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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.

SUBMITTER: Boulin T

PROVIDER: S-EPMC3221097 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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

Boulin T T Fauvin A A Charvet C L CL Cortet J J Cabaret J J Bessereau J-L JL Neveu C C

British journal of pharmacology 20111101 5

<h4>Background and purpose</h4>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 ...[more]

PMID: 21486278

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Project description:Acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels involved in the neurotransmission of both vertebrates and invertebrates. A number of anthelmintic compounds like levamisole and pyrantel target the AChRs of nematodes producing spastic paralysis of the worms. The muscle AChRs of nematode parasites fall into three pharmacological classes that are preferentially activated by the cholinergic agonists levamisole (L-type), nicotine (N-type) and bephenium (B-type), respectively. Despite a number of studies of the B-type AChR in parasitic species, this receptor remains to be characterized at the molecular level. Recently, we have reconstituted and functionally characterized two distinct L-AChR subtypes of the gastro-intestinal parasitic nematode Haemonchus contortus in the Xenopus laevis oocyte expression system by providing the cRNAs encoding the receptor subunits and three ancillary proteins (Boulin et al. in Br J Pharmacol 164(5):1421-1432, 2011). In the present study, the effect of the bephenium drug on Hco-L-AChR1 and Hco-L-AChR2 subtypes was examined using the two-microelectrode voltage-clamp technique. We demonstrate that bephenium selectively activates the Hco-L-AChR1 subtype made of Hco-UNC-29.1, Hco-UNC-38, Hco-UNC-63, Hco-ACR-8 subunits that is more sensitive to levamisole than acetylcholine. Removing the Hco-ACR-8 subunit produced the Hco-L-AChR2 subtype that is more sensitive to pyrantel than acetylcholine and partially activated by levamisole, but which was bephenium-insensitive indicating that the bephenium-binding site involves Hco-ACR-8. Attempts were made to modify the subunit stoichiometry of the Hco-L-AChR1 subtype by injecting five fold more cRNA of individual subunits. Increased Hco-unc-29.1 cRNA produced no functional receptor. Increasing Hco-unc-63, Hco-unc-38 or Hco-acr-8 cRNAs did not affect the pharmacological characteristics of Hco-L-AChR1 but reduced the currents elicited by acetylcholine and the other agonists. Here, we provide the first description of the molecular composition and functional characteristics of any invertebrate bephenium-sensitive receptor.

Project description:The mechanism of resistance to the anthelmintic levamisole in parasitic nematodes is poorly understood, although there is some evidence implicating changes in expression of nicotinic acetylcholine receptor (nAChR) subunit genes. Hence, in order to define levamisole resistance mechanisms in some Australian field-derived isolates of Haemonchus contortus we examined gene expression patterns and SNPs in nAChR subunit genes, as well as expression levels for P-glycoprotein (P-gp) and receptor ancillary protein genes, in various life stages of one levamisole-sensitive and three levamisole-resistant isolates of this species. Larvae of two isolates showed high-level resistance to levamisole (resistance ratios at the IC50 > 600) while the third isolate showed a degree of heterogeneity, with a resistance factor of only 1.1-fold at the IC50 alongside the presence of a resistant subpopulation. Transcription patterns for nAChR subunit genes showed a great degree of variability across the different life stages and isolates. The most consistent observation was the down-regulation of Hco-unc-63a in adults of all resistant isolates. Transcription of this gene was also reduced in the L3 stage of the two most resistant isolates, highlighting its potential as a resistance marker in the readily accessible free-living stages. There was down regulation of all four Hco-unc-29 paralogs in adults of one resistant isolate. There were no consistent changes in expression of P-gps or ancillary protein genes across the resistant isolates. The present study has demonstrated a complex pattern of nAChR subunit gene expression in H. contortus, and has highlighted several instances where reduced expression of subunit genes (Hco-unc-63a, Hco-unc-29) may be associated with the observed levamisole resistance. The data also suggests that it will be difficult to detect resistance using gene transcription-based methods on pooled larval samples from isolates containing only a resistant subpopulation due to the averaging of gene expression data across the whole population.

Dataset Information

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

Publications

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

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