The nicotinic acetylcholine receptor EAT-2, a novel target for mitigating parasitic nematode infections

Plant parasitic nematodes (PPNs) are worms that infect all major crops and cause yield declines valued above US$150 billion annually. To mitigate the damage caused by these parasites, control strategies have relied on synthetic chemical nematicides that are effective in keeping infection below disease thresholds. The determinants of the nervous system that underpin key parasitic behaviours have been successful targets for pest mitigation. This includes nicotinic acetylcholine receptors (nAChRs), fast acting ligand gated ion channels that underpin key nerve and nerve-muscle physiology. They offer selective targets for naturally occurring and synthetic chemicals that effectively control pests in agriculture and veterinary medicine. In this study I investigated EAT-2, a nAChR that regulates feeding in Caenorhabditis elegans as a target to mitigate PPNs. EAT-2 plays an important role, it has a non-classical agonist binding site that makes it unique among members of the acetylcholine gated cation channels, and it has a unique dependence for association with the EAT-18 auxiliary protein for function. Thus, I hypothesized that EAT-2 is potentially a selective pharmacophore for mitigating PPNs. I initiated the study by establishing the presence of EAT-2 in PPNs and then utilized pharmacological evidence to suggest that EAT-2 regulates stylet thrusting in the PPN Globodera rostochiensis. Stylet thrusting is a parasitic behaviour required for hatching of infective juveniles (J2s), root invasion, and the establishment of feeding sites. This highlights that pharmacological modulation of EAT-2 affords a route to disrupt the parasitic behaviour and potentially break the lifecycle. I used C. elegans as an experimental platform to identify EAT-2 modulators by screening 192 compounds. This identified monepantel and 3 analogues, an analogue of epibatidine and rotenone as inhibitors of pharyngeal function. I validated this using functional expression of the EAT-2/EAT18 receptor in Xenopus oocytes and showed that these hits were antagonists of the receptor. Moreover, when exposed to J2s of G. rostochiensis, these hits inhibited stylet thrusting. Although these lead compounds bind EAT-2 they have broader efficacy across sub-types of nAChRs. However, the hits resolved by the screen and subsequent validation are new EAT-2 compounds that occupy this unique agonist binding site. Overall, the screening approach identified compounds that suggest that EAT-2 is a bona fide target for PPN mitigation. EAT-2 pharmacology holds promise for novel nematicidal potential.