Using RNA interference to manipulate endogenous gene expression in Schistosoma mansoni sporocysts.
Direct assessments of gene function in parasitic flatworms have been hampered by the lack of effective tools to alter gene expression. The aim of the present study was to use RNA-interference (RNAi) to achieve targeted gene knockdown in larval stages of the human blood fluke, Schistosoma mansoni. We selected two S. mansoni genes for RNAi experiments: SGTP1, a facilitated diffusion glucose transporter and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). When S. mansoni larvae were treated in vitro for 6 days with dsRNA specific to one of these two genes, targeted transcript levels were reduced by 70-80% as determined by quantitative PCR (qPCR), while non-targeted transcripts were unaffected. Parasite exposure to SGTP1 dsRNA, but not GAPDH dsRNA, reduced larval glucose-uptake capacity by 40%, demonstrating that SGTP1 transcript knockdown results in the functional phenotype of reduced glucose transport activity. The effect of dsRNA treatment on transcript level was evident for up to 28 days after an initial dsRNA treatment. Interestingly, dsRNA treatment was effective only when miracidia were allowed to undergo the transition to sporocysts in its presence, while treatment of fully transformed sporocysts was ineffective. Fluorescence patterns in larvae exposed to rhodamine-labeled dsRNA as miracidia and sporocysts were similar, suggesting that the difference in susceptibility to dsRNA treatment between the two life stages may not be due to differences in dsRNA entry. Overall, this technology will enable direct assessment of the roles of individual genes in physiological processes of larval stages of S. mansoni, a crucial step in the identification of novel intervention targets for this important human pathogen.
Boyle
Jon P
JP
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, 2115 Observatory Drive, Madison, WI 53706, USA.
Wu
Xiao-Jun
XJ
Shoemaker
Chuck B
CB
Yoshino
Timothy P
TP
D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics; D003470:Culture Media; D005947:Glucose / Q000378:metabolism; D033661:Oocysts / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional; D012330:RNA, Double-Stranded / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D012550:Schistosoma mansoni / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development*
D000947:Antigens, Helminth; D003470:Culture Media; D012330:RNA, Double-Stranded; D012235:Rhodamines; D005947:Glucose