Dataset Information

Molecular and pharmacological characterization of an acetylcholine-gated chloride channel (ACC-2) from the parasitic nematode Haemonchus contortus.

ABSTRACT: Nematode cys-loop ligand-gated ion channels (LGICs) have been shown to be attractive targets for the development of novel anti-parasitic drugs. The ACC-1 family of receptors are a unique group of acetylcholine-gated chloride channels present only in invertebrates, and sequence analysis suggests that they contain a novel binding site for acetylcholine. We have isolated a novel member of this family, Hco-ACC-2, from the parasitic nematode Haemonchus contortus and using site-directed mutagenesis, electrophysiology and molecular modelling examined how two aromatic amino acids in the binding site contributed to agonist recognition. It was found that instead of a tryptophan residue in binding loop B, which essential for ligand binding in mammalian nAChRs, there is a phenylalanine (F200) in Hco-ACC-2. Amino acid changes at F200 to either a tyrosine or tryptophan were fairly well tolerated, where a F200Y mutation resulted in a channel hypersensitive to ACh and nicotine as well as other cholinergic agonists such as carbachol and methacholine. In addition, both pyrantel and levamisole were partial agonists at the wild-type receptor and like the other agonists showed an increase in sensitivity at F200Y. On the other hand, in Hco-ACC-2 there is a tryptophan residue at position 248 in loop C that appears to be essential for receptor function, as mutations to either phenylalanine or tyrosine resulted in a marked decrease in agonist sensitivity. Moreover, mutations that swapped the residues F200 and W248 (ie. F200W/W248F) produced non-functional receptors. Overall, Hco-ACC-2 appears to have a novel cholinergic binding site that could have implications for the design of specific anthelmintics that target this family of receptors in parasitic nematodes.

SUBMITTER: Habibi SA

PROVIDER: S-EPMC6287471 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Publications

Molecular and pharmacological characterization of an acetylcholine-gated chloride channel (ACC-2) from the parasitic nematode Haemonchus contortus.

Habibi Sarah A SA Callanan Micah M Forrester Sean G SG

International journal for parasitology. Drugs and drug resistance 20180922 3

Nematode cys-loop ligand-gated ion channels (LGICs) have been shown to be attractive targets for the development of novel anti-parasitic drugs. The ACC-1 family of receptors are a unique group of acetylcholine-gated chloride channels present only in invertebrates, and sequence analysis suggests that they contain a novel binding site for acetylcholine. We have isolated a novel member of this family, Hco-ACC-2, from the parasitic nematode Haemonchus contortus and using site-directed mutagenesis, e ...[more]

PMID: 30266440

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Project description:Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance.

Project description:CircRNAs, a novel class of ncRNA family, are endogenous transcriptional products involved in various biological and physiological processes in plants and animals. However, almost no information is available for circRNAs of parasitic helminths. In the present study, the circRNAs repertoire was comprehensively explored in Haemonchus contortus, a blood-sucking parasitic nematode of ruminants. In total, 20073 circRNAs were identified and annotated from three key developmental stages/genders of H. contortus including the free-living infective third-stage larvae (L3, 18883), parasitic adult female (Af, 3491), and male worms (Am, 2550) via deep-sequencing technology and bioinformatic analysis. Among these identified circRNAs, 71% were derived from exonic regions of protein-coding genes. The number of circRNAs transcribed from the X chromosome (4704) was higher than that from Chromosome I-V (3143, 3273, 3041, 3030, 2882). The amount of highly expressed circRNAs in third-stage larvae was significantly more abundant than that in adult stage. 15948 and 16847 circRNAs were differentially expressed between Af and L3s and between Am and L3, respectively. Among them, 13409 circRNAs existed in both comparisons. Furthermore, 1119 circRNAs were differentially expressed between Af_and_Am. GO enrichment analysis indicated that source genes of circRNAs differentially expressed between Am and L3 as well as between Af and L3 were significantly enriched in many biological processes, primarily including signaling, signal transduction and cell communication terms. KEGG analysis revealed that parental genes of differentially expressed circRNAs were mainly related to metabolism (pyruvate metabolism, glycerophospholipid metabolism, and carbon metabolism), MAPK signaling pathway, and phosphatidylinositol signaling system. Moreover, many circRNAs contained one or more miRNA potential binding sites, suggesting that they could regulate gene expression at the post-transcriptional level. Furthermore, the correctness of head-to-tail back splicing site and alternative circularization events were verified by Sanger sequencing using both divergent and convergent primers. Finally, the reliability of RNA-Seq data and the resistance of circRNAs to RNase R digestion were confirmed by quantitative RT-PCR. Taken together, our findings provide a foundation for elucidating the regulatory mechanisms of circRNAs in H. contortus, which will advance the understanding of circRNAs in parasitic nematodes.

Dataset Information

Molecular and pharmacological characterization of an acetylcholine-gated chloride channel (ACC-2) from the parasitic nematode Haemonchus contortus.

Publications

Molecular and pharmacological characterization of an acetylcholine-gated chloride channel (ACC-2) from the parasitic nematode Haemonchus contortus.

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