Nervous system guides behavioral immunity in Caenorhabditis elegans

Recognition of pathogen-derived compounds

To elicit behavioral immunity, C. elegans must recognize the encountered bacteria as a pathogen. This recognition process initially relies on sensing pathogen-generated compounds through its nervous system. According to previous studies, several bacterial species such as PA14 (Ha et al., 2010; Zhang, Lu & Bargmann, 2005), S. marcescens (Pradel et al., 2007), B. thuringiensis (Peng et al., 2018), M. nematophilum (Filipowicz, Lalsiamthara & Aballay, 2021), E. faecalis (Filipowicz, Lalsiamthara & Aballay, 2021), S. aureus (Irazoqui et al., 2010), Streptomyces (Tran et al., 2017), and Oomycetes (Fasseas et al., 2021) have been reported as C. elegans pathogens. Some of pathogen-derived compounds (Fasseas et al., 2021; Hao et al., 2018; Park, Meisel & Kim, 2020; Pradel et al., 2007; Prakash et al., 2021; Tran et al., 2017) have been identified (Table 1). These compounds can be categorized into two classes: non-gaseous organic compounds (e.g., phenazine-1-carboxamide, pyochelin, 1-undecene, Serrawettin W2, dodecanoic acid) and the gaseous inorganic compound nitric oxide (NO, Table 1). To sense non-gaseous organic compounds, C. elegans employs chemosensory neurons such as AWB, ASJ, ASH, ADL, ADF, ASK, PHA, and PHB. Interestingly, most of these neurons are known for sensing aversive stimuli (Liu et al., 2023; Tanimoto et al., 2017; Zou et al., 2017). For sensing the gaseous inorganic compound NO, ASJ sensory neurons are utilized (Hao et al., 2018). These findings highlight the importance of ASJ neurons, as they are currently the only pair of neurons known to respond to both types of pathogen-derived compounds.

Different neurons utilize different molecules to process signals from pathogen-derived compounds. For example, phenazine-1-carboxamide can induce rapid transcription of the daf-7 gene, encoding a TGF-β ligand, in ASJ neurons through a G-protein-dependent signaling pathway (Park, Meisel & Kim, 2020). Increased levels of DAF-7 may act in conjunction with infection-associated modulation of internal state to facilitate subsequent avoidance behavior of P. aeruginosa and aid in reducing bacterial load by regulating oxygen sensing-behavior in C. elegans (Meisel et al., 2014; Singh & Aballay, 2019a). Additionally, ASJ neurons can sense PA14-derived NO through two kinds of receptor-type guanylate cyclase, DAF-11 and GCY-27 (Hao et al., 2018). These results suggest that neurons can respond to pathogen-derived compounds through either pre-existing molecules or newly induced molecules. Unfortunately, our knowledge of the receptors for these compounds is limited. Tran et al. (2017) identified SRB-6 as a potential receptor for the streptomyces-derived dodecanoic acid. Our previous work showed that DAF-11 and GCY-27 may be involved in NO-sensing in ASJ neurons, although we did not have direct data to support them as receptors for NO (Hao et al., 2018). Thus, it remains largely elusive how the C. elegans nervous system detects pathogen-derived compounds.

Sensing pathogen-induced cellular damage or stress

Apart from releasing harmful compounds, pathogen infection can cause cellular damage or stress, including mitochondrial stress and disruptions in protein biosynthesis, which C. elegans can detect. This process, sometimes referred to as “surveillance immunity”, involves monitoring core cellular processes for disruptions (Pukkila-Worley, 2016). It represents a sophisticated mechanism for C. elegans to identify pathogen invasion by sensing disturbances in essential cellular functions. The major regulators of stress responses in C. elegans, such as the p38 MAPK, DAF-16/FOXO, SKN-1/Nrf2, and HSF-1, play crucial roles in recognizing pathogen-induced damage-associated molecular patterns (DAMPs) and mounting immune responses against pathogens (Hajdú, Szathmári & Sőti, 2024; Yang et al., 2022a).

C. elegans intestinal epithelial cells function as a major pathogen recognition site for cellular damage or stress (Fig. 1). Some pathogens, such as P. aeruginosa, can disrupt host protein biosynthesis. For example, exotoxin A from P. aeruginosa can enter C. elegans intestinal epithelial cells and inhibit protein translation (Dunbar et al., 2012; McEwan, Kirienko & Ausubel, 2012). This inhibition leads to an increase in the level of the ZIP-2 bZIP transcription factor, which may regulate C. elegans immune response genes, including irg-1 (Dunbar et al., 2012), pmk-1 (Taouktsi et al., 2023), and skn-1 (Grover et al., 2024).

Mitochondria, central organelles for energy production and metabolism, are common targets of pathogen attack in intestinal epithelial cells (Fig. 1) (Chen et al., 2021; Liao et al., 2022). Exposure to pathogens like PA14 can cause mitochondrial dysfunction and activate the mitochondrial unfolded protein response, UPR(mt) (Chen et al., 2021; Liao et al., 2022; Pellegrino et al., 2014). C. elegans monitors the import efficiency of the transcription factor ATFS-1, which mediates UPR(mt), to sense potential pathogen infection. Inhibition of mitochondrial functions induces bacterial avoidance (Liao et al., 2022; Liu et al., 2014; Pellegrino et al., 2014). During mitochondrial stress, ATFS-1 induces not only mitochondrial protective genes to mediate UPR(mt) but also innate immune genes, including a secreted lysozyme and antimicrobial peptides, as well as two C-type lectins involved in pathogen recognition (Fig. 2) (Pellegrino et al., 2014).

