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A Deep Exploration of the Transcriptome and “Excretory/Secretory” Proteome of Adult Fascioloides magna*

  • Cinzia Cantacessi
    Footnotes
    Affiliations
    Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia;

    Queensland Tropical Health Alliance, James Cook University, Cairns, Queensland 4878, Australia;
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  • Jason Mulvenna
    Correspondence
    To whom correspondence should be addressed: R. B. Gasser,; and J. Mulvenna,
    Footnotes
    Affiliations
    Queensland Institute of Medical Research, Brisbane, Queensland 4006, Australia;
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  • Neil D. Young
    Footnotes
    Affiliations
    Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia;
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  • Martin Kasny
    Footnotes
    Affiliations
    Faculty of Science, Charles University in Prague, Prague, Czech Republic;
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  • Petr Horak
    Affiliations
    Faculty of Science, Charles University in Prague, Prague, Czech Republic;
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  • Ammar Aziz
    Affiliations
    Queensland Tropical Health Alliance, James Cook University, Cairns, Queensland 4878, Australia;
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  • Andreas Hofmann
    Affiliations
    Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Queensland 4111, Australia.
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  • Alex Loukas
    Affiliations
    Queensland Tropical Health Alliance, James Cook University, Cairns, Queensland 4878, Australia;
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  • Robin B. Gasser
    Correspondence
    To whom correspondence should be addressed: R. B. Gasser,; and J. Mulvenna,
    Affiliations
    Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia;
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  • Author Footnotes
    * Currently, the Gasser Lab is supported by funding from the Australian Research Council (ARC), Melbourne Water Corporation, and the National Health and Medical Research Council (NHMRC) (R. B. G.). Support from the Victorian Life Sciences Computation Initiative (VLSCI) and IBM Collaboratory is also acknowledged (R. B. G.). The Hofmann and Loukas Labs are also supported by grants from the NHMRC and ARC. This study was also supported by the Linkage Infrastructure, Equipment and Facilities (LIEF) scheme from ARC (J. M.), the Czech Science Foundation (grant nos. P502/10/P248 and 206/09/H026), the Czech Ministry of Education (grant nos. MSM LC06009 and MSM 0021620820), and (partially) UNCE of the Charles University (grant no. 204017) (M. K.). J. M. holds a Career Development Award (CDA1), N. D. Y. holds an Early Career Researcher (ECR) Fellowship, and A. L. holds a Principal Research Fellowship from NHMRC.
    This article contains supplemental Figure S1 and Tables S1 to S4.
    ¶ These authors contributed equally to this work.
Open AccessPublished:August 16, 2012DOI:https://doi.org/10.1074/mcp.M112.019844
      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.
      Parasitic liver flukes (Platyhelminthes: Trematoda) of livestock, such as Fasciola hepatica and Fasciola gigantica, are responsible for major economic losses worldwide, estimated at USD ∼3 billion, due to morbidity, mortality, and decreased productivity (
      • Boray J.C.
      Chemotherapy of infections with Fasciolidae.
      ,
      • Kaplan R.M.
      Fasciola hepatica: a review of the economic impact in cattle and considerations for control.
      ,
      • Young N.D.
      • Hall R.S.
      • Jex A.R.
      • Cantacessi C.
      • Gasser R.B.
      Elucidating the transcriptome of Fasciola hepatica——a key to fundamental and biotechnological discoveries for a neglected parasite.
      ). The giant liver fluke, Fascioloides magna, infects a range of wild and domestic ruminants (e.g. cervids and bovids), primarily in North America and Europe (
      • Pybus M.J.
      Liver flukes.
      ). The life cycle of F. magna is indirect; eggs are released by mature flukes and excreted in the feces of the mammalian host. In aerated water, miracidia hatch from the eggs and penetrate the body of a susceptible, aquatic intermediate snail host (e.g. Fossaria parva or Fo. modicella in North America; Galba truncatula in Europe) within ∼2 h (
      • Coil W.H.
      The penetration of Fascioloides magna miracidia into the snail host Fossaria bulimoides. A scanning electron microscope study.
      ). In the intermediate host, the parasite develops through the stages of sporocysts, rediae, and cercariae; the latter larval stage emerges from the snail within ∼40–58 days (
      • Pybus M.J.
      Liver flukes.
      ). The cercariae encyst as metacercariae on submerged or emergent vegetation and are then ingested by a mammalian host. Once in the host, the metacercariae excyst and the juvenile flukes penetrate the intestinal walls and migrate to the liver, where they are encapsulated (in pairs) in pseudocysts formed by the hepatic parenchyma and then develop into mature flukes (
      • Pybus M.J.
      Liver flukes.
