Analysis of P-glycoprotein gene transcripts in Fasciola hepatica and its relation with resistance to triclabendazole

Abstract

Fasciolosis is a parasitic disease caused by the trematode Fasciola hepatica. Its control ismainly based on the use of triclabendazole (TCBZ), a halogenated benzimidazole with excellent efficacy against juvenile and adult stages. Resistance to TCBZ in F. hepatica has been reported in many parts of the world. Benzimidazole resistance in nematodes is mainly caused by specific amino acid substitutions in β-tubulin resulting from a nucleotide substitution in the gene.However, the resistance to TCBZ in F. hepatica is not associated with changes in this molecule. The increased cellular efflux of TCBZ in F. hepatica is related to P-glycoproteins (PGP) and a T687G single nucleotide polymorphism (SNP) was initially suggested as responsible for the resistant phenotype. In the present work, a complete PGP sequence has been assembled based on sequences available in databases. We analyzed transcripts from this gene region in TCBZ resistant strains from Peru and Argentina and in susceptible strains from Mexico and Ireland. We observed that the T687G SNP is not associated with resistance to TCBZ. Instead, we identified other SNPs, including variants in the coding region and in introns. Two SNPs A617T and T267G, which fall in a PGP intron, are shared by all resistant strains but also are present in some susceptible ones. RT-PCR from adult RNA showed no evidence of alternative splicing. Our results highlight the lack of clear association of SNPs in this PGP gene with resistance to TCBZ. The recent release of two assemblies of the F. hepatica genome revealing the presence of multiple ABC transporters and multidrug resistance genes opens new avenues for investigating variants associated with the resistance of F. hepatica to TCBZ.