Dataset Information

Transgenically expressed Parascaris P-glycoprotein-11 can modulate ivermectin susceptibility in Caenorhabditis elegans.

ABSTRACT: P-glycoproteins (Pgps) are suspected to mediate drug extrusion in nematodes contributing to macrocyclic lactone resistance. This association was recently shown for Parascaris Pgp-11. Ivermectin resistance was correlated with the presence of three pgp-11 single nucleotide polymorphisms and/or increased pgp-11 mRNA levels. In the present study, the ability of Pgp-11 to modulate ivermectin susceptibility was investigated by its expression in a pgp-11-deficient Caenorhabditis elegans strain. Expression of Parascaris pgp-11 in two transgenic lines significantly decreased ivermectin susceptibility in a motility (thrashing) assay conducted in liquid medium. The EC50 values increased by 3.2- and 4.6-fold in the two lines relative to a transgenic control strain. This is the first report on the successful functional analysis of a parasitic nematode Pgp in the model organism C.?elegans.

SUBMITTER: Janssen IJ

PROVIDER: S-EPMC4401813 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Transgenically expressed Parascaris P-glycoprotein-11 can modulate ivermectin susceptibility in Caenorhabditis elegans.

Janssen I Jana I IJ Krücken Jürgen J Demeler Janina J von Samson-Himmelstjerna Georg G

International journal for parasitology. Drugs and drug resistance 20150408 2

P-glycoproteins (Pgps) are suspected to mediate drug extrusion in nematodes contributing to macrocyclic lactone resistance. This association was recently shown for Parascaris Pgp-11. Ivermectin resistance was correlated with the presence of three pgp-11 single nucleotide polymorphisms and/or increased pgp-11 mRNA levels. In the present study, the ability of Pgp-11 to modulate ivermectin susceptibility was investigated by its expression in a pgp-11-deficient Caenorhabditis elegans strain. Express ...[more]

PMID: 25905032

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Project description:Macrocyclic lactones (MLs) represent the major drug class for control of parasitic infections in humans and animals. However, recently reports of treatment failures became more frequent. In addition to human and ruminant parasitic nematodes this also is the case for the horse-nematode Parascaris equorum. Nevertheless, to date the molecular basis of ML resistance is still not understood. Unspecific resistance mechanisms involving transporters such as P-glycoproteins (Pgps) are expected to contribute to ML resistance in nematodes. Here, complete sequences of two P. equorum Pgps were cloned and identified as orthologs of Caenorhabditis elegans Ppg-11 and an unnamed Caenorhabditis briggsae Pgp designated as Pgp-16 using phylogenetic analysis. Quantitative real-time PCR was used to compare expression between tissues. Significantly higher PeqPgp-11 expression was found in the gut for both genders, whereas for PeqPgp-16 the body wall was identified as predominant expression site. Furthermore, Pgps were analyzed regarding their participation in resistance development. Using SeqDoC analyses, Pgp-sequences of P. equorum populations with different ML susceptibility were compared. This approach revealed three single nucleotide polymorphisms (SNPs) causing missense mutations in the PeqPgp-11 sequence which correlated with decreased ML susceptibility. However, no resistance associated differences in mRNA expression levels were detected between embryonated eggs of these populations. In contrast, comparison of two pre-adult groups with different ivermectin (IVM) susceptibility revealed the presence of the three SNPs and in addition statistically significant PeqPgp-11 overexpression in the group of worms with reduced susceptibility. These results indicate that Pgp-11 might be involved in IVM resistance in P. equorum as it shows increased expression in an IVM exposed life-cycle stage of an IVM resistant population as well as occurrence of putatively resistance associated SNPs in populations with reduced IVM susceptibility. These SNPs are promising diagnostic candidates for detection of ML resistance with potential also for other parasitic nematode species.

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.

Dataset Information

Transgenically expressed Parascaris P-glycoprotein-11 can modulate ivermectin susceptibility in Caenorhabditis elegans.

Publications

Transgenically expressed Parascaris P-glycoprotein-11 can modulate ivermectin susceptibility in Caenorhabditis elegans.

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