      ). The migration of the immature stages through the hepatic tissues, together with the large number of pseudocysts and the size of mature flukes (up to 8 cm in length (
      • Pybus M.J.
      Liver flukes.
      )), can result in liver fibrosis. Clinical signs associated with infection by F. magna include lethargy, anorexia, depression, and weight loss, with sudden death occurring in heavily infected animals (
      • Pybus M.J.
      Liver flukes.
      ).
      In livestock, the control of liver fluke infections has relied predominantly on treatment with anthelmintic drugs, such as closantel, oxyclozanide, and triclabendazole (
      • Fairweather I.
      Triclabendazole: new skills to unravel an old(ish) enigma.
      ). Triclabendazole is considered the drug of choice against both juvenile and adult stages of liver flukes in the definitive (mammalian) host, whereas other compounds affect only the adult stage (
      • Maes L.
      • Vanparijs O.
      • Lauwers H.
      • Deckers W.
      Comparative efficacy of closantel and triclabendazole against Fasciola hepatica in experimentally infected sheep.
      ,
      • Keiser J.
      • Utzinger J.
      Chemotherapy for major food-borne trematodes: a review.
      ). Thus, triclabendazole is widely and often excessively used for the treatment of trematodiases in livestock (
      • Fairweather I.
      Triclabendazole: new skills to unravel an old(ish) enigma.
      ,
      • Flynn R.J.
      • Mulcahy G.
      • Elsheikha H.M.
      Coordinating innate and adaptive immunity in Fasciola hepatica infection: implications for control.
      ), and this leads to a significant risk that drug resistance will develop. Indeed, there are recently published reports of triclabendazole resistance in Fa. hepatica populations in Australia (
      • Overend D.J.
      • Bowen F.L.
      Resistance of Fasciola hepatica to triclabendazole.
      ) and in Western European countries (
      • Coles G.C.
      • Rhodes A.C.
      • Stafford K.A.
      Activity of closantel against adult triclabendazole-resistant Fasciola hepatica.
      ,
      • Moll L.
      • Gaasenbeek C.P.H.
      • Vellema P.
      • Borgsteede F.H.M.
      Resistance of Fasciola hepatica against triclabendazole in cattle and sheep in the Netherlands.
      ,
      • Thomas I.
      • Coles G.C.
      • Duffus K.
      Triclabendazole-resistant Fasciola hepatica in southwest Wales.
      ,
      • Fairweather I.
      Triclabendazole progress report, 2005–2009: an advancement of learning?.
      ,
      • Gaasenbeek C.P.H.
      • Moll L.
      • Cornelissen J.
      • Vellema P.
      • Borgsteede F.H.M.
      An experimental study on triclabendazole resistance of Fasciola hepatica in sheep.
      ). In addition, despite major efforts in studies aimed at developing novel intervention strategies against liver flukes (
      • Spithill T.W.
      • Dalton J.P.
      Progress in development of liver fluke vaccines.
      ,
      • Piacenza L.
      • Acosta D.
      • Basmadjian I.
      • Dalton J.P.
      • Carmona C.
      Vaccination with cathepsin L proteinases and with leucine aminopeptidase induces high levels of protection against fascioliasis in sheep.
      ,
      • Dalton J.P.
      • Mulcahy G.
      Parasite vaccines—a reality?.
      ,
      • Dalton J.P.
      • Brindley P.J.
      • Knox D.P.
      • Brady C.P.
      • Hotez P.J.
      • Donnelly S.
      • O'Neill S.M.
      • Mulcahy G.
      • Loukas A.
      Helminth vaccines: from mining genomic information for vaccine targets to systems used for protein expression.
      ,
      • Dalton J.P.
      • O'Neill S.
      • Stack C.
      • Collins P.
      • Walshe A.
      • Sekiya M.
      • Doyle S.
      • Mulcahy G.
      • Hoyle D.
      • Khaznadji E.
      • Moire N.
      • Brennan G.
      • Mousley A.
      • Kreshchenko N.
      • Maule A.G.
      • Donnelly S.M.
      Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines.
      ,
      • McManus D.P.
      • Dalton J.P.
      Vaccines against the zoonotic trematodes Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica.
      ,
      • Acosta D.
      • Cancela M.
      • Piacenza L.
      • Roche L.
      • Carmona C.
      • Tort J.F.
      Fasciola hepatica leucine aminopeptidase, a promising candidate for vaccination against ruminant fasciolosis.
      ,
      • Jayaraj R.
      • Piedrafita D.
      • Dynon K.
      • Grams R.
      • Spithill T.W.
      • Smooker P.M.
      Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens.
      ), there is still a paucity of information on host–parasite interactions at the molecular level. Recent studies (
      • Young N.D.
      • Hall R.S.
      • Jex A.R.
      • Cantacessi C.
      • Gasser R.B.
