Table 1.
Comparison of the number of CYP450 and CYP450 reductase genes from different species compiled from Nelson et al. [15]
Fig 1.
Chemical structures of CYP450 inhibitors used in this study.
Table 2.
List of primers used for PCR, RT-PCR and qRT-PCR.
Fig 2.
Comparison of Schistosoma mansoni CYP450 protein (Sman) with CYP450 proteins from other species.
Multiple alignment of CYP450 proteins from S. mansoni (csm305A); rabbit CYP450 2C5 (1nr6_a); human CYP450 2C9 (1r9o_a); human CYP450 2C19 (4gqs_a); human CYP450 1A1 (4i8v_a); and human CYP450 2b6 (4rrt_a). The residues are shown in one letter code and colored by type: red- negatively charged, blue—positively charged, yellow—Cys, green—hydrophobic, cyan—Gly, ochre—Pro, purple—aromatic. The residues are shown in brighter colors for conserved positions. The ‘P450-signature’ sequence, which forms a channel for electron transfer, and the CYP450 consensus motif responsible for heme-binding and interaction with molecular oxygen and the relevant substrates are boxed. Predicted helices in the secondary structure based on homology modelling of SmCYP450 are indicated by the bold letters A-L based on rabbit CYP450 2C5 [38].
Fig 3.
Structural modeling of S. mansoni CYP450 (CYP3050A1) and comparison to the structure determined for rabbit CYP450 2C5 (1nr6_a) [38].
The heme is shown is each model as a space-filling projection. The J and J’ helices in rabbit CYP450 2C5, which are absent in S. mansoni CYP450, are highlighted in yellow.
Fig 4.
CYP450 messenger RNA abundance during the lifecycle of Schistosoma mansoni.
Whole RNA was extracted from different stages of S. mansoni (cercariae, 1-day old schistosomula; juvenile liver worms (23 days post infection), adult males (49 days post infection), adult females (49 days post infection) and eggs) using TRIzol reagent and chloroform/ethanol extraction protocol. cDNA was synthesized from whole RNA and used for qRT-PCR, with reactions done in triplicate. Adult males (= 1) were used as calibrator stage and mRNA abundance was normalized to α-tubulin. Error bars indicate standard error of the mean with n ≥ 3 biological replicates. Numbers indicate fold change relative to adult males and all values are significantly different from adult males; p < 0.05; student t-test. The results indicate that S. mansoni CYP450 is expressed in all stages investigated and that its expression is developmentally regulated.
Fig 5.
Effect of silencing Schistosoma mansoni CYP450 in cultured larval worms.
Freshly prepared schistosomula (300–400) were placed in each well containing 1 ml Basch’s Media in a 24-well plate and overnight in a 37°C with 5% CO2. The following day schistosomula were treated with 10 or 30 μg/ml S. mansoni CYP450 dsRNA or 30 μg/ml negative control dsRNA. Over several days worms were observed for dead (dark, granular appearance) or alive (translucent). (A) mRNA expression patterns in schistosomula treated with S. mansoni CYP450 specific dsRNA or negative control dsRNA control after 3 days of treatment (Experiments 1 and 2) or 2 days treatment (Experiment 3). The control gene for cDNA input is S. mansoni glyceraldehyde 3-phosphate dehydrogenase (GAPDH). C, schistosomula treated with 30 μg/mL irrelevant dsRNA; 10, schistosomula treated with 10 μg/mL SmCYP450 dsRNA; 30, schistosomula treated with 30 μg/mL SmCYP450 dsRNA. (B) Effect of S. mansoni CYP450 dsRNA on schistosomula survival in cultures with 30 μg/mL negative control dsRNA (black square), 10 μg/mL S. mansoni CYP450-specific ds RNA (open triangle), and 30 μg/mL S. mansoni CYP450-specific ds RNA (black triangle). Treatments were done in triplicate and repeated 3 times. Error bars indicate standard error of the mean; *, p < 0.05; student t-test.
Fig 6.
Activity of anti-fungal imidazole CYP450 inhibitors on larval and adult Schistosoma mansoni worms.
Survival of schistosomula (A) after 2 d culture and adult worms (B) after 5 d culture for miconazole (black diamond), clotrimazole (black square), and ketoconazole (black triangle). (C) In house SAR on known miconazole analogs against adult worms. Numbers in the parenthesis are survival (%) of adult worms on day 7 in 10 μM of respective compound.
Table 3.
Results with selected cytochrome P450 inhibitors used in this study.
Fig 7.
Combinations of miconazole and RNAi have increased killing activity, suggesting that they function through inhibition of the same target.
(A) Schistosomula cultured with 10 μg/ml S. mansoni CYP450 dsRNA (black diamond); 2.5 μM miconazole (open circle); or 10 μg/ml S. mansoni CYP450 dsRNA and 2.5 μM miconazole (black circle). (B) Schistosomula cultured with 10 μg/ml S. mansoni CYP450 dsRNA (black diamond); 5 μM miconazole (black square); 5 μM miconazole plus 30 μg/ml irrelevant dsRNA and 5 μM miconazole (open circle); or 5 μM miconazole plus 10 μg/ml S. mansoni CYP450 dsRNA and 5 μM miconazole (black circle). All experiments were done in triplicate. Error bars indicate standard error of mean; *, p < 0.05; student t-test).
Fig 8.
Effect of miconazole on egg development.
(A) Example of egg development scoring scheme. Upper panel shows fluorescent images of eggs representative of each developmental stage scored; the bottom panel shows brightfield images of the same eggs. (B) Scoring of egg development in cultured eggs treated with 0, 5, or 10 μM miconazole. The percentage of eggs scored at developmental stages I-III (black bars) and eggs scored at developmental stages IV-V (gray bars) are indicated. For 0 μM miconazole, n = 55 eggs scored; for 5 μM miconazole, n = 56 eggs scored; for 10 μM miconazole, n = 62 eggs scored.