Bacterial peptidoglycan acts as a digestive signal mediating host adaptation to diverse food resources in *C. elegans*

Nematodes comprise about 80% of multicellular animals and play major ecosystem roles, with Caenorhabditis elegans thriving in microbe-rich niches such as rotting plant matter. Efficient digestion of diverse foods enhances survival and adaptation, yet the specific food-derived signals that activate host digestive processes are unclear. Prior work established a C. elegans digestion assay in which the inedible bacterium Staphylococcus saprophyticus (SS) cannot support growth, heat-killed E. coli (HK–E. coli) is low-quality but digestible, and the combination HK–E. coli + SS enables animals to digest SS and grow. The study asks whether a common bacterial signal activates the digestive system to broaden edible food range. The authors hypothesize that a conserved bacterial component, potentially peptidoglycan (PGN), serves as a cue sensed by host intestinal factors to trigger digestion through mitochondrial and neuroendocrine pathways, thereby improving adaptation to varied food conditions.

- Animals have evolved sophisticated neural mechanisms to sense food and regulate intake (refs 2–6). - The authors’ previous work showed HK–E. coli can activate digestion of SS and implicated bacterial membrane proteins and host neural/innate immune pathways in enabling digestion of inedible food. - PGN is a ubiquitous bacterial cell wall component composed of β(1–4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) crosslinked by peptides. - Prior studies reported PGN muropeptides promote mitochondrial homeostasis by acting as ATP synthase agonists and inhibiting the mitochondrial unfolded protein response (UPRmt), and in mice PGN can promote growth via NOD2 signaling (Schwarzer et al.). - In C. elegans, PGN-related molecules (e.g., MurNAc-L-Ala) can enhance pathogen tolerance, underscoring beneficial PGN effects beyond immunity. These works motivated testing PGN as a generalized digestion-activating signal.

- Food categorization and growth assays: Defined good food (E. coli OP50), inedible food (Staphylococcus saprophyticus, SS), and low-quality food (heat-killed E. coli, HK–E. coli). Assessed developmental progression by relative body length at set times and temperatures (typically 20–25°C), comparing SS, HK–E. coli, and HK–E. coli + SS. - Bacterial genetics screen: Screened HK–E. coli mutants unable to promote SS digestion. Identified ΔycbB (L,D-transpeptidase for PGN maturation) and ΔygeR (PGN hydrolase) as failing to support growth with SS. - PGN supplementation and structural dissection: Extracted PGN from bacteria (e.g., E. coli, Bacillus subtilis, Enterococcus faecalis) using boiling, enzymatic digestions (DNase, RNase, trypsin), washes, and storage in HEPES. Supplemented purified PGN to SS lawns and assessed growth and intestinal lumen bloating. Enzymatically treated PGN with lysozyme, NagZ (β-hexosaminidase), AmiD (amidase), or proteinase K to map active structural units. - Candidate receptor identification: Intersected previously identified E. coli-binding and PGN-binding host proteins, selecting 23 with intestinal expression for RNAi screening (feeding RNAi) under HK–E. coli + SS to find factors required for digestion. - Genetic tools and strains: Used bcf-1 RNAi; bcf-1 mutants (ok2599, ylf1); intestinal-specific RNAi; UPRmt reporter hsp-6p::GFP; dve-1p::dve-1::GFP; atfs-1(et18) (gain-of-function), atfs-1(gk3094) (loss-of-function), pmk-1(km25), double mutants bcf-1(ok2599);pmk-1(km25), and atfs-1(et18);pmk-1(km25). Tested wild isolates ED3077 and JU2513. - PGN–protein interaction: Performed pulldown assays incubating worm lysates from bcf-1p::bcf-1::gfp::flag animals with PGN, pelleting PGN complexes, and probing with anti-FLAG. Tested effect of proteinase K on PGN’s ability to pull down BCF-1. - Expression analyses: Measured BCF-1::GFP induction by PGN (microscopy and Western blot). Quantified UPRmt activation by hsp-6p::GFP fluorescence. Monitored DVE-1 nuclear localization under bcf-1 knockdown. Performed qRT-PCR to quantify hsp-6 expression. - Behavior and physiology: Food avoidance assays (avoidance index N_off/N_total). Measured intestinal lumen width to assess bacterial accumulation and digestion efficiency. - Innate immunity readouts: Western blot for phosphorylated PMK-1 (p-PMK-1) under various genetic and dietary conditions. - Adaptation/population assays: Seeded single L1 on mixed-bacteria plates (e.g., SS + E. coli, SS + B. subtilis, SS + Enterococcus faecalis; and complex mixes OP50+SS+B.s+E.f), counted total animals after 9–10 days to estimate adaptation via digestion-supported population expansion. - Statistics: Multiple unpaired two-tailed t-tests across figures; data presented as mean ± SD with n from ≥3 independent experiments; significance thresholds reported (e.g., ****p < 0.0001).