      Elucidating the transcriptome of Fasciola hepatica——a key to fundamental and biotechnological discoveries for a neglected parasite.
      ,
      • Robinson M.W.
      • Menon R.
      • Donnelly S.M.
      • Dalton J.P.
      • Ranganathan S.
      An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.
      ,
      • Young N.D.
      • Jex A.R.
      • Cantacessi C.
      • Hall R.S.
      • Campbell B.E.
      • Spithill T.W.
      • Tangkawattana S.
      • Tangkawattana P.
      • Laha T.
      • Gasser R.B.
      A portrait of the transcriptome of the neglected trematode, Fasciola gigantica—biological and biotechnological implications.
      ) have provided the first insights into the molecular biology of fasciolids by exploring the transcriptomes of the adult stages of both Fa. hepatica and Fa. gigantica (
      • Young N.D.
      • Hall R.S.
      • Jex A.R.
      • Cantacessi C.
      • Gasser R.B.
      Elucidating the transcriptome of Fasciola hepatica——a key to fundamental and biotechnological discoveries for a neglected parasite.
      ,
      • Young N.D.
      • Jex A.R.
      • Cantacessi C.
      • Hall R.S.
      • Campbell B.E.
      • Spithill T.W.
      • Tangkawattana S.
      • Tangkawattana P.
      • Laha T.
      • Gasser R.B.
      A portrait of the transcriptome of the neglected trematode, Fasciola gigantica—biological and biotechnological implications.
      ) and of the composition of the excretory/secretory (ES)
      The abbreviations used are:
      BLAST
      Basic Local Alignment Search Tool
      ES
      excretory/secretory
      GO
      gene ontology
      KEGG
      Kyoto Encyclopedia of Genes and Genomes
      DTT
      dithiothreitol
      IAA
      iodoacetamide
      NCBI
      National Center for Biotechnology Information
      OGE
      OFF-GEL electrophoresis
      PBS
      phosphate-buffered saline
      SDS-PAGE
      sodium dodecyl-sulfate polyacrylamide gel electrophoresis.
      1The abbreviations used are:BLAST
      Basic Local Alignment Search Tool
      ES
      excretory/secretory
      GO
      gene ontology
      KEGG
      Kyoto Encyclopedia of Genes and Genomes
      DTT
      dithiothreitol
      IAA
      iodoacetamide
      NCBI
      National Center for Biotechnology Information
      OGE
      OFF-GEL electrophoresis
      PBS
      phosphate-buffered saline
      SDS-PAGE
      sodium dodecyl-sulfate polyacrylamide gel electrophoresis.
      products from both the juvenile and adult stages of Fa. hepatica (
      • Robinson M.W.
      • Menon R.
      • Donnelly S.M.
      • Dalton J.P.
      • Ranganathan S.
      An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.
      ). In these studies, proteolytic enzymes (e.g. cathepsins, asparaginyl endopeptidase cysteine proteases, and trypsin-like serine proteases) were identified as key molecules in both the transcriptome and ES products, which are likely to play key roles in parasite migration through tissues and in the modulation of immune responses in the mammalian host. Given the potential of proteolytic enzymes as vaccine candidates against trematodiases (
      • Dalton J.P.
      • Mulcahy G.
      Parasite vaccines—a reality?.
      ,
      • Dalton J.P.
      • Brindley P.J.
      • Knox D.P.
      • Brady C.P.
      • Hotez P.J.
      • Donnelly S.
      • O'Neill S.M.
      • Mulcahy G.
      • Loukas A.
      Helminth vaccines: from mining genomic information for vaccine targets to systems used for protein expression.
      ,
      • Dalton J.P.
      • O'Neill S.
      • Stack C.
      • Collins P.
      • Walshe A.
      • Sekiya M.
      • Doyle S.
      • Mulcahy G.
      • Hoyle D.
      • Khaznadji E.
      • Moire N.
      • Brennan G.
      • Mousley A.
      • Kreshchenko N.
      • Maule A.G.
      • Donnelly S.M.
      Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines.
      ,
      • Kasny M.
      • Mikes L.
      • Hampl V.
      • Dvorak J.
      • Caffrey C.R.
      • Dalton J.P.
      • Horak P.
      Chapter 4. Peptidases of trematodes.
      ), comparative analyses of the transcriptomes and ES products of liver flukes is of critical importance for an improved understanding of their molecular biology, as well as for developing new treatment and control strategies against them.
      Advances in “-omic” and computational technologies for the preprocessing, assembly, and annotation of sequence data (
      • DeMarco R.
      • Verjovski-Almeida S.
      Schistosomes—proteomics studies for potential novel vaccines and drug targets.
      ,
      • Chuan J.
      • Feng Z.
      • Brindley P.J.
