Characterisation of P-glycoprotein-9.1 in Haemonchus contortus.

Pablo Godoy,Robin N Beech,Roger K Prichard,Hua Che

doi:10.1186/s13071-016-1317-8

Abstract

BackgroundThe existence nematodes of veterinary importance such as Haemonchus contortus resistant to anthelmintic drugs, including the macrocyclic lactones, has become a major concern in animal health. Macrocyclic lactone resistance in H. contortus seems to be multigenic including the active efflux of these drugs by P-glycoproteins, members of the ABC transporter family, present in this parasite. The goals of the present work were to determine the activity of H. contortus P-glycoprotein 9.1 (Hco-PGP-9.1) and its interaction with the avermectins, ivermectin, abamectin, and also the milbemycin, moxidectin. Additionally, the localisation of Hco-PGP-9.1 was sought in adult worms.MethodsHco-Pgp-9.1 was cloned and expressed in mammalian cells and its expression profile was determined at the transcriptional and protein level by qRT-PCR and Western-blot, respectively. The nematode transport activity was assessed using the tracer dye Rhodamine 123. A ligand competition assay between different macrocyclic lactones and Rhodamine 123 was used to establish whether or not there was interaction between Hco-PGP-9.1 and the avermectins (abamectin and ivermectin) or moxidectin. In addition, immunostaining was carried out to localise Hco-PGP-9.1 expression in the transgenic cells and in adult female parasites.ResultsHco-PGP-9.1 was expressed in the cell membrane of the transfected host cells and was able to extrude Rhodamine 123. Ivermectin and abamectin, but not moxidectin, had a pronounced inhibitory effect on the ability of Hco-PGP-9.1 to transport Rhodamine 123. Antibodies raised against Hco-PGP-9.1 epitopes localised to the uterus of adult female H. contortus.ConclusionsThese results suggest a strong interaction of the avermectins with Hco-PGP-9.1. However, possibly due to its physico-chemical properties, moxidectin had markedly less effect on Hco-PGP-9.1. Because of the greater interaction of the avermectins than moxidectin with this transporter, it is more likely to contribute to avermectin resistance than to moxidectin resistance in H. contortus. Possible over expression of Hco-PGP-9.1 in the female reproductive system in resistant worms could reduce paralysis of the uterus by macrocyclic lactones, allowing continued egg release in drug challenged resistant worms.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1317-8) contains supplementary material, which is available to authorized users.

Highlights

The existence nematodes of veterinary importance such as Haemonchus contortus resistant to anthelmintic drugs, including the macrocyclic lactones, has become a major concern in animal health
Regarding orthologs in other trichostrongylids from ruminants, an up-regulation of Tcipgp-9 in all life-cycle stages of the sheep nematode Teladorstagia circumcinta in IVM resistant isolates has been described [25]. In another helminth from cattle, Cooperia onchophora an up-regulation of the ortholog Con-pgp-9 in an IVM selected strain was described [26]. Due to this data showing an over-expression of HcoPgp-9.1, and its orthologs, in H. contortus and other nematodes with veterinary importance resistant to Macrocyclic lactone (ML), as well as the strong interaction described between HcoPGP-2 and the avermectins, suggesting a nematode transporter involvement in ML resistance in H. contortus [27], the aim of the present work was to characterise the Hco-PGP-9.1 transport activity and its interaction with the MLs, in order to ascertain its possible involvement in the resistance to these drugs
The expression of the Hco-Pgp-9.1 transcript appeared to be decreasing by the third passage of the same culture, but the difference compared with the endogenous pig mdr1 was still large

Summary

Introduction

The existence nematodes of veterinary importance such as Haemonchus contortus resistant to anthelmintic drugs, including the macrocyclic lactones, has become a major concern in animal health. Macrocyclic lactone resistance in H. contortus seems to be multigenic including the active efflux of these drugs by P-glycoproteins, members of the ABC transporter family, present in this parasite. The primary means of parasite control in animal health is through anthelmintic drugs, of which, the macrocyclic lactones (MLs) are widely used, with activity. Regarding orthologs in other trichostrongylids from ruminants, an up-regulation of Tcipgp-9 in all life-cycle stages of the sheep nematode Teladorstagia circumcinta in IVM resistant isolates has been described [25] In another helminth from cattle, Cooperia onchophora an up-regulation of the ortholog Con-pgp-9 in an IVM selected strain was described [26]

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