Characterisation of P-glycoprotein-9.1 in Haemonchus contortus

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.

Acquired Tolerance to Ivermectin and Moxidectin after Drug Selection Pressure in the Nematode Caenorhabditis elegans

“…The cross-resistance phenotype observed in ML-selected strains is certainly based, at least partly, on the overexpression of genes encoding multidrug ABC transporters. Given that the different MLs are structurally related and that they all interact with Pgps of parasitic nematodes (62)(63)(64)(65), it is expected that the overexpression of Pgps in worms will lead to cross-resistance to IVM, MOX, EPR, and eventually other MLs and drugs that are Pgp substrates. Interestingly, MOX interacts weakly with nematode Pgps (62)(63)(64)(65)(66), and this is in agreement with its higher level of toxicity in ML-resistant strains.…”

“…Given that the different MLs are structurally related and that they all interact with Pgps of parasitic nematodes (62)(63)(64)(65), it is expected that the overexpression of Pgps in worms will lead to cross-resistance to IVM, MOX, EPR, and eventually other MLs and drugs that are Pgp substrates. Interestingly, MOX interacts weakly with nematode Pgps (62)(63)(64)(65)(66), and this is in agreement with its higher level of toxicity in ML-resistant strains. Moreover, the cross-resistance phenotype between ML compounds in parasitic nematodes was associated with the overexpression of several Pgps (10,13,43,44).…”

“…Third, IVM and MOX differentially interact with Pgps and other multidrug resistance (MDR) transporters. Indeed, it was clearly demonstrated that the interaction of MOX with mammalian Pgp (68), but also with H. contortus Pgp-2, Pgp-9, and Pgp-16 (62)(63)(64); Cylicocylus elongatus Pgp-9 (65); and Dirofilaria immitis Pgp-11 (66), is much weaker than that of IVM. Moreover, it was suggested that the MRPs may play less of a role in protecting C. elegans from MOX toxicity than they do in protecting the nematode from IVM toxicity (22).…”

In silico analysis of the binding of anthelmintics to Caenorhabditis elegans P-glycoprotein 1

“…Also, induction of expression of some Pgp genes after selection under IVM pressure is associated with IVM resistance in C. elegans and in several parasitic nematodes, which can be partly reversed by using mammalian Pgp inhibitors (James and Davey, 2009, Lespine et al., 2012, Menez et al., 2016). In addition, ML were shown to inhibit parasitic nematode Pgp-mediated drug transport in heterologous recombinant systems overexpressing Hco-Pgp-2, Hco-Pgp-9.1, Hco-Pgp-16, Cylicocylus elongatus ( Ceg )-Pgp-9 or Dirofilaria immitis ( Dim) -Pgp-11 (Godoy et al., 2015a, Godoy et al., 2015b, Godoy et al., 2016, Kaschny et al., 2015, Mani et al., 2016). Nevertheless, all these data give only indirect evidence that ML can be substrates of nematode Pgps.…”

The role of drug efflux systems in anthelmintic resistance in parasitic nematodes

“…However, it was recently described that IVM markedly inhibited rhodamine-123 (R-123; a fluorescent substrate) transport through pgp-2, pgp-9.1 and pgp-16 from adult worms of H. contortus expressed in mammalian cells (Godoy et al, 2015a;Godoy et al, 2015b;Godoy et al, 2016), whereas moxidectin caused less inhibition of R-123 efflux as compared to IVM. The authors concluded that IVM is a better substrate of nematode P-gps than moxidectin and this may help to explain the slower rate of development of resistance in H. contortus to moxidectin compared with the avermectins.…”