Repertoire of P-glycoprotein drug transporters in the zoonotic nematode Toxocara canis

Abstract

Toxocara canis has a complex lifecycle including larval stages in the somatic tissue of dogs that tolerate macrocyclic lactones. In this study, we investigated T. canis permeability glycoproteins (P-gps, ABCB1) with a putative role in drug tolerance. Motility experiments demonstrated that while ivermectin failed to abrogate larval movement, the combination of ivermectin and the P-gp inhibitor verapamil induced larval paralysis. Whole organism assays revealed functional P-gp activity in larvae which were capable of effluxing the P-gp substrate Hoechst 33342 (H33342). Further investigation of H33342 efflux demonstrated a unique rank order of potency for known mammalian P-gp inhibitors, suggesting that one or more of the T. canis transporters has nematode-specific pharmacological properties. Analysis of the T. canis draft genome resulted in the identification of 13 annotated P-gp genes, enabling revision of predicted gene names and identification of putative paralogs. Quantitative PCR was used to measure P-gp mRNA expression in adult worms, hatched larvae, and somatic larvae. At least 10 of the predicted genes were expressed in adults and hatched larvae, and at least 8 were expressed in somatic larvae. However, treatment of larvae with macrocyclic lactones failed to significantly increase P-gp expression as measured by qPCR. Further studies are needed to understand the role of individual P-gps with possible contributions to macrocyclic lactone tolerance in T. canis.

Strain-level variations of Dirofilaria immitis microfilariae in two biochemical assays

“…This property makes the dye convenient for fluorescence-based assays. The disposition and interaction between Hoechst 33342 and nematode P-glycoprotein has been demonstrated in heterologous expression systems [ 39 ] and in whole worm larval stages [ 40 ]. We had also previously shown that basal levels of P-gp efflux activity varied between D .…”

“…immitis , three P-glycoprotein genes (ABCB1) and three haf transporters (ABCB and ABCC) have been described [ 34 ]. This number is lower that the repertoire of P-gps in filarial nematode– 8 in Brugia malayi [ 43 ], and P-gp gene repertoires in other Clade III nematodes– 10 in Parascaris univalens [ 44 ] and 10 in Toxocara canis [ 40 ]. The detection of only a few transporters is likely to be artefactual due to the incomplete and fragmented nature of all publicly available D .…”

“…immitis [ 34 ]. Since other nematodes in clade III have larger ABCB repertoires (16 in Brugia malayi [ 43 ], 10 in Parascaris univalens [ 44 ] and 13 in Toxocara canis [ 40 ]), it is expected that other transporters in D . immitis will be identified when the genome quality and annotation improves.…”