Suppression of mRNAs encoding tegument tetraspanins from Schistosoma mansoni results in impaired tegument turnover - PubMed (original) (raw)

Suppression of mRNAs encoding tegument tetraspanins from Schistosoma mansoni results in impaired tegument turnover

Mai H Tran et al. PLoS Pathog. 2010.

Abstract

Schistosomes express a family of integral membrane proteins, called tetraspanins (TSPs), in the outer surface membranes of the tegument. Two of these tetraspanins, Sm-TSP-1 and Sm-TSP-2, confer protection as vaccines in mice, and individuals who are naturally resistant to S. mansoni infection mount a strong IgG response to Sm-TSP-2. To determine their functions in the tegument of S. mansoni we used RNA interference to silence expression of Sm-tsp-1 and Sm-tsp-2 mRNAs. Soaking of parasites in Sm-tsp dsRNAs resulted in 61% (p = 0.009) and 74% (p = 0.009) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in adult worms, and 67%-75% (p = 0.011) and 69%-89% (p = 0.004) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in schistosomula compared to worms treated with irrelevant control (luciferase) dsRNA. Ultrastructural morphology of adult worms treated in vitro with Sm-tsp-2 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. Schistosomula exposed in vitro to Sm-tsp-2 dsRNA had a significantly thinner and more vacuolated tegument, and morphology consistent with a failure of tegumentary invaginations to close. Injection of mice with schistosomula that had been electroporated with Sm-tsp-1 and Sm-tsp-2 dsRNAs resulted in 61% (p = 0.005) and 83% (p = 0.002) reductions in the numbers of parasites recovered from the mesenteries four weeks later when compared to dsRNA-treated controls. These results imply that tetraspanins play important structural roles impacting tegument development, maturation or stability.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Expression of _Sm_-tsp-1 and Sm-tsp-2 at different stages of the S. mansoni life cycle.

RNA levels of Sm-tsp-1 (vertical bars) and Sm-tsp-2 (horizontal bars) relative to _Sm_-α-tubulin were analyzed by qRT-PCR. Data are representative of mean±S.E. from three separate experiments.

Figure 2

Figure 2. Expression of _Sm_-TSP-1 or _Sm_-TSP-2 on the surface of live schistosomula.

Immunofluorescent labelling of live 3 h schistosomula with antisera raised against _Sm_-TSP-1 (A) and _Sm_-TSP-2 (B), and control pre-immune serum (C) followed by anti-mouse Ig-FITC. Schistosomula are shown in bright-field and FITC stained. Scale = 50 µm.

Figure 3

Figure 3. Suppression of Sm-tsp mRNAs in adult parasites by RNAi.

Sm-tsp-1 (A) and Sm-tsp-2 (B) transcript levels relative to _Sm_-paramyosin (mean±S.E.) in adult parasites soaked for 7 days with 1 µg/ml of Sm-tsp or luciferase control dsRNAs.

Figure 4

Figure 4. Suppression of Sm-tsp mRNAs in schistosomula by RNAi.

Sm-tsp-1 (A) and Sm-tsp-2 (B) transcript levels relative to Sm-paramyosin (mean±S.E.) in schistosomula soaked for 7, 14 and 21 days with 1 µg/ml of Sm-tsp or luciferase control dsRNAs.

Figure 5

Figure 5. Protein expression levels of parasites treated with Sm-tsp-2 dsRNA.

Protein extracts from adult parasites (A) and schistosomula (B) treated with Sm-tsp-2 or luciferase dsRNAs for 7 days were loaded onto a 12% SDS-PAGE gel at 2, 1, 0.5 and 0.25 µg. Proteins were transferred onto nitrocellulose and immunoblotted with anti-_Sm_-TSP2 or anti-_Sm_-Pmy monoclonal antibodies. The intensity of paramyosin expression was evaluated to determine equal protein loading.

Figure 6

Figure 6. Ultrastructure of the tegument of parasites treated with Sm-tsp-2 dsRNA RNA observed using transmission electron microscopy.

A. Tegument of adult female treated with luciferase dsRNA. B. Tegument of schistosomulum incubated for 7 days with luciferase dsRNA. C and E. Tegument of adult female incubated with Sm-tsp-2 dsRNA. The tegument is more highly vacuolated (C) and thinner (E) compared with controls. D and F. Tegument of schistosomula incubated for 7 days with Sm-tsp-2 dsRNA. Digitate extensions (arrows) are more abundant on the surface of the tegument. Abbreviations: Mus-muscles; teg-surface layer of tegument. The tegument of schistosomula were thinner, p<0.001 (G) and more dense, p = 0.014 (H) in Sm-tsp-2 dsRNA treated schistosomula.

Figure 7

Figure 7. Infection of mice with Sm-tsp dsRNA treated schistosomula.

Schistosomula were electroporated with 100 µg/ml of Sm-tsp-1, Sm-tsp-2 or luciferase dsRNAs, washed and counted. C57BL/6 female mice were immunized intramuscularly with 2,000 dsRNA treated schistosomula and were perfused 4 weeks later to determine parasite numbers (A). Expression of Sm-tsp-1 and Sm-tsp-2 mRNA transcript levels of parasites harvested from Experiment 1 (B). The Sm-tsp-1 and Sm-tsp-2 transcript levels of schistosomula that were electroporated and concurrently cultured in vitro for 4 weeks were also determined (C).

References

    1. Fenwick A, Rollinson D, Southgate V. Implementation of human schistosomiasis control: Challenges and prospects. Adv Parasitol. 2006;61:567–622. - PubMed
    1. Gryseels B, Polman K, Clerinx J, Kestens L. Human schistosomiasis. Lancet. 2006;368:1106–1118. - PubMed
    1. Steinmann P, Keiser J, Bos R, Tanner M, Utzinger J. Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect Dis. 2006;6:411–425. - PubMed
    1. Miller P, Wilson RA. Migration of the schistosomula of Schistosoma mansoni from the lungs to the hepatic portal system. Parasitology. 1980;80:267–288. - PubMed
    1. Jones MK, Gobert GN, Zhang L, Sunderland P, McManus DP. The cytoskeleton and motor proteins of human schistosomes and their roles in surface maintenance and host-parasite interactions. Bioessays. 2004;26:752–765. - PubMed

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