Fig 1.
Parasite tissue damage and death following meclonazepam exposure.
(A) Juvenile, liver-stage parasites (four week infections, left) and adult, intestinal stage parasites (seven week infections, right) exposed to meclonazepam (MCLZ 5 µM, 14 hours). Top - Brightfield images of worms fixed after drug treatment. Bottom - Worms were then sectioned and processed for imaging by transmission electron microscopy (TEM), showing damage to parasite tissues. Note vacuoles (arrowed) forming in the tegument layer (T), as well as sub-tegumental tissues at and below the body-wall muscle (BWM), of MCLZ treated worms. (B) In vitro concentration (0 - 10 µM) response for MCLZ treated adult parasites measuring contraction (open symbols, expressed as percent of DMSO control treatment) and activation of pro-apoptotic caspases 3/7 (solid symbols). Symbols denote mean ± standard error of n = 13 adult male worms for length measurements and n = 6 replicates of caspase activity assays for each treatment, each replicate consisting of ≥ 5 worms per drug concentration that were pooled into a single tube and homogenized. (C) Time course of pro-apoptotic caspase activation in adult parasites following in vitro MCLZ treatment (n = 6 replicates as in (B), ** p < 0.01 significance of difference relative to t = 0 hr treatment time point). (D) Time course of MCLZ effects on parasites following in vivo administration of 30 mg/kg dose of drug to mice harboring seven week infections. Mice (n = 3 per time point) were euthanized at various intervals (ranging from 15 minutes to 48 hours) to measure shift of worms from the mesenteric vasculature to the liver (open symbols) and caspase activation (solid symbols). All timepoints were assessed as significantly different from the t = 0 hr control ANOVA and Dunnett’s multiple comparisons test, * p < 0.05 and ** p < 0.01.
Fig 2.
Transcriptional response of parasites to in vivo meclonazepam treatment.
(A) Volcano plots of transcript expression in juvenile, four week parasites (left) and (B) adult, seven week parasites (right) harvested from mice treated with either DMSO control or MCLZ (30 mg/kg). Cut-offs for differentially expressed genes shown at log2 fold change > 1 and adjusted p-value < 0.01. (C) Venn diagram summarizing overlap between differentially expressed gene lists (↑ = upregulated, ↓ = downregulated) in four and seven week parasites. (D) Hierarchical clustering of differentially expressed, parasite divergent gene products across the four and seven week DMSO control and MCLZ treated cohorts. (E & F) Analysis showing that transcripts from genes that are more divergent in parasitic flatworms are also enriched in the extremes of up and downregulated rankings of (E) four week, juvenile and (F) seven week, adult parasites following MCLZ treatment. Gene ranking on the x-axis reflects ordering from positive (left) to negative (right) log2 fold change with MCLZ treatment. Each closed symbol on the scatter plot represents a window of 100 transcripts, and the y-axis reflects the proportion of these transcripts that are categorized as parasite divergent (BLAST e-value > 0.01 when querying free-living flatworm genomes or transcriptomes).
Fig 3.
Time course of gene expression changes following different anthelmintic treatments.
(A) Hierarchical clustering of seven week, adult S. mansoni harvested from mice treated with either a curative dose of MCLZ (30 mg/kg) or PZQ (400 mg/kg). Heatmap reflects z-score of gene expression (transcripts per million) across MCLZ and PZQ drug treatment cohorts. (B) Comparative trajectories of transcripts with similar and different short and long term expression changes following drug exposure. Blue = MCLZ treated samples. Brown = PZQ treated samples.
Fig 4.
Praziquantel-resistant parasites are susceptible to meclonazepam.
Pro-apoptotic caspase activation in seven week, adult S. mansoni treated in vitro with various concentrations of MCLZ (0 - 5 µM) for 24 hours (n = 30 worms per concentration for each schistosome population). Open bars reflect the average ± standard error response of the PZQ sensitive population SmLE-PZQ-ES, and solid bars indicate response of the PZQ-resistant population SmLE-PZQ-ER.
Fig 5.
Potential mechanisms of meclonazepam and praziquantel.
MCLZ and PZQ have nearly identical phenotypes on adult parasites (left), such as contractile paralysis and tegument damage, as well as similar transcriptional responses. Both compounds are agonists at heterologously expressed, homomeric TRP channels (right); MCLZ on Smp_333650 (purple) and PZQ on Smp_246790 (blue) [18,19]. Within the parasite, these TRP subunits could conceivably form either homomeric or heteromeric channels consisting of multiple subunits expressed in the same cell type. Analysis of single cell transcriptomic data [35] (bottom) for Smp_333650 and Smp_246790. Muscle clusters 1 - 8 are ordered left to right, as are neuron clusters 1 - 31. Dot diameter denotes the percentage of cells within the cell type cluster expressing the gene of interest. Dot color indicates the average expression of the indicated gene by cluster.