Cellular mechanism underlying pathogen-mediated aversive olfactory learning

When P. aeruginosa or E. faecalis infects the intestine of C. elegans, it triggers the development of a learned aversive response (backward locomotion). This reflexive behavior requires the involvement of a four-layer aversive learning circuit in the nematode (Filipowicz, Lalsiamthara & Aballay, 2022). The olfactory signal from P. aeruginosa and E. faecalis is detected by AWB neurons and transmitted sequentially through electrical synapses to second-layer AUA and RMG neurons, chemical synapses to third-layer AVA, AVD, and AVE command interneurons, and finally to fourth-layer motor neurons, namely the VA, DA, VD, DD, and AS neurons, which are crucial for backward locomotion (Filipowicz, Lalsiamthara & Aballay, 2022). These motor neurons ultimately facilitate the backward movement necessary for avoiding the detected odor. Additionally, aversive learning to P. aeruginosa requires AWC, ADF, AIY, AIB, RIA neurons (Ha et al., 2010), while aversive learning to E. faecalis requires ASE and AWC neurons (Filipowicz, Lalsiamthara & Aballay, 2021).

Despite the intricate and bacteria-dependent nature of the neurons involved, both P. aeruginosa and E. faecalis engage the AWB neurons, which can respond to both attractive and aversive olfactory cues (Yang et al., 2022b), as well as the AWC neurons, which generally respond to attractive olfactory stimuli, for initial olfactory recognition. The signal transmission from these pathogens appears to activate a series of neurons at each layer of signal propagation, suggesting that the circuit likely intersects in a complex manner. This intricate interconnectivity makes it challenging to dissect the complete aversive learning circuit specific to a particular pathogen.

Molecular mechanism underlying pathogen-mediated aversive olfactory learning

1) Biogenic amine signaling.

The tyramine produced by the intestinal bacteria Providencia can be converted to octopamine, which then acts on the octopamine receptor OCTR-1 present in the ASH neurons (O’Donnell et al., 2020). The ASH neurons are a pair of neurons primarily known for sensing aversive stimuli. This interaction between octopamine and the ASH neurons leads to altered food choices in the nematode.

Serotonin signaling plays a crucial role in aversive olfactory learning to PA14 and S. marcescens. Prolonged exposure to either of them upregulates TPH-1, a serotonin biosynthetic enzyme, in ADF neurons. Serotonin secreted from ADF acts through MOD-1, a serotonin-gated chloride channel expressed in interneurons AIY/AIZ, to promote aversive learning (Zhang, Lu & Bargmann, 2005). When exposed to PA14, a redistribution of LGC-50, a serotonin-gated cation channel, occurs within RIA neuronal processes. This redistribution is crucial for the aversive olfactory learning associated with PA14 (Morud et al., 2021). These findings suggest that the intracellular trafficking and synaptic localization of LGC-50 may represent a molecular cornerstone underlying the learning mechanism. Furthermore, RIA and AIZ neurons, acting as postsynaptic targets of the serotonergic ADF neurons, express serotonin-gated chloride and cation channels, respectively. This suggests that when pathogen stimulates the ADF neurons to release serotonin, it may simultaneously suppress RIA while activating AIZ. This provides a model for how serotonin finely modulates the downstream neuronal state.

2) Insulin and insulin-like peptide (ILP) signaling.

ILP pathway plays significant roles in regulating aversive olfactory learning, developmental processes, lifespan, reproduction, and neural plasticity across various species (Chen et al., 2013; Cornils et al., 2011; Hwangbo et al., 2004). INS-6 and INS-7, members of the type-beta class of the C. elegans ILP superfamily, contribute antagonistically to aversive olfactory learning to PA14 through an ILP-to-ILP signaling pattern (Chen et al., 2013). INS-6, produced by the ASI sensory neurons, can suppress the transcription of ins-7 in the URX neurons, thereby facilitating olfactory learning (Chen et al., 2013; Liu et al., 2018). High levels of INS-7, secreted by the URX neurons, disrupt olfactory learning by counteracting the function of DAF-2 in the postsynaptic RIA neurons (Chen et al., 2013). Additionally, INS-4 and INS-16 provide balanced signaling from the bacteria-sensing neuron AWA and the pheromone-sensing neuron ADL, respectively. This balanced signaling is critical for aversive olfactory learning of PA14 (Wu et al., 2019). These findings suggest that ILPs may interfere with each other, allowing for fine-tuning of neuronal functions. Furthermore, exposure to PA14 induces the expression of INS-11 in the intestine, which may contribute to aversive learning behavior by inhibiting the expression of TPH-1 in the ADF neurons and INS-6 in the ASI neurons (Lee & Mylonakis, 2017). This implies that the ILP pathway can interact with the serotonergic pathway as well.

3) TGF-β signaling.

DBL-1, a well-characterized C. elegans TGF-β ligand, has been implicated in aversive olfactory learning to PA14. Elevated expression of DBL-1 in AVA command interneurons may facilitate aversive olfactory learning through the type I TGF-β receptor SMA-6 in the adult hypodermis (Zhang & Zhang, 2012).

4) RNAi-mediated learning.

An intriguing mechanism of aversive learning involves the ingestion of small RNAs (sRNAs) from pathogenic bacteria by the C. elegans gut. These sRNAs operate through the RNA interference (RNAi) pathway in the gut, leading to signaling that involves the piRNA pathway and epigenetic modifiers. This signaling upregulates the expression of daf-7 in the ASI neurons, ultimately inducing avoidance of PA14 in a manner independent of innate immunity. Remarkably, this sRNA-mediated memory can be transmitted across generations (Fig. 2) (Kaletsky et al., 2020).