Proton signaling links epithelial sensing to neural control of host defense in C. elegans

Domain: biology
Published: 2026-03-27
Editorial outcome: minor_revision

Abstract
Effective host defense against pathogens requires coordinated behavioral and immune responses, yet the mechanisms that couple epithelial sensing to these systemic defenses remain poorly understood. Here we identify a proton-mediated gut-to-neuron signaling pathway that orchestrates host defense in C. elegans. Intestinal pathogens stimulate mechanosensitive Ca2+ influx into intestinal epithelial cells (IECs) through the TRP channel GON-2, activating the Na+/H+ exchanger NHX-6 via the calmodulin CMD-1 to drive basolateral proton release. These protons activate cholinergic motor neurons through the acid-sensing ion channel ASIC-1, enhancing cholinergic transmission to promote both pathogen avoidance and intestinal innate immunity. Notably, mouse NHE1 and ASICS1a can functionally substitute for their nematode counterparts. Together, these findings demonstrate a role for proton signaling in gut-to-neuron communication, revealing a potentially conserved mechanism that links epithelial sensing to neuroimmune defense.

Introduction
Animals rely on both behavioral and immune mechanisms to defend against pathogen infections1-3. While immune responses eliminate invading pathogens, behavioral responses help prevent encounters, promote escape, and reduce further ingestion1-4. The coordination of these two mechanisms is essential for effective pathogen defense, with the nervous system serving as a key integrative regulator5-7. Specifically, the initiation of avoidance behavior relies on neural detection of sensory cues from external and ingested pathogens1,2,4, while neuroimmune communication fine-tunes immune responses and maintains systemic homeostasis5-7. This bidirectional crosstalk can also influence neuronal functions, leading to behavioral changes7-9. However, the molecular mechanisms that coordinate behavioral and immune defenses via this bidirectional communication remain poorly defined.

Intestinal epithelial cells (IECs) form a critical anatomical and functional barrier against ingested pathogens while closely interacting with the nervous system9-11. In mammals, IECs consist of specialized subtypes that communicate with the luminal environment11. For example, Paneth cells sense intestinal pathogens and release antimicrobial substances, contributing to innate immune defenses12,13. Enteroendocrine cells (EECs), acting as chemosensors, detect luminal contents and secrete neuroendocrine molecules10,14,15. In the mucosa, the vagus nerve is positioned near EECs and expresses receptors for gut-derived molecules, allowing basolaterally released signals to reach the brain and influence behavior4,10,14,15. Conversely, the vagus nerve releases acetylcholine (ACh)7,11. Activation of muscarinic ACh receptors (mAChRs) induces secretion from Paneth cells and EECs11. This apical-basolateral dichotomy and bidirectional neuroepithelial communication position IECs to coordinate intestinal immune responses with organism-wide behavioral defenses. However, the specific molecular pathways by which IECs communicate with neurons to coordinate host defense remain poorly understood.

The nematode C. elegans provides a powerful model for dissecting such inter-tissue signaling and neuroimmune communication at molecular, single-cell, and whole-animal levels2,5,16,17. In the laboratory, C. elegans feeds on non-pathogenic E. coli OP50, and intestinal infections can be readily introduced with human pathogens, facilitating mechanistic studies17. Despite its simple anatomy, C. elegans is capable of behaviorally avoiding and immunologically eliminating ingested pathogens18-25. Its innate immune pathways, including PMK-1/p38 MAPK and canonical Wnt signaling, are evolutionarily conserved21,26,27. Like mammals, its nervous system regulates intestinal immunity, and intestine-to-neuron signaling can modulate pathogen avoidance16,19,21,28-31. Several neuronal regulators of behavioral and immune responses have been identified20,30,32-36, yet whether a conserved intestine-to-neuron signaling pathway exists to coordinate host defense remains unknown.

Here, we identify a proton-mediated gut-to-neuron signaling pathway that links epithelial sensing to both behavioral and immune defenses in C. elegans. Intestinal pathogens stimulate mechanosensitive Ca2+ influx into IECs via the TRP channel GON-2, activating the Na+/H+ exchanger (NHE) NHX-6 through CMD-1/calmodulin to drive basolateral proton release. These protons activate cholinergic ventral nerve cord (VNC) motor neurons via the acid-sensing ion channel (ASIC) ASIC-1, enhancing cholinergic transmission to promote pathogen avoidance. Concurrently, cholinergic signaling acts back on IECs to activate canonical Wnt and PMK-1/p38 MAPK pathways via the mAChR GAR-2;GAR-3, enhancing intestinal immunity. Using the C. elegans intestine-motor neuron unit as a platform, we show that mouse NHE1 and ASIC1a can functionally substitute for their nematode counterparts.

Together, these findings demonstrate a role for protons as gut-derived signaling molecules that couple epithelial stress to neuronal and immune responses, revealing a potentially conserved mechanism that coordinates host defense against intestinal pathogens.

Results
ASIC-1 mediates the acid sensitivity of VA and VB motor neurons in C. elegans
Acid-sensing ion channels (ASICs), members of the degenerin/epithelial Na+ channel (DEG/ENaC) family, are evolutionarily conserved from C. elegans to mammals, including humans and mice37,38. We previously found that C. elegans ASIC-1 functions as a mechanoreceptor in VA and VB motor neurons39. However, mammalian ASICs are primarily recognized as proton-gated cation channels that respond to extracellular acidic pH38. Consistent with this, C. elegans ASIC-1 can form proton-gated homomeric channels in Xenopus oocytes40.

To assess whether VA and VB neurons are acid-sensitive, we applied acidic pH solutions directly to the VNC of dissected worms and monitored neuronal responses by calcium imaging. We used a transgenic strain expressing GCaMP6 under the unc-17Δ1 promoter39 (Punc-17Δ1), which drives expression in multiple types of cholinergic VNC motor neurons, including A-type (VA and DA), B-type (VB and DB), and AS-type neurons. Upon acidic pH stimulation, all VA and VB in the imaging field showed robust calcium responses (Fig. 1a,b). Unexpectedly, DA and DB also exhibited acid-evoked responses, although weaker than those observed in VA and VB (Fig. 1b). In contrast, AS neurons showed no detectable response (Fig. 1b). Acid-evoked responses in A- and B-type motor neurons were pH-dependent, becoming detectable at pH 6 and plateauing at pH 4 (Fig. 1a,b). Thus, we selected pH 4 for further analysis.

Since A- and B-type motor neurons are synaptically interconnected and receive inputs from other neurons41, we next investigated whether their responses were intrinsic. Disruption of gap junction proteins expressed in VA/VB (INX-3, INX-12, and UNC-9)42 or in DA/DB (INX-3, INX-7, and UNC-9)42 had no effect on acid-evoked responses (Supplementary Fig. 1a). Responses in VA and VB also persisted in both unc-13(e51) and unc-31(e169) mutants (Fig. 1c and Supplementary Fig. 1b), which are defective in neurotransmitter and neuropeptide release, respectively43,44. In contrast, responses in DA and DB were abolished in unc-13(e51) mutants (Fig. 1c and Supplementary Fig. 1b). Furthermore, inhibiting synaptic release from VA and VB using tetanus toxin (TeTx) under Pdel-139 significantly reduced acid-evoked responses in DA and DB (Fig. 1c and Supplementary Fig. 1b). Although additional innexins may be expressed in these neurons45,46, and RNAi efficiency and cell-type specificity were not directly assessed, the data do not support a major contribution of gap junctions. Together, these results suggest that VA and VB are intrinsically acid-sensitive, whereas the responses observed in DA and DB are likely due to synaptic input from VA and VB.

We then examined whether ASIC-1 mediates the acid sensitivity of VA and VB. In asic-1(ok415) deletion mutants, acid-evoked responses in A- and B-type motor neurons were nearly absent (Fig. 1d and Supplementary Fig. 1c). Expression of wild-type asic-1 in VA and VB under Pdel-1 fully rescued acid-evoked responses in VA and VB, as well as in DA and DB (Fig. 1d and Supplementary Fig. 1c). In contrast, asic-1(ok415) had no effect on the voltage-dependent whole-cell currents of VA and VB (Supplementary Fig. 1d,e), consistent with our previous findings39. Together, these results demonstrate that ASIC-1 mediates the acid sensitivity of VA and VB without generally altering their ion channel function, and further support the notion that acid-evoked responses in DA and DB arise from VA and VB synaptic input.

Intestinal pathogens induce proton secretion from IECs through NHX-6
Next, we investigated the source of protons that may act on VA and VB in vivo. The C. elegans intestine runs alongside the VNC across the body cavity (Fig. 2a) and is surrounded by the commissures of A- and B-type motor neurons47. In mammals, intestinal pathogens or inflammation can cause acidosis in the basolateral space of IECs48,49. To test this possibility in C. elegans, we visualized the extracellular pH of A- and B-type motor neurons during pathogen infections using pHluorin, a pH-sensitive GFP reporter whose fluorescence decreases as pH reduces50. We fused pHluorin to the extracellular side of the mouse CD8 transmembrane domain51 and expressed the fusion protein in cholinergic VNC motor neurons using Punc-17Δ1 (Fig. 2a). Two paradigmatic human pathogenic bacteria, the Gram-positive S. aureus and the Gram-negative P. aeruginosa, were used to infect the transgenic worms. Compared to control worms fed on E. coli, those infected with S. aureus or P. aeruginosa displayed significantly reduced pHluorin fluorescence, indicating decreased extracellular pH (Fig. 2b). Pathogen infections induced extracellular acidification at multiple motor neuron locations along VNC (Fig. 2b and Supplementary Fig. 3a). In contrast, neither heat-killed S. aureus nor the nonpathogenic mutant P. aeruginosa-gacA(-) affected pHluorin fluorescence (Fig. 2b), arguing against contributions from bacterial identity or dietary differences. Although these experiments do not directly determine whether pathogen infections cause global pseudocoelomic acidification, they demonstrate that the extracellular space surrounding motor neurons at widespread VNC sites becomes acidified during pathogen infections. Across these conditions, the co-expressed, pH-insensitive mStrawberry signal remained unchanged during pathogen exposure (Supplementary Fig. 4a), indicating stable reporter expression.

We next examined the role of NHEs in proton secretion, as these transporters are known to acidify the extracellular environment by releasing protons50,52. Among the nine C. elegans NHEs, NHX-1, NHX-2, NHX-6, and NHX-7 are expressed predominantly in the intestine, with NHX-6 and NHX-7 localized to the basolateral membrane of IECs52. Previous studies have shown that NHX-7 is necessary for phasic proton release from posterior IECs to stimulate posterior body muscle contraction during defecation50,53. Nevertheless, we tested all NHEs using either available mutants or IEC-specific RNAi worms. Only nhx-6(ok609) deletion mutants exhibited significantly increased pHluorin fluorescence during pathogen infections (Fig. 2c and Supplementary Fig. 2 and 3b). This result aligns with the broader expression of NHX-6 in the intestine compared to the posterior restriction of NHX-750,52. The increased pHluorin fluorescence in nhx-6(ok609) mutants was rescued by expressing wild-type nhx-6 specifically in IECs using Pges-154 (Fig. 2c and Supplementary Fig. 3b). mStrawberry fluorescence was unaffected by pathogen exposure across these genotypes (Supplementary Fig. 4b). Together, these results suggest that intestinal pathogens stimulate tonic proton release from IECs via NHX-6, leading to acidification of the extracellular environment surrounding VNC motor neurons.