      • McManus D.P.
      • Han Z.
      • Jianxin P.
      • Hu W.
      Our wormy world: genomics, proteomics and transcriptomics in East and southeast Asia.
      ,
      • Cantacessi C.
      • Campbell B.E.
      • Gasser R.B.
      Key strongylid nematodes of animals—impact of next-generation transcriptomics on systems biology and biotechnology.
      ) are substantially assisting studies of the transcriptomes and proteomes of parasitic helminths (
      • Young N.D.
      • Hall R.S.
      • Jex A.R.
      • Cantacessi C.
      • Gasser R.B.
      Elucidating the transcriptome of Fasciola hepatica——a key to fundamental and biotechnological discoveries for a neglected parasite.
      ,
      • Chuan J.
      • Feng Z.
      • Brindley P.J.
      • McManus D.P.
      • Han Z.
      • Jianxin P.
      • Hu W.
      Our wormy world: genomics, proteomics and transcriptomics in East and southeast Asia.
      ,
      • Cantacessi C.
      • Campbell B.E.
      • Gasser R.B.
      Key strongylid nematodes of animals—impact of next-generation transcriptomics on systems biology and biotechnology.
      ,
      • Verjovski-Almeida S.
      • DeMarco R.
      Current developments in Schistosoma proteomics.
      ,
      • Mulvenna J.
      • Hamilton B.
      • Nagaraj S.H.
      • Smyth D.
      • Loukas A.
      • Gorman J.J.
      Proteomics analysis of the excretory/secretory component of the blood-feeding stage of the hookworm, Ancylostoma caninum.
      ,
      • Young N.D.
      • Campbell B.E.
      • Hall R.S.
      • Jex A.R.
      • Cantacessi C.
      • Laha T.
      • Sohn W.M.
      • Sripa B.
      • Loukas A.
      • Brindley P.J.
      • Gasser R.B.
      Unlocking the transcriptomes of two carcinogenic parasites, Clonorchis sinensis and Opisthorchis viverrini.
      ). In the present study we explored, for the first time on a large scale, the transcriptome of adult F. magna, using Illumina-based sequencing technology and bioinformatic analyses of sequence data, and we characterized the protein components of the ES products from this developmental stage. This insight into the molecular biology of F. magna offers unprecedented opportunities for comparative investigations of various economically important liver flukes and the design of new interventions against these parasites.

      DISCUSSION

      The present study provides a comprehensive snapshot of the transcriptome and the 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 (
      • Young N.D.
      • Hall R.S.
      • Jex A.R.
      • Cantacessi C.
      • Gasser R.B.
      Elucidating the transcriptome of Fasciola hepatica——a key to fundamental and biotechnological discoveries for a neglected parasite.
      ,
      • Young N.D.
      • Jex A.R.
      • Cantacessi C.
      • Hall R.S.
      • Campbell B.E.
      • Spithill T.W.
      • Tangkawattana S.
      • Tangkawattana P.
      • Laha T.
      • Gasser R.B.
      A portrait of the transcriptome of the neglected trematode, Fasciola gigantica—biological and biotechnological implications.
      ), which is indicative of the biological similarities among members of the family Fasciolidae. Most abundant in the transcriptome of F. magna were molecules containing a predicted signal peptide; this finding is in accordance with the results of a previous study of the transcriptome of Fa. gigantica (
      • Young N.D.
      • Jex A.R.
      • Cantacessi C.
      • Hall R.S.
      • Campbell B.E.
      • Spithill T.W.
      • Tangkawattana S.
      • Tangkawattana P.
      • Laha T.
      • Gasser R.B.
      A portrait of the transcriptome of the neglected trematode, Fasciola gigantica—biological and biotechnological implications.
      ) and likely reflects the crucial role(s) that secreted proteins play in the biology of these organisms (
      • Robinson M.W.
      • Corvo I.
      • Jones P.M.
      • George A.M.
      • Padula M.P.
      • To J.
      • Cancela M.
      • Rinaldi G.
      • Tort J.F.
      • Roche L.
      • Dalton J.P.
      Collagenolytic activities of the major secreted cathepsin L peptidases involved in the virulence of the helminth pathogen, Fasciola hepatica.
      ). Of the 835 predicted proteins with a signal peptide in F. magna, 80 were identified in the ES products from this organism at a confidence level of 99%, in accordance with previous proteomic analyses of the ES products of other liver flukes (i.e. 20 to 90 proteins identified) (
      • Robinson M.W.
      • Menon R.
      • Donnelly S.M.
      • Dalton J.P.
      • Ranganathan S.
      An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.
      ,
      • Wilson R.A.
      • Wright J.M.
      • de Castro-Borges W.
      • Parker-Manuel S.J.
      • Dowle A.A.
