Genetic manipulation of Schistosoma haematobium, the neglected schistosome

Abstract

Background: Minimal information on the genome and proteome of Schistosoma haematobium is available, in marked contrast to the situation with the other major species of human schistosomes for which draft genome sequences have been reported. Accordingly, little is known about functional genomics in S. haematobium, including the utility or not of RNA interference techniques that, if available, promise to guide development of new interventions for schistosomiasis haematobia.

Methods/findings: Here we isolated and cultured developmental stages of S. haematobium, derived from experimentally infected hamsters. Targeting different developmental stages, we investigated the utility of soaking and/or square wave electroporation in order to transfect S. haematobium with nucleic acid reporters including Cy3-labeled small RNAs, messenger RNA encoding firefly luciferase, and short interfering RNAs (siRNAs). Three hours after incubation of S. haematobium eggs in 50 ng/µl Cy3-labeled siRNA, fluorescent foci were evident indicating that labeled siRNA had penetrated into miracidia developing within the egg shell. Firefly luciferase activity was detected three hours after square wave electroporation of the schistosome eggs and adult worms in 150 ng/µl of mRNA. RNA interference knockdown (silencing) of reporter luciferase activity was seen following the introduction of dsRNA specific for luciferase mRNA in eggs, schistosomules and mixed sex adults. Moreover, introduction of an endogenous gene-specific siRNA into adult schistosomes silenced transcription of tetraspanin 2 (Sh-tsp-2), the apparent orthologue of the Schistosoma mansoni gene Sm-tsp-2 which encodes the surface localized structural and signaling protein Sm-TSP-2. Together, knockdown of reporter luciferase and Sh-tsp-2 indicated the presence of an intact RNAi pathway in S. haematobium. Also, we employed laser scanning confocal microscopy to view the adult stages of S. haematobium.

Conclusions: These findings and approaches should facilitate analysis of gene function in S. haematobium, which in turn could facilitate the characterization of prospective intervention targets for this neglected tropical disease pathogen.

Figure 1. Eggs of Schistosoma haematobium.

These schistosome eggs were obtained from experimentally infected hamsters, and thereafter were maintained in culture. Eggs were recovered from small intestines (panels A and B) or liver (panels C and D). Scale bars, 100 µm.

Figure 2. Adults and micrograph showing the characteristic longitudinal disposition of the eggs along the body of the schistosomules of Schistosoma haematobium.

Panel A: micrograph illustrating a population of mixed sex adults obtained by portal perfusion from infected hamsters and maintained in culture. Panel B: high magnification micrograph showing the characteristic longitudinal disposition of the eggs along the body of the female. Panel C: images of cercariae released from infected snails. Panel D: images of representative schistosomules in culture 3 hours after cercarial transformation. Scale bars, 500 µm (A) and 100 µm (B, C and D).

Figure 3. Labeled short interfering RNA enters cultured eggs of Schistosoma haematobium.

Representative images of schistosome eggs 3 hours after soaking in Cy3-siRNA; panel A: no Cy3-siRNA treatment control, bright field; panel B: no Cy3-siRNA treatment control, fluorescence field, panel C: soaked eggs in medium containing 50 ng/µl of Cy3-siRNA, bright field, panel D: soaked eggs in medium containing 50 ng/µl of Cy3-siRNA, fluorescence field. Scale bar, 50 µm.

Figure 4. Luciferase activity measured in Schistosoma haematobium.

Panel A: S. haematobium eggs transfected with 150 ng/µl of firefly luciferase mRNA. Detection of luciferase activity in mock control (mock) and in mLuc treated eggs, measured three (3 h) and 20 (20 h) hours after electroporation. Panel B: Luciferase activity measured in extracts of adult worms 3 h after electroporation, (mock) adult worms treated with no molecule, (intact) intact worms treated with 150 ng/µl mRNA, and (fragmented) worms diced into three or more pieces and treated with 150 ng/µl mRNA.

Figure 5. Suppression of exogenous luciferase activity in transfected eggs, chopped/diced adults and schistosomules of Schistosoma haematobium.

Panel A: schematic representation of the experimental designs. Panel B: Luciferase activity measured in the indicated groups three hours after electroporation.

Figure 6. Silencing of the gene encoding the tetraspanin 2 antigen of Schistosoma haematobium.

Quantitative RT-PCR analysis of the mRNAs from adult S. haematobium worms at 48 h after transfection by electroporation with siRNA specific for Sh-tsp-2. >80% silencing of the Sh-tsp-2 (siShTSP2) evident when compared to the control group treated with siRNA scrambled control (siScrambled). Sh-tsp-2 expression was normalized to a control mRNA encoding tropomyosin.

Figure 7. Representative three dimensional (3D) renderings from laser scanning confocal images of adult forms of Schistosoma haematobium.

Panel A: 3D rendering from the female (left) and male (right) S. haematobium. Images were captured with 5× objective as tile-scan to cover the entire worm in addition to z-stacks. Propidium iodide (PI) was used to label the nuclei (red). B–E: High power (20×/1.0) 3D rendering from a female S. haematobium capturing the anterior of the worm. Three channels were extracted after applying a linear spectral unmixing algorithm to a lambda stack confocal images. B, The surface of the female S. haematobium visualized using reflected light scattering from the 488 nm laser line. C, PI –labeling, D, ‘autofluorescence’ from the schistosome eggs, E, merge of B–D, showing the structure of the anterior of the worm, with a semi-transparent visualization. In D and E, arrows indicate location of schistosome eggs. F–H, 3D rendering from the anterior of a male worm visualized with a similar approach used in B–E. F, reflected light channel, G, PI channel, H, merge of F and G using semi-transparent visualization. Scale bars, 500 µm (A), 400 µm (B–E), 200 µm (F–H).