Pathogen-induced IEC proton secretion requires TRP channel GON-2-mediated Ca2+ influx and CMD-1/calmodulin
In mammals, NHE activity is regulated by Ca2+/calmodulin signaling50,55. Many innate immune cells express the mechanosensitive ion channels TRPV4, which mediates Ca2+ influx upon activation56. In C. elegans, the TRP channel GTL-2 mediates Ca2+ influx in response to membrane damage in epidermal cells57. To determine whether pathogens alter Ca2+ dynamics in IECs, we monitored cytosolic Ca2+ using a transgenic strain expressing GCaMP6 under Pges-1. In wild type fed E. coli, we observed frequent spontaneous Ca2+ transients in IECs (Fig. 2d). Infection with either S. aureus or P. aeruginosa significantly increased the frequency and amplitude of Ca2+ transients and shortened inter-peak intervals, whereas heat-killed S. aureus or P. aeruginosa-gacA(-) had no effect (Fig. 2d). Among the three TRP channels expressed in IECs, TRPA-1, GTL-1, and GON-258,59, only gon-2(q362) mutants exhibited a strong reduction in pathogen-induced Ca2+ transients, which was rescued by IEC-specific expression of wild-type gon-2 under Pges-1 (Fig. 2e and Supplementary Fig. 3c). In contrast, trpa-1(ok999) mutants and IEC-specific gtl-1 RNAi worms responded similarly to wild type (Fig. 2e and Supplementary Fig. 3c). These results demonstrate that GON-2 is required for pathogen-enhanced Ca2+ influx in IECs.

We next investigated how GON-2 is activated in IECs. In wild type fed E. coli, either physical immobilization using glue or pharmacological paralysis by levamisole significantly suppressed spontaneous Ca2+ activity, as reflected by reduced transient frequency and prolonged inter-peak intervals (Supplementary Fig. 3d). gon-2(q362) mutants exhibited similarly reduced Ca2+ activity, consistent with previous findings60, and this defect was rescued by IEC-specific gon-2 expression (Supplementary Fig. 3d). Notably, immobilization did not further suppress Ca2+ activity in gon-2(q362) mutants (Supplementary Fig. 3d), suggesting that GON-2 mediates mechanosensitive activation of IECs during body bending. Given that both S. aureus and P. aeruginosa damage IEC membranes and cause intestinal distention61, we asked whether pathogen infections enhance GON-2-dependent mechanosensation. Indeed, glue immobilization suppressed pathogen-induced Ca2+ transients in wild type to levels observed in gon-2(q362) mutants, and immobilizing infected gon-2(q362) mutants produced no further suppression (Fig. 2e and Supplementary Fig. 3c). In gon-2(q362) mutants, pathogen exposure failed to alter spontaneous Ca2+ activity (Supplementary Fig. 3e). However, artificially inducing intestinal bloating in E. coli-fed worms via aex-5 or nhx-2 RNAi19 failed to increase Ca2+ transients (Supplementary Fig. 3f), suggesting that gross tissue distention alone is insufficient to enhance GON-2 activation. Thus, IECs are mechanosensitive in a GON-2-dependent manner, and pathogen infections enhance this mechanosensory activation, likely through membrane damage that perturbs membrane tension. Consistently, pHluorin fluorescence in VNC motor neurons was significantly increased in gon-2(q362) mutants compared to wild type, and this defect was rescued by IEC-specific gon-2 expression (Fig. 2f and Supplementary Fig. 3g). pHluorin fluorescence in nhx-6;gon-2 double mutants was indistinguishable from that of nhx-6(ok609) single mutants (Fig. 2f and Supplementary Fig. 3g), suggesting that GON-2 is required for NHX-6-mediated proton secretion. The calmodulin ortholog cmd-162 was also required, as IEC-specific cmd-1 RNAi significantly increased pHluorin fluorescence in VNC motor neurons during pathogen infections, phenocopying nhx-6(ok609) mutants (Fig. 2g and Supplementary Fig. 3h). In nhx-6(ok609) mutants, IEC-specific cmd-1 RNAi had no additional effect (Fig. 2g and Supplementary Fig. 3h). Furthermore, in nhx-6(ok609), gon-2(q362), nhx-6;gon-2, cmd-1 RNAi, and nhx-6;cmd-1 RNAi worms, pathogen infections failed to alter pHluorin fluorescence in VNC motor neurons (Supplementary Fig. 4e). mStrawberry fluorescence was unaffected by pathogen exposure across these genotypes (Supplementary Fig. 4c,d). Together, these results demonstrate that pathogen-induced proton secretion from IECs is driven by mechanosensitive Ca2+ influx through GON-2 and is mediated by CMD-1/calmodulin-dependent activation of NHX-6.

Intestinal pathogens activate motor neurons via NHX-6-H+-ASIC-1 signaling
To investigate the physiological impact of extracellular acidification on motor neurons, we recorded their spontaneous activity using GCaMP6 in worms fed on different bacteria. Compared to E. coli, both S. aureus and P. aeruginosa significantly increased the basal GCaMP6 fluorescence in motor neurons and the amplitude of spontaneous Ca2+ transients, without affecting their frequency (Fig. 3a,b). In contrast, heat-killed S. aureus or P. aeruginosa-gacA(-) had no such effect (Fig. 3a,b). Notably, these pathogen-induced effects were markedly diminished in nhx-6(ok609) mutants and fully rescued by expressing wild-type nhx-6 specifically in IECs using Pges-1 (Fig. 3c,d and Supplementary Fig. 5a,b). Similarly, gon-2(q362) mutants exhibited reduced Ca2+ activity in motor neurons, which was rescued by IEC-specific gon-2 expression. Moreover, the gon-2(q362) mutation did not further reduce Ca2+ activity in nhx-6(ok609) mutants (Supplementary Fig. 5c-f), consistent with GON-2 and NHX-6 functioning in the same pathway. These results suggest that intestinal pathogens enhance the spontaneous activity of cholinergic motor neurons through a GON-2-NHX-6 signaling axis in IECs.

We next tested whether this enhanced motor neuron activity in response to pathogens requires ASIC-1. As expected, the pathogen-induced effects were significantly reduced in asic-1(ok415) mutants, and rescued by expressing wild-type asic-1 in VA and VB using Pdel-1 (Fig. 3c,d and Supplementary Fig. 5a,b), supporting the conclusion that ASIC-1 functions in VA/VB and activates DA/DB non-autonomously. Furthermore, asic-1;nhx-6 double mutants exhibited decreased GCaMP6 signals comparable to either single mutant (Fig. 3c,d and Supplementary Fig. 5a,b). These results suggest that intestinal pathogens regulate motor neuron activity though NHX-6-H+-ASIC-1 signaling.

Supporting this signaling axis, NHX-6 in IECs and ASIC-1 in VA/VB are anatomically adjacent (Fig. 3e), positioning VA/VB to directly sense protons released from IECs via paracrine signaling. NHX-6 was expressed throughout the intestine and localized to both the basolateral and apical membranes of IECs, while ASIC-1 was localized to VA/VB somas and projected along their commissures (Fig. 3e), consistent with previous reports39,52. This spatial organization provides a structural basis for pathogen-induced motor neuron activation via NHX-6-H+-ASIC-1 signaling.

To determine whether ASIC-1 functions as an acid-sensor, rather than a mechanoreceptor, in activating motor neurons during pathogen infections, we eliminated body bending-induced mechanical stimuli by fully paralyzing worms express HisCl1 in body-wall muscles using 50 mM histamine39. To ensure sufficient bacterial ingestion despite immobility, worms were raised in liquid cultures. Under these conditions, S. aureus was still able to activate motor neurons in wild type, as evidenced by increased basal GCaMP6 fluorescence and spontaneous Ca2+ amplitude, both of which were substantially diminished in asic-1(ok415) mutants (Supplementary Fig. 6a,b). In contrast, deletion of del-1, which encodes another mechanoreceptor in VA and VB39, did not affect S. aureus-induced motor neuron activation (Supplementary Fig. 6a,b). As controls, neither histamine treatment alone nor HisCl1 expression altered motor neuron activity (Supplementary Fig. 6c,d). These results support ASIC-1’s role as an acid-sensor that activates motor neurons in response to pathogen infections.

NHX-6-H+-ASIC-1 signaling promotes pathogen avoidance
During pathogen infections, C. elegans actively evades infectious bacteria to minimize exposure and enhance survival19,20,24,25. A- and B-type motor neurons are critical for generating the forward and backward locomotion underlying this behavior, with DA and DB innervating dorsal muscles and VA and VB innervating ventral muscles63,64. Given the pathogen-induced activation of motor neurons, we wondered whether NHX-6-H+-ASIC-1 signaling could promote pathogen avoidance by enhancing locomotion. To test this, we conducted a series of behavioral assays.

First, we quantified the locomotion speeds of wild-type worms fed different bacteria. Compared to E. coli, both S. aureus and P. aeruginosa significantly increased average speed, forward speed, and backward speed (Fig. 4a). In contrast, this pathogen-induced increase in locomotion speed was abolished in asic-1(ok415), nhx-6(ok609), and asic-1;nhx-6 mutants, in which worms raised on pathogens exhibited locomotion speeds indistinguishable from those raised on E. coli (Fig. 4b and Supplementary Fig. 7a). Expressing wild-type asic-1 in VA/VB or nhx-6 in IECs restored pathogen-induced speed increases in the corresponding mutants (Fig. 4b and Supplementary Fig. 7a).

Second, we assessed pathogen avoidance behavior on partial lawns of bacteria. While none of wild type, asic-1(ok415), nhx-6(ok609), or asic-1;nhx-6 mutants left the E. coli lawn within 24 hours (Supplementary Fig. 7b), they exhibited avoidance of S. aureus and P. aeruginosa shortly after ingestion (Fig. 4c and Supplementary Fig. 7c). Notably, both asic-1(ok415) and nhx-6(ok609) mutants showed significantly reduced pathogen avoidance compared to wild type, which was rescued by expressing the corresponding wild-type genes in VA/VB and IECs, respectively (Fig. 4c and Supplementary Fig. 7c). asic-1;nhx-6 double mutants again resembled either single mutant (Fig. 4d and Supplementary Fig. 7d). Similarly, both gon-2(q362) mutants and worms with IEC-specific cmd-1 RNAi displayed reduced avoidance, which was rescued in gon-2(q362) mutants by IEC-specific gon-2 expression (Fig. 4e,f and Supplementary Fig. 7e,f). Notably, the asic-1(ok415) mutation did not further exacerbate the avoidance defects of gon-2(q362) mutants or cmd-1 RNAi worms (Fig. 4e,f and Supplementary Fig. 7e,f), consistent with the role of GON-2 and CMD-1 in activating NHX-6-H+-ASIC-1 signaling.

Third, we tested whether exogenous H+ could bypass the requirement for NHX-6. H+ was supplemented to nematode growth medium (NGM) plates by adjusting pH to a range of 7 to 4. In nhx-6(ok609) mutants, acidic pH enhanced pathogen avoidance, reaching wild-type levels at pH 4 (Supplementary Fig. 7g), indicating that extracellular H+ is sufficient to trigger avoidance in the absence of NHX-6. In contrast, asic-1(ok415) mutants failed to respond to pH 4 (Fig. 4g), demonstrating the necessity of ASIC-1 for H+ function. Although altering the growth medium pH is an indirect manipulation, these findings are consistent with ASIC-1-dependent proton sensing.

Finally, we expressed the mammalian capsaicin-gated cation channel TRPV165 in VA/VB of asic-1(ok415) mutants and exposed the animals to S. aureus in the presence of varying concentrations of capsaicin. Capsaicin had no effect on avoidance behavior in wild type or in asic-1 mutants, and expression of TRPV1 in VA/VB in the absence of capsaicin did not alter avoidance. In contrast, capsaicin significantly increased pathogen avoidance in the transgenic mutants in a concentration-dependent manner (Supplementary Fig. 7h), suggesting that direct activation of VA/VB is sufficient to promote avoidance behavior in the absence of NHX-6-H+-ASIC-1 signaling.

Together, these results demonstrate that NHX-6-H+-ASIC-1 signaling between the intestine and motor neurons is both necessary and sufficient for efficient pathogen avoidance during intestinal infections.

