Fig 1.
In vitro effects of GSK343 on the motility, pairing, viability and ultrastructure of S. mansoni adult worms.
(A) Percentage of relative motility of adult worms treated with different concentrations of GSK343 and controls (0.1% DMSO) at different times of exposure from 3 to 48 h. Mean ± SD of three experiments, each with 10 worm pairs. (B) Monitoring the pairing of controls and treated couples with different concentrations of GSK343 at different times of exposure from 3 to 48 h. Mean ± SD of three experiments, each with 10 worm pairs. (C) Viability was estimated by the total amount of ATP available in the parasites, using a luminescent assay. Pairs of adult worms were treated for 48 h with GSK343 at the different concentrations indicated or with vehicle (0.1% DMSO). The viability was expressed as % the luminescence values relative to the control (DMSO). Mean ± SD from three replicate experiments, each with 10 worm pairs. For panels (A), (B) and (C): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with control. (D) Scanning electron microscopy of worms exposed for 48 h to 20 μM GSK343 (note that the size bar is shown within the black thin line below each image); panels 1 and 2: Male and female control (Bar = 500 μm; 200 μm) presenting the couple fully paired; panels 3 and 4: Medial dorsal region of the male presenting tegument without structural alterations (Bar = 100 μm; 10 μm); panel 5: Male posterior region without morphological alterations (Bar = 50 μm). panels 6 and 7: Adult worm treated with GSK343 and its anterior region (Bar = 500 μm; 100 μm); panels 8 and 9: Enlarged view of dorsal region of adult worm treated with GSK343 showing lesions of tegument around the tubercles; panel 10: Posterior region of male adult worm treated with GSK343 (Bar = 100 μm). f: female; os: oral sucker; vs: ventral sucker; b, blebs; tb: tubercles; la: lesion area.
Fig 2.
Damaged dorsal surface of S. mansoni adult female worms exposed to 20 μM GSK343 for 24 and 48 h.
Scanning electron microscopy of parasites; note that the size bar is shown within the black thin line below each image. (A, B and C) Images of dorsal region of female control worms, incubated with DMSO (0.1%) for 24 h, without structural alterations (Bar = 30 μm; 10 μm and 10 μm).; (D) Anterior region of female control worm incubated for 24 h with vehicle (Bar = 50 μm); (E, F and G) Middle dorsal region of females incubated with GSK343 for 24 h presenting apparent surface damage of the membrane with the presence of several blisters in the tegument (Bar = 20 μm; 20 μm and 5 μm); (H) Anterior region of female worm treated with GSK343 for 24 h without structural alterations (Bar = 50 μm); (I, J and K) Dorsal region of female control worms, incubated with DMSO (0.1%) for 48 h presenting the entire tegument without changes (Bar = 50 μm; 5 μm and 2 μm); (L) Anterior region of female control worm incubated for 48 h with 0.1% DMSO showing no alterations. (Bar = 50 μm); (M, N and O) Middle dorsal region of females incubated with GSK343 for 48h showing peeling of the tegument and presence of blisters (Bar = 50 μm; 10 μm and 5 μm); (P) Anterior region of female worm treated with GSK343 for 48 h presenting peeling of the tegument and the ventral sucker (Bar = 50 μm). os: oral sucker; vs: ventral sucker; b: blisters; p: peeling.
Fig 3.
Decrease in egg size and oviposition in S. mansoni females exposed to 20 μM GSK343 for 24 or 48 h.
Scanning electron microscopy of eggs; note that the size bar is shown within the black thin line below each image. (A) Micrographs of eggs from control females not exposed to the compound (0.1% DMSO); panels 1 and 3: Oval egg with normal lateral spine (s) at 24 and 48 h (Bar = 40 μm); panels 2 and 4: the surface of the eggs shows normal micro spicules at 24 and 48 h incubation (Bar = 2 μm). Micrographs of eggs from females exposed to GSK343 (20 μM); panels 5 and 7: Reduced egg size with lateral spine (s) at 24 and 48 h of treatment (Bar = 40 μm); panels 6 and 8: the surface of the eggs show fissures at 24 and 48 h (Bar = 2 μm; 3 μm). (B and C) Total area dimension of S. mansoni eggs and number of eggs released by females incubated with GSK343 (20 μM) or vehicle (0.1% DMSO), respectively. *** p < 0.001.
Fig 4.