      • Ashton P.D.
      • Young N.D.
      • Gasser R.B.
      • Spithill T.W.
      Exploring the Fasciola hepatica tegument proteome.
      ,
      • Zheng M.
      • Hu K.
      • Liu W.
      • Hu X.
      • Hu F.
      • Huang L.
      • Wang P.
      • Hu Y.
      • Huang Y.
      • Li W.
      • Liang C.
      • Yin X.
      • He Q.
      • Yu X.
      Proteomic analysis of excretory secretory products from Clonorchis sinensis adult worms: molecular characterization and serological reactivity of excretory-secretory antigen-fructose-1,6-bisphosphatase.
      ,
      • Mulvenna J.
      • Sripa B.
      • Brindley P.J.
      • Gorman J.
      • Jones M.K.
      • Colgrave M.L.
      • Jones A.
      • Nawaratna S.
      • Laha T.
      • Suttiprapa S.
      • Smout M.J.
      • Loukas A.
      The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini.
      ). This finding suggests that, in addition to peptides that were undetectable because of their low molecular weight and concentration and/or possible “endogenous” secretion, most proteins in ES products of F. magna were identified. In a previous study of the transcriptome and secreted proteome of Fa. hepatica, Robinson et al. (
      • Robinson M.W.
      • Menon R.
      • Donnelly S.M.
      • Dalton J.P.
      • Ranganathan S.
      An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.
      ) also observed a discrepancy between the number of secreted proteins predicted from transcriptomic data and that of proteins identified in the ES products from this species. In the present study, a total of 42 transcripts encoding cathepsins (e.g. cathepsins B and L) were detected in the transcriptome of F. magna, of which 7 were predicted to contain a signal peptide indicative of secretion (see supplemental Table S2), and 8 were identified in the ES products (see Fig. 3C). A possible explanation for the absence of a predicted signal peptide in transcripts encoding proteins identified in the ES products is that some members of the cathepsin family might be excreted/secreted via a “nonclassical” pathway that does not involve signal peptide cleavage (
      • Nickel W.
      The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes.
      ). Conversely, the difference between the number of cathepsin-encoding transcripts predicted from transcriptomic data and the number of cathepsins identified in the ES products supports the hypothesis that the transcriptome of fasciolids encodes “endogenous” cathepsins that function in key biological pathways, such as egg production, protein turnover, and molecular remodelling (
      • Robinson M.W.
      • Colhoun L.M.
      • Fairweather I.
      • Brennan G.P.
      • Waite J.H.
      Development of the vitellaria of the liver fluke, Fasciola hepatica, in the rat host.
      ), and “exogenous” cathepsins with roles that appear to relate to the digestion of host molecules (
      • Chen J.M.
      • Dando P.M.
      • Stevens R.A.
      • Fortunato M.
      • Barrett A.J.
      Cloning and expression of mouse legumain, a lysosomal endopeptidase.
      ,
      • Turk D.
      • Guncar G.
      Lysosomal cysteine proteases (cathepsins): promising drug targets.
      ).
      In parasitic trematodes, some cathepsins (e.g. cathepsins B and L) are known to be encoded by multigene families (
      • Robinson M.W.
      • Dalton J.P.
      • Donnelly S.
      Helminth pathogen cathepsin proteases: it's a family affair.
      ), which poses a challenge for both the de novo assembly of transcripts encoding different, particularly closely related isoforms and the identification of such isoforms using mass spectroscopy. The accurate characterization of isoforms via spectrometry is highly dependent on their sequence similarity and their abundance in the matrix subjected to analysis (in this case, ES products). Thus, it is possible that the high sequence similarity of the cathepsin L proteins identified in the transcriptome impaired mass spectral identification of low-abundant cathepsins L, which, in some cases, relied on the specific detection of variation of only two or three peptides. The presence of multiple isoforms of secreted cathepsin L has been reported previously for both Fa. hepatica (
      • Robinson M.W.
      • Menon R.
      • Donnelly S.M.
      • Dalton J.P.
      • Ranganathan S.
      An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.
      ,
      • Wilson R.A.
      • Wright J.M.
      • de Castro-Borges W.
      • Parker-Manuel S.J.
      • Dowle A.A.
      • Ashton P.D.
      • Young N.D.
      • Gasser R.B.
      • Spithill T.W.
      Exploring the Fasciola hepatica tegument proteome.
      ) and Fa. gigantica (
      • Young N.D.
      • Jex A.R.
      • Cantacessi C.
      • Hall R.S.
      • Campbell B.E.
      • Spithill T.W.
      • Tangkawattana S.
      • Tangkawattana P.
      • Laha T.
      • Gasser R.B.
      A portrait of the transcriptome of the neglected trematode, Fasciola gigantica—biological and biotechnological implications.
