Genetic knockdown and pharmacological inhibition of parasite multidrug resistance transporters disrupts egg production in Schistosoma mansoni

Abstract

P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRPs) are ATP-dependent transporters involved in efflux of toxins and xenobiotics from cells. When overexpressed, these transporters can mediate multidrug resistance (MDR) in mammalian cells, and changes in Pgp expression and sequence are associated with drug resistance in helminths. In addition to the role they play in drug efflux, MDR transporters are essential components of normal cellular physiology, and targeting them may prove a useful strategy for development of new therapeutics or of compounds that enhance the efficacy of current anthelmintics. We previously showed that expression of Schistosoma mansoni MDR transporters increases in response to praziquantel (PZQ), the current drug of choice against schistosomiasis, and that reduced PZQ sensitivity correlates with higher levels of these parasite transporters. We have also shown that PZQ inhibits transport by SMDR2, a Pgp orthologue from S. mansoni, and that PZQ is a likely substrate of SMDR2. Here, we examine the physiological roles of SMDR2 and SmMRP1 (the S. mansoni orthologue of MRP1) in S. mansoni adults, using RNAi to knock down expression, and pharmacological agents to inhibit transporter function. We find that both types of treatments disrupt parasite egg deposition by worms in culture. Furthermore, administration of different MDR inhibitors to S. mansoni-infected mice results in a reduction in egg burden in host liver. These schistosome MDR transporters therefore appear to play essential roles in parasite egg production, and can be targeted genetically and pharmacologically. Since eggs are responsible for the major pathophysiological consequences of schistosomiasis, and since they are also the agents for transmission of the disease, these results suggest a potential strategy for reducing disease pathology and spread.

Figure 1. Knockdown of SMDR2 and SmMRP1 expression in adult parasites.

Adult parasites were perfused at 6–7 weeks post infection and electroporated with 3 µg of siRNAs or water. Following electroporation, pooled adult worms (males and females) were incubated as described in Materials and Methods, and the expression of SMDR2 and SmMRP1 analyzed for changes in RNA and protein abundance (A, B). Western blot analysis of anti-Pgp (A) or anti-MRP1 (B) cross-reactive proteins (upper panel) isolated from worms treated with SMDR2 siRNA (A, lane 2), SmMRP1 siRNA (B, lane 2), or water (Control, lane 1). Note the decrease in immunoreactivity for both target sequences. Anti-β-tubulin was used as a loading control. (C, D) Relative expression of SMDR2 (n = 6–7) or SmMRP1 (n = 3–4) RNA in adult worms treated with water (H₂O, white bars), luciferase siRNA (grey bars), SMDR2 siRNA or SmMRP1 siRNA (black bars), or both SMDR2 and SmMRP1 (hatched bars). SMDR2 and SmMRP1 siRNAs efficiently knock down the mRNA expression levels of SMDR2 by ≥50% and SmMRP1 by ≥70%, respectively. The fold changes were determined by quantitative RT-PCR using 18S RNA as the reference gene. *, ** indicate P<0.05 and P<0.01, respectively, compared to the water control, ANOVA.

Figure 2. Knockdown of SMDR2 or SmMRP1 in adult schistosomes disrupts parasite egg production.

Adult schistosomes were electroporated with H₂O or 3 µg siRNAs and incubated in RPMI medium for 48 h. Following electroporation, 2–3 adult pairs (n = 4–7) were cultured in 16-well plates for 4–5 days and the number of eggs counted. RNAi treatments were luciferase siRNA (grey bar), SmMRP1 siRNA (black bar), SMDR2 siRNA (hatched bar), or both SmMRP1 and SMDR2 siRNA (dotted bar). Egg counts within each experiment were normalized to the corresponding worms treated with H₂O (white bar). Treatment with the MDR transporter siRNAs significantly reduced egg production by ≥ 60%, but no significant change in egg production was found for worms electroporated with luciferase siRNA or H₂O. *, *** indicate P<0.05 and P<0.001, respectively, ANOVA.

Figure 3. Exposure of S. mansoni adult worms in culture to MDR inhibitors disrupts egg production.

Adult worm pairs (n = 3–4) were incubated in different concentrations of MDR inhibitors (black bars) for 48 h. Cumulative egg counts were normalized to those of control worms (white bars), which were exposed to DMSO carrier. Addition of the Pgp inhibitors C-4 (A), dexverapamil (B), cyclosporin A (C), tariquidar (D), or the MRP-1 inhibitor MK 571 (E) significantly disrupts egg production. (F) The effect of the Pgp inhibitors on egg production by females is independent of presence of male worms. Adult females were incubated in the absence of males for 48 h in the culture media alone (Control), or in the presence of 10 µM cyclosporin A (CSA), tariquidar, dexverapamil, or C-4. Shown are the cumulative egg counts per female, n = 3–5 for each treatment. *, **, and *** indicate P<0.05, P<0.01, and P<0.001 respectively, ANOVA.

Figure 4. Eggs produced by worms exposed to MDR inhibitors show morphological abnormalities.

Micrographs of S. mansoni eggs collected from adult worm pairs cultured in the absence (A) or presence (B) of tariquidar for 48 h. Control eggs appear normal and oval shaped with a lateral spine, in contrast to malformed, necrotic, and disintegrated eggs from worms exposed to tariquidar.

Figure 5. Administration of MDR inhibitors to S. mansoni-infected mice reduces host liver egg burden.

(A). Mean egg burden/g of liver (n = 3–5) from mice at 6–7 weeks post infection with approximately 200 cercariae, normalized to Control within each experiment. Mice were treated with 3 doses on alternating days of: diluted DMSO/Cremophore EL carrier (Control; white bar, n = 8); C-4 (50 mg/kg, n = 6); tariquidar (15 mg/kg, n = 3); dexverapamil (60 mg/kg, n = 3), or cyclosporin A (CSA, 60 mg/kg, n = 6). (B) Granulomas/cm² found in livers of infected mice (n = 6) treated with carrier or drugs, as in A. (C) Mean ex vivo egg production (n = 4–6) from 3 pairs of adult parasites perfused from mice that were treated with the MDR inhibitors dexverapamil (60 mg/kg), C-4 (50 mg/kg), or cyclosporin A (CSA; 60 mg/kg) and subsequently cultured in RPMI for 48 h. Control represents eggs from parasites perfused from mice treated with carrier alone (diluted DMSO/Cremophore EL). Only CSA continues to disrupt egg production through the culture period. *, **, ***, and **** indicate P<0.05, P<0.01, P<0.001, and P<0.0001, respectively, unpaired, two-tailed t-tests.