Predominant ultrastructural changes on the dorsal surface of S. mansoni juvenile worms exposed for 48 h to different concentrations of GSK343.
(A) Scanning electron microscopy of juvenile parasites; note that the size bar is shown within the black thin line below each image. (panel 1) Low magnification image of control juvenile worm (0.1% DMSO) (Bar = 100 μm); (panels 2 and 3) Oral and ventral sucker of control juvenile worm presenting tegument without structural alterations (Bar = 30 μm and 20 μm); (panels 4 and 5) Medial dorsal region of control juvenile worms showing intact tegument (Bar = 20 μm and 5 μm); (panel 6) Low magnification image of juvenile worm treated with 20 μM GSK343 (Bar = 100 μm); (panels 7 and 8) Non-detectable changes to oral and ventral sucker of juvenile worm treated with 20 μM GSK343 (Bar = 40 μm and 30 μm); (panels 9 and 10) Medial dorsal region of juvenile worms showing enlarged view showing blisters and focal lesions on the tegument (Bar = 10 μm and 5 μm); (panel 11) Low magnification image of juvenile worm treated with 50 μM GSK343 shows altered morphology (Bar = 200 μm); (panels 12 and 13) Oral and ventral sucker of juvenile worm treated with 50 μM GSK343 presenting areas of cracking (Bar = 40 μm and 30 μm); (panels 14 and 15) Medial dorsal region of juvenile worms treated with 50 μM GSK343 showing erosion of tegument (Bar = 10 μm and 5 μm). os: oral sucker; vs: ventral sucker; b: blisters. (B) Viability of juvenile worms was estimated by the total amount of ATP available in the parasites, using a luminescent assay. Juvenile worms were treated for 24 h (black bars) and 48 h (gray bars) with GSK343 at the different concentrations indicated or with vehicle (0.1% DMSO). The viability was expressed as % of the luminescence values relative to the control (DMSO). Mean ± SD from three replicate experiments, each with 10 juvenile worms. *p < 0.05, ** p< 0.01, *** p< 0.001.
Fig 5.
Morphological changes and increased apoptosis in schistosomula exposed to 20 μM GSK343.
(A) Scanning electron microscopy of schistosomula; note that the size bar is shown within the black thin line below each image. (A, panels 1 and 2) Control schistosomula micrographs at the same magnification (Bar = 40 μm) showing the ventral sucker (vs) of typical schistosomula maintained for 16 h in culture medium (panel 1) or further incubated for 24 h in culture medium plus vehicle (0.1% DMSO) (panel 2); the lateral and ventral aspects are normal, and the schistosomula underwent normal development and lengthening after 24 h incubation in vehicle-containing medium (compare panels 2 and 1). (A, panel 3) Higher magnification of control schistosomulum from panel 2 showing that the entire body surface is covered by spines with no ultrastructural changes (Bar = 3 μm); (A, panels 4 and 5) Longer incubation (48 h) of control schistosomula in vehicle-containing medium shows further development and growth (panel 4, Bar = 50 μm) compared with panel 2; and a higher magnification (panel 5, Bar = 3 μm) confirms that the surface is covered by spines with no ultrastructural changes. (A, panels 6 and 7) Schistosomula micrographs at the same magnification (Bar = 30 μm) showing the ventral sucker (vs) of typical schistosomula maintained for 16 h in culture medium (panel 6) or further incubated for 24 h in culture medium plus 20 μM GSK343 (panel 7); after 24 h in the presence of GSK343 the schistosomulum showed no apparent development or change in size, with an extremely contracted body region and the presence of numerous folds and small blisters (panel 7). (A, panel 8) Higher magnification of schistosomulum from panel 7 is an example showing that the teguments of parasites exposed to 20 μM GSK343 for 24 h have a high degree of changes including peeling and blister formation (Bar = 3 μm). (A, panels 9 and 10) Longer incubation of schistosomula (48 h) in culture medium plus 20 μM GSK343 showed no further development (panel 9) compared with the initial schistosomulum (panel 6), with the presence of a greater number of blisters when compared with panel 7; panel 10 shows a higher magnification of the schistosomulum from panel 9, with intense peeling of tegument and loss of numerous spines (Bar = 5 μm). (B) Total length of schistosomula incubated with vehicle (0.1% DMSO) or with 20 μM GSK343 for 24 and 48h. Mean ± SD from three replicate experiments, each with 20 schistosomula. (C) Increase in caspases 3/7 activity after 48 h treatment with 20 μM GSK343. Mean ± SD from three replicate experiments, each with 20,000 schistosomula. (D, panels 1 to 4) TUNEL assay of schistosomula incubated for 72 h with 0.1% DMSO (panels 1 and 2) or with 20μM GSK343 (panels 3 and 4). Green parasites indicate DNA fragmentation. In panel 1, DAPI nuclear DNA staining of control parasites (0.1% DMSO). In panel 2, TUNEL staining of control parasites from panel 1 (0.1% DMSO). In panel 3, DAPI nuclear DNA staining of GSK343 treated parasites. In panel 4, TUNEL staining of GSK343 treated parasites from panel 3. (vs), ventral sucker; (s), spines; (b), blisters. ***p < 0.001.