      ). In Fa. hepatica, these proteins have been shown to be crucial for parasite survival, mediating essential processes such as the digestion of host macromolecules and the suppression of the host immune response (
      • Dalton J.P.
      • O'Neill S.
      • Stack C.
      • Collins P.
      • Walshe A.
      • Sekiya M.
      • Doyle S.
      • Mulcahy G.
      • Hoyle D.
      • Khaznadji E.
      • Moire N.
      • Brennan G.
      • Mousley A.
      • Kreshchenko N.
      • Maule A.G.
      • Donnelly S.M.
      Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines.
      ). In Fa. hepatica, 24 different cathepsin L isoforms have been identified and classified into five clades (designated Clades 1–5; FhCL1–5) (
      • Robinson M.W.
      • Tort J.F.
      • Lowther J.
      • Donnelly S.M.
      • Wong E.
      • Xu W.
      • Stack C.M.
      • Padula M.
      • Herbert B.
      • Dalton J.P.
      Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulence-associated factors.
      ); members of Clades 1, 2, and 5 were shown to be present among ES products from adult worms, whereas members of Clades 3 and 4 were detected exclusively in ES products from juvenile worms, suggesting that different isoforms play distinct roles in molecular mechanisms linked to the invasion of and survival in the vertebrate host (
      • Robinson M.W.
      • Tort J.F.
      • Lowther J.
      • Donnelly S.M.
      • Wong E.
      • Xu W.
      • Stack C.M.
      • Padula M.
      • Herbert B.
      • Dalton J.P.
      Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulence-associated factors.
      ). The expansion of the cathepsin family of peptidases in Fa. hepatica is the result of a series of gene duplication events (
      • Robinson M.W.
      • Tort J.F.
      • Lowther J.
      • Donnelly S.M.
      • Wong E.
      • Xu W.
      • Stack C.M.
      • Padula M.
      • Herbert B.
      • Dalton J.P.
      Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulence-associated factors.
      ,
      • Irving J.A.
      • Spithill T.W.
      • Pike R.N.
      • Whisstock J.C.
      • Smooker P.M.
      The evolution of enzyme specificity in Fasciola spp.
      ). The sequence diversity displayed by members of the cathepsin protein family, together with their broad range of substrate specificities, has been hypothesized to play a role in the ability of parasitic trematodes to infect a wide range of mammalian hosts (
      • Dalton J.P.
      • O'Neill S.
      • Stack C.
      • Collins P.
      • Walshe A.
      • Sekiya M.
      • Doyle S.
      • Mulcahy G.
      • Hoyle D.
      • Khaznadji E.
      • Moire N.
      • Brennan G.
      • Mousley A.
      • Kreshchenko N.
      • Maule A.G.
      • Donnelly S.M.
      Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines.
      ,
      • Irving J.A.
      • Spithill T.W.
      • Pike R.N.
      • Whisstock J.C.
      • Smooker P.M.
      The evolution of enzyme specificity in Fasciola spp.
      ,
      • Tort J.
      • Brindley P.J.
      • Knox D.
      • Wolfe K.H.
      • Dalton J.P.
      Proteinases and associated genes of parasitic helminths.
      ). In the present study, transcripts encoding cathepsins with significant homology to FhCL3 were identified in the transcriptome of adult F. magna; however, the corresponding proteins were not detected in the ES products of this trematode by proteomic means. This finding contrasts with a previous analysis of Fa. hepatica, in which molecules encoding FhCL3 were not detected in the transcriptome of the adult worm (
      • Robinson M.W.
      • Tort J.F.
      • Lowther J.
      • Donnelly S.M.
      • Wong E.
      • Xu W.
      • Stack C.M.
      • Padula M.
      • Herbert B.
      • Dalton J.P.
      Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulence-associated factors.
      ). Five distinct cathepsin L isoforms were identified in the ES proteome of F. magna, including four isoforms and a cathepsin L-like protease, all significant homologues of FhCL1. However, some S2 active site residues of the F. magna cathepsin L (see Fig. 4) peptidases were similar to those of cathepsins expressed specifically by the juvenile stages of Fa. hepatica (
      • Robinson M.W.
      • Tort J.F.
      • Lowther J.
      • Donnelly S.M.
      • Wong E.
      • Xu W.
      • Stack C.M.
      • Padula M.
      • Herbert B.
      • Dalton J.P.
      Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulence-associated factors.
      ). The developmental regulation of the expression of cathepsin L is likely to represent a potential adaptation of different developmental stages of the parasite to the diverse environmental conditions encountered throughout its life cycle. Based on these observations, it is tempting to speculate that if developmental regulation of cathepsin L peptidases occurs in F. magna, it will differ from that in Fa. hepatica. Given the potential of cathepsins as vaccine targets against fascioliasis (
      • Dalton J.P.
