Kill two birds with one stone: novel levamisole and pyrantel receptors and mechanisms of resistance in Haemonchus contortus

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英语. Levamisole and pyrantel are important drugs of the anthelmintic arsenal used in livestock and humans. Anthelmintic resistance is a growing issue and we do believe that unravelling anthelmintic target sites of action constitutes a pre-requisite step towards the identification of drug-resistance mechanisms. Levamisole-sensitive acetylcholine receptors (L-AChRs), are hetero-pentameric ligandgated ion-channels that mediate fast excitatory neurotransmission at the nematode neuromuscular junctions. The canonical Caenorhabditis elegans L-AChR is made up of five different subunits, the three α-type UNC-63, UNC-38, LEV-8 and the two non-α UNC-29 and LEV-1. In contrast to C. elegans, three main subunit changes occurred in the gastrointestinal nematode of small ruminants Haemonchus contortus: the UNC-29 subunit duplicated in four copies, the LEV-1 subunit had no signal peptide and there was no orthologue of LEV-8. We previously characterized several L-AChR subtypes of H. contortus using the Xenopus oocyte expression system. The L-AChR subtype 1.1 is composed of the UNC-29.1, UNC-38, UNC-63 and ACR-8 subunits and is more sensitive to levamisole than acetylcholine. The replacement of UNC-29.1 by the UNC-29.3 or UNC-29.4 copies gave rise to the respective L-AChR subtypes 1.3 and 1.4, which both exhibited dissimilar pharmacology patterns than the L-AChR subtype 1.1. Interestingly, the omission of ACR-8 led to the L-AChR subtype 2.1 which was more sensitive to pyrantel than levamisole. However the UNC-29.2 copy failed to replace UNC-29.1 and LEV-1 was never proven to be functional. In addition, we identified the expression of a truncated form of UNC-63 (Hco-UNC-63b) in the levamisole- and pyrantel-resistant isolate RHS6 evidencing a strong dominant negative effect on the functioning of the L-AChR. Taking the C. elegans L-AChR as a reference receptor led us to assemble new subunit combinations including both Hco-UNC-29.2 and Hco-LEV-1 subunits. Hence, we reconstituted two novel subtypes of H. contortus L-AChRs showing distinct pharmacologies. These data reinforced the critical role for Hco-ACR-8 in levamisole sensitivity. Furthermore, we identified a truncated spliced form of Hco-LEV-1 in the Kokstadt multidrug-resistant isolate and showed that it hampered the function of L-AChRs when co-expressed with its full-length counterpart. Altogether, these studies highlight the wide repertoire of L-AChR subtypes in parasitic nematodes and bring new insight to our understanding of levamisole resistance.