Examining Gut-Brain Axis Interactions Through Entomopathogenic Nematodes and Their Bacterial Endosymbionts

Nematodes are emerging as important models for understanding the molecular basis of animal-microbe interactions, including how bacterial products impact animal behaviors. Entomopathogenic nematodes, like Steinernema, are especially attractive models because of their symbiotic interactions with single Xenorhabdus bacterial partners. Previous studies demonstrated how S. carpocapsae nematodes grown on X. nematophila with mutations in the gene encoding the stress-response transcription factor rpoS showed differences in egg-laying behavior when compared to wild-type controls. The RpoS regulon includes the hpaBC locus, encoding homoprotocatechuate pathway enzymes that can act on intermediates in dopamine metabolism. We hypothesize that this egg-laying phenotype may result from the modulation of nematode dopamine levels through the action of HpaBC. To test this hypothesis, we aimed to create a Xenorhabdus strain with tunable hpaBC expression using X. griffiniae, the endosymbiont of S. hermaphroditum, a species for which genetic tools are rapidly emerging. We created plasmids to modify the X. griffiniae hpaBC locus by cloning 200 bp, 300 bp, and 500 bp regions of hpaBC into an existing arabinose-inducible expression system, the native hpaBC promoter with a pBAD arabinose-inducible promoter. After conjugation of the plasmid into X. griffiniae, exconjugates were confirmed by PCR and will be used in future in vivo studies to examine potential links between symbiont-derived HpaBC and dopamine synthesis within the host, including whether HpaBC activity regulates typical neurodevelopment and dopamine-mediated behaviors like egg-laying. Since HpaBC regulates the homoprotocatechuate pathway in numerous bacterial strains, future investigations may highlight HpaBC homologs as dopamine regulators in other animal hosts.