Fig 6.
Quantitation of the decrease in viability of schistosomula caused by GSK343 treatment at different concentrations and incubation times.
(A) Schistosomula treated with the indicated concentrations of GSK343 or with vehicle (0.1% DMSO) for 24, 48 and 72 h were visualized by staining with propidium iodide (marker of dead cells; 572 nm emission filter microscope) and with fluorescein diacetate (marker of living cells; 492 nm emission filter microscope). The bottom row shows a positive control, namely exposure to 70% ethanol, which kills all parasites. For each time point (indicated at the top), the left panel shows a light microscopy image and the right panel shows the image of the same field with differential fluorescence detection of PI-positive dead and FDA-positive live schistosomula by superimposition of 536nm and 494nm epifluorescence spectra. (B) Quantitation of viability of treated schistosomula. Percentage of viable schistosomula (non-stained with propidium iodide) over three days of treatment. For each condition tested, about 3600 schistosomula were used, divided into four biological replicates and three time points analyzed. Mean ± SD from four replicate experiments. (C) ATP quantitation using a luminescent assay to assess schistosomula survival under GSK343 exposure. Schistosomula (100-120/well) were incubated with the indicated concentrations of GSK343 or with vehicle (0.1% DMSO) for up to 5 days. The viability was expressed as % the luminescence values relative to the control (DMSO). Mean ± SD from three replicate experiments. *p < 0.05; **p < 0.01 (two-way ANOVA).
Fig 7.
Histone H3 post-translational modification profile in S. mansoni after treatment with GSK343.
Five different histone marks in adult worms (A to E) and in schistosomula (F to J) were detected and quantified by western blotting with (A and F) anti-H3K27me3, (B and G) anti-H3K9ac, (C and H) anti-H3K27ac, (D and I) anti-H3K4me3, (E and J) anti-H3K27me1 antibodies. Each panel consists of two parts: the upper part shows the lanes of a typical western blot of nuclear extract from untreated control parasites (C) or parasites treated for 48 h with 20 μM GSK343 (T) which were developed either with the specific antibody against the indicated histone mark or with an antibody anti-H3, used as a sample loading normalizer. A molecular weight marker protein (MW, 15 kDa) is indicated. The lower part of each panel shows the mean intensity of the bands for three biological replicates, obtained by extracting the intensity values of scanned images; for each sample, the intensity of the modified histone band was normalized by the intensity of histone H3. Mean ± SD is shown; t-test was applied and statistically significant p-values are indicated: *p < 0.05; **p < 0.01.
Fig 8.
Heatmap of differentially expressed genes detected by RNA-seq in adult S. mansoni female and male worms treated with GSK343.
The heatmaps show the hierarchical clustering of differentially expressed genes (lines) in three biological replicates (columns) of female (A) and male (B) adult worm samples, either for controls or for treated parasites, as indicated at the top of the heatmaps. Parasites were exposed for 48 h in vitro to vehicle (control) or to 20 μM GSK343. Gene expression levels were measured by RNA-seq and are shown as Z-scores, which are the number of standard deviations below (blue, downregulated) or above (red, upregulated) the mean expression value among treated and control samples for each gene; the expression level Z-scores are color-coded as indicated on the scale at the bottom.
Fig 9.
Heatmap of differentially expressed genes detected by RNA-seq in schistosomula treated with GSK343.
The heatmap shows the hierarchical clustering of differentially expressed genes (lines) in four biological replicates (columns) of schistosomula samples, either for controls or for treated parasites, as indicated at the top of the heatmap. Parasites were exposed for 48 h in vitro to vehicle (control) or to 20 μM GSK343. Gene expression levels were measured by RNA-seq and are shown as Z-scores, which are the number of standard deviations below (blue, downregulated) or above (red, upregulated) the mean expression value among treated and control samples for each gene; the expression level Z-scores are color-coded as indicated on the scale at the bottom.
