title: Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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
  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.
authors.affiliation: Institut de Biologie de l'École Normale Supérieure, Biology Department, Paris, France. boulin@biologie.ens.fr
authors.forename: T
authors.initials: T
authors.lastname: Boulin
authors.forename: A
authors.initials: A
authors.lastname: Fauvin
authors.forename: C L
authors.initials: CL
authors.lastname: Charvet
authors.forename: J
authors.initials: J
authors.lastname: Cortet
authors.forename: J
authors.initials: J
authors.lastname: Cabaret
authors.forename: J-L
authors.initials: JL
authors.lastname: Bessereau
authors.forename: C
authors.initials: C
authors.lastname: Neveu
mesh_terms: D000018:Abomasum / Q000469:parasitology; D000818:Animals; D000969:Antinematodal Agents / Q000494:pharmacology*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance / Q000187:drug effects / Q000235:genetics; D005260:Female; D005753:Gastric Mucosa / Q000469:parasitology; D017238:Genes, Helminth; D006188:Haemonchiasis / Q000188:drug therapy / Q000469:parasitology / Q000662:veterinary; D006189:Haemonchus / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D007978:Levamisole / Q000494:pharmacology*; D008297:Male; D009865:Oocytes / Q000378:metabolism; D021122:Protein Subunits; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D012756:Sheep / Q000469:parasitology; D012757:Sheep Diseases / Q000188:drug therapy / Q000469:parasitology; D014982:Xenopus laevis / Q000235:genetics
chemical_list: D000969:Antinematodal Agents; D029742:Caenorhabditis elegans Proteins; D015801:Helminth Proteins; D021122:Protein Subunits; D011950:Receptors, Cholinergic; D007978:Levamisole