NHX-6-H+-ASIC-1 signaling enhances intestinal innate immunity via cholinergic activation of the mAChR GAR-2;GAR-3 in IECs
Innate immune responses in C. elegans IECs trigger antimicrobial production to combat ingested pathogens18,21,22. Previous studies have shown that neuronally released ACh enhances host resistance to S. aureus and P. aeruginosa, but the neuronal source of this cholinergic signal remains unknown21,22. This prompted us to investigate whether NHX-6-H+-ASIC-1 signaling promotes intestinal immunity by activating cholinergic VA/VB motor neurons.

To eliminate confounding effects of avoidance behavior, we performed full-lawn survival assays32. Both asic-1(ok415) and nhx-6(ok609) mutants exhibited significantly reduced survival during S. aureus or P. aeruginosa infection, and asic-1;nhx-6 double mutants phenocopied the single mutants (Fig. 5a and Supplementary Fig. 8a). These defects were fully rescued by expressing wild-type nhx-6 in IECs and asic-1 in VA/VB, but not by expressing asic-1 in dopaminergic neurons using Pdat-139, where ASIC-1 is also expressed37,39 (Fig. 5a and Supplementary Fig. 8a,b), supporting a gut-to-motor neuron axis. Furthermore, nhx-6(ok609), but not asic-1(ok415) mutants, exhibited improved survival on pH 4 NGM plates during infections (Fig. 5b and Supplementary Fig. 8c), confirming a pH-dependent activation of ASIC-1 downstream of NHX-6. These results suggest that NHX-6-H+-ASIC-1 signaling across IECs and VA/VB promotes host defense.

Consistent with previous findings21,22, deletion of cwn-2 (Wnt ligand) or pmk-1 (p38 MAPK) reduced survival during pathogen infections (Fig. 5c and Supplementary Fig. 8d). These defects were greatly rescued by IEC-specific expression and recapitulated by IEC-specific RNAi (Fig. 5c and Supplementary Fig. 8d). asic-1;cwn-2 and asic-1;pmk-1 double mutants resembled cwn-2(ok895) and pmk-1(km25) single mutants, respectively (Fig. 5c and Supplementary Fig. 8d). During S. aureus infection, both asic-1(ok415) and nhx-6(ok609) mutants showed reduced mRNA levels of cwn-2 and five Wnt-dependent immune genes (ilys-2, ilys-3, lys-5, cpr-2, and clec-60)21, as well as decreased expression of the Pclec-60::GFP reporter21 (Fig. 5d,e). Similarly, during P. aeruginosa infection, both mutants exhibited reduced PMK-1 phosphorylation, lower mRNA levels of six PMK-1-dependent immune genes (lys-8, K08D8.5, irg-4, sysm-1, F55G11.4, and irg-5)22, and weaker Pirg-5::GFP reporter28 expression (Supplementary Fig. 8e-g). The defective immune reporter expression was rescued by expressing wild-type nhx-6 in IECs and asic-1 in VA/VB, respectively (Fig. 5e and Supplementary Fig. 8f). asic-1;nhx-6 double mutants phenocopied the single mutants (Fig. 5d,e and Supplementary Fig. 8e-g). Notably, the immune defects of nhx-6(ok609), but not asic-1(ok415), were rescued by pH 4 (Fig. 5f,g and Supplementary Fig. 8h-j), further supporting the role of proton-mediated ASIC-1 activation in host defense. These results suggest that NHX-6-H+-ASIC-1 signaling enhances intestinal immunity through the Wnt and PMK-1/p38 MAPK pathways.

Notably, asic-1(ok415), nhx-6(ok609), and asic-1;nhx-6 double mutants showed normal pharyngeal pumping rates, defecation cycle durations, and intestinal fluorescence of ingested E. coli OP50-GFP in 24 hours (Supplementary Fig. 9a-c). However, these mutants accumulated significantly more S. aureus in the intestine, as indicated by increased colony-forming units (CFUs) at 16 and 24 hours (Supplementary Fig. 9d). These results suggest that while the mutants can normally ingest, process, and expel bacteria, they are defective in eliminating ingested pathogens, reinforcing the immune-promoting role of NHX-6-H+-ASIC-1 signaling.

In cholinergic neurons, ACh is synthesized by the choline acetyltransferase CHA-1 and loaded into synaptic vesicles by the transporter UNC-1766. We found that VA/VB-targeted cha-1 RNAi worms and unc-17(e245) mutants both showed reduced survival during pathogen infections (Fig. 5h and Supplementary Fig. 10a). The defect of unc-17(e245) mutants was greatly rescued by expressing wild-type unc-17 in VA/VB and phenocopied by VA/VB-targeted unc-17 RNAi (Fig. 5h and Supplementary Fig. 10a). Importantly, asic-1(ok415) did not further reduce survival in VA/VB-targeted unc-17 RNAi worms (Fig. 5h and Supplementary Fig. 10a). VA/VB-targeted unc-17 RNAi did not further decrease survival in cwn-2(ok895) or pmk-1(km25) mutants (Fig. 5h and Supplementary Fig. 10a). During pathogen infections, unc-17(e245) mutants exhibited reduced mRNA levels of cwn-2, Wnt- and PMK-1-dependent immune genes, and decreased PMK-1 phosphorylation (Fig. 5i and Supplementary Fig. 10b,c). VA/VB-targeted unc-17 RNAi did not further suppress immune reporter expression in asic-1(ok415) mutants (Fig. 5j and Supplementary Fig. 10d). These results suggest that ACh released from VA/VB acts upstream of Wnt and PMK-1/p38 MAPK signaling to mediate the immune-promoting effects of NHX-6-H+-ASIC-1 signaling.

It was previously demonstrated that ACh induced by S. aureus infection activates the mAChR GAR-2;GAR-3 in IECs, triggering canonical Wnt signaling and antimicrobial gene expression21. To determine whether P. aeruginosa-induced ACh release also signals through GAR-2;GAR-3 to activate PMK-1, we analyzed gar-2(ok520);gar-3(gk305) double mutants. These mutants exhibited decreased survival during both S. aureus and P. aeruginosa infections, which was rescued by IEC-specific expression of wild-type genes and recapitulated by IEC-specific RNAi (Supplementary Fig. 10e,f). In contrast, deletion of gar-1, encoding another mAChR, did not affect survival during pathogen infections (Supplementary Fig. 10g). During P. aeruginosa infection, gar-2;gar-3;pmk-1 triple mutants showed similarly reduced survival to pmk-1(km25) single mutants (Supplementary Fig. 10h). gar-2;gar-3 mutants also exhibited reduced PMK-1 phosphorylation, lower mRNA levels of PMK-1-dependent immune genes, and diminished immune reporter expression (Supplementary Fig. 10i-k). Furthermore, VA/VB-targeted unc-17 RNAi did not further reduce immune reporter expression and survival in gar-2;gar-3 mutants (Supplementary Fig. 10k,l). Pharmacological treatment with oxotremorine, a muscarinic agonist21, significantly increased PMK-1 phosphorylation and improved survival in unc-17(e245) mutants during P. aeruginosa infection (Supplementary Fig. 10m,n). These results suggest that GAR-2;GAR-3 acts downstream of ACh released from cholinergic motor neurons to activate both the Wnt and PMK-1/p38 MAPK pathways in IECs during pathogen infections.

Finally, asic-1;gar-2;gar-3 triple mutants exhibited reduced survival and immune reporter expression similar to gar-2;gar-3 double mutants during pathogen infections (Fig. 5k,l and Supplementary Fig. 10o,p). Together, these results suggest that NHX-6-H+-ASIC-1 signaling enhances intestinal immunity by promoting cholinergic activation of GAR-2;GAR-3 in IECs.

Mouse ASIC1a and NHE1 can functionally substitute for their counterparts in C. elegans
C. elegans ASIC-1 shares extensive sequence and overall structure similarities with mammalian ASIC1a, which forms proton-gated homomeric channels expressed in peripheral sensory neurons, including vagal afferents38,49. Among mammalian NHEs, NHE1 is known to localize to the basolateral membrane of IECs67.

To test whether the NHX-6-ASIC-1 signaling axis is functionally conserved, we ectopically expressed mouse ASIC1a (mASIC1a) in VA/VB and mouse NHE1 (mNHE1) in IECs of C. elegans, respectively. In asic-1(ok415) mutants, pH 4-evoked calcium responses in A- and B-type motor neurons were rescued by mASIC1a expression (Fig. 6a and Supplementary Fig. 11a). In nhx-6(ok609) mutants during pathogen infections, expression of mNHE1 normalized pHluorin fluorescence in motor neurons to wild-type levels (Fig. 6b and Supplementary Fig. 11b). mStrawberry fluorescence was unaffected across these rescue conditions (Supplementary Fig. 11c). Additionally, spontaneous motor neuron activity was rescued by expressing mASIC1a in asic-1(ok415) or mNHE1 in nhx-6(ok609) (Fig. 6c,d and Supplementary Fig. 11d,e). Correspondingly, mutants expressing the respective mouse genes displayed wild-type levels of pathogen avoidance and survival against pathogens (Fig. 6e,f and Supplementary Fig. 11f,g). Together, these results demonstrate that mouse ASIC1a and NHE1 can functionally substitute for C. elegans ASIC-1 and NHX-6 in vivo, supporting the potential evolutionary conservation of this epithelial-neural signaling pathway.

Methods
Bacterial strains and culture
Bacterial strains E. coli OP50, S. aureus NCTC8325, and P. aeruginosa PA14 were cultured under conditions optimized for their growth and virulence90. E. coli was cultured in LB medium. P. aeruginosa and S. aureus were cultured in LB and tryptic soy broth (Hopebio) containing 10 μg/ml nalidixic acid (Aladdin), respectively, with gentle shaking. All bacteria were grown overnight at 37 °C and subsequently seeded onto 3.5-cm agar plates. Specifically, E. coli was seeded on NGM plates and incubated at 37 °C for 20 h. P. aeruginosa was seeded on modified NGM plates containing 0.35% peptone (Aobox) and 2 mg/ml tryptophan (Aladdin), followed by incubation at 37 °C for 24 hours and 25 °C for an additional 6 h. A 1:5 dilution of S. aureus was seeded on tryptic soy agar (Hopebio) plates with 10 μg/ml nalidixic acid and incubated at 37 °C for 6-8 h. Before use, all seeded plates were equilibrated at 25 °C for 1-2 h.

C. elegans strains and culture
C. elegans strains were raised on NGM plates seeded with E. coli OP50 at 22 °C. For worm culture plates of defined pH, the pH of nematode growth medium (NGM) and tryptic soy agar was adjusted with HCl prior to solidification. Transgenic strains were generated by microinjecting plasmid DNA, and at least two independent transgenic lines were tested to confirm the results. Integrated transgenic strains were created using gamma irradiation and were outcrossed at least three times before use. The strains used in this study are listed in Supplementary Data 1. All experiments were performed using young adult hermaphrodites unless specified otherwise.

Mutant rescue and gene knockdown
Mutants were rescued by expressing wild-type cDNAs. Gene knockdown was achieved via RNAi by co-expressing two plasmids that encoded complementary sense and antisense mRNA fragments of the target gene. The promoters used for cell-specific rescue and RNAi included Punc-17Δ1 (cholinergic VNC motor neurons)39, Pdel-1 (VA and VB motor neurons)39, Pges-1 (IECs)54, and Pdat-1 (CEP, ADE, and PDE neurons)39. Intestinal-specific RNAi of aex-5 and nhx-2 was done by feeding C. elegans strain VP303 with E. coli HT115 carrying L4440 plasmids containing target gene fragments19. All C. elegans cDNAs and target mRNA fragments were cloned from a Bristol N2 cDNA library. Mouse Asic1a and Nhe1 cDNAs were amplified from a mouse kidney cDNA library. Primers (Tsingke Biotechnology Co., Ltd.) are listed in Supplementary Table 1.

