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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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Detection of a nonsense mutation in Hc-Howick AADM worms.(A) Direct sequencing of RT-PCR products revealed a transversion in exon 6 from G to T (arrow) in the Hco-mptl-1 gene that leads to a premature stop at codon 93 (TAA; shown in red) in about 80% of Hc-Howick AADM mutants as estimated from the electropherogram. (B) The point mutation creates a restriction site for the endonuclease BfrI (CTTAAG; underlined). Only the product amplified from cDNA of the Hc-Howick AADM mutant could be digested.
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ppat-1000380-g006: Detection of a nonsense mutation in Hc-Howick AADM worms.(A) Direct sequencing of RT-PCR products revealed a transversion in exon 6 from G to T (arrow) in the Hco-mptl-1 gene that leads to a premature stop at codon 93 (TAA; shown in red) in about 80% of Hc-Howick AADM mutants as estimated from the electropherogram. (B) The point mutation creates a restriction site for the endonuclease BfrI (CTTAAG; underlined). Only the product amplified from cDNA of the Hc-Howick AADM mutant could be digested.

Mentions: No obvious mutations such as mis-spliced exons were detected in the Hc-Howick AADM isolates. When sequencing the Hco-mptl-1 coding regions (SL1 and Hco-mptl-1_rev6) from both susceptible and AAD-1566-mutant Howick isolates, a transversion from G277 to T in exon 6 of the Hco-mptl-1 gene was observed that led to a premature stop codon (E93*; Figure 6). Direct sequencing of RT-PCR products (using Hco-mptl-1_frw4 and Hco-mptl-1_rev1 primers) revealed that about 80% of the Hc-Howick AADM cDNAs, as estimated from the electropherogram [27], carried a T at position 277 (Figure 6A). The point mutation underlying E93* creates a restriction site for the endonuclease BfrI (recognition site: CTTAAG) that lent itself for RFLP analysis. Only the PCR product amplified from cDNA of Hc-Howick AADM was digested by BfrI (Figure 6B). As expected from the sequencing, a small proportion (about 20%) of the product was not cut, indicating that not all of the Hco-mptl-1 genes from Hc-Howick AADM population carried the G277T mutation. When this BfrI-unrestricted product from Hc-Howick AADM was excised from an agarose gel, cloned and sequenced, a further polymorphism was detected that led to skipping of exon 8 (Figure 4, Hco-MPTL-1-m6). As this exon is very short (22 bases), it was impossible to discriminate between mutant and parental wild type PCR products (Figure 3). Loss of exon 8 causes a frame-shift leading to a premature stop codon and a predicted Hco-MPTL-1 protein truncated at amino acid 166 (Figure 4). A minority of the Hco-mptl-1 PCR products obtained from Hc-Howick AADM did not contain any major mutations. These sequences could come from AAD-susceptible individuals within the H. contortus Howick AADM populations or from AAD-mutant individuals that carry other, yet to be identified mutations.


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)

Detection of a nonsense mutation in Hc-Howick AADM worms.(A) Direct sequencing of RT-PCR products revealed a transversion in exon 6 from G to T (arrow) in the Hco-mptl-1 gene that leads to a premature stop at codon 93 (TAA; shown in red) in about 80% of Hc-Howick AADM mutants as estimated from the electropherogram. (B) The point mutation creates a restriction site for the endonuclease BfrI (CTTAAG; underlined). Only the product amplified from cDNA of the Hc-Howick AADM mutant could be digested.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000380-g006: Detection of a nonsense mutation in Hc-Howick AADM worms.(A) Direct sequencing of RT-PCR products revealed a transversion in exon 6 from G to T (arrow) in the Hco-mptl-1 gene that leads to a premature stop at codon 93 (TAA; shown in red) in about 80% of Hc-Howick AADM mutants as estimated from the electropherogram. (B) The point mutation creates a restriction site for the endonuclease BfrI (CTTAAG; underlined). Only the product amplified from cDNA of the Hc-Howick AADM mutant could be digested.
Mentions: No obvious mutations such as mis-spliced exons were detected in the Hc-Howick AADM isolates. When sequencing the Hco-mptl-1 coding regions (SL1 and Hco-mptl-1_rev6) from both susceptible and AAD-1566-mutant Howick isolates, a transversion from G277 to T in exon 6 of the Hco-mptl-1 gene was observed that led to a premature stop codon (E93*; Figure 6). Direct sequencing of RT-PCR products (using Hco-mptl-1_frw4 and Hco-mptl-1_rev1 primers) revealed that about 80% of the Hc-Howick AADM cDNAs, as estimated from the electropherogram [27], carried a T at position 277 (Figure 6A). The point mutation underlying E93* creates a restriction site for the endonuclease BfrI (recognition site: CTTAAG) that lent itself for RFLP analysis. Only the PCR product amplified from cDNA of Hc-Howick AADM was digested by BfrI (Figure 6B). As expected from the sequencing, a small proportion (about 20%) of the product was not cut, indicating that not all of the Hco-mptl-1 genes from Hc-Howick AADM population carried the G277T mutation. When this BfrI-unrestricted product from Hc-Howick AADM was excised from an agarose gel, cloned and sequenced, a further polymorphism was detected that led to skipping of exon 8 (Figure 4, Hco-MPTL-1-m6). As this exon is very short (22 bases), it was impossible to discriminate between mutant and parental wild type PCR products (Figure 3). Loss of exon 8 causes a frame-shift leading to a premature stop codon and a predicted Hco-MPTL-1 protein truncated at amino acid 166 (Figure 4). A minority of the Hco-mptl-1 PCR products obtained from Hc-Howick AADM did not contain any major mutations. These sequences could come from AAD-susceptible individuals within the H. contortus Howick AADM populations or from AAD-mutant individuals that carry other, yet to be identified mutations.

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