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

Nematodes, crucial for soil ecosystem function, exhibit remarkable dietary diversity. Food availability and usage are major evolutionary forces, yet the specific signals initiating digestion remain unclear. Understanding these signals is key to understanding nematode success in diverse environments. *Caenorhabditis elegans*, a model organism, feeds on various microbes. A previous study established a food digestion system using inedible *Staphylococcus saprophyticus* (SS) and low-quality heat-killed *E. coli* (HK-*E. coli*). *C. elegans* grows only when both are present, indicating a signal from HK-*E. coli* activates digestion. This research explores the hypothesis that a common signal from bacterial food triggers nematode digestion, allowing for broader dietary range and improved survival in varied environments. The study aims to identify this common signal and elucidate its underlying mechanism. This study hypothesizes that there is a common signal from bacterial food activating the digestive system, enabling breakdown of inedible food and promoting nematode success in nature. Understanding this mechanism will provide crucial insights into the vital roles nematodes play in various ecosystems.

Nematodes represent approximately 80% of multicellular animals and play crucial roles in various ecosystems. Their ability to consume a diverse array of food sources is critical to their success. Previous studies have identified the importance of sophisticated nervous systems in food sensing and intake in various animals, but the specific signals that trigger digestion in nematodes remain poorly understood. The free-living nematode *C. elegans* serves as a valuable model for studying food sensing and behavior. Prior work demonstrated a system in which feeding *C. elegans* inedible *S. saprophyticus* (SS) along with heat-killed *E. coli* (HK-*E. coli*) allowed for growth, suggesting a digestion-activating signal from HK-*E. coli*. Bacterial membrane proteins have also been implicated in this process, alongside neural and innate immunity pathways. This builds upon the understanding that peptidoglycan (PGN) is involved in regulating innate immunity and mitochondrial homeostasis, making it a potential candidate for the digestion signal.

The study employed several key methodologies. First, a previously established food digestion system in *C. elegans* was used, focusing on the growth difference between animals fed inedible *S. saprophyticus* (SS) versus those fed SS along with HK-*E. coli*. This system allowed assessment of the ability to digest inedible food. *E. coli* mutants were screened to identify genes responsible for the digestion activation signal, focusing on mutants affecting peptidoglycan (PGN) synthesis and degradation (*ΔycbB* and *ΔygeR*). The effect of purified PGN extracted from various bacterial species (*E. coli*, *Bacillus subtilis*, *Enterococcus faecalis*) on animal growth was assessed. Different enzymes were used to treat PGN to identify the minimal active unit needed to stimulate digestion. To identify the host protein interacting with PGN, an RNAi screen was performed on *E. coli*-binding and PGN-binding proteins expressed in the intestine. *bcf-1*, encoding a glycosylated protein, was identified as a key player. In vitro binding assays were performed to confirm the interaction between PGN and BCF-1, and proteinase K was used to probe the role of PGN's peptide component. To assess the role of the mitochondrial unfolded protein response (UPRmt), expression levels of UPRmt markers (*hsp-6*) were measured in wild-type and *bcf-1* mutant worms under various feeding conditions. RNA sequencing data of *bcf-1* mutants were analyzed to identify pathways potentially involved in UPRmt regulation. RNAi knockdown was employed for neuropeptide genes to test the neuronal involvement in UPRmt activation. The role of innate immunity (PMK-1 pathway) in digestion defects was investigated using double mutants (*bcf-1;pmk-1*) and quantifying phosphorylated PMK-1 (p-PMK-1). Finally, a nematode adaptation assay was conducted by monitoring population growth of worms on different food combinations over multiple days.

The study revealed that bacterial PGN serves as a key signal activating food digestion in *C. elegans*. Specifically, PGN interacts with the intestinal glycosylated protein BCF-1. This interaction inhibits the mitochondrial unfolded protein response (UPRmt), promoting digestion and animal growth. The UPRmt pathway was shown to negatively regulate food digestion. The *bcf-1* gene is essential for PGN-mediated activation of digestion and is highly conserved in bacterial-feeding nematodes. Activation of the UPRmt in *bcf-1* mutants was shown to be cell non-autonomous and dependent on the neuropeptide NLP-3. Inhibition of the innate immune PMK-1 pathway could partially rescue the digestion defects observed in *bcf-1* mutants. The study found that animals with a functional PGN-BCF-1 system had significantly higher population numbers under complex feeding conditions. The findings are also validated in wild type nematodes isolated from natural environment. The results suggest that PGN-BCF-1 interaction acts as a "good-food signal", enhancing food digestion and promoting animal adaptation to diverse food sources.

The findings address the research question by identifying bacterial PGN as a critical signal for activating food digestion in *C. elegans*. The mechanism involves the conserved intestinal protein BCF-1, which interacts with PGN to suppress UPRmt and enhance digestion. This interaction has significant implications for nematode survival and adaptation, particularly their capacity to utilize a wide range of food sources. The study highlights a novel connection between the gut microbiota, innate immunity, and the nervous system in regulating digestion and adaptation. This inter-organ communication involves the release of neuropeptide NLP-3 from neurons in response to PGN-BCF-1 interaction. The results offer a new perspective on the complex interplay between host physiology and microbiota, highlighting the functional importance of bacterial cell wall components in maintaining host homeostasis and adaptation. This discovery has important ecological implications, considering the ecological roles of nematodes and the widespread presence of PGN in bacterial cell walls.

This study demonstrates that bacterial peptidoglycan (PGN) acts as a 'good-food signal' in *C. elegans*, activating digestion through interaction with the intestinal protein BCF-1. This interaction inhibits UPRmt via a neuropeptide signaling pathway involving NLP-3, promoting adaptation to diverse food resources. The results provide critical insights into nematode dietary adaptation and the role of bacterial cell wall components in host-microbe interactions. Further research should explore the broader implications of this PGN-BCF-1 interaction across other nematode species and examine the specific mechanisms involved in PMK-1-mediated suppression of digestion.

The study primarily uses *C. elegans* as a model organism, limiting the generalizability of findings to other nematode species. While the study suggests that the PGN-BCF-1 interaction is conserved among bacterial-feeding nematodes, further research is needed to verify this across a wider range of species. The study focused on the role of UPRmt and PMK-1 pathways in digestion, and additional pathways might contribute to the observed effects. Finally, the specific structural features of PGN required for BCF-1 interaction and subsequent digestion activation remain to be fully characterized.