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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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Expression of DEG-3 subfamily members in Haemonchus contortus.Hco-mptl-1, Hco-des-2H and Hco-deg-3H are expressed in adult as well as L3-larvae stages of both the Hc-CRA and Hc-Howick parental reference isolates (AAD naïve) as determined by reverse transcriptase PCR. Genomic DNA (gDNA) was included as a control.
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ppat-1000380-g002: Expression of DEG-3 subfamily members in Haemonchus contortus.Hco-mptl-1, Hco-des-2H and Hco-deg-3H are expressed in adult as well as L3-larvae stages of both the Hc-CRA and Hc-Howick parental reference isolates (AAD naïve) as determined by reverse transcriptase PCR. Genomic DNA (gDNA) was included as a control.

Mentions: As determined by PCR with gene-specific primers on genomic DNA, Hco-mptl-1 (Hco-mptl-1_frw6 and Hco-mptl-1_rev6), Hco-des-2H (Hco-des2_frw8 and Hco-des2_rev8) and Hco-deg-3H (Hco-deg3_frw1 and Hco-deg3_rev1) are present in the Hc-CRA and Hc-Howick parental isolates (Figure 2). Of the 3 products obtained for the Hco-mptl-1 gene, the smallest one (1478 bp) corresponded to Hco-mptl-1. The same primers were used for reverse transcriptase PCR on total RNA, showing that all 3 genes were expressed and spliced in L3-larvae as well as in adult nematodes (Figure 2).


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)

Expression of DEG-3 subfamily members in Haemonchus contortus.Hco-mptl-1, Hco-des-2H and Hco-deg-3H are expressed in adult as well as L3-larvae stages of both the Hc-CRA and Hc-Howick parental reference isolates (AAD naïve) as determined by reverse transcriptase PCR. Genomic DNA (gDNA) was included as a control.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000380-g002: Expression of DEG-3 subfamily members in Haemonchus contortus.Hco-mptl-1, Hco-des-2H and Hco-deg-3H are expressed in adult as well as L3-larvae stages of both the Hc-CRA and Hc-Howick parental reference isolates (AAD naïve) as determined by reverse transcriptase PCR. Genomic DNA (gDNA) was included as a control.
Mentions: As determined by PCR with gene-specific primers on genomic DNA, Hco-mptl-1 (Hco-mptl-1_frw6 and Hco-mptl-1_rev6), Hco-des-2H (Hco-des2_frw8 and Hco-des2_rev8) and Hco-deg-3H (Hco-deg3_frw1 and Hco-deg3_rev1) are present in the Hc-CRA and Hc-Howick parental isolates (Figure 2). Of the 3 products obtained for the Hco-mptl-1 gene, the smallest one (1478 bp) corresponded to Hco-mptl-1. The same primers were used for reverse transcriptase PCR on total RNA, showing that all 3 genes were expressed and spliced in L3-larvae as well as in adult nematodes (Figure 2).

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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