Ivermectin resistance in nematodes may be caused by alteration of P-glycoprotein homolog¹

Abstract

Resistance to ivermectin and related drugs is an increasing problem for parasite control. The mechanism of ivermectin resistance in nematode parasites is currently unknown. Some P-glycoproteins and multidrug resistance proteins have been found to act as membrane transporters which pump drugs from the cell. A disruption of the mdr1a gene, which encodes a P-glycoprotein in mice, results in hypersensitivity to ivermectin. Genes encoding members of the P-glycoprotein family are known to exist in nematodes but the involvement of P-glycoprotein in nematode ivermectin-resistance has not been described. Our data suggest that a P-glycoprotein may play a role in ivermectin resistance in the sheep nematode parasite Haemonchus contortus. A full length P-glycoprotein cDNA from H. contortus has been cloned and sequenced. Analysis of the sequence showed 61–65% homology to other P-glycoprotein/multidrug resistant protein sequences, such as mice, human and Caenorhabditis elegans. Expression of P-glycoprotein mRNA was higher in ivermectin-selected than unselected strains of H. contortus. An alteration in the restriction pattern was also found for the genomic locus of P-glycoprotein derived from ivermectin-selected strains of H. contortus compared with unselected strains. P-glycoprotein gene structure and/or its transcription are altered in ivermectin-selected H. contortus. The multidrug resistance reversing agent, verapamil, increased the efficacy of ivermectin and moxidectin against a moxidectin-selected strain of this nematode in jirds (Meriones unguiculatus). These data indicate that a P-glycoprotein may be involved in resistance to ivermectin and other macrocyclic lactones in H. contortus.

Introduction

Resistance to ivermectin (IVM), a macrocyclic lactone anthelmintic, in nematode parasites of livestock has become a serious problem in many parts of the world 1, 2and the increasing use of anthelmintics in animals and humans is likely to increase the incidence of resistance. The mechanisms underlying this resistance have not yet been established. Detection of field resistance is currently dependent on the analysis of drug efficacy involving post mortem worm counts, nematode egg-count reduction or the use of in vitro assays of the development of immature stages. These methods are insufficiently sensitive to monitor the development of resistance before it is overt. Understanding the molecular basis of resistance to IVM and that of other macrocyclic lactone anthelmintics should assist in the development of more sensitive methods for monitoring the development of resistance and possibly for reversing it.

IVM is thought to bind to an alpha subunit of a glutamate-gated chloride channel in nematode cell membranes 3, 4resulting in the hyperpolarization of neuromuscular cells and pharyngeal paralysis. The possibility that IVM resistance is due to altered binding to its receptor has been investigated in IVM resistant and sensitive Haemonchus contortus [5]. These workers concluded that resistance was not due to an alteration in its binding to the glutamate-gated chloride channel receptor.

P-glycoproteins have been considered to cause drug resistance in mammalian tumor cells 6, 7. P-glycoproteins in Leishmania appear to be involved in drug resistance 8, 9, 10, 11. In some drug-resistant strains of the malaria parasite Plasmodium falciparum, amplification of a P-glycoprotein gene (pfmdr1) is observed 12, 13. In the free-living nematode Caenorhabditis elegans, a P-glycoprotein has been observed to provide protection against toxic compounds made by plants and microbes in the rhizosphere [14]. The nematode C. elegans has multiple proteins, homologues of mammalian proteins involved in cellular resistance to chemotherapeutic drugs, that protect the worms from the toxic effects of heavy metals [15]. It has been observed that the disruption of the mdr1a P-glycoprotein (P-gp), a membrane transport glycoprotein [6], in the mouse leads to a marked increase in central nervous system sensitivity to IVM [16], and it has been shown that IVM is a potent P-gp ligand in mammalian cells 17, 18. In view of the mammalian evidence that IVM may bind to and be transported by P-gp, we investigated the possibility that a P-gp might mediate ivermectin resistance in this parasite.