      • O'Neill S.
      • Stack C.
      • Collins P.
      • Walshe A.
      • Sekiya M.
      • Doyle S.
      • Mulcahy G.
      • Hoyle D.
      • Khaznadji E.
      • Moire N.
      • Brennan G.
      • Mousley A.
      • Kreshchenko N.
      • Maule A.G.
      • Donnelly S.M.
      Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines.
      ), further studies aimed at elucidating the expression profiles of cathepsin-encoding transcripts in different stages of the F. magna life cycle, as well as the presence of members of this protein family in the ES products of the juvenile stages, will be crucial.
      In addition to the cathepsin L isoforms, six other proteases were detected in the ES products from F. magna, namely, two isoforms of cathepsin B2, a cathepsin A, a legumain, two isoforms of lysosomal Pro-Xaa carboxypeptidase and a leucine aminopeptidase (see Fig. 3C). Like cathepsin L, cathepsin B is thought to be expressed as an inactive zymogen and is activated following the removal of a pre-pro-region by an asparaginyl endopeptidase (legumain) (
      • Dalton J.P.
      • Brindley P.J.
      • Donnelly S.
      • Robinson M.W.
      The enigmatic asparaginyl endopeptidase of helminth parasites.
      ), also present in the ES products from F. magna. In the present study, predominantly activated forms of cathepsin L were identified, as evidenced by the lack of peptides assigned to the pre-pro-region. In a single case, the amino acid sequence of one of the cathepsin B isoforms identified included three pre-pro-region peptides, typical of the inactive form of this protein; however, each of the peptides from this region was represented by only one spectrum, whereas spectral counts of peptides from the mature sequence were higher (e.g. up to 23 for one of the peptides). Therefore, it is likely that inactivated proteases constituted a small proportion of the total number of cathepsins B detected in the ES products. In addition, for the second isoform of cathepsin B2 identified herein (Table III), the Asn in the region of the amino acid sequence, which precedes the first residue of the mature protein (typical of all cathepsin L peptidases from Fa. hepatica and proposed to act as a substrate for possible activation of these proteins by legumain proteases) (
      • Dalton J.P.
      • Brindley P.J.
      • Donnelly S.
      • Robinson M.W.
      The enigmatic asparaginyl endopeptidase of helminth parasites.
      ), was substituted for by a Glu residue. The same substitution was observed in three of the cathepsins L of F. magna (Fig. 4). Several of the proteases identified in the F. magna ES products were of lysosomal origin, including cathepsin A, the two Pro-Xaa carboxypeptidases, and, possibly, legumain (see Fig. 3C). On the basis of PSORT and the presence of mannose 6-phosphate glycosylation sites, 12 lysosomal proteins were identified in the F. magna ES products, which constituted ∼15% of the total identifications; after cystatin and the cathepsin proteases, lysosomal proteins were most abundant in the ES products. Recent proteomic studies of S. mansoni (blood fluke) and Fa. hepatica vomitus also identified a number of lysosomal proteins as enzymes putatively involved in digestive processes (
      • Wilson R.A.
      • Wright J.M.
      • de Castro-Borges W.
      • Parker-Manuel S.J.
      • Dowle A.A.
      • Ashton P.D.
      • Young N.D.
      • Gasser R.B.
      • Spithill T.W.
      Exploring the Fasciola hepatica tegument proteome.
      ,
      • Hall S.L.
      • Braschi S.
      • Truscott M.
      • Mathieson W.
      • Cesari I.M.
      • Wilson R.A.
      Insights into blood feeding by schistosomes from a proteomic analysis of worm vomitus.
      ). In S. mansoni, this observation led to the hypothesis that lysosomal proteases might be actively secreted into the gut lumen, the pH of which facilitates the activity of these proteins, in order to enable the digestion of plasma components as well as hemoglobin (
      • Hall S.L.
      • Braschi S.
      • Truscott M.
      • Mathieson W.
      • Cesari I.M.
      • Wilson R.A.
      Insights into blood feeding by schistosomes from a proteomic analysis of worm vomitus.
      ). A legumain, a pro-X carboxylpeptidase, a beta-glucosidase, several isoforms of ferritin, and the Niemann-Pick C2 protein were also identified here as constituents of the ES products from F. magna. The presence of these proteins in the F. magna ES products, as well as serpin and kunitz-type protease inhibitors, is likely a consequence of the fact that, in vitro, F. magna readily regurgitates the contents of its digestive tract into the culture medium, as previously observed for Fa. hepatica (
      • Wilson R.A.
      • Wright J.M.
      • de Castro-Borges W.
      • Parker-Manuel S.J.
      • Dowle A.A.