Imaging
NHX-6::mStrawberry and ASIC-1::GFP translational fusion protein imaging was performed using an Olympus IXplore SpinSR confocal microscope with a 60×/1.42 NA oil immersion objective. All other fluorescence images were acquired using a Nikon Ts2R inverted microscope equipped with an Mshot MD60 CCD camera and the MShot Image Analysis System (version 1.1). Acquisition settings were kept constant throughout each experiment. For calcium and pHluorin imaging, young adult hermaphrodites were immobilized on coverslips using Vetbond Tissue Adhesive (3M Company). Worms were bathed in a solution containing 140 mM NaCl, 5 mM KCl, 5 mM CaCl2, 5 mM MgCl2, 11 mM dextrose, and 5 mM HEPES (pH 7.2, 320 mOsm) and imaged at 1 frame per second for 2 min. For infection assays, young adult worms were cultivated on S. aureus for 4-6 hours or P. aeruginosa for 8-12 hours prior to imaging. For acid-evoked calcium imaging, because VNC motor neurons were inaccessible to bath-applied acid, a longitudinal incision was made along the glued region to expose neurons in the anterior part of the animal. Acidic solutions were prepared by adjusting the pH with HCl, and the osmolarity was adjusted to 320 mOsm. Acidic solutions were applied to the VNC via pressure ejection (2 psi for 5 s) at a 90° angle using an Eppendorf FemtoJet 4i microinjector.

Whole-cell current recording
VA5 and VB6 motor neuron were identified based on their anatomical locations. For whole-cell current recordings91, borosilicate glass pipettes (tip resistance ~20 MΩ) were used as electrodes. Classical whole-cell configuration was obtained by applying negative pressure to the recording pipette. Recordings were performed on a Nikon FN1 microscope with a MultiClamp 700B amplifier (Molecular Devices) using Clampex 10 (Molecular Devices). A series of voltage steps (-60 to +70 mV at 10-mV intervals and 1.2-s duration) was applied from a holding voltage of -60 mV. Data were sampled at a rate of 10 kHz after filtering at 2 kHz.

Bacterial lawn avoidance assay
For avoidance assays29, a 10 μl drop of overnight bacterial cultures was placed at the center of a 3.5-cm agar plate to make partial-lawn plates. Twenty L4 worms grown on E. coli OP50 were transferred to the center of each bacterial lawn and incubated at 25 °C. The number of worms on the bacterial lawns (Non) was counted at designated time points after exposure. Percent avoidance = (Ntotal-Non)/Ntotal x 100%. All experiments were independently repeated three times.

Survival assay
For survival assays90, 10 µl of overnight bacterial culture was spread to cover the entire surface of a 3.5-cm NGM plate to make full-lawn plates. To prevent progeny production and bagging, 5-Fluoro-2’-deoxyuridine (FUdR, 50 μg/ml; Aladdin) was included. Synchronized L4 worms (100 per plate) were transferred onto the plates and cultivated at 25 °C. Worm viability was assessed every 12 hours, and those unresponsive to nose and tail touch were scored as dead. Worms that died due to bursting vulva or crawled off the plate were censored from analysis. Live worms were transferred to fresh plates daily. Percent survival = (Nlive/Ntotal) x 100%. For oxotremorine treatment, 1 mM oxotremorine (Aladdin) was added to NGM plates. All experiments were independently repeated three times.

Behavioral analysis
All behavioral assays were performed at 22 °C. Locomotion was recorded and analyzed using an automated Track-A-Worm system (version 2.0)92. A single young adult worm was transferred to the center of a 6-cm NGM plate seeded with a thin layer of designated bacteria. After 30 s for recovery, the worm was imaged for 1 min at 15 frames per second. Pharyngeal pumping, the rhythmic contraction and relaxation of the pharynx that drives bacterial ingestion, was manually quantified under a Nikon SMZ800N stereomicroscope. Pumping rate was calculated as the number of contractions of the pharyngeal terminal bulb in 60 s93, serving as a quantitative measure of feeding activity. Counting was performed in triplicate and averaged for each worm. Defecation cycles were observed under the same stereomicroscope. The duration between contractions of the posterior body muscles was recorded 10 times and averaged for each worm20.

qRT-PCR analysis
Worms were collected in M9 buffer and lysed using TRIzol (TransGen Biotech, ET111-01). Total RNA was extracted by chloroform separation followed by isopropanol precipitation, washed with 75% ethanol, and resuspended in RNase-free water. cDNA was then synthesized using the HiScript II Q RT SuperMix kit (Vazyme, R233-01). qRT-PCR was conducted using SYBR qPCR Master Mix (Biosharp, BL697A) on a CFX Connect Real-Time PCR Detection System (Bio-Rad). mRNA levels were normalized using β-actin mRNA, and relative fold changes were calculated using the ΔΔCt method. All experiments were independently repeated three times. Primers (Tsingke Biotechnology Co., Ltd.) are listed in Supplementary Table 2.

Western blot
Day 1 adult worms were collected in M9 buffer and lysed by sonication in RIPA buffer (Beyotime, P0013C) supplemented with 1 mM PMSF (Biosharp, BL507A) and a protease/phosphatase inhibitor cocktail (NCM Biotech, P002). Protein levels of phosphorylated PMK-1, total PMK-1, and β-actin were analyzed by standard western blotting using rabbit anti-phospho-p38 MAPK monoclonal antibody (1:1000; ABclonal, AP0526), rabbit anti-p38 MAPK monoclonal antibody (1:1000; Abcam, ab170099), and mouse anti-β-actin monoclonal antibody (1:5000; ABclonal, AC004). Immunoblots were visualized using the ChemiScope 6000 system (Clinx), and band intensities were quantified using ImageJ (FIJI, version 1.50i). All experiments were independently repeated three times.

Quantification of intestinal bacteria accumulation
To visualize and quantify intestinal accumulation of bacteria, full-lawn plates were prepared by spreading an overnight culture of OP50-GFP94. Synchronized L4 worms grown on E. coli OP50 were transferred to the plates and incubated at 25 °C for the indicated time. After washed with M9 buffer to remove any external bacteria, worms were imaged using the Nikon Ts2R inverted microscope.

Intestinal accumulation of S. aureus was quantified using colony-forming unit (CFU) assays22. L4 worms grown on E. coli OP50 were transferred to full-lawn S. aureus plates and incubated at 25 °C for the designated time. Fifty worms were picked into M9 containing 0.1 M sodium azide to be paralyzed. After washing three times with M9 containing 1 mg/ml ampicillin (Biosharp) and 1 mg/ml gentamicin (Biosharp), worms were incubated for 30 min in antibiotic M9 to remove any external bacteria. Worms were then lysed with a motorized pestle, serially diluted, and spread on TSA plates containing 10 μg/ml nalidixic acid. The plates were incubated overnight at 37 °C to determine CFUs.

Data analysis
Calcium and pHluorin imaging data were analyzed using ImageJ, with individual cells assigned as separate regions of interest (ROIs). For VNC cholinergic motor neuron imaging, all neurons in the visual field were analyzed. For IEC imaging, the int2 cells were selected. For calcium imaging, fluorescence intensity (F) in each ROI were plotted as absolute intensity over time and then converted to a bleach-corrected ΔF/F0 values using a custom MATLAB module39. Spontaneous calcium transients were defined as events with ΔF/F0 ≥ 0.05 lasting ≥ 3 s in neurons and ΔF/F0 ≥ 0.1 lasting ≥ 10 s in IECs, and inter-peak intervals were calculated from recordings containing at least two detectable events. For pHluorin imaging, the co-expressed, pH-insensitive mStrawberry signal was used as an internal expression control to assess extracellular pH changes independently of potential differences in reporter expression.

Whole-cell current data were analyzed using Clampfit 10 (Molecular Devices). Current amplitudes were calculated from the mean current during the last 100 ms of each 1.2-s voltage pulse.

