title: Development of functional genomic tools in trematodes: RNA interference and luciferase reporter gene activity in Fasciola hepatica.
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.
authors.affiliation: Department of Tropical Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Rinaldi
authors.forename: Maria E
authors.initials: ME
authors.lastname: Morales
authors.forename: Martín
authors.initials: M
authors.lastname: Cancela
authors.forename: Estela
authors.initials: E
authors.lastname: Castillo
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brindley
authors.forename: José F
authors.initials: JF
authors.lastname: Tort
mesh_terms: D000818:Animals; D005210:Fasciola hepatica / Q000201:enzymology / Q000235:genetics*; D005211:Fascioliasis / Q000469:parasitology*; D017930:Genes, Reporter; D023281:Genomics / Q000379:methods*; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007931:Leucyl Aminopeptidase / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000469:parasitology; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012908:Snails
chemical_list: D015801:Helminth Proteins; D049409:Luciferases, Firefly; D007931:Leucyl Aminopeptidase