In vitro and in vivo evaluation of small interference RNA-mediated gynaecophoral canal protein silencing in Schistosoma japonicum

Abstract

Background: Schistosomiasis causes liver and intestinal damage and can be very debilitating. The pairing of a male worm with a female worm residing in the gynaecophoral canal of male plays a critical role in the development of female parasite. Because the male specific gynaecophoral canal protein of Schistosoma japonicum (SjGCP) is found in significant quantities in the adult female worm after pairing, it could play an important role in parasite pairing.

Methods: In the present study, three small interfering (si)RNA duplexes targeting the SjGCP gene were designed, synthesized and the silencing effects were evaluated in vitro as well as in mice infected with S. japonicum in vivo.

Results: In vitro studies using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR revealed the reduction of SjGCP at the transcript level. Similarly, western blotting and immunofluorescence studies showed its reduction at the protein level after treatment of parasites with siRNAs. At a concentration of 200 nM, two siRNAs totally abolished the parasite pairing. To evaluate such a pairing inhibitory effect in vivo, mice infected with S. japonicum were treated with siRNA and both parasite pairing and burden were evaluated. In vivo tests confirmed the in vitro silencing effect of SjGCP siRNA and revealed that the systemic delivery of siRNA significantly inhibited early parasite pairing and the associated burden.

Conclusions: Our preliminary results demonstrated that the SjGCP plays an important role in pairing and subsequent development in S. japonicum, and its silencing might have potential as a therapeutic approach for controlling schistosomiasis.

Figure 1

Soaking the parasite with fluorescent siRNA. Parasites were cultured for 3 h in culture medium containing unlabeled siRNA and fluoresceine‐labeled siRNA at a final concentration of 200 nM. Whole‐mount parasites treated with unlabeled siRNA (a, b) and fluoresceine‐labeled (c, d) were examined using a Zeiss confocal microscope. The parasite treated with unlabeled siRNA (a) was first used to adjust the parameters to remove autofluorescence. Os, oral sucker; Vs, ventral sucker

Figure 2

Effect of in vitro RNAi on SjGCP at the transcript level. (a) Semi‐Q RT‐PCR analysis at 3 days post‐treatment. (b) Image from (a) analyzed by Smartview software. Each value in the column is the ratio of the optical density of SjGCP and beta tubulin. (c) Semi‐Q RT‐PCR analysis of at 7 days post‐treatment. (d) Image from (c) analyzed by Smartview software. (e) Real‐time RT‐PCR analysis of SjGCP s1 siRNA at 3 and 7 days post‐treatment. Data are expressed as the mean ± SD of triplicate experiments

Figure 3

Effect of RNAi on SjGCP at the protein level. (a) Western blot analysis at 3 days post‐treatment. (b) Immunofluorescence patterns in the male gynaecophoral canal at 7 days post‐treatment. The arrow shows the side of the gynaecophoral canal in schistosomes; data are the representative results of 20 male worms examined in each treatment

Figure 4

RNAi effect of SjGCP s1 siRNA at the transcript and protein levels in parasites isolated from mice infected with S. japonicum that were administered s1 siRNA. (a) Semi‐Q RT‐PCR analysis at 19 days post‐infection. (b) Immunofluorescence patterns in the male gynaecophoral canal at 19 days post‐infection, where the arrow shows the side of the gynaecophoral canal of schistosomes; data are the representative results obtained from ten male worms examined in each treatment. (c) Real‐time RT‐PCR analysis at 32 days post‐infection; data are expressed as the mean ± SD of triplicate experiments. (d) Western blot analysis at 32 days post‐infection