Estela Castillo

RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs

Schistosoma mansoni leucine aminopeptidase (LAP) is thought to play a central role in hatching of the miracidium from the schistosome egg. We identified two discrete LAPs genes in the S. mansoni genome, and their orthologs in S. japonicum. The similarities in sequence and exon/intron structure of the two genes, LAP1 and LAP2, suggest that they arose by gene duplication and that this occurred before separation of the mansoni and japonicum lineages. The SmLAP1 and SmLAP2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP2 expression was higher in free living larval (miracidia) and in parasitic intra-snail (sporocysts) stages. We investigated the role of each enzyme in hatching of schistosome eggs and the early stages of schistosome development by RNA interference (RNAi). Using RNAi, we observed marked and specific reduction of mRNAs, along with a loss of exopeptidase activity in soluble parasite extracts against the diagnostic substrate l-leucine-7-amido-4-methylcoumarin hydroxide. Strikingly, knockdown of either SmLAP1 or SmLAP2, or both together, was accompanied by >or=80% inhibition of hatching of schistosome eggs showing that both enzymes are important to the escape of miracidia from the egg. The methods employed here refine the utility of RNAi for functional genomics studies in helminth parasites and confirm these can be used to identify potential drug targets, in this case schistosome aminopeptidases.

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

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.

Developmental expression of high molecular weight tropomyosin isoforms in Mesocestoides corti

Tropomyosins are a family of actin-binding proteins with diverse roles in actin filament function. One of the best characterized roles is the regulation of muscle contraction. Tropomyosin isoforms can be generated from different genes, and from alternative promoters and alternative splicing from the same gene. In this work, we have isolated sequences for tropomyosin isoforms from the cestode Mesocestoides corti, and searched for tropomyosin genes and isoforms in other flatworms. Two genes are conserved in the cestodes M. corti and Echinococcus multilocularis, and in the trematode Schistosoma mansoni. Both genes have the same structure, and each gene gives rise to at least two different isoforms, a high molecular weight (HMW) and a low molecular weight (LMW) one. Because most exons are duplicated and spliced in a mutually exclusive fashion, isoforms from one gene only share one exon and are highly divergent. The gene duplication preceded the divergence of neodermatans and the planarian Schmidtea mediterranea. Further duplications occurred in Schmidtea, coupled to the selective loss of duplicated exons, resulting in genes that only code for HMW or LMW isoforms. A polyclonal antibody raised against a HMW tropomyosin from Echinococcus granulosus was demonstrated to specifically recognize HMW tropomyosin isoforms of M. corti, and used to study their expression during segmentation. HMW tropomyosins are expressed in muscle layers, with very low or absent levels in other tissues. No expression of HMW tropomyosins is present in early or late genital primordia, and expression only begins once muscle fibers develop in the genital ducts. Therefore, HMW tropomyosins are markers for the development of muscles during the final differentiation of genital primordia.

RNA interference in Fasciola hepatica newly excysted juveniles: Long dsRNA induces more persistent silencing than siRNA

In trematodes RNA interference is the current tool of choice for functional analysis of genes since classical reverse genetic approaches remain unavailable. Whereas this approach has been optimized in schistosomes, few reports are available for other trematodes, likely reflecting the difficulties in the establishment of the technology. Here we standardized conditions for RNAi in the liver fluke Fasciola hepatica, the causative agent of fasciolosis, one of the most problematic infections affecting livestock worldwide. Targeting a single copy gene, encoding leucine aminopeptidase (LAP) as a model, we refined delivery conditions which identified electro-soaking, i.e. electroporation and subsequent incubation as efficient for introduction of small RNAs into the fluke. Knock down of LAP was achieved with as little as 2.5 μg/ml dsRNA concentrations, which may reduce or obviate off-target effects. However, at these concentrations, tracking incorporation by fluorescent labeling was difficult. While both long dsRNA and short interfering RNA (siRNA) are equally effective at inducing a short-term knock down, dsRNA induced persistent silencing up to 21 days after treatment, suggesting that mechanisms of amplification of the interfering signal can be present in this pathogen. Persistent silencing of the invasive stage for up to 3 weeks (close to what it takes for the fluke to reach the liver) opens the possibility of using RNAi for the validation of putative therapeutic targets.

Characterization of a putative hsp70 pseudogene transcribed in protoscoleces and adult worms of Echinococcus granulosus.

Searching for hsp70 genes in Echinococcus granulosus, a divergent cytoplasmic hsp70-like sequence (EgpsiHsp70) was isolated, possessing a small truncation in the region coding for the C-terminal glycine-rich linker and EEVD-Ct motif. Southern Blot analyses of E. granulosus, and in silico analyses of E. multilocularis indicate that this truncated sequence is repeated several times in both genomes, in some cases containing clear cut features of pseudogenization. Phylogenetic analyses and comparison of surrounding regions indicate that all these copies originated by successive genomic duplications of one originally truncated copy. These copies are diverging at an increased rate compared to functional cytoplasmic hsp70 genes, and ratios of non-synonymous over synonymous substitutions rates (dN/dS) point to a relaxation of sequence constraint, suggesting that these sequences are pseudogenes. Interestingly, RT-PCR demonstrates that EgpsiHsp70 is transcribed in protoscoleces and adult individuals of E. granulosus. We suggest that this sequence does not code for a functional polypeptide, although some features are unexpected for a sequence evolving under a strictly neutral mode. Transcription could either be vestigial or have a specific, non-coding function.

A new approach for the characterization of proliferative cells in cestodes

Cestodes show a remarkable proliferative capability that sustains the constant growth and differentiation of proglottids essential for their lifestyle. It is believed that a separate population of undifferentiated stem cells (the so-called germinative cells) are the only cells capable of proliferation during growth and development. The study of this particular cell subpopulation is hampered by the current lack of methods to isolate it. In this work, we developed a reproducible flow cytometry and cell sorting method to quantify and isolate the proliferating cells in the tetrathyridia larvae of the model cestode Mesocestoides corti, based on the DNA content of the cells. The isolated cells display the typical germinative cell morphology, and can be used for RNA isolation with a yield in the ng to μg range. We expect that this approach may facilitate the characterization of the germinative cells in M. corti and other model tapeworms.