Limits...
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.

Show MeSH
Phylogenetic analysis of the DEG-3 subfamily of nAChR.ClustalW dendrogram [49] of nAChRs subunits of the DEG-3 subfamily (amino acid sequences) from B. malayi (Bma; grey), C. briggsae (Cbr; green), C. elegans (Cel; blue), and H. contortus (Hco; orange). Two isoforms (a and b) of Cel-ACR-20 are shown. The scale bar indicates the number of amino acid substitutions per site, bootstrapping values are shown in percent positives of 1000 rounds. Tree construction and bootstrapping was initially performed on full-length sequences only; the partial sequences (dashed lines, thin characters) were added subsequently based on a second ClustalW guide tree.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2662886&req=5

ppat-1000380-g001: Phylogenetic analysis of the DEG-3 subfamily of nAChR.ClustalW dendrogram [49] of nAChRs subunits of the DEG-3 subfamily (amino acid sequences) from B. malayi (Bma; grey), C. briggsae (Cbr; green), C. elegans (Cel; blue), and H. contortus (Hco; orange). Two isoforms (a and b) of Cel-ACR-20 are shown. The scale bar indicates the number of amino acid substitutions per site, bootstrapping values are shown in percent positives of 1000 rounds. Tree construction and bootstrapping was initially performed on full-length sequences only; the partial sequences (dashed lines, thin characters) were added subsequently based on a second ClustalW guide tree.

Mentions: The putative target of the AADs in C. elegans, ACR-23, is a member of the nematode-specific DEG-3 family of nAChR alpha subunits. A tblastn search [19] with DEG-3 members against the (incomplete) H. contortus genome database (http://www.sanger.ac.uk/Projects/H_contortus) returned strong hits from different contigs, coding for a total of 6 different DEG-3 subfamily nAChR subunit homologues. However, the lack of overlap between the different contigs precluded the assembly of full length coding sequences. The predicted H. contortus proteins were named Hco-MPTL-1 (accession number: contig_0024907; contig_0033952; contig_0079482; haem-240m02.q1k; contig_0053297; contig_069357), Hco-DES-2H (contig_0064641), Hco-DEG-3H (contig_0075200; contig_0075201), Hco-ACR-24H (contig_0003482; contig_0064300), Hco-ACR-5H (contig_0106281; contig_0023143) and Hco-ACR-17H (contig_0101516; contig_0101514). For Hco-MPTL-1, Hco-DES-2H and Hco-DEG-3H, full-length coding sequences were obtained by cDNA library screening or RACE-PCR, respectively (see below). Figure 1 shows the position of the H. contortus sequences in a phylogenetic tree of the DEG-3 subfamily nAChR from C. elegans, C. briggsae and Brugia malayi. Note that an incomplete sequence of Hco-MPTL-1 was previously named Hc-ACR-23H [12].


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)

Phylogenetic analysis of the DEG-3 subfamily of nAChR.ClustalW dendrogram [49] of nAChRs subunits of the DEG-3 subfamily (amino acid sequences) from B. malayi (Bma; grey), C. briggsae (Cbr; green), C. elegans (Cel; blue), and H. contortus (Hco; orange). Two isoforms (a and b) of Cel-ACR-20 are shown. The scale bar indicates the number of amino acid substitutions per site, bootstrapping values are shown in percent positives of 1000 rounds. Tree construction and bootstrapping was initially performed on full-length sequences only; the partial sequences (dashed lines, thin characters) were added subsequently based on a second ClustalW guide tree.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000380-g001: Phylogenetic analysis of the DEG-3 subfamily of nAChR.ClustalW dendrogram [49] of nAChRs subunits of the DEG-3 subfamily (amino acid sequences) from B. malayi (Bma; grey), C. briggsae (Cbr; green), C. elegans (Cel; blue), and H. contortus (Hco; orange). Two isoforms (a and b) of Cel-ACR-20 are shown. The scale bar indicates the number of amino acid substitutions per site, bootstrapping values are shown in percent positives of 1000 rounds. Tree construction and bootstrapping was initially performed on full-length sequences only; the partial sequences (dashed lines, thin characters) were added subsequently based on a second ClustalW guide tree.
Mentions: The putative target of the AADs in C. elegans, ACR-23, is a member of the nematode-specific DEG-3 family of nAChR alpha subunits. A tblastn search [19] with DEG-3 members against the (incomplete) H. contortus genome database (http://www.sanger.ac.uk/Projects/H_contortus) returned strong hits from different contigs, coding for a total of 6 different DEG-3 subfamily nAChR subunit homologues. However, the lack of overlap between the different contigs precluded the assembly of full length coding sequences. The predicted H. contortus proteins were named Hco-MPTL-1 (accession number: contig_0024907; contig_0033952; contig_0079482; haem-240m02.q1k; contig_0053297; contig_069357), Hco-DES-2H (contig_0064641), Hco-DEG-3H (contig_0075200; contig_0075201), Hco-ACR-24H (contig_0003482; contig_0064300), Hco-ACR-5H (contig_0106281; contig_0023143) and Hco-ACR-17H (contig_0101516; contig_0101514). For Hco-MPTL-1, Hco-DES-2H and Hco-DEG-3H, full-length coding sequences were obtained by cDNA library screening or RACE-PCR, respectively (see below). Figure 1 shows the position of the H. contortus sequences in a phylogenetic tree of the DEG-3 subfamily nAChR from C. elegans, C. briggsae and Brugia malayi. Note that an incomplete sequence of Hco-MPTL-1 was previously named Hc-ACR-23H [12].

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