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.

Figure 1

Ivermectin-induced paralysis of larvae is only observed following P-gp inhibition. Bars represent (A) mean area occupied or (B) mean wavelength ± SE of larvae following exposure to ivermectin with or without verapamil. (Asterisks indicate groups that are significantly different, ***p < 0.001, **p < 0.01, *p < 0.05).

Figure 2

Representative bright-field overlays and fluorescence images of T. canis larvae stained with Hoechst 33,342 in the absence of drugs (A, B) and presence of inhibitors-ivermectin (C,D), cyclosporine A (E, F), loperamide (G, H), reserpine (I, J), verapamil (K, L) and tariquidar (M, N) (400x). The brightfield image was used to annotate the outline of the larvae which was overlaid on the fluorescent image. The area stained was quantitated using image analysis software (Fig. 3).

Figure 3

Increased H33342 staining of T. canis larvae exposed to P-gp inhibitors. Mean ± SE are shown, asterisks indicate P-gp inhibition relative to no drug control. (Asterisks indicate groups that are significantly different, ****p < 0.0001, **p < 0.01, *p < 0.05).

Figure 4

Maximum likelihood phylogenetic tree of conceptually translated P-gp genes. Pgp genes predicted in the genome (o) and cloned ($\bullet$) are highlighted along with assigned names for T. canis P-gp genes.

Figure 5

Fold change ± SE in expression of Pgp genes in adult T. canis worms compared to hatched larvae. Fold change was calculated using the efficiency corrected ΔCt method using T. canis 18S as the reference gene. Bars above the dotted line represent increased transcription in adults and bars below the line represent increased transcription in larvae. (Asterisks represent statistically significant differences, ***p < 0.001, **p < 0.01, *p < 0.05).

Figure 6

Mean fold change ± SE in expression of P-gp genes following (A) ivermectin or (B) milbemycin oxime treatment of larvae hatched in vitro. Fold change was obtained using the efficiency corrected ΔCt method using T. canis 18S as the reference gene. (Asterisk represents statistical difference from untreated controls ****p <  < 0.0001, *p < 0.05).

Figure 7

Mean fold change ± SE in expression of P-gp genes in larvae from mice treated with (A) ivermectin or (B) moxidectin compared to untreated controls. Fold change was calculated using the efficiency corrected ΔCt method using T. canis 18S as the reference gene. (Asterisk represents statistical difference from untreated controls **p < 0.01).

Figure 8

Expression of P-gp mRNA in T. canis larvae in situ detected by RNAscope assay. Small box indicates location of magnified inset. Hybridization of probe (green punctates) is indicated by arrow heads. T. canis β-tubulin (positive control) and Bacillus subtilis dapB (negative control) were probed in parallel. Scale bar = 50 μm.