Fig 10.
Gene Ontology terms enrichment analysis of downregulated genes detected by RNA-seq in adult S. mansoni female and male worms treated with GSK343.
Top 20 enriched GO terms for differentially expressed downregulated genes in female (A) and in male (B) adult worms. The three major GO term categories, namely Biological Process, Cellular Component and Molecular Function are separately represented in each panel. The size of the circles is proportional to the number of genes in each significantly enriched category, as indicated at the lower part scales; the colors show the statistical significance of the enrichment, as indicated by the -log10 FDR values that appear in the color-coded scales at the bottom. A GO enrichment significance cutoff of FDR ≤ 0.05 was used.
Fig 11.
Gene Ontology terms enrichment analysis of upregulated genes detected by RNA-seq in adult S. mansoni male worms treated with GSK343.
Top 20 enriched GO terms for differentially expressed upregulated genes in male adult worms. The three major GO term categories, namely Biological Process, Cellular Component and Molecular Function are separately represented in each panel. The size of the circles is proportional to the number of genes in each significantly enriched category, as indicated at the lower part scales; the colors show the statistical significance of the enrichment, as indicated by the -log10 FDR values that appear in the color-coded scales at the bottom. A GO enrichment significance cutoff of FDR ≤ 0.05 was used; no significantly enriched GO terms were found among the female upregulated genes.
Fig 12.
Genes related to DNA replication that were downregulated in GSK343 treated female adult worms.
(A) Heatmap of 32 genes related to DNA replication that were downregulated in treated females. Genes are shown on the lines, and three biological replicates are shown in the columns, for control (turquoise bar at the top of columns) or GSK343-treated female samples (yellow bar at the top of columns). Parasites were exposed for 48 h in vitro to vehicle (control) or to 20 μM GSK343. Gene expression levels were measured by RNA-seq and are shown as Z-scores, which are the number of standard deviations below (blue, downregulated) or above (red, upregulated) the mean expression value among treated and control samples for each gene; the expression level Z-scores are color-coded as indicated on the scales at the bottom. The heatmap was obtained with unsupervised clustering of samples and genes. (B) Principal component analysis (PCA) of expression of the 32 genes from (A) that were measured by RNA-seq in control and treated females, males and schistosomula, as indicated by the legend at the bottom.
Fig 13.
Genes related to ncRNA metabolism, egg biosynthesis and cell differentiation that were downregulated in GSK343 treated female adult worms.
(A) Heatmap of 51 genes related to ncRNA metabolism that were downregulated in treated females. The heatmap was obtained with unsupervised clustering of samples and genes. (B) Principle component analysis (PCA) of expression of the 51 genes from (A) that were measured by RNA-seq in control and treated females, males and schistosomula, as indicated by the legend at the bottom. (C) Heatmap of 9 selected genes, related to egg biosynthesis and cell differentiation, whose expression was significantly affected in GSK343-treated females. Heatmap obtained with unsupervised clustering of genes. In the heatmaps of (A) and (C), genes are shown on the lines, and three biological replicates are shown in the columns, for control (turquoise bar at the top of columns) or GSK343-treated female samples (yellow bar at the top of columns). Parasites were exposed for 48 h in vitro to vehicle (control) or to 20 μM GSK343. Gene expression levels were measured by RNA-seq and are shown as Z-scores, which are the number of standard deviations below (blue, downregulated) or above (red, upregulated) the mean expression value among treated and control samples for each gene; the expression level Z-scores are color-coded as indicated on the scales at the bottom.
Fig 14.
Gene Ontology terms enrichment analysis of differentially expressed genes detected by RNA-seq in schistosomula treated with GSK343.
Enriched GO terms for differentially expressed genes in schistosomula, separated by downregulated genes (A) and upregulated genes (B). The three major GO term categories, namely Biological Process, Cellular Component and Molecular Function are separately represented in each panel. The size of the circles is proportional to the number of genes in each significantly enriched category, as indicated at the lower part scales; the colors show the statistical significance of the enrichment, as indicated by the -log10 FDR values that appear in the color-coded scales at the bottom. A GO enrichment significance cutoff of FDR ≤ 0.05 was used.