Haemonchus contortus acetylcholine receptors of the DEG-3 subfamily and their role in sensitivity to monepantel.
Gastro-intestinal nematodes in ruminants, especially Haemonchus contortus, are a global threat to sheep and cattle farming. The emergence of drug resistance, and even multi-drug resistance to the currently available classes of broad spectrum anthelmintics, further stresses the need for new drugs active against gastro-intestinal nematodes. A novel chemical class of synthetic anthelmintics, the Amino-Acetonitrile Derivatives (AADs), was recently discovered and the drug candidate AAD-1566 (monepantel) was chosen for further development. Studies with Caenorhabditis elegans suggested that the AADs act via nicotinic acetylcholine receptors (nAChR) of the nematode-specific DEG-3 subfamily. Here we identify nAChR genes of the DEG-3 subfamily from H. contortus and investigate their role in AAD sensitivity. Using a novel in vitro selection procedure, mutant H. contortus populations of reduced sensitivity to AAD-1566 were obtained. Sequencing of full-length nAChR coding sequences from AAD-susceptible H. contortus and their AAD-1566-mutant progeny revealed 2 genes to be affected. In the gene monepantel-1 (Hco-mptl-1, formerly named Hc-acr-23H), a panel of mutations was observed exclusively in the AAD-mutant nematodes, including deletions at intron-exon boundaries that result in mis-spliced transcripts and premature stop codons. In the gene Hco-des-2H, the same 135 bp insertion in the 5' UTR created additional, out of frame start codons in 2 independent H. contortus AAD-mutants. Furthermore, the AAD mutants exhibited altered expression levels of the DEG-3 subfamily nAChR genes Hco-mptl-1, Hco-des-2H and Hco-deg-3H as quantified by real-time PCR. These results indicate that Hco-MPTL-1 and other nAChR subunits of the DEG-3 subfamily constitute a target for AAD action against H. contortus and that loss-of-function mutations in the corresponding genes may reduce the sensitivity to AADs.
Novartis Centre de Recherche Santé Animale, St Aubin (FR), Switzerland.
Lucien
L
Rufener
Pascal
P
Mäser
Isabel
I
Roditi
Ronald
R
Kaminsky
D000595:Amino Acid Sequence; D000607:Aminoacetonitrile / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000871:Anthelmintics / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics*; D015723:Gene Library; D017238:Genes, Helminth; D006189:Haemonchus / Q000235:genetics* / Q000378:metabolism; D015801:Helminth Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D011978:Receptors, Nicotinic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000871:Anthelmintics; D015801:Helminth Proteins; D011978:Receptors, Nicotinic; D000607:Aminoacetonitrile; C533978:monepantel