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Haemonchus contortus acetylcholine receptors of the DEG-3 subfamily and their role in sensitivity to monepantel.

Rufener L, Mäser P, Roditi I, Kaminsky R - PLoS Pathog. (2009)

Bottom Line: Using a novel in vitro selection procedure, mutant H. contortus populations of reduced sensitivity to AAD-1566 were obtained.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.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.

View Article: PubMed Central - PubMed

Affiliation: Novartis Centre de Recherche Santé Animale, St Aubin (FR), Switzerland.

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.

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Hc-CRA AADM mutants lack the splice acceptor site of intron 14.Sequencing of PCR products amplified from genomic DNA revealed a 10 bp deletion in the Hc-CRA AADM mutant that encompasses the predicted splice acceptor site (bold). The blue box corresponds to the end of intron 14 and the yellow box to the start of exon 15. Asterisks denote bases identical throughout all 12 sequenced clones.
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ppat-1000380-g005: Hc-CRA AADM mutants lack the splice acceptor site of intron 14.Sequencing of PCR products amplified from genomic DNA revealed a 10 bp deletion in the Hc-CRA AADM mutant that encompasses the predicted splice acceptor site (bold). The blue box corresponds to the end of intron 14 and the yellow box to the start of exon 15. Asterisks denote bases identical throughout all 12 sequenced clones.

Mentions: To understand the molecular basis of exon loss in the Hc-CRA AADM isolate, PCR primers Hco-mptl-1_frw8 and Hco-mptl-1_rev6 (Table S1) were designed to flank the mis-spliced exon 15. PCR was performed using genomic DNA as a template. Sequencing of cloned PCR products revealed a 10 bp deletion upstream of exon 15 in the Hc-CRA AADM mutant that encompasses the predicted splice acceptor site (UUUCAG; Figure 5). Presumably, the splicing machinery is not able to identify the end of intron 14 and uses the next splice acceptor site (intron 15). This would explain why exon 15 is skipped (Figure 4, Hco-MPTL-1-m3). Joining of exon 14 to exon 16 causes a frame-shift leading to a premature stop codon. With primers flanking exon 4 (Hco-mptl-1_frw10/gDNA and Hco-mptl-1_rev8; Table S1), a 323 bp deletion was detected consisting of the end of intron 3 (206 bp) and most of exon 4 (117 bp). Again, loss of the predicted splice acceptor site at the end of intron 3 may explain the observed loss of exon 4 (Figure 4, Hco-MPTL-1-m2), since the splicing machinery will use the next available splice acceptor site (intron 4), joining exon 3 and exon 5. The resulting frame-shift causes a premature stop at codon 19 (TGA), terminating translation after the signal peptide (Figure 4, Hco-MPTL-1-m2).


Haemonchus contortus acetylcholine receptors of the DEG-3 subfamily and their role in sensitivity to monepantel.

Rufener L, Mäser P, Roditi I, Kaminsky R - PLoS Pathog. (2009)

Hc-CRA AADM mutants lack the splice acceptor site of intron 14.Sequencing of PCR products amplified from genomic DNA revealed a 10 bp deletion in the Hc-CRA AADM mutant that encompasses the predicted splice acceptor site (bold). The blue box corresponds to the end of intron 14 and the yellow box to the start of exon 15. Asterisks denote bases identical throughout all 12 sequenced clones.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2662886&req=5

ppat-1000380-g005: Hc-CRA AADM mutants lack the splice acceptor site of intron 14.Sequencing of PCR products amplified from genomic DNA revealed a 10 bp deletion in the Hc-CRA AADM mutant that encompasses the predicted splice acceptor site (bold). The blue box corresponds to the end of intron 14 and the yellow box to the start of exon 15. Asterisks denote bases identical throughout all 12 sequenced clones.
Mentions: To understand the molecular basis of exon loss in the Hc-CRA AADM isolate, PCR primers Hco-mptl-1_frw8 and Hco-mptl-1_rev6 (Table S1) were designed to flank the mis-spliced exon 15. PCR was performed using genomic DNA as a template. Sequencing of cloned PCR products revealed a 10 bp deletion upstream of exon 15 in the Hc-CRA AADM mutant that encompasses the predicted splice acceptor site (UUUCAG; Figure 5). Presumably, the splicing machinery is not able to identify the end of intron 14 and uses the next splice acceptor site (intron 15). This would explain why exon 15 is skipped (Figure 4, Hco-MPTL-1-m3). Joining of exon 14 to exon 16 causes a frame-shift leading to a premature stop codon. With primers flanking exon 4 (Hco-mptl-1_frw10/gDNA and Hco-mptl-1_rev8; Table S1), a 323 bp deletion was detected consisting of the end of intron 3 (206 bp) and most of exon 4 (117 bp). Again, loss of the predicted splice acceptor site at the end of intron 3 may explain the observed loss of exon 4 (Figure 4, Hco-MPTL-1-m2), since the splicing machinery will use the next available splice acceptor site (intron 4), joining exon 3 and exon 5. The resulting frame-shift causes a premature stop at codon 19 (TGA), terminating translation after the signal peptide (Figure 4, Hco-MPTL-1-m2).

Bottom Line: Using a novel in vitro selection procedure, mutant H. contortus populations of reduced sensitivity to AAD-1566 were obtained.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.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.

View Article: PubMed Central - PubMed

Affiliation: Novartis Centre de Recherche Santé Animale, St Aubin (FR), Switzerland.

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.

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