Statistical analyses and data graphing were performed using Origin 2019 (OriginLab), Prism 8 (GraphPad), or IBM SPSS Statistics 21 (IBM). One-way ANOVA with Tukey’s post hoc test was used for multiple group comparisons, two-sided unpaired t test for two group comparisons, and log-rank (Kaplan-Meier) test for survival analyses. Behavioral avoidance and whole-cell current data were analyzed using two-way repeated-measures ANOVA. When a significant main effect or interaction was detected in the omnibus model, post hoc pairwise comparisons were performed using Sidak’s multiple-comparisons correction. The figure legends provide detailed statistical information, including statistical methods, n numbers, and p values. Statistical significance was set at p < 0.05. No data were excluded from analyses, and all data were shown as mean ± SEM.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this comprehensive and well-written manuscript, Lei et al provide convincing evidence for an important mechanism of pathogen detection by the intestinal epithelium in C. elegans. Prior work had shown that a cholinergic signal produced from the nervous system during infection triggered Wnt signaling in the intestinal epithelium, ultimately driving the expression of antimicrobial genes that promote survival of infection. However, the upstream signal that triggers the acetylcholine signal from neurons was unknown. The present work provides a plausible mechanism for at least part of the story.\nThe overall model proposed here is that intestinal distention and/or membrane disruption of the IECs produces a mechanical stimulus that drives GON-2-mediated Ca2+ influx. This influx activates NHX-6 via CMD-1 to export H+ through the basolateral side, acidifying the area surrounding the epithelium. Cholinergic motor neurons that surround the epithelium in close proximity use ASIC-1 to sense the pH in this space. Upon acidification, ASIC-1 triggers Ca2+ transients in these neurons, resulting in acetylcholine release, which through previously identified muscarinic receptors triggers both Wnt signaling and the p38 MAPK pathway in the intestinal epithelium.\nOverall, the data appear compelling and the interpretation is mostly warranted. However, a few concerns should be addressed:\nMAJOR\n• The proposed model hinges on mechanosensation, but this is not proven or likely. Heat-killed Saureus triggers a similar transcriptional response of live S. aureus (Irazoqui PNAS 2008), but the authors show that HK S. aureus does not trigger the GON-2 pathway. Pseudomonas does not induce gut distention until later in the infection (Aballay, Kim, others), which places that at odds with the kinetics observed here. Furthermore, the authors do not use latex beads, which distend the gut but are inert, to test this hypothesis. At least, the proposal for mechanosensation as THE trigger (or with membrane damage, see below) are merely speculative and should be couched with much more caution. Experiments to validate one or the other are preferred.\n• No description of microfluidic device experiments for neuron GCAMP measurements. Without this description, it is not possible to interpret the signals from the different microbial triggers.\n• No description of how IEC GCAMP was acquired. Similar to above.\n• No description of pH control/rescue method. The lack of detail of how the pH was controlled and how the worms were exposed precludes any interpretation.\n• They posit that membrane damage triggers GON-2, but if the membrane is damaged, cation chemical gradients with the extracellular space would be expected to dissipate. How do they reconcile? This is especially important for H+, which diffuse rapidly.\n• Does the entire pseudocoelom acidify under conditions of infection?\nMINOR\n• Fig. 3b – the data for VB6 don’t follow the color convention?\n• Wrong statistics for Avoidance vs Time assays (Two-Way Repeated Measures ANOVA. If they wish to show differences at specific times (e.g., 24h), they should perform post-hoc pairwise comparisons (e.g., Bonferroni or Sidak corrected) only after finding a significant main effect or interaction in the omnibus model.)\n• Discuss the benefit of having this IEC   neuron   IEC loop instead of cell-autonomous IEC signaling alone. Is it just for behavior?\n• More discussion that the effects are partial – other missing parts/inputs?\n• The transcriptional responses to S.aureus and P. aeruginosa are very different, yet both trigger this pathway. Discuss how specificity may be achieved.", "reviewer_2": "Host organisms must combat pathogens to ensure individual survival and fitness. Consequently, they have evolved a suite of defense strategies to systemically maintain physiological homeostasis and protect vital organ functions—a process fundamental to biological integrity and species stability. While the nervous system is known to play a critical role in coordinating host responses to infection, the underlying mechanisms remain poorly understood. In this study, the authors employ C. elegans as a model to elucidate a novel signaling pathway mediated by protons and acetylcholine that integrates behavioral avoidance with immune defense against pathogens such as S. aureus and P. aeruginosa. The authors demonstrate that ingested pathogens trigger proton release from intestine cells, which subsequently activates VA/VB motor neurons via the acid-sensing ion channel ASIC-1. This neuronal activation not only drives avoidance behavior but also initiates a feedback signal that potentiates intestinal immune-defense by activating the Wnt and p38/PMK-1 MAPK pathways. The study is supported by extensive data generated through multiple complementary techniques and the manuscript is well-organized, all of which make their findings robust and compelling.\nMajor:\n1) What is the source of protons released from intestinal cells during infection? Specifically, does the release originate from lysosomal compartments, or is it generated through specific biochemical reactions triggered by ingested pathogens?\n2) How does pathogen alter the calcium influx mediated by GON-2? Is the observed calcium influx increase a direct consequence of pathogen-induced mechanical stress? Is GON-2 mechanosensitive?\n3) How do GON-2 and CMD-1 specifically regulate proton release from intestine cells?\n4) What is the specific motor program executed by VA and AB neurons to drive pathogen avoidance, and is this circuit modulated by other neurons or neuroendocrine signals?\n5) Does intestinal GON-2 mediate the pathogen-evoked shifts in basal calcium levels (measured by GCaMP6) in VA/VB sensory neurons?\nMinor:\n1) Is there any possibility that Calcium/Calmodulin might regulate PMK-1/P38 MAPK pathway cell-autonomously?\n2) Is there any possibility that NHX-6 — H+ — ASIC-1 signaling pathway would regulate immune-defense temporally?", "reviewer_3": "The authors identify a role for a proton-mediated gut-to-neuron signaling pathway in the integration of behavioural and immune defenses against pathogen infection. They use an elegant combination of experimental approaches to provide substantial physiological and mechanistic insight into the components of this response and where and how they contribute. Importantly, they also show that the mammalian counterparts of the two key components of the pathway, an Na+/H+ exchanger and an acid sensing ion channel, can substitute for the C. elegans genes, underlining the high level of conservation and the relevance of a nematode model for studying gut-nervous system interactions. This is an important and topical area of research and of general interest. The research is of high quality, using innovative experimental approaches, well-founded on a thorough consideration of the current state of the field. It would thus be highly suitable for publication in this journal.\nHowever, I have a number of serious concerns with missing controls, missing statistical comparisons and inappropriate analysis of the data, with the result that the conclusions are not always supported by the data. This must be addressed before it is suitable for publication. In most cases, this would be rectified by small changes to the way in which the current data is presented or analyzed, or by ensuring that their statements take account of the caveats.\nI therefore strongly recommend publication, subject to revision to address these points.\nMajor comments\nThe authors use a pHluorin-mStrawberry fusion, quantifying fluorescence as a readout for pH in the extracellular environment of the motor neurons. The data appear to show pHluorin fluorescence, without, for example, use of the mStrawberry fluorescence as a control. The possibility thus remains that the observed change results from a change in expression levels of the pH indicator. Indeed, the authors use an unc-17 promoter to drive its expression, but later show that unc-17 is itself implicated in this pathway.\nFor the intestinal calcium imaging experiments (fig. 2d, e), the individual data points are discrete values, multiples of 0.5. This suggests that the data was analyzed by simply counting the number of peaks or full cycles within the 2-minute recording time. This presumably explains why the mean frequency implies a surprisingly short interval length (approx. 1.7 per minute for wt = 35 seconds!). The data would be more powerful, and the comparisons more meaningful, if the authors calculated the mean interval between peaks. The short recording interval, coupled with the small number of replicates, means that the number of data points is small, particularly given that we know from previous studies that gon-2 loss of function results in increased variance of interval length (Xing et al. 2008, Kwan et al. 2008).\nFigure 2e and related supplemental: We know from previous research that gon-2 loss of function results in an increase in cycle length, for both calcium and behavior (Xing et al. 2008). To convincingly say that gon-2 is required for the pathogen-induced change in calcium transient frequency and amplitude, animals grown on E. coli and pathogen must be compared directly. Given the role of gon-2 in contributing to the calcium events that play such a critical central role in both rhythmicity and overall function of the gut, care should be taken in determining whether the role identified here is specific to gon-2 or an overall contribution of intracellular calcium signaling.\nFigure 2c, f, g: To convincingly say that these genes are required for the pathogen-induced change in pHluorin fluorescence, animals grown on E. coli and pathogen must be compared directly.\nFor the motorneuron silencing experiments using a histamine-gated chloride channel (supp. fig. 5), a control for the effects of histamine alone (i.e. in the absence of His-Cl1 expression) is required.\nThe authors show that, compared with E. coli, exposure to the pathogenic bacteria resulted in an increase in locomotion speed. They also show that on the pathogens asic-1 and nhx-6 mutants show a lower speed than wild type. To assert that the pathogen effect on speed is disrupted in these mutants, they need to show evidence that the change is disrupted, i.e. directly compare E. coli-grown with pathogen-grown.\nAltering the growth medium pH is an appealing experiment and may support the conclusions about the role of ASIC-1. However, the observed effects could be due to any number of off-target effects unrelated to the extracellular environment of the motorneurons. Indeed, is not clear how a change in pH in the medium would be translated into a corresponding change in an internal compartment, particularly given the important roles proton signaling/pH oscillations in multiple locations. The conclusions should not be overstated.\nWhen using TRPV1 expression as a means to activate the motorneurons in an ASIC-1-independent manner (supp. Fig. 6h), the authors do not show any controls for the effects of capsaicin itself (i.e. whether in an asic-1 loss of function background capsaicin has analogous effects, to exclude the possibilities that the observed effect is due to a C. elegans TRPV channel, which could be acting elsewhere.\nMinor points\nLine 111/Figure 1: The authors investigate the role of innexins expressed in the motor neurons, using data from Altun et al. 2009. More recent evidence (e.g. Bhattacharya et al. 2019; RNA seq data in the CeNGEN project (Taylor et al. 2021)) indicates a larger number of innexin genes are expressed in these cells. This should, at the very least, be acknowledged in the text. The authors also present no evidence that the RNAi actually knocks down the target genes or that it is expressed in the desired cells. Again, in the absence of controls, this should at least be acknowledged.\nLine 145: “hours” does not seem to fit the sentence.\nFigure 2e: gtl-1 is incorrectly written as glt-1.\nFigure 2d, e: n values are given for some genotypes but not for all.\nFigure 4: misspelling of rescue.\nLine 349: “pumping rates” requires more explanation for a non-specialist reader. Likewise, “increased colony-forming units” (or simply “CFUs” in the figure legend) – a better explanation of the experiment would be helpful."}, "author_response": "Response to Reviewers’ Comments\nWe thank the reviewers for their careful evaluation of our manuscript and for their\nconstructive and insightful comments. We have addressed each point in detail,\nperformed additional experiments, and revised the manuscript accordingly to\nstrengthen the study. Below, we provide point-by-point responses. For clarity,\nreviewer comments are reproduced in italics, followed by our responses, with\nreference to the relevant text and figures in the revised manuscript. All changes have\nbeen incorporated into the revised manuscript and are highlighted in blue.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this comprehensive and well-written manuscript, Lei et al provide convincing\nevidence for an important mechanism of pathogen detection by the intestinal\nepithelium in C. elegans. Prior work had shown that a cholinergic signal produced\nfrom the nervous system during infection triggered Wnt signaling in the intestinal\nepithelium, ultimately driving the expression of antimicrobial genes that promote\nsurvival of infection. However, the upstream signal that triggers the acetylcholine\nsignal from neurons was unknown. The present work provides a plausible\nmechanism for at least part of the story.