An amino acid substitution in Fasciola hepatica P-glycoprotein from triclabendazole-resistant and triclabendazole-susceptible populations

Abstract

Control of fasciolosis is threatened by the development of anthelmintic resistance. Enhanced triclabendazole (TCBZ) efflux by ABC transporters such as P-glycoprotein (Pgp) has been implicated in this process. A putative full length cDNA coding for a Pgp expressed in adult Fasciola hepatica has been constructed and used to design a primer set capable of amplifying a region encoding part of the second nucleotide binding domain of Pgp when genomic DNA was used as a template. Application of this primer set to genomic DNA from TCBZ-resistant and -susceptible field populations has shown a significant difference in the alleles present. Analysis of an allele occurring at a three-fold higher frequency in the "resistant" population revealed that it was characterised by a serine to arginine substitution at residue 1144. Homology modelling studies have been used to locate this site in the Pgp structure and hence assess its potential to modify functional activity.

The T687G SNP in a P-glycoprotein gene of Fasciola hepatica is not associated with resistance to triclabendazole in two resistant Australian populations

“…In our sample set, we observed seven different haplotypic groups termed A to G within the same region of the Pgp gene sequenced by Wilkinson et al [14] with all identified SNPs located within the intronic region. Haplotype A corresponded to haplotype 1 of Wilkinson et al [14] but the other haplotypes are unique to our populations.…”

“…A previous study proposed that a T687G single nucleotide polymorphism (SNP) in the exon of the second nucleotide binding domain of a Pgp gene, encoding a S1144R substitution, was associated with TCBZ resistance in F. hepatica [14]. However, this analysis was performed on F. hepatica populations that were not well defined with respect to TCBZ resistance and the T687G SNP was only observed in DNA from 3 flukes from TCBZ-"resistant" populations and 1 fluke from TCBZ-"susceptible" populations.…”

“…All SNPs occurred with an intron. The reference sequence is haplotype 1 of Wilkinson et al [14] which corresponds to haplotype A in this study. The genomic sequence is D915DFT Contig3365 from the F hepatica 1.0.allpaths.pg assembly of F. hepatica available at the parasite.wormbase.org site.…”

“…* This indel occurred within a poly A region and could have occurred between positions 347 and 352 but was designated 352 here for simplicity. [14]. Sequencing of PCR fragments was performed at the Australian Genome Research Facility (AGRF, Melbourne, Parkville, VIC) using a 3730xl DNA Analyzer (Life Technologies, USA).…”

“…However, the mechanism of resistance to TCBZ is not known. A T687G single nucleotide polymorphism (SNP) in a P-glycoprotein gene was proposed as a molecular marker for TCBZ resistance in F. hepatica (Wilkinson et al, 2012). We analyzed this Pgp gene from TCBZ-susceptible and TCBZ-resistant populations from Australia to determine if the SNP was a marker for TCBZ resistance.…”