Figure 1B) or E. faecalis (Figure 1C); however, animals exposed to bacteria prior to testing showed reflexive aversion to the same bacteria.…”

“…However, intestinal distention on E. faecalis showed no such correlation, perhaps due to the fact that distention was much more prevalent and severe in animals exposed to E. faecalis (Figure S5C and D). The association of intestinal distention and E. faecalis cues requires the TRPM channels GON-2 and GTL-2 (10). We found that these channels were also required for the learned reflexive aversion to E. faecalis , as animals with loss-of-function mutations in the genes encoding for GON-2 and GTL-2 displayed mitigated responses to E. faecalis after training (Figure 4C).…”

“…The following C. elegans strains used in this study were obtained from the CGC: N2 (WT strain), CZ9957 gtl-2(n2618) , EJ1158 gon-2(q388) , JN1713 peIs1713 [ sra-6p::mCasp-1 + unc-122p::mCherry ], JN1715 peIs1715 [ str-1p::mCasp-1 + unc-122p::GFP ], NY2078 ynIs78 [ flp-8p::GFP ], NY2087 ynIs87 [ flp-21p::GFP ], PR680 che-1(p680) , PY7502 oyIs85 [ ceh-36p::TU#813 + ceh-36p::TU#814 + srtx-1p::GFP + unc-122p:DsRed ], and PY7505 oyIs84 [ gpa-4p::TU#813 + gcy-27p::TU#814 + gcy-27p::GFP + unc-122p::DsRed ]. The following transgenic strains were generated for this study using standard microinjection protocols (10): AY178 ynIs78 [ flp-8p::GFP ]; flp-8p::ced-3 (p15)::nz + flp-32::cz::ced-3 (p17) + unc-122p::rfp and AY179 ynIs87 [ flp-21p::GFP ]; flp-21p::ced-3 (p15)::nz + ncs-1p::cz::ced-3 (p17) + unc-122p::rfp. C. elegans hermaphrodites were maintained on E. coli OP50 at 20°C.…”

“…Lawn avoidance assays were carried out as previously described (10). Briefly, bacterial cultures were grown by inoculating P. aeruginosa or E. faecalis colonies into 2 mL of either LB or BHI broth, respectively, and growing them for 5-6 hr on a shaker at 37°C.…”