\nThe overall model proposed here is that intestinal distention and/or membrane\ndisruption of the IECs produces a mechanical stimulus that drives GON-2-mediated\nCa2+ influx. This influx activates NHX-6 via CMD-1 to export H+ through the\nbasolateral side, acidifying the area surrounding the epithelium. Cholinergic motor\nneurons that surround the epithelium in close proximity use ASIC-1 to sense the pH\nin this space. Upon acidification, ASIC-1 triggers Ca2+ transients in these neurons,\nresulting in acetylcholine release, which through previously identified muscarinic\nreceptors triggers both Wnt signaling and the p38 MAPK pathway in the intestinal\nepithelium.\nOverall, the data appear compelling and the interpretation is mostly warranted.\nHowever, a few concerns should be addressed:\nResponse: We thank the reviewer for the overall positive assessment of our work.\nMAJOR\n• The proposed model hinges on mechanosensation, but this is not proven or likely.\nHeat-killed Saureus triggers a similar transcriptional response of live S. aureus\n(Irazoqui PNAS 2008), but the authors show that HK S. aureus does not trigger the\nGON-2 pathway. Pseudomonas does not induce gut distention until later in the\ninfection (Aballay, Kim, others), which places that at odds with the kinetics observed\nhere. Furthermore, the authors do not use latex beads, which distend the gut but are\ninert, to test this hypothesis. At least, the proposal for mechanosensation as THE\ntrigger (or with membrane damage, see below) are merely speculative and should be\ncouched with much more caution. Experiments to validate one or the other are\npreferred.\nResponse: We thank the reviewer for this important critique and agree that\nmechanosensation should not be presented as a single or definitive trigger of\nintestinal epithelial activation. Rather, our data support that pathogen infections\nenhance GON-2-dependent and mechanically sensitive Ca2+ signaling in intestinal\nepithelial cells (IECs).\nOur conclusions regarding mechanical sensitivity are based on direct experimental\nevidence. In wild-type animals fed E. coli, suppressing body movement, either by\nphysical immobilization with glue or by pharmacological paralysis with levamisole,\nsignificantly reduced spontaneous IEC Ca2+ transients, phenocopying gon-2 mutants.\nImmobilization did not further suppress Ca2+ activity in gon-2 mutants, and IEC-\nspecific expression of gon-2 rescued the defects (Supplementary Fig. 3d),\nsuggesting that GON-2 is required for mechanically sensitive Ca2+ signaling\nassociated with body bending.\nDuring pathogen infections, infection-enhanced Ca2+ influx in IECs was similarly\nsuppressed by immobilization in wild type, and immobilization of infected gon-2\nmutants produced no additional effect (Fig. 2e and Supplementary Fig. 3c). Together,\nthese epistasis experiments suggest that pathogens potentiate an existing GON-2-\ndependent mechanosensitive signaling pathway.\nThe reviewer notes that heat-killed S. aureus induces transcriptional responses\nsimilar to live S. aureus (Irazoqui, J. E. et al., Plos Pathog, 2010), whereas we\nobserve that heat-killed S. aureus fails to induce GON-2-dependent Ca2+ signaling.\nThe transcriptional responses described by Irazoqui and colleagues are interpreted\nas PAMP-driven host responses. Our findings therefore suggest that live pathogens\nengage an additional layer of epithelial signaling, namely enhanced Ca2+ influx, likely\nreflecting infection-associated epithelial stress or damage rather than PAMP\nrecognition alone.\nWe agree that bulk intestinal distention alone is unlikely to explain our\nobservations. Consistently, genetically induced intestinal bloating in E. coli-fed\nanimals via aex-5 or nhx-2 RNAi did not increase IEC Ca2+ transients\n(Supplementary Fig. 3f). Although we did not use inert latex beads, these genetic\nmanipulations provide an independent means of uncoupling intestinal distention from\ninfection-induced Ca2+ signaling.\nTogether, our data support a model in which pathogen infections enhance GON-2-\ndependent mechanosensitive Ca2+ influx in IECs, likely reflecting infection-associated\nepithelial stress or membrane perturbation. This interpretation is consistent with\ncurrent models of mechanosensitive channel gating, in which channel activity can be\nmodulated by membrane tension, curvature, or lipid perturbations (Kefauver, J. M.,\nWard, A. B. & Patapoutian, A., Nature, 2020). However, we do not directly measure\nmembrane tension or other biophysical parameters in this study. Accordingly, we\nhave revised Fig. 7, its legend, and the Discussion (the 9th paragraph) to clarify that\ninfection-associated epithelial stress is proposed to engage GON-2 indirectly, rather\nthan invoking a defined or exclusive mechanical trigger.\n• No description of microfluidic device experiments for neuron GCAMP\nmeasurements. Without this description, it is not possible to interpret the signals from\nthe different microbial triggers.\nResponse: We thank the reviewer for raising this point. We did not use microfluidic\ndevices for neuronal GCaMP imaging in this study. Instead, calcium and pHluorin\nimaging were performed on immobilized animals under defined bath conditions.\nTo clarify this, we have expanded the Methods (“Imaging”) to explicitly describe the\nimaging setup, immobilization procedure, bath composition, acquisition parameters,\nand infection conditions used for neuronal and intestinal imaging, as follows: “All\nother fluorescence images were acquired using a Nikon Ts2R inverted microscope\nequipped with an Mshot MD60 CCD camera and the MShot Image Analysis System\n(version 1.1). Acquisition settings were kept constant throughout each experiment.\nFor calcium and pHluorin imaging, young adult hermaphrodites were immobilized on\ncoverslips using Vetbond Tissue Adhesive (3M Company). Worms were bathed in a\nsolution containing 140 mM NaCl, 5 mM KCl, 5 mM CaCl , 5 mM MgCl , 11 mM\n2 2\ndextrose, and 5 mM HEPES (pH 7.2, 320 mOsm) and imaged at 1 frame per second\nfor 2 min. For infection assays, young adult worms were cultivated on S. aureus for\n4-6 hours or P. aeruginosa for 8-12 hours prior to imaging.”\nIn addition, we have clarified ROI selection and data analysis in the Methods\n(“Data analysis”) as follows: “Calcium and pHluorin imaging data were analyzed\nusing ImageJ, with individual cells assigned as separate regions of interest (ROIs).\nFor VNC cholinergic motor neuron imaging, all neurons in the visual field were\nanalyzed. For IEC imaging, the int2 cells were selected. For calcium imaging,\nfluorescence intensity (F) in each ROI were plotted as absolute intensity over time\nand then converted to a bleach-corrected ΔF/F0 values using a custom MATLAB\nmodule. Spontaneous calcium transients were defined as events with ΔF/F0 ≥ 0.05\nlasting ≥ 3 s in neurons and ΔF/F0 ≥ 0.1 lasting ≥ 10 s in IECs, and inter-peak\nintervals were calculated from recordings containing at least two detectable events.”\n• No description of how IEC GCAMP was acquired. Similar to above.\nResponse: We thank the reviewer for this comment. As noted above, we have\nexpanded the Methods (“Imaging” and “Data analysis”) to provide a detailed\ndescription of the calcium imaging protocol used for both neuronal and IEC GCaMP\nimaging.\n• No description of pH control/rescue method. The lack of detail of how the pH was\ncontrolled and how the worms were exposed precludes any interpretation.\nResponse: We thank the reviewer for this comment. To test motor neuron acid\nsensitivity, acidic solutions were applied directly to ventral nerve cord (VNC) motor\nneurons in dissected worms.\nThese procedures are now described in the revised Methods as follows “For\ncalcium and pHluorin imaging, young adult hermaphrodites were immobilized on\ncoverslips using Vetbond Tissue Adhesive (3M Company). Worms were bathed in a\nsolution containing 140 mM NaCl, 5 mM KCl, 5 mM CaCl , 5 mM MgCl , 11 mM\n2 2\ndextrose, and 5 mM HEPES (pH 7.2, 320 mOsm) and imaged at 1 frame per second\nfor 2 min.” and “For acid-evoked calcium imaging, because VNC motor neurons were\ninaccessible to bath-applied acid, a longitudinal incision was made along the glued\nregion to expose neurons in the anterior part of the animal. Acidic solutions were\nprepared by adjusting the pH with HCl, and the osmolarity was adjusted to 320\nmOsm. Acidic solutions were applied to the VNC via pressure ejection (2 psi for 5 s)\nat a 90° angle using an Eppendorf FemtoJet 4i microinjector.”\nTo examine the effects of exogenous H+ on pathogen avoidance and survival,\nnematode growth medium (NGM) or tryptic soy agar plates were acidified by HCl\nadjustment prior to solidification, generating plates with defined pH values (pH 4-7).\nThese procedures are now described in the revised Methods as follows “For worm\nculture plates of defined pH, the pH of nematode growth medium (NGM) and tryptic\nsoy agar was adjusted with HCl prior to solidification.”\n• They posit that membrane damage triggers GON-2, but if the membrane is\ndamaged, cation chemical gradients with the extracellular space would be expected\nto dissipate. How do they reconcile? This is especially important for H+, which\ndiffuse rapidly.\nResponse: We thank the reviewer for this thoughtful critique and agree that\nwidespread or sustained membrane rupture would be expected to dissipate ionic\ngradients, particularly for highly diffusible ions such as H+. In our model, however, by\n“membrane perturbation” or “epithelial stress”, we refer to localized or sublethal\nalterations in membrane tension or integrity that can arise during pathogen infections,\nrather than gross membrane rupture.\nConsistently, our data show that pathogen infections enhance GON-2-dependent\nCa2+ influx in IECs without causing indiscriminate Ca2+ entry or baseline\ndepolarization under our experimental conditions, arguing against nonspecific\nmembrane leakage. We therefore propose that GON-2 activation occurs within a\nphysiological regime in which localized membrane perturbation or epithelial stress\nenhances mechanosensitive Ca2+ influx while overall ionic homeostasis is preserved,\nlikely supported by the rapid repair capacity of the intestinal epithelium. Such\nsignaling is likely to be spatially and temporally restricted, occurring prior to severe\nepithelial compromise.\nWe have revised the Discussion as follows: “This infection-associated\nenhancement likely reflects localized epithelial stress or membrane perturbation\narising during infections, operating within a spatially and temporally restricted\nphysiological regime that precedes severe epithelial compromise.”\n• Does the entire pseudocoelom acidify under conditions of infection?\nResponse: We thank the reviewer for this important question. In our initial\nexperiments, we recorded pHluorin signals from VNC motor neurons in the mid-\nanterior region of the animal, due to the limited field of view required for high-\nresolution imaging. To address whether infections induce widespread\npseudocoelomic acidification, we extended our analysis to 6 additional VNC motor\nneurons located more distally, including anterior (DB3, DA2, VA3, VB4) and posterior\n(VA10, VB11) neurons.\nAlthough these experiments do not directly determine whether the entire\npseudocoelom becomes acidified, they reveal extracellular acidification at\nwidespread VNC locations during pathogen infections (new Supplementary Fig. 3a).\nWe have incorporated these new findings into the revised Results as follows:\n“Although these experiments do not directly determine whether pathogen infections\ncause global pseudocoelomic acidification, they demonstrate that the extracellular\nspace surrounding motor neurons at widespread VNC sites becomes acidified during\npathogen infections.”\nMINOR\n• Fig. 3b – the data for VB6 don’t follow the color convention?\nResponse: Corrected, thanks.\n• Wrong statistics for Avoidance vs Time assays (Two-Way Repeated Measures\nANOVA. If they wish to show differences at specific times (e.g., 24h), they should\nperform post-hoc pairwise comparisons (e.g., Bonferroni or Sidak corrected) only\nafter finding a significant main effect or interaction in the omnibus model.)\nResponse: We thank the reviewer for this clarification. We have reanalyzed the\navoidance data using two-way repeated-measures ANOVA. When a significant main\neffect or interaction was detected in the omnibus model, post hoc pairwise\ncomparisons were performed using Sidak’s multiple-comparisons correction. The\nupdated statistical analyses are now reported in revised Fig. 4c-g, Fig. 6e,\nSupplementary Fig. 7b-h, and Supplementary Fig. 11f. The corresponding figure\nlegends and Methods have been updated accordingly.\n• Discuss the benefit of having this IEC  neuron  IEC loop instead of cell-\nautonomous IEC signaling alone. Is it just for behavior?\nResponse: We thank the reviewer for this important suggestion. We propose that the\nIEC-neuron-IEC loop provides advantages beyond cell-autonomous IEC signaling by\nenabling rapid coordination of behavioral and immune responses. By engaging the\nnervous system, intestinal epithelial sensing can simultaneously promote pathogen\navoidance behavior, thereby limiting pathogen ingestion, while enhancing intestinal\ndefense. In addition, this circuit architecture likely enables signal amplification,\nflexibility, and integration with internal state, features that would be difficult to achieve\nthrough IEC-intrinsic signaling alone. We have incorporated this discussion into the\nrevised Discussion as follows: “By routing epithelial sensing signals through the\nnervous system, the IEC-neuron-IEC loop enables rapid coupling of behavioral\navoidance with intestinal immunity, while affording amplification, flexibility, and\nintegration with internal state that cannot be achieved by cell-autonomous IEC\nsignaling alone.”\n• More discussion that the effects are partial – other missing parts/inputs?\nResponse: We thank the reviewer for this insightful comment and agree that the\nNHX-6-H+-ASIC-1 pathway represents a partial component of host defense and does\nnot act in isolation. Our data indicate that this pathway enhances, rather than solely\ntriggers, pathogen avoidance and intestinal innate immunity. Additional inputs are\nlikely to contribute, including cholinergic signaling from additional neurons, other\nneuronal pathways, established pathogen-sensing and immune signaling\nmechanisms, and parallel epithelial defense pathways. We have clarified in the\nrevised Discussion as follows: “These findings define a form of damage-associated\nimmune surveillance that links epithelial sensing to neural control of host defense,\nfunctioning alongside established pathogen-sensing pathways, other neuronal inputs,\nand parallel epithelial immune mechanisms.”