Identification of microbe-derived olfactory signals and their receptors in Caenorhabditis elegans

The acquisition of essential nutrients through diet is crucial for the survival of animals. Animals utilise their sensory abilities to locate food and evade threats in their surroundings. However, the molecular details of the olfactory basis of foraging remain understudied. In this study, using the nematode Caenorhabditis elegans and its native microbiota, we have investigated the olfactory bases of foraging and its molecular underpinnings. C. elegans is a bacterivorous worm found in rotten fruit and compost. This environment is rich in bacterial diversity, offering worms a myriad of dietary options. However, the role of specific bacterial odours in C. elegans’ foraging behaviour remains unclear. In the first part of my thesis, we asked if C. elegans exploits olfactory cues to forage for essential amino acid-rich (EAA) diets. Using the native microbiome of C. elegans, we show that worms rely on olfaction to select leucine-enriched bacteria. Using gas chromatography-mass spectrometry, we find that leucine-enriched bacteria produce isoamyl alcohol (IAA) in the highest abundance in their odour bouquet. We show that leucine enrichment diverts the bacterial metabolic flux into IAA biosynthesis. C. elegans primarily uses AWA and AWC neurons for sensing odours. Using odour sensory mutant lines of worms, we find that attraction to the majority of the attractive odours produced by preferred bacteria, as well as foraging for a leucine-enriched diet, is mediated via the AWC neurons. Using adaptation assays, we find that prior exposure to IAA diminishes the diet preference of worms for preferred diets, suggesting IAA is the most relevant foraging signal for C. elegans. Finally, we find that several wild isolates of C. elegans display robust responses to IAA, emphasising its ecological relevance. In the second part of my thesis, we investigated the function of G-protein-coupled receptors (GPCRs) in sensing microbial odours. C. elegans genome encodes for ~1300 GPCRs with a large repertoire (~335) expressed in odour sensory neurons. However, until now, only six receptors have been deorphanised. We hypothesised that the ligand for these receptors may be found in the headspace of the C. elegans microbiome. By testing the chemotaxis response of CRISPR-deletion mutants of specific GPCRs to microbial odours, we uncovered SRD-12 as a receptor for IAA. By performing neuron-specific rescue of srd-12, we find that SRD-12 is a cognate receptor for IAA and a mediator of dietary decisions in worms. By extensive genetic analysis, we find that worms can utilise more than one receptor to sense IAA at high concentrations. Furthermore, by performing in silico analysis and site-directed mutagenesis, we demonstrate that the tyrosine residue at position 253 is crucial for the activation of SRD-12 by IAA. Overall, we discover an odour-receptor pair essential for foraging in worms. Finally, we explored the potential of using C. elegans for heterologous expression of mammalian olfactory receptors. Several studies have shown that in humans, the expression of olfactory receptors (ORs) is not limited to the nasal epithelium, but rather expands to organs like the gut, lungs, kidneys, etc. ORs in the gut regulate host physiology. However, most of these ORs are orphaned. C. elegans, a genetically amenable system with GPCRs expressing machinery, can serve as an excellent model for the heterologous expression of mammalian ORs. Heterologous expression of mammalian ORs in C. elegans can enable quick identification of their ligand by employing chemotaxis assays.