Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance

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.

Is anthelmintic resistance a concern for the control of human soil-transmitted helminths?

“…Polymorphism in b-tubulin isotype 1 seems to be most important for BZ resistance in H. contortus (Mottier and Prichard, 2008). Recently, our understanding of LEV resistance has greatly advanced with Boulin et al (2011) reporting that a number of changes in several genes, which together encode the acetylcholine receptor, result in loss of the LEV-sensitive acetylcholine receptor and thereby may cause resistance in veterinary nematodes. These data together with the findings of Kopp et al (2009) suggest that resistance to the nicotinic drugs is based on changes in the expression of the subunits that together form the receptor.…”

Ion channels and receptor as targets for the control of parasitic nematodes

“…(D) A nicotine-sensitive, but levamisole-insensitive receptor is formed by expression of the C. elegans ACR-16 subunit (Raymond et al, 2000). (E) Omission of Hco-ACR-8 from the receptor shown in panel B results in a nAChR with reduced sensitivity to levamisole, but increased sensitivity to pyrantel (Boulin et al, 2011). (F).…”

“…The H. contortus levamisole-sensitive receptor can be reconstituted in Xenopus oocytes by expressing the ACR-8, UNC-29, UNC-38 and UNC-63 subunits (Fig. 3) (Boulin et al, 2011). Removing the ACR-8 subunit from the H. contortus receptor produces a version which gives a smaller response to levamisole and acetylcholine, but is more sensitive to pyrantel, which is frequently thought to act at the same site at levamisole (Martin et al, 1991;Martin and Robertson, 2007).…”

“…(A) A levamisole-sensitive, but nicotine-insensitive nAChR was reconstituted in the Xenopus oocyte by co-expression of the C. elegans LEV-1, LEV_8, UNC-29, UNC-38 and UNC-63 subunits (Boulin et al, 2008). (B) A levamisole-sensitive nAChR reconstituted by co-expression of the indicated subunits from H. contortus (Boulin et al, 2011). (C) A levamisole-and oxantel-sensitive, but pyrantel-insensitive, nAChR reconstituted by co-expression of the indicated subunits from A. suum (Williamson et al, 2009).…”

The role of drug efflux systems in anthelmintic resistance in parasitic nematodes

“…Studies conducted on the pig nematode, Oesophagostomum dentatum, involving different members of this class including levamisole and pyrantel showed that there was a lower percentage of opened ionchannels in resistant worms compared to the counterparts, suggesting an increased desensitization of drug receptors in resistant isolates (Robertson et al, 1999(Robertson et al, , 2000. Neveu et al (2010) (Boulin et al, 2011). Further research showed that resistance to levamisole is associated with a spliced variant of the acr-8 gene, referred to as Hco-acr-8b which showed elevated expression in resistant worms and was undetectable in susceptible worms (Williamson et al, 2011).…”