Abstract
Parasitic liver flukes of the family Fasciolidae are responsible for major socioeconomic losses worldwide. However, at present, knowledge of the fundamental molecular biology of these organisms is scant. Here, we characterize, for the first time, the transcriptome and secreted proteome of the adult stage of the "giant liver fluke," Fascioloides magna, using Illumina sequencing technology and one-dimensional SDS-PAGE and OFFGEL protein electrophoresis, respectively. A total of ∼54,000,000 reads were generated and assembled into ∼39,000 contiguous sequences (contigs); ∼20,000 peptides were predicted and classified based on homology searches, protein motifs, gene ontology, and biological pathway mapping. From the predicted proteome, 48.1% of proteins could be assigned to 384 biological pathway terms, including "spliceosome," "RNA transport," and "endocytosis." Putative proteins involved in amino acid degradation were most abundant. Of the 835 secreted proteins predicted from the transcriptome of F. magna, 80 were identified in the excretory/secretory products from this parasite. Highly represented were antioxidant proteins, followed by peptidases (particularly cathepsins) and proteins involved in carbohydrate metabolism. The integration of transcriptomic and proteomic datasets generated herein sets the scene for future studies aimed at exploring the potential role(s) that molecules might play at the host-parasite interface and for establishing novel strategies for the treatment or control of parasitic fluke infections.
Highlights
From the ‡Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia; §Queensland Tropical Health Alliance, James Cook University, Cairns, Queensland 4878, Australia; ʈQueensland Institute of Medical Research, Brisbane, Queensland 4006, Australia; ‡‡Faculty of Science, Charles University in Prague, Prague, Czech Republic; §§Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Queensland 4111, Australia
The present study provides a comprehensive snapshot of the transcriptome and the excretory/ secretory (ES) proteome of the adult stage of F. magna and represents an invaluable resource for fundamental investigations of the molecular biology of liver flukes that is of both veterinary and public health importance
Almost two-thirds of F. magna transcripts sequenced in the present study were similar to molecules identified in the transcriptomes of Fa. hepatica and Fa. gigantica, respectively [3, 25], which is indicative of the biological similarities among members of the family Fasciolidae
Summary
Procurement of Parasite Material, RNA Isolation, and Illumina Sequencing—Adult F. magna were collected from naturally infected livers of red deer (Cervus elaphus) (from the Brdy mountains, Czech Republic) and washed in 0.1 M phosphate-buffered saline (PBS), pH 7.2, at 37 °C. Proteins were conceptually translated from the predicted coding domains of individual transcriptomic sequences and their functions were predicted using InterProScan [41], employing the default search parameters Based on their homology to conserved domains and protein families, proteins predicted for F. magna were assigned parental (i.e. level 2) gene ontology (GO) terms Bioinformatic Analyses of Proteomic Sequence Data—Protein sequences from mass spectrometric analyses were compared to peptide sequences predicted from transcriptomic data and to proteins available from the SwissProt database (May 2011) using Mascot v.2.3.02 (http://www.matrixscience.com), employing the following search parameters: enzyme ϭ trypsin; precursor ion mass tolerance ϭ Ϯ0.1; fixed modifications ϭ methionine oxidation; variable modifications ϭ carbamidomethylation; number of missed cleavages allowed ϭ 2; charge states ϭ ϩ2 and ϩ3. Putative mannose 6-phosphate glycosylation sites were identified using the NetNGlyc server [56]
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