- Low-quality food triggers digestion of inedible food: HK–E. coli alone does not support growth but, when mixed with SS, activates digestion of SS and supports growth (Fig. 1a,b; SS vs. HK–E. coli + SS: ****p < 0.0001). - PGN is a digestion-activating signal: E. coli PGN supplementation to SS rescues growth and reduces intestinal lumen bloating compared to SS alone (Fig. 1d,e; lumen width reduction ****p < 0.0001). PGN from E. coli or Bacillus subtilis promotes digestion of SS, while PGN from Enterococcus faecalis does not (strain-specific effect). - Active PGN moiety: Lysozyme-treated PGN retains activity, whereas PGN treated with AmiD, NagZ, or proteinase K loses activity, indicating the digestion signal corresponds to 5′ NAG–NAM disaccharide muropeptides with an amino acid peptide on NAM. - BCF-1 is required for PGN-induced digestion: From 23 intestinal candidate genes, bcf-1 RNAi caused slow growth on HK–E. coli + SS; bcf-1 mutant strains (ok2599, ylf1) also showed slow growth and intestinal distension under HK–E. coli + SS (Fig. 2b–d). bcf-1 mutants failed to benefit from SS + PGN supplementation (Fig. 3g). - PGN binds and induces BCF-1: PGN pulldown from worm lysates captured BCF-1 in a concentration-dependent manner; proteinase K abolished binding, implicating PGN peptide stems in interaction (Fig. 3c,d). PGN feeding induced BCF-1::GFP expression (~6-fold by Western; Fig. 3f) and higher fluorescence (****p < 0.0001; Fig. 3e). - PGN inhibits UPRmt via BCF-1: ΔycbB (PGN-deficient) feeding activates UPRmt (hsp-6p::GFP) in WT, which is suppressed by PGN add-back; suppression fails in bcf-1 mutants (Fig. 4a). bcf-1 RNAi/mutants show UPRmt activation and increased food avoidance (Fig. 4b,c). - UPRmt activation inhibits digestion: atfs-1(et18) (constitutively active UPRmt) animals show significantly reduced growth on HK–E. coli + SS (****p < 0.0001; Fig. 4d). Loss of atfs-1 rescues digestion defects from bcf-1 RNAi (Fig. 4e), linking excessive UPRmt to impaired digestion. - Neuropeptide NLP-3 mediates bcf-1 effects: bcf-1 knockdown leads to DVE-1 nuclear accumulation; RNAi of atfs-1, dve-1, or ubl-5 suppresses UPRmt in bcf-1 mutants (Fig. 5a,b). Knockdown of neuropeptide processing genes egl-3/egl-21 or nlp-3 reduces UPRmt activation and partially rescues growth defects and food avoidance in bcf-1 mutants (Fig. 5c–e), indicating cell non-autonomous regulation via NLP-3. - PMK-1 innate immunity pathway contributes to digestion inhibition: bcf-1 mutants show elevated p-PMK-1 under OP50 and HK–E. coli + SS; pmk-1 mutation rescues bcf-1 growth defects and reduces food avoidance (Fig. 6a–c). atfs-1(et18) increases p-PMK-1; pmk-1 does not significantly impact UPRmt marker hsp-6; atfs-1(et18);pmk-1 double mutants partially rescue growth defects (Fig. 6d–f), suggesting UPRmt impairs digestion partly via PMK-1-dependent innate immunity. - Adaptation in complex food environments: Adding E. coli or B. subtilis to SS increases population size after ~9 days; Enterococcus faecalis does not. In a complex mix (OP50+SS+B.s+E.f), bcf-1 mutants produce fewer animals than WT, indicating PGN–BCF-1 signaling enhances adaptation by enabling digestion of varied foods (Fig. 7a–c). Wild isolates ED3077 and JU2513 also grow on SS + PGN (****p < 0.0001; Fig. 7d).

The study identifies bacterial peptidoglycan as a specific signal that activates C. elegans to digest otherwise inedible food. Mechanistically, PGN is sensed by the intestinal glycoprotein BCF-1, which suppresses UPRmt at least partly via neuropeptide signaling through NLP-3. Excessive UPRmt activation compromises digestion and elevates food avoidance, with the innate immune PMK-1 pathway mediating part of this inhibition. Thus, a gut–neuron axis integrates PGN–BCF-1 cues to balance mitochondrial stress responses and innate immunity, enabling digestion. The findings explain how bacterial-feeding nematodes adapt to diverse and fluctuating food sources: PGN–BCF-1 acts as a "good-food signal" that promotes digestive activation and growth, thereby enhancing population expansion in complex microbial environments. The work also connects mitochondrial proteostasis (UPRmt) to digestive capacity and links PGN signaling to both neuronal and immune pathways in vivo.

This work reveals a conserved digestive activation mechanism in bacterial-feeding nematodes whereby bacterial PGN engages the intestinal glycoprotein BCF-1 to inhibit UPRmt via neuropeptide NLP-3, promoting digestion of inedible food (SS), growth, and population expansion. It establishes that UPRmt activation suppresses digestion partly through p38 MAPK/PMK-1 innate immunity. The PGN–BCF-1 axis thus acts as a "good-food signal" enabling adaptation to diverse bacterial diets. Future work should define the precise PGN structural ligands and binding determinants on BCF-1, delineate BCF-1-independent PGN effects (e.g., via ATP synthase), identify additional neuropeptides or tissues in the gut–neuron circuit, and test conservation of PGN-driven digestive regulation in other nematode species and animals.

- PGN effects are strain-specific; not all bacterial PGN (e.g., from Enterococcus faecalis) promoted digestion, limiting generalizability across microbes. - Evidence suggests additional, BCF-1-independent PGN pathways (e.g., ATP synthase) influencing UPRmt and digestion; these were not fully dissected. - NLP-3 knockdown only partially rescued bcf-1 digestive defects, indicating other neuropeptides/signals are involved but unidentified. - The exact PGN muropeptide structures and BCF-1 binding interfaces remain to be biochemically defined. - Innate immunity (PMK-1) explains only part of UPRmt-mediated digestion inhibition; PMK-1-independent mechanisms are unresolved. - Experiments are performed under laboratory conditions on agar plates; ecological complexity in natural habitats may introduce additional variables not captured here.