Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model <i>Caenorhabditis elegans</i> and parasitic species

<div><p>Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode <i>Caenorhabditis elegans</i>, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: <i>unc-38</i>, <i>unc-63</i>, <i>unc-29</i>, <i>lev-1</i> and <i>lev-8</i>. These subunits form a functional L-AChR when heterologously expressed in <i>Xenopus laevis</i> oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to <i>C</i>. <i>elegans lev-8</i>. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the <i>C</i>. <i>elegans</i> L-AChR when expressed in <i>Xenopus</i> oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a ‘model hopping’ approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode <i>Haemonchus contortus</i> to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of <i>C</i>. <i>elegans lev-8</i> null mutants. The critical role of <i>acr-8</i> to <i>in vivo</i> drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for <i>Hco-acr-8</i> which reduced the sensitivity of <i>H</i>. <i>contortus</i> larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus <i>C</i>. <i>elegans</i>. More broadly, this highlights the limits of <i>C</i>. <i>elegans</i> as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds.</p>