      • Ashton P.D.
      • Young N.D.
      • Gasser R.B.
      • Spithill T.W.
      Exploring the Fasciola hepatica tegument proteome.
      ). Indeed, the same proteins were also identified in vomitus from both of the latter trematodes and S. mansoni (
      • Wilson R.A.
      • Wright J.M.
      • de Castro-Borges W.
      • Parker-Manuel S.J.
      • Dowle A.A.
      • Ashton P.D.
      • Young N.D.
      • Gasser R.B.
      • Spithill T.W.
      Exploring the Fasciola hepatica tegument proteome.
      ,
      • Hall S.L.
      • Braschi S.
      • Truscott M.
      • Mathieson W.
      • Cesari I.M.
      • Wilson R.A.
      Insights into blood feeding by schistosomes from a proteomic analysis of worm vomitus.
      ).
      Table IIIFascioloides magna excretory/secretory proteins identified using tandem mass spectrscopy
      IDSCCODescriptionSPTMLONFhCsOv
      Proteases
      399261838Cathepsin B2+-Ex++++-
      39928411Cathepsin B2+-Ex+++--
      399311744Cathepsin L1+-Ex-+--
      399321337Cathepsin L1+-Ex-+--
      39929929Cathepsin L1+-Ex++--
      39933327Cathepsin L1+-Ex++--
      39930430Cathepsin L-like proteinase+-Ex/Pl++--
      128337Cathepsin A (carboxypeptidase C)+-Ex/Ly++---
      3216915Calpain--Cy-+-+
      22081329Legumain-1+-Ly/Ex-+--
      1118137Lysosomal Pro-Xaa carboxypeptidase (S28 family)+-Ly++++--
      1246211Lysosomal Pro-X carboxypeptidase--Ly-+--
      106325Leucine aminopeptidase 1--Cy/Mi-+--
      Oxidation/reduction
      22571125Ferritin-like protein--Cy-+-+
      24226755Yolk ferritin--Cy-+-+
      6813417Yolk ferritin--Cy-+-+
      3565418Yolk ferritin--Ex-+-+
      3449516Ferritin heavy chain--Cy-+-+
      1228722Glutathione S-transferase--Cy-+-+
      23322265Mu-glutathione transferase--Cy/Ex-+-+
      1482410Dihydrolipoamide dehydrogenase--Mi--+-
      22000454Peroxiredoxin--Ex-+--
      21268221Thioredoxin--Cy/Ex-+-+
      4552337Fatty-acid-binding protein Fh15--Cy-+-+
      22680336Fatty-acid-binding protein Fh15--Cy-+-+
      21349219Fatty-acid-binding protein type 3--Cy/Ex-+-+
      21954221Fatty-acid-binding protein type 3--Cy/Ex/Nu-+-+
      543029Short chain dehydrogenase--Cy-++-
      Carbohydrate metabolism
      1498616Hexokinase--Ex-+--
      11097620Alpha-glucosidase--Ly+---
      11549626Lysosomal alpha-mannosidase+-Ly+++---
      10957615Lysosomal alpha-mannosidase+-Ly----
      11712410Beta-galactosidase+-Ly++---
      1121410Alpha-galactosidase+-Ly+---
      12521417Lysosomal alpha-glucosidase+-Ly+++---
      177247Beta-hexosaminidase B+-Ly+++---
      1164927Enolase--Cy-+++
      Protease inhibitor
      87673832Cystatin--Cy-+-+
      2249818Leukocyte elastase inhibitor--Cy----
      762329Leukocyte elastase inhibitor--Cy----
      2998621Serpin--Mi-+--
      467127Serpin B--Pl-+--
      22726430Kunitz-type proteinase inhibitor+-Ex-+--
      22727217Kunitz-type protease inhibitor–like protein+-Ex-+--
      Structural
      1276817Plastin-2--Cy----
      430354Myoferlin-7Pl---+
      5968434Ribosomal protein L40-like isoform 1--Cy/Nu----
      2802311Tegumental protein 20.8 kDa (dynein light chain)--Cy---+
      9449220Transgelin--Cy----
      1108223Cyclophilin A--Ex----
      12338216CD63 antigen (Tetraspanin)-3Pl-+--
      21424220Tetraspanin-CD63 receptor-3Pl-+--
      6044210Actin--Cy-+++
      Transporters
      126144Multidrug resistance protein 1, 2, 3--Pl---
      1105138Glucose transporter+10Pl-+-
      1122237Solute carrier family 22 member 5-5Pl-+-
      214725ATP synthase, H+ transporting--Pl++-
      Miscellaneous
      116721029Annexin A7--Cy-+--
      2360513Annexin A6--Cy-+--
      3162410Annexin A7--Cy-+--
      3411