Graphical abstract

BACKGROUND AND PURPOSE The cholinergic agonist levamisole is widely used to treat parasitic nematode infestations. This anthelmintic drug paralyses worms by activating a class of levamisole‐sensitive acetylcholine receptors (L‐AChRs) expressed in nematode muscle cells. However, levamisole efficacy has been compromised by the emergence of drug‐resistant parasites, especially in gastrointestinal nematodes such as Haemonchus contortus. We report here the first functional reconstitution and pharmacological characterization of H. contortus L‐AChRs in a heterologous expression system. EXPERIMENTAL APPROACH In the free‐living nematode Caenorhabditis elegans, five AChR subunit and three ancillary protein genes are necessary in vivo and in vitro to synthesize L‐AChRs. We have cloned the H. contortus orthologues of these genes and expressed them in Xenopus oocytes. We reconstituted two types of H. contortus L‐AChRs with distinct pharmacologies by combining different receptor subunits. KEY RESULTS The Hco‐ACR‐8 subunit plays a pivotal role in selective sensitivity to levamisole. As observed with C. elegans L‐AChRs, expression of H. contortus receptors requires the ancillary proteins Hco‐RIC‐3, Hco‐UNC‐50 and Hco‐UNC‐74. Using this experimental system, we demonstrated that a truncated Hco‐UNC‐63 L‐AChR subunit, which was specifically detected in a levamisole‐resistant H. contortus isolate, but not in levamisole‐sensitive strains, hampers the normal function of L‐AChRs, when co‐expressed with its full‐length counterpart. CONCLUSIONS AND IMPLICATIONS We provide the first functional evidence for a putative molecular mechanism involved in levamisole resistance in any parasitic nematode. This expression system will provide a means to analyse molecular polymorphisms associated with drug resistance at the electrophysiological level.

SummaryBackground and PurposeControl of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR‐16, from a nematode parasite.Experimental ApproachUsing RT‐PCR, molecular cloning and two‐electrode voltage clamp electrophysiology, we localized acr‐16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr‐16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested.Key ResultsAmino acid sequence comparison with vertebrate nAChR subunits revealed ACR‐16 to be most closely related to α7 receptors, but with some striking distinctions. acr‐16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, bephenium, tribendimidine and pyrantel) did not activate Asu‐ACR‐16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3‐bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu‐ACR‐16 was insensitive to α‐bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu‐ACR‐16 had lower calcium permeability than α7 receptors.Conclusions and ImplicationsWe suggest that ACR‐16 has diverse tissue‐dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.

Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important “classical” anthelmintics like levamisole and pyrantel, as well as “novel” anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.