RESUMO
OBJECTIVES: The molecular mechanisms of levamisole (LEV) activity and expression of resistance remain largely unknown in parasitic nematodes. In contrast, genetic screens for mutants that survive exposure to LEV in the free-living nematode Caenorhabditis elegans have led to the identification of five genes (unc-38, unc-63, unc-29, lev-1 and lev-8) that encode a LEV-sensitive acetylcholine receptor (L-AChR). Loss of these genes leads to LEV resistance. In this study, orthologues of these genes were identified in three species of trichostrongylid nematodes that have a major impact on small ruminants: Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis. Polymorphism associated with LEV resistance have been investigated by comparing transcripts of these subunits in LEV susceptible and LEV-resistant isolates of the three strongylid species. BASIC METHODS: Partial sequences were identified by PCR experiments and full-length cDNA sequences corresponding to AChR subunits in the three trichostrongylid species were obtained using 3'-rapid amplification of cDNA ends-PCR and 5' rapid amplification of cDNA ends anchored with the spliced leader sequence, SL1. Expression of L-AChR subunits was investigated in LEV-resistant and LEV-susceptible isolates of H. contortus, T. circumcincta and T. colubriformis using reverse transcription PCR. MAIN RESULTS: We have identified a total of 20 full-length cDNA sequences corresponding to L-AChR subunits in three parasitic trichostrongylid species of which 14 correspond to novel sequences. Genes orthologous to unc-29, unc-63, unc-38 and lev-1 were found in each trichostrongylid species, whereas no gene corresponding to lev-8 has yet been identified. We have found 11 distinct paralogous sequences corresponding to the C. elegans unc-29 gene clustered in four groups revealing an unexpected diversity of unc-29-like genes. Complete coding sequences of the L-AChR subunits in two LEV-resistant and three susceptible isolates of H. contortus, T. circumcincta and T. colubriformis were essentially unchanged, but abbreviated transcripts of the unc-63 subunit were specifically expressed in resistant isolates of all three species. CONCLUSION: The candidate gene strategy developed in this study revealed an unexpectedly high diversity of L-AChR subunits specific to the trichostrongylid parasites that are a principal target for the drug LEV. Abbreviated variants, predicted to produce nonfunctional unc-63, were associated with LEV resistance. This study contributes significantly to a better understanding of LEV receptor constitution in parasitic nematodes and highlights the putative role of aberrant mRNA encoding L-AChR subunits in LEV resistance.
Assuntos
Resistência a Medicamentos/genética , Variação Genética , Levamisol/farmacologia , Nematoides/genética , Parasitos/genética , Subunidades Proteicas/genética , Receptores Colinérgicos/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Resistência a Medicamentos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes de Helmintos/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Dados de Sequência Molecular , Nematoides/efeitos dos fármacos , Parasitos/efeitos dos fármacos , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Colinérgicos/química , Receptores Colinérgicos/metabolismoRESUMO
The cDNA-AFLP (cDNA-amplified fragment length polymorphism) method comparing transcripts from levamisole-resistant and susceptible Haemonchus contortus isolates has led to the successful identification of a number of potentially useful levamisole-resistance markers. In the present study, we report the characterization of the transcript-derived fragment (TDF) named HAX which was confirmed to be specifically expressed in three levamisole-resistant isolates by RT-PCR experiments. Cloning and sequencing of the full-length cDNA sequence of HAX revealed high similarity to the Caenorhabditis elegans acr-8 gene and its putative H. contortus orthologue encoding a nicotinic acetylcholine receptor subunit. This Hco-acr-8b short isoform corresponded to a spliced variant of Hco-acr-8 mRNA containing the two first exons and a part of intron 2. As nicotinic acetylcholine receptors constitute the pharmacological target of levamisole, Hco-acr-8b may potentially be involved in the molecular mechanisms leading to levamisole-resistance acquisition in H. contortus.