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.
Highlights
Parasitic diseases are a major problem worldwide being a health issue and, when affecting productive species, an important factor in the economy
The reduction of the mRNA levels of FhLAP, and the absence of variations in three other F. hepatica genes indicated that the silencing was specific for the target F. hepatica leucine aminopeptidase (FhLap) gene, excluding off-targeting effects. These findings demonstrated RNA interference (RNAi) silencing of an endogenous gene at mRNA level, following on from our initial demonstration of knock-down of the exogenous reporter gene encoding firefly luciferase at mRNA and at protein level
Concluding remarks Because we are interested to transfer reverse genetics tools developed for Schistosoma to less well studied flukes or other helminths, we designed a reporter system for RNAi using an exogenous gene, and tested it in the liver fluke Fasciola hepatica
Summary
Parasitic diseases are a major problem worldwide being a health issue and, when affecting productive species, an important factor in the economy. The validation of the role of these molecules is hampered in many cases by the absence of appropriate tools of analysis, in particular functional genomics approaches to address the importance of a target gene in the pathogen. While functional genomics of parasitic nematodes have benefited from the advancements in the model species Caenorhabditis elegans, in parasitic flatworms, this avenue has been explored almost exclusively in species of the genus Schistosoma, but the techniques are still in their infancy (reviewed in [1,2,3,4]). Transfection with reporter genes using endogenous promoters has been attempted successfully in adults, miracidia and sporocysts of S. mansoni by biolistic approaches [5,6,7,8,9,10,11]. Transient transfection by electroporation was achieved in schistosomules and adults of S. mansoni [12] and S. japonicum [13]. Electroporation was tested as a delivery method for dsRNA
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