Research Article: Development of Functional Genomic Tools in Trematodes: RNA Interference and Luciferase Reporter Gene Activity in Fasciola hepatica

Date Published: July 9, 2008

Publisher: Public Library of Science

Author(s): Gabriel Rinaldi, Maria E. Morales, Martín Cancela, Estela Castillo, Paul J. Brindley, José F. Tort, Alex Loukas

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.

Partial Text: Parasitic diseases are a major problem worldwide being not only a health issue but also, when affecting productive species, an important factor in the economy. Advances in biochemistry and molecular biology of parasites have made possible to identify at the molecular level several candidate mediators in the parasite-host interaction. However, 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]–[4]). Transfection with reporter genes using endogenous promoters has been attempted successfully in adults, miracidia and sporocysts of S. mansoni by biolistic approaches [5]–[11]. Transient transfection by electroporation was achieved in schistosomules and adults of S. mansoni[12] and S. japonicum[13]. More recently advancements towards stable transduction using retroviral and transposon retroposon sequences have been made [14],[15]. Gene silencing by RNA interference was first demonstrated in schistosomules by soaking worms in double stranded RNA [16] and later extended to sporocysts [17]. Electroporation was also tested as a delivery method for dsRNA providing for stable and long term effects [18]. These initial reports paved the ground for the use of the methodology in the analysis of gene function [19]–[23]. Although the possibilities of this techniques in parasitic flatworms are still far away from the systematic use of RNAi at genomic scale as initiated in free living planarians [24]–[27], the existence of extensive genomic and expression information for S. mansoni and S. japonicum[28]–[32] provide opportunities for advances in the discovery of new anti-schistosome interventions.

Source:

http://doi.org/10.1371/journal.pntd.0000260

 

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