Haemonchus contortus Acetylcholine Receptors of the DEG-3 Subfamily and Their Role in Sensitivity to Monepantel

Abstract

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.

Monepantel Irreversibly Binds to and Opens Haemonchus contortus MPTL-1 and Caenorhabditis elegans ACR-20 Receptors

“…RNA extraction, cDNA synthesis, and PCR amplification of mptl-1 were performed, as described previously in Rufener et al (2009). In brief, total RNA was extracted from a pool of adult nematodes, and 1 mg total RNA (DNase-treated) was reverse-transcribed to cDNA using a (dT)30 primer and SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA).…”

“…The same procedure was used to obtain the mptl-1 sequence with a 127-bp deletion (removing the entire exon 15) from the H. contortus AAD-mutant nematodes. The Hco-ric-3 gene was amplified using the same primers as described in Rufener et al (2009). The selected inserts were subcloned into a pT7-TS transcription vector (that introduces Xenopus laevis b-globin untranslated DNA to the 59 and 39 end of the gene) via the restriction sites inserted in the primers.…”

“…The development of nematodes resistant to monepantel has led to identification of the acr-23 gene in the nonparasitic species Caenorhabditis elegans and a homologous gene, mptl-1, in the parasitic nematode H. contortus (Kaminsky et al, 2008;Rufener et al, 2009). Both genes belong to the nematode-specific DEG-3 subfamily of ionotropic acetylcholine receptors (AChR).…”

Monepantel Allosterically Activates DEG-3/DES-2 Channels of the Gastrointestinal NematodeHaemonchus contortus

“…A molecular genetic approach with the nematode Caenorhabditis elegans indicated that the AADs, too, acted via ligand-gated ion channels: in mutagenized worms subjected to sublethal doses of AAD, loss of sensitivity was mapped to a gene encoding a putative nicotinic acetylcholine receptor (nAChR) ␣ subunit, ACR-23 (Kaminsky et al, 2008a). Related genes were found to be involved in H. contortus, in which loss-of-sensitivity mutants carried a panel of nonsense mutations and mis-splicing mutations in the two nAChR genes Hco-des-2 and Hco-mptl-1 (Rufener et al, 2009).…”

Acquired resistance to monepantel in C. elegans – what about parasitic nematodes?

“…1 Similarly, mutations in the homolog mptl-1 were isolated from AAD-resistant H. contortus species. 13 Since monepantel does not kill C. elegans, one can easily imagine that under constant selective pressure, the more tolerant individuals are given the possibility to escape and to reproduce. With parasitic nematodes such as H. contortus, paralysis would result in its elimination through the feces.…”