Development of functional genomic tools in trematodes: RNA interference and luciferase reporter gene activity in Fasciola hepatica - PubMed (original) (raw)
Development of functional genomic tools in trematodes: RNA interference and luciferase reporter gene activity in Fasciola hepatica
Gabriel Rinaldi et al. PLoS Negl Trop Dis. 2008.
Abstract
The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite-host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi) reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC). We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA) specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP), and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth parasites. These could facilitate the study of gene function and the identification of relevant targets for intervention in organisms that are by other means intractable. More specifically, these results open new perspectives for functional genomics of F. hepatica, which hopefully can lead to the development of new interventions for fascioliasis.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. Suppression of exogenous luciferase activity in transfected schistosomules of Schistosoma mansoni.
Panel A. Five experiments with two sequential electroporations are summarized in the diagram, and the corresponding luciferase activity assays are depicted below. Each experiment consisted of two groups of aproximately 10,000 schistosomules. The treated groups were sequentially electroporated at the indicated times after cercarial transformation (CT, black arrow) whith dsRNA (dsLuc, red) and mRNA (mLuc, green), while the paired control groups were treated only with mLuc at the corresponding time. Three hours after the last electroporation worms were harvested and luciferase activity measured. Activity is expressed as percent of the respective control group that was considered as 100%. For simplicity one bar represents all control groups. Panel B. Relative luciferase activity tested in one day old schistosomules transformed simultaneously with mLuc and dsLuc in a single electroporation. The activity of the control group treated only with mLuc was considered as 100%. Panel C. Relative luciferase activity in fourteen day old schistosomules transformed sequentially with dsLuc and one day later with plasmid encoding firefly luciferase (pLuc, orange), and harvested two days after to measure luciferase activity. Activity is expressed as percent of the control group treated only with pLuc considered as 100%.
Figure 2. Luciferase activity in transfected Fasciola hepatica newly excysted juveniles.
Panel A. Detection of luciferase activity at the indicated times post-electroporation of the mLuc. Differences between the mLuc treated groups and non treated group (control) were significant (p<0.05). Panel B. Juvenile worms at the indicated times post excystement were electroporated with 50 ng/µl of luciferase mRNA (mLuc), incubated for three hours, and the luciferase activity in their tissue was measured. A mock electroporation, control group was included (electroporated in the absence of exogenous mRNA). Difference between 1, 2 or 3 day old NEJs and the control group are strongly significant (p<0.01). Panel C. Localization of mLuc in juvenile worms. Two day old NEJs were mock electroporated or with 50 ng/µl of mLuc, fixed three hours later and processed for whole mount hybridization. Digoxigenin labeled antisense probe detected mLuc in the digestive tract of treated worms (lower panel) while no signal was detected in mock electroporated worms (upper panel). Scale bar represent 50 microns. Panel D. Two day old juvenile worms electroporated with 0, 50 or 100 ng/µl of Silencer Cy3 fluorescent siRNA, and followed by fluorescent microscopy. Representative images of live worms maintained in culture for 24 hours after treatment are shown, with normal field in the upper panels and fluorescence in the lower panels.
Figure 3. Suppression of exogenous luciferase activity in transformed F. hepatica newly excysted juveniles (NEJ).
Panel A. One day old NEJ were transformed by electroporation with dsLuc or an irrelevant dsRNA, dsMalE. One day later, they were again transformed by electroporation, this time with mLuc. Untreated worms indicate the absence of luciferase activity in F. hepatica NEJ. There was a significant reduction in the luciferase activity in worms treated with dsLuc+mLuc (p<0.05). Unexpectedly, the activity was higher in the worms treated with the irrelevant dsMalE than in the control treated only with mLuc. Panel B. mLuc amplified by RT-PCR from serial dilutions of total RNA obtained on specimens fixed in formalin 48 hours after the electroporation.
Figure 4. RNA interference of the endogenous Fasciola hepatica leucine aminopeptidase (_Fh_LAP).
Panel A. Schematic representation of the _Fh_LAP gene, and the regions targeted by the dsRNA and amplified by PCR to test the effect. The region encoding the conserved carboxy domain of LAP is indicated by darker shading, with the signature active site regions indicated by white lines. Panel B. RT-PCR products amplified from serial dilutions of total RNA obtained 48 hours after the electroporation of NEJ with dsLap or dsLuc as an irrelevant dsRNA control. Reductions of mRNA were established by comparison to the amplification of the conserved GAPDH, and the juvenile specific CathB2 and CathL3 genes.
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