\n• The transcriptional responses to S.aureus and P. aeruginosa are very different, yet\nboth trigger this pathway. Discuss how specificity may be achieved.\nResponse: We thank the reviewer for raising this insightful comment. Although S.\naureus and P. aeruginosa elicit substantially different transcriptional immune\nresponses, our data suggest that proton-mediated gut-to-neuron signaling reflects a\nshared physiological readout of intestinal epithelial stress rather than pathogen-\nspecific recognition. We propose that this pathway functions as a common, rapid\nalert signal that coordinates behavioral avoidance and immune responses, while\npathogen specificity is achieved through parallel signaling pathways that engage\ndistinct pattern- or damage-associated cues and drive pathogen-biased\ntranscriptional responses. We have incorporated these into the revised Discussion as\nfollows: “Although S. aureus and P. aeruginosa elicit divergent transcriptional\nresponses, proton-mediated gut-to-neuron signaling likely represents a shared\nphysiological readout of intestinal epithelial stress, while pathogen specificity is\nconferred by parallel signaling pathways that drive distinct transcriptional and\nimmune programs.”\nReviewer #2 (Remarks to the Author):\nHost organisms must combat pathogens to ensure individual survival and fitness.\nConsequently, they have evolved a suite of defense strategies to systemically\nmaintain physiological homeostasis and protect vital organ functions—a process\nfundamental to biological integrity and species stability. While the nervous system is\nknown to play a critical role in coordinating host responses to infection, the\nunderlying mechanisms remain poorly understood. In this study, the authors employ\nC. elegans as a model to elucidate a novel signaling pathway mediated by protons\nand acetylcholine that integrates behavioral avoidance with immune defense against\npathogens such as S. aureus and P. aeruginosa. The authors demonstrate that\ningested pathogens trigger proton release from intestine cells, which subsequently\nactivates VA/VB motor neurons via the acid-sensing ion channel ASIC-1. This\nneuronal activation not only drives avoidance behavior but also initiates a feedback\nsignal that potentiates intestinal immune-defense by activating the Wnt and\np38/PMK-1 MAPK pathways. The study is supported by extensive data generated\nthrough multiple complementary techniques and the manuscript is well-organized, all\nof which make their findings robust and compelling.\nResponse: We thank the reviewer for the overall positive assessment of our work.\nMajor:\n1) What is the source of protons released from intestinal cells during infection?\nSpecifically, does the release originate from lysosomal compartments, or is it\ngenerated through specific biochemical reactions triggered by ingested pathogens?\nResponse: We thank the reviewer for this insightful question. Our data demonstrate\nthat pathogen infections induce NHX-6-dependent proton release from intestinal\nepithelial cells (IECs). However, the immediate intracellular source of these protons\ncannot be conclusively resolved by our experiments.\nC. elegans IECs maintain dynamic proton pools associated with rhythmic intestinal\npH oscillations, driven in part by proton movement across the apical membrane from\nthe intestinal lumen (Pfeiffer et al., 2008). Pathogen exposure may therefore promote\nbasolateral release of pre-existing proton gradients. As the reviewer noted, it is also\npossible that intracellular acidic compartments contribute. Notably, lysosome-related\norganelles in IECs are disrupted by pathogen-derived toxins such as pyocyanin from\nP. aeruginosa (Tse-Kang et al., 2024), raising the possibility that infection-associated\nepithelial stress could influence intracellular proton handling.\nHowever, lysosomal proton efflux mechanisms remain poorly defined in C.\nelegans, and key lysosomal proton channels such as TMEM175 lack clear nematode\northologs (Hu et al., 2022). Thus, our findings define the signaling and transport\nmachinery required for pathogen-induced proton release, while leaving open the\nprecise subcellular origin of the protons involved.\nWe have clarified this point in the revised Discussion as follows “While our data\ndemonstrate that pathogen infections trigger NHX-6-dependent proton release from\nIECs, the immediate intracellular source of these protons cannot be conclusively\nresolved by our experiments. IECs in C. elegans maintain dynamic proton pools\nassociated with rhythmic intestinal pH oscillations, which are driven in part by proton\nmovement across the apical membrane from the intestinal lumen. Pathogen\nexposure may therefore promote basolateral release of pre-existing proton gradients.\nIn addition, intracellular acidic compartments could contribute to this response.\nLysosome-related organelles in IECs are disrupted by pathogen-derived toxins such\nas pyocyanin from P. aeruginosa, raising the possibility that infection-associated\nstress alters intracellular proton handling. However, lysosomal proton efflux\nmechanisms remain poorly defined in C. elegans, and key lysosomal proton\nchannels such as TMEM175 lack clear nematode orthologs. Thus, our findings define\nthe signaling and transport machinery required for pathogen-evoked proton release,\nwhile leaving open the precise subcellular origin of the protons involved.”\n2) How does pathogen alter the calcium influx mediated by GON-2? Is the observed\ncalcium influx increase a direct consequence of pathogen-induced mechanical\nstress? Is GON-2 mechanosensitive?\nResponse: We thank the reviewer for this important question. Our data show that\npathogen infections enhance IEC Ca2+ influx in a GON-2-dependent manner. While\nthis enhancement is consistent with increased mechanosensitive signaling, we do not\nclaim it as a direct or single mechanical stimulus. Rather, we propose that infection-\nassociated epithelial stress, such as localized changes in membrane tension or\nintegrity, enhances mechanosensitive Ca2+ influx. This interpretation is consistent\nwith current models of mechanosensitive channel regulation, in which channel\nactivity can be modulated by membrane tension, curvature, or lipid perturbations\n(Suchyna, T. M., Prog Biophys Mol Biol, 2017), However, we do not directly measure\nmembrane tension or other biophysical parameters in this study. We have revised\nFig. 7, its legend, and the Discussion (the 9th paragraph) to reflect this limitation and\nto avoid overinterpretation.\nMoreover, although our genetic and epistasis analyses demonstrate that GON-2\nmediates mechanosensitive Ca2+ influx in IECs during body bending, they do not\nestablish whether GON-2 itself is the pore-forming mechanosensitive channel. GON-\n2 may instead function as an essential component or regulator of a\nmechanosensitive channel complex, or influence channel expression, assembly, or\nlocalization. We have explicitly acknowledged this point in the revised Discussion as\nfollows: “However, whether GON-2 itself constitutes the mechanosensitive pore-\nforming channel remains an open question. GON-2 may instead function as an\nessential component or regulator of a mechanosensitive channel complex, or\ninfluence channel expression, assembly, or localization.”\n3) How do GON-2 and CMD-1 specifically regulate proton release from intestine\ncells?\nResponse: We thank the reviewer for this important question. Our data indicate that\nGON-2-dependent Ca2+ influx and the downstream Ca2+ sensor CMD-1 are required\nto engage NHX-6-dependent proton release from IECs during pathogen infections.\nHowever, we do not propose that GON-2 and CMD-1 function exclusively in proton\nsignaling. Rather, they may act as upstream epithelial stress sensors that couple\nCa2+ signaling to multiple downstream responses relevant to host defense, with\nproton release representing one physiologically important output that enables gut-to-\nneuron communication.\nWe have highlighted this in the revised Discussion as follows: “In parallel,\nCa2+/calmodulin signaling may also directly engage PMK-1/p38 MAPK or other\nimmune signaling pathways cell-autonomously in IECs, as has been demonstrated\nfor p38 MAPK regulation in AWC olfactory sensory neurons, providing an additional\nlayer of regulation that complements gut-to-neuron communication.” and “These\nfindings define a form of damage-associated immune surveillance that links epithelial\nsensing to neural control of host defense, functioning alongside established\npathogen-sensing pathways, other neuronal inputs, and parallel epithelial immune\nmechanisms.”\n4) What is the specific motor program executed by VA and AB neurons to drive\npathogen avoidance, and is this circuit modulated by other neurons or\nneuroendocrine signals?\nResponse: We thank the reviewer for this important question. A-type (including VA\nand DA) and B-type (including VB and DB) ventral nerve cord motor neurons are\ncholinergic neurons that drive backward (reversal) and forward locomotion,\nrespectively, with VA/VB innervating ventral muscles and DA/DB innervating dorsal\nmuscles. These motor neurons receive inputs from command interneurons that\nintegrate sensory and internal-state information to bias locomotor output.\nPathogen avoidance involves changes in reversal frequency, turning, and\nlocomotion speed, reflecting shifts in motor patterns. Our data indicate that pathogen-\ninduced activation of VA and VB promotes pathogen avoidance by increasing\naverage, forward, and backward speeds, and that these effects require NHX-6 in\nIECs and ASIC-1 in VA/VB (Fig. 4a,b; Supplementary Fig. 7a).\nVA and VB neurons are embedded within the locomotor circuit and are likely\nmodulated by upstream interneurons and neuromodulatory or neuroendocrine\nsignals during pathogen infections. Dissecting these additional circuit-level\ncontributions will be an important direction for future work. We have clarified in the\nrevised Discussion as follows: “Within this framework, proton-mediated activation of\nmotor neurons enhances locomotor dynamics associated with pathogen avoidance,\nwhile remaining embedded within a broader motor circuit subject to modulation by\nupstream interneurons and neuromodulatory signals.”\n5) Does intestinal GON-2 mediate the pathogen-evoked shifts in basal calcium levels\n(measured by GCaMP6) in VA/VB sensory neurons?\nResponse: We thank the reviewer for this thoughtful question. To directly address\nthis, we performed additional GCaMP6 imaging. We found that gon-2(q362) mutants\nexhibited significantly reduced basal GCaMP6 fluorescence and spontaneous Ca2+\ntransient amplitude in VA/VB during pathogen infections, and these defects were\nrescued by IEC-specific gon-2 expression. Moreover, the gon-2(q362) mutation did\nnot further reduce VA/VB Ca2+ activity in nhx-6(ok609) mutants (new Supplementary\nFig. 5c-f), consistent with GON-2 and NHX-6 acting in the same pathway. Together,\nthese results suggest that intestinal GON-2 is required to couple pathogen infections\nto elevated VA/VB activity. We have incorporated these new findings into the revised\nResults.\nMinor:\n1) Is there any possibility that Calcium/Calmodulin might regulate PMK-1/P38 MAPK\npathway cell-autonomously?\nResponse: We thank the reviewer for this insightful question. Ca2+/calmodulin-\ndependent regulation of p38 MAPK signaling has been described in other contexts.\nFor example, in AWC olfactory neurons, Ca2+ influx through the UNC-2/UNC-36\ncalcium channel activates the Ca2+/calmodulin-dependent kinase UNC-43, which in\nturn engages p38 MAPK signaling during asymmetric cell fate specification (Sagasti\net al., Cell, 2001).\nThese findings raise the possibility that Ca2+/calmodulin could also regulate PMK-\n1/p38 MAPK or other immune signaling pathways cell-autonomous in IECs. Although\nour data do not directly test this possibility, we have acknowledged it in the revised\nDiscussion as follows: “In parallel, Ca2+/calmodulin signaling may also directly\nengage PMK-1/p38 MAPK or other immune signaling pathways cell-autonomously in\nIECs, as has been demonstrated for p38 MAPK regulation in AWC olfactory sensory\nneurons, providing an additional layer of regulation that complements gut-to-neuron\ncommunication.”\n2) Is there any possibility that NHX-6 — H+ — ASIC-1 signaling pathway would\nregulate immune-defense temporally?\nResponse: We thank the reviewer for this thoughtful question. Yes, our data support\nthe idea that the NHX-6-H+-ASIC-1 pathway could function in a temporally restricted\nmanner. We propose that this pathway is engaged early during pathogen infections,\nwhen IECs experience localized or sublethal epithelial stress. In contrast, widespread\nor sustained membrane rupture would be expected to dissipate ionic gradients,\nparticularly for highly diffusible ions such as H+, thereby limiting this mode of\nsignaling\nWe have clarified in the revised Discussion as follows: “This infection-associated\nenhancement likely reflects localized epithelial stress or membrane perturbation\narising during infections, operating within a spatially and temporally restricted\nphysiological regime that precedes severe epithelial compromise.”\nReviewer #3 (Remarks to the Author):\nThe authors identify a role for a proton-mediated gut-to-neuron signaling pathway in\nthe integration of behavioural and immune defenses against pathogen infection. They\nuse an elegant combination of experimental approaches to provide substantial\nphysiological and mechanistic insight into the components of this response and\nwhere and how they contribute. Importantly, they also show that the mammalian\ncounterparts of the two key components of the pathway, an Na+/H+ exchanger and\nan acid sensing ion channel, can substitute for the C. elegans genes, underlining the\nhigh level of conservation and the relevance of a nematode model for studying gut-\nnervous system interactions. This is an important and topical area of research and of\ngeneral interest. The research is of high quality, using innovative experimental\napproaches, well-founded on a thorough consideration of the current state of the\nfield. It would thus be highly suitable for publication in this journal.