Towards an understanding of the mechanism of action of praziquantel

Abstract

Although praziquantel (PZQ) has been used to treat schistosomiasis for over 20 years its mechanism of action remains unknown. We have developed an assay based on the transcriptional response of Schistosoma mansoni PR-1 to heat shock to confirm that while 6-week post-infection (p.i.) schistosomes are sensitive to PZQ, 4-week p.i. schistosomes are not. Further, we have used this assay to demonstrate that in mice this sensitivity develops between days 37 and 40 p.i. When PZQ is linked to the fluorophore BODIPY to aid microscopic visualization, it appears to enter the cells of intact 4 and 6-week p.i. schistosomes as well as mammalian NIH 3T3 cells with ease suggesting that the differential effects of PZQ is not based on cell exclusion. A transcriptomal analysis of gene expression between 4 and 6 weeks p.i. revealed 607 up-regulated candidate genes whose products are potential PZQ targets. A comparison of this gene list with that of genes expressed by PZQ sensitive miracidia reduced this target list to 247 genes, including a number involved in aerobic metabolism and cytosolic calcium regulation. Finally, we also report the effect of an in vitro sub-lethal exposure of PZQ on the transcriptome of S. mansoni PR-1. Annotation of genes differentially regulated by PZQ exposure suggests that schistosomes may undergo a transcriptomic response similar to that observed during oxidative stress.

Fig. 1

Quantitative RT-PCR assays of the transcriptomal response of S. mansoni PR-1 to heat shock after treatment with PZQ. The effect of 30 min exposure to increasing concentrations of PZQ on the transcriptomal response of (A) a 6-week and (B) a 4-week p.i. mixed sex population. (C) The effect of 80 μg/mL PZQ for 30min on the transcriptomal response of schistosomes harvested every 3 days from day 28 to 43 p.i. The fold change for each of the five transcripts indicated in the key was calculated using the 2^−ΔΔCT method [21]. Error bars represent the standard deviation of three measurements and a fold change >1 indicates an increase in transcript abundance.

Fig. 2

Analysis of the transcriptome of 6-week p.i. S. mansoni exposed to a sub-lethal dose (50 μg/mL) of PZQ for up to 4 h. Gene ontology annotation of (A) 251 array elements up-regulated in response to PZQ and (B) 131 array elements down-regulated in response to PZQ. Transcripts were evaluated that had a 2-fold increase or decrease in abundance after PZQ exposure and were present in at least 80% of the data points. S. japonicum array elements were excluded from gene ontology analysis. Substances with multiple annotated functions were included and could be in more than one category. (C) qRT-PCR results plotted as fold change calculated using the 2^−ΔΔCT method [21] as a function of time after PZQ exposure. Error bars represent the standard deviation of three measurements. Fold change >1 indicates an increase in transcript abundance.

Fig. 3

A–D: Six-week p.i. male schistosomes at 88× magnification stained with (A) DAPI (B) DAPI and BODIPY, (C) mPZQ-BODIPY and DAPI and (D) mPZQ-BODIPY with DAPI staining optically removed. E–H: Six-week p.i. female schistosomes at 353× magnification stained with (E) DAPI, (F) DAPI and BODIPY, (G) mPZQ-BODIPY and DAPI and (H) mPZQ-BODIPY with DAPI staining optically removed. I–L: Four-week p.i. mixed sex schistosomes at 353× magnification stained with (I) DAPI, (J) DAPI and BODIPY, (K) mPZQ-BODIPY and DAPI and (L) mPZQ-BODIPY with DAPI staining optically removed. DAPI stain is blue; BODIPY and mPZQ-BODIPY are green.

Fig. 4

Mammalian NIH 3T3 cells at 40× magnification stained with (A) DAPI, (B) DAPI and BODIPY, (C) mPZQ-BODIPY and DAPI and (D) mPZQ-BODIPY with DAPI staining optically removed. DAPI stain is blue; BODIPY and mPZQ-BODIPY are green.

Fig. 5

Geneontology annotation of 272microarray elements that were up-regulated at day 42 compared to day 28 p.i. S. japonicum elements were excluded from this analysis. Elements with multiple annotated functions were included and could be in more than one category.