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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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The Hco-mptl-1 locus, mRNA and protein (top) and mis-splicing mutations in the AAD mutants (bottom).Exons are represented by boxes, start codons by arrows. The 5′ region of the genomic DNA is not drawn to scale (double parallel bars). No hits were found in the H. contortus genome project for the exons and introns shown in clear grey. The spliced leader is shown in violet and mis-spliced exons in red. The signal peptide is shown in yellow and the predicted transmembrane domains (TM) in blue.
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ppat-1000380-g004: The Hco-mptl-1 locus, mRNA and protein (top) and mis-splicing mutations in the AAD mutants (bottom).Exons are represented by boxes, start codons by arrows. The 5′ region of the genomic DNA is not drawn to scale (double parallel bars). No hits were found in the H. contortus genome project for the exons and introns shown in clear grey. The spliced leader is shown in violet and mis-spliced exons in red. The signal peptide is shown in yellow and the predicted transmembrane domains (TM) in blue.

Mentions: In order to compare the Hco-mptl-1 sequences from the AAD-susceptible isolates and their AAD-mutant progeny, primers were designed at each extremity of the ORF (Hco-mptl-1_5′_frw3 and Hco-mptl-1_3′end_rev1) and the full length Hco-mptl-1 coding sequences amplified from cDNA from adults. A product of about 1800 bp was obtained for all isolates apart from the Hc-CRA AADM, which produced a shorter product of 1650 bp (Figure 3B). Sequencing clones of the latter revealed that they lacked either exon 4 or exon 15 (Figure 4, Hco-MPTL-1-m2 and m3). This was confirmed with primers flanking either exon 4 (Hco-mptl-1_5′_frw2 and Hco-mptl-1_rev8; Figure 3C) or exon 15 (Hco-mptl-1_frw6 and Hco-mptl-1_rev6; Figure 3D). PCR with a SL1 forward primer and a reverse primer in the Hco-mptl-1 coding sequence (Hco-mptl-1_rev1, product of about 1200 bp; Figure 3A) also produced shorter products (1000 bp and 850 bp; Figure 3A) from Hc-CRA AADM. The 850 bp product turned out to lack both exon 2 and exon 3 while the 1 kb product lacked exon 4 (Figure 4, Hco-MPTL-1-m1 and m2). The 1200 bp product was cloned from Hc-CRA AADM but contained only silent mutations compared to Hc-CRA. Loss of exon 4 caused a frame-shift leading to a premature stop of translation and a predicted Hco-MPTL-1 protein truncated at amino acid 19 (Figure 4). Loss of exon 15 also led to a premature stop codon that truncated the Hco-MPTL-1 protein at amino acid 448 (Figure 4). The mutation Hco-MPTL-1-m1 (loss of exon 2 and 3) did not cause a frame-shift but the loss of the signal peptide and the first 39 amino acids of the extracellular loop.


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)

The Hco-mptl-1 locus, mRNA and protein (top) and mis-splicing mutations in the AAD mutants (bottom).Exons are represented by boxes, start codons by arrows. The 5′ region of the genomic DNA is not drawn to scale (double parallel bars). No hits were found in the H. contortus genome project for the exons and introns shown in clear grey. The spliced leader is shown in violet and mis-spliced exons in red. The signal peptide is shown in yellow and the predicted transmembrane domains (TM) in blue.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000380-g004: The Hco-mptl-1 locus, mRNA and protein (top) and mis-splicing mutations in the AAD mutants (bottom).Exons are represented by boxes, start codons by arrows. The 5′ region of the genomic DNA is not drawn to scale (double parallel bars). No hits were found in the H. contortus genome project for the exons and introns shown in clear grey. The spliced leader is shown in violet and mis-spliced exons in red. The signal peptide is shown in yellow and the predicted transmembrane domains (TM) in blue.
Mentions: In order to compare the Hco-mptl-1 sequences from the AAD-susceptible isolates and their AAD-mutant progeny, primers were designed at each extremity of the ORF (Hco-mptl-1_5′_frw3 and Hco-mptl-1_3′end_rev1) and the full length Hco-mptl-1 coding sequences amplified from cDNA from adults. A product of about 1800 bp was obtained for all isolates apart from the Hc-CRA AADM, which produced a shorter product of 1650 bp (Figure 3B). Sequencing clones of the latter revealed that they lacked either exon 4 or exon 15 (Figure 4, Hco-MPTL-1-m2 and m3). This was confirmed with primers flanking either exon 4 (Hco-mptl-1_5′_frw2 and Hco-mptl-1_rev8; Figure 3C) or exon 15 (Hco-mptl-1_frw6 and Hco-mptl-1_rev6; Figure 3D). PCR with a SL1 forward primer and a reverse primer in the Hco-mptl-1 coding sequence (Hco-mptl-1_rev1, product of about 1200 bp; Figure 3A) also produced shorter products (1000 bp and 850 bp; Figure 3A) from Hc-CRA AADM. The 850 bp product turned out to lack both exon 2 and exon 3 while the 1 kb product lacked exon 4 (Figure 4, Hco-MPTL-1-m1 and m2). The 1200 bp product was cloned from Hc-CRA AADM but contained only silent mutations compared to Hc-CRA. Loss of exon 4 caused a frame-shift leading to a premature stop of translation and a predicted Hco-MPTL-1 protein truncated at amino acid 19 (Figure 4). Loss of exon 15 also led to a premature stop codon that truncated the Hco-MPTL-1 protein at amino acid 448 (Figure 4). The mutation Hco-MPTL-1-m1 (loss of exon 2 and 3) did not cause a frame-shift but the loss of the signal peptide and the first 39 amino acids of the extracellular loop.

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.

Show MeSH