\nHowever, I have a number of serious concerns with missing controls, missing\nstatistical comparisons and inappropriate analysis of the data, with the result that the\nconclusions are not always supported by the data. This must be addressed before it\nis suitable for publication. In most cases, this would be rectified by small changes to\nthe way in which the current data is presented or analyzed, or by ensuring that their\nstatements take account of the caveats.\nI therefore strongly recommend publication, subject to revision to address these\npoints.\nResponse: We thank the reviewer for the overall positive assessment of our work.\nMajor comments\nThe authors use a pHluorin-mStrawberry fusion, quantifying fluorescence as a\nreadout for pH in the extracellular environment of the motor neurons. The data\nappear to show pHluorin fluorescence, without, for example, use of the mStrawberry\nfluorescence as a control. The possibility thus remains that the observed change\nresults from a change in expression levels of the pH indicator. Indeed, the authors\nuse an unc-17 promoter to drive its expression, but later show that unc-17 is itself\nimplicated in this pathway.\nResponse: We thank the reviewer for raising this important point. To address this\nconcern, we performed additional control experiments by recording mStrawberry\nfluorescence in the same Punc-17-driven pHluorin-mStrawberry transgenic worms\nunder the relevant infection and genetic conditions.\nAcross these conditions, mStrawberry fluorescence remained unchanged,\nindicating stable reporter expression. We have presented these control data in new\nSupplementary Fig. 4a-d and Supplementary Fig. 11c and described in the revised\nResults as follows:\n“Across these conditions, the co-expressed, pH-insensitive mStrawberry signal\nremained unchanged during pathogen exposure (Supplementary Fig. 4a), indicating\nstable reporter expression.”\n“mStrawberry fluorescence was unaffected by pathogen exposure across these\ngenotypes (Supplementary Fig. 4b).”\n“mStrawberry fluorescence was unaffected by pathogen exposure across these\ngenotypes (Supplementary Fig. 4c,d).”\n“mStrawberry fluorescence was unaffected across these rescue conditions\n(Supplementary Fig. 11c).”\nWe have also clarified this approach in the revised Methods (“Imaging”) as follows:\n“For pHluorin imaging, the co-expressed, pH-insensitive mStrawberry signal was\nused as an internal expression control to assess extracellular pH changes\nindependently of potential differences in reporter expression.”\nFor the intestinal calcium imaging experiments (fig. 2d, e), the individual data points\nare discrete values, multiples of 0.5. This suggests that the data was analyzed by\nsimply counting the number of peaks or full cycles within the 2-minute recording time.\nThis presumably explains why the mean frequency implies a surprisingly short\ninterval length (approx. 1.7 per minute for wt = 35 seconds!). The data would be\nmore powerful, and the comparisons more meaningful, if the authors calculated the\nmean interval between peaks. The short recording interval, coupled with the small\nnumber of replicates, means that the number of data points is small, particularly\ngiven that we know from previous studies that gon-2 loss of function results in\nincreased variance of interval length (Xing et al. 2008, Kwan et al. 2008).\nResponse: We thank the reviewer for this insightful and constructive comment. The\nreviewer is correct that, in our original analysis, Ca2+ activity in intestinal epithelial\ncells (IECs) was quantified over a 2-min recording at 1 frame per second, resulting in\ndiscrete values that reflect the number of Ca2+ transients detected within the imaging\nwindow. In response to this suggestion, we have reanalyzed the data by calculating\nthe mean inter-peak interval for each animal, cited previous studies, and have\nrevised the figures and text accordingly (revised Fig. 2d,e and Supplementary Fig.\n3c-f). This analysis confirms our conclusions regarding GON-2-dependent\nenhancement of IEC mechanosensitive activation during pathogen infections.\nFigure 2e and related supplemental: We know from previous research that gon-2 loss\nof function results in an increase in cycle length, for both calcium and behavior (Xing\net al. 2008). To convincingly say that gon-2 is required for the pathogen-induced\nchange in calcium transient frequency and amplitude, animals grown on E. coli and\npathogen must be compared directly. Given the role of gon-2 in contributing to the\ncalcium events that play such a critical central role in both rhythmicity and overall\nfunction of the gut, care should be taken in determining whether the role identified\nhere is specific to gon-2 or an overall contribution of intracellular calcium signaling.\nResponse: We thank the reviewer for this important comment. Consistent with\nprevious findings (Xing et al., 2008), gon-2(q362) mutants exhibited reduced\nintestinal Ca2+ activity, which was rescued by IEC-specific expression of gon-2\n(Supplementary Fig. 3d). As suggested, we directly compared gon-2(q362) mutants\ngrown on E. coli or exposed to either S. aureus or P. aeruginosa. In contrast to wild-\ntype animals (Fig. 2d), pathogen exposure did not significantly alter Ca2+ transient\nfrequency, amplitude, or inter-peak interval in gon-2(q362) mutants (new\nSupplementary Fig. 3e). These results suggest that pathogen-induced enhancement\nof IEC Ca2+ signaling requires GON-2, while not excluding potential contributions\nfrom other sources of intracellular Ca2+ signaling.\nWe have incorporated these analyses and cited previous studies in the revised\nResults (“Pathogen-induced IEC proton secretion requires TRP channel GON-2-\nmediated Ca2+ influx and CMD-1/calmodulin”). We have also clarified this point in the\nrevised Discussion as follows “we find that these phylogenetically distinct pathogens\nenhance IEC mechanosensitive Ca2+ influx via the TRP channel GON-2, while not\nexcluding contributions from other sources of intracellular Ca2+ signaling”.\nFigure 2c, f, g: To convincingly say that these genes are required for the pathogen-\ninduced change in pHluorin fluorescence, animals grown on E. coli and pathogen\nmust be compared directly.\nResponse: We thank the reviewer for this insightful suggestion. To address this,\nwe performed additional pHluorin imaging using animals raised on E. coli, including nhx-\n6, gon-2, nhx-6;gon-2, cmd-1(RNAi), and nhx-6;cmd-1(RNAi). These new data\nenable direct within-genotype comparisons and show that loss of nhx-6, gon-2, or\ncmd-1 abolishes the pathogen-induced reduction in pHluorin fluorescence (new\nSupplementary Fig. 4e). We have incorporated these new findings into the revised\nResults.\nFor the motorneuron silencing experiments using a histamine-gated chloride channel\n(supp. fig. 5), a control for the effects of histamine alone (i.e. in the absence of His-\nCl1 expression) is required.\nResponse: We thank the reviewer for this insightful suggestion. To address this\nconcern, we performed additional control experiments comparing wild-type animals\nwith and without histamine, as well as animals expressing HisCl1 in body-wall\nmuscles in the absence of histamine. These new data show that neither histamine\ntreatment alone nor HisCl1 expression alters motor neuron activity (new\nSupplementary Fig. 6c,d). We have incorporated these new findings into the revised\nResults.\nThe authors show that, compared with E. coli, exposure to the pathogenic bacteria\nresulted in an increase in locomotion speed. They also show that on the pathogens\nasic-1 and nhx-6 mutants show a lower speed than wild type. To assert that the\npathogen effect on speed is disrupted in these mutants, they need to show evidence\nthat the change is disrupted, i.e. directly compare E. coli-grown with pathogen-grown.\nResponse: We thank the reviewer for this important suggestion. To address this,\nwe performed additional recordings using animals of each genotype raised on E. coli.\nThese new data enable direct within-genotype comparisons and show that the\npathogen-induced increase in locomotion speed observed in wild type is absent in\nasic-1, nhx-6, and asic-1;nhx-6 mutants (revised Fig. 4b and Supplementary Fig. 7a).\nWe have incorporated these new findings into the revised Results.\nAltering the growth medium pH is an appealing experiment and may support the\nconclusions about the role of ASIC-1. However, the observed effects could be due to\nany number of off-target effects unrelated to the extracellular environment of the\nmotorneurons. Indeed, is not clear how a change in pH in the medium would be\ntranslated into a corresponding change in an internal compartment, particularly given\nthe important roles proton signaling/pH oscillations in multiple locations. The\nconclusions should not be overstated.\nResponse: We thank the reviewer for raising this important point and agree that\naltering the pH of the growth medium represents an indirect manipulation and may\ninfluence internal proton dynamics through multiple physiological processes.\nAccordingly, we interpret these experiments as supportive, correlative evidence\nconsistent with a role for ASIC-1 in proton sensing, rather than as a precise\nmanipulation of the motor neuron extracellular environment. We have clarified this\ninterpretation in the revised Results as follows: “Although altering the growth medium\npH is an indirect manipulation, these findings are consistent with ASIC-1-dependent\nproton sensing.”\nWhen using TRPV1 expression as a means to activate the motorneurons in an ASIC-\n1-independent manner (supp. Fig. 6h), the authors do not show any controls for the\neffects of capsaicin itself (i.e. whether in an asic-1 loss of function background\ncapsaicin has analogous effects, to exclude the possibilities that the observed effect\nis due to a C. elegans TRPV channel, which could be acting elsewhere.\nResponse: We thank the reviewer for this important suggestion. Our existing data\nshow that capsaicin treatment alone does not affect avoidance behavior in wild-type\nanimals. To further exclude nonspecific effects of capsaicin, activation of endogenous\nC. elegans TRPV channels, or TRPV1 expression, we performed additional control\nexperiments.\nWe find that capsaicin treatment alone also has no effect on avoidance in asic-1\nmutants, and that expression of TRPV1 in VA/VB neurons in the absence of\ncapsaicin does not alter avoidance behavior (revised Supplementary Fig. 7h).\nTogether, these controls demonstrate that the observed effects are specific to\ncapsaicin-dependent activation of heterologously expressed TRPV1 in motor\nneurons, rather than off-target effects of capsaicin, endogenous channels, or TRPV1\nexpression. We have incorporated these new findings into the revised Results.\nMinor points\nLine 111/Figure 1: The authors investigate the role of innexins expressed in the motor\nneurons, using data from Altun et al. 2009. More recent evidence (e.g. Bhattacharya\net al. 2019; RNA seq data in the CeNGEN project (Taylor et al. 2021)) indicates a\nlarger number of innexin genes are expressed in these cells. This should, at the very\nleast, be acknowledged in the text. The authors also present no evidence that the\nRNAi actually knocks down the target genes or that it is expressed in the desired\ncells. Again, in the absence of controls, this should at least be acknowledged.\nResponse: We thank the reviewer for this important and constructive comment. We\nagree that more recent studies, including Bhattacharya et al. (2019) and the\nCeNGEN project (Taylor et al., 2021), indicate that ventral nerve cord motor neurons\nexpress a broader set of innexin genes than originally reported by Altun et al. (2009).\nWe now explicitly acknowledge this in the revised Results.\nWe also acknowledge that RNAi knockdown efficiency and cell-type specificity\nwere not directly assessed for the innexin RNAi experiments, and this limitation is\nnow explicitly stated in the Results. Nevertheless, disruption of multiple gap junction\ncomponents did not affect acid-evoked responses, VA/VB responses persisted in\nneurotransmission-defective mutants, and DA/DB responses were abolished when\nsynaptic transmission was impaired. Collectively, these observations argue against a\nmajor contribution of gap-junctional coupling. We now explicitly note this in the\nrevised Results as follows: “Although additional innexins may be expressed in these\nneurons, and RNAi efficiency and cell-type specificity were not directly assessed, the\ndata do not support a major contribution of gap junctions.”\nLine 145: “hours” does not seem to fit the sentence.\nResponse: Deleted, thanks.\nFigure 2e: gtl-1 is incorrectly written as glt-1.\nResponse: Corrected, thanks.\nFigure 2d, e: n values are given for some genotypes but not for all.\nResponse: Corrected, thanks.\nFigure 4: misspelling of rescue.\nResponse: Corrected, thanks.\nLine 349: “pumping rates” requires more explanation for a non-specialist reader.\nLikewise, “increased colony-forming units” (or simply “CFUs” in the figure legend) – a\nbetter explanation of the experiment would be helpful.\nResponse: We thank the reviewer for this helpful suggestion. We have revised the\nlegend of Supplementary Fig. 9 to briefly explain both measurements. In the revised\nMethods, we now describe pharyngeal pumping as the rhythmic contraction and\nrelaxation of the pharynx that drives bacterial ingestion, with pumping rate serving as\na quantitative measure of feeding activity. We also clarify that colony-forming units\n(CFUs) represent the number of viable bacteria recovered from the intestine following\nworm lysis, serial dilution, and plating on selective media. Detailed experimental\nprocedures are provided in the Methods."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have appropriately addressed all my concerns.", "reviewer_2": "The authors have done a nice job in addressing my questions. It is ready for publication. Congratulations!", "reviewer_3": "All of my concerns have been addressed. I congratulate the authors on an excellent piece of work and have no further comments."}, "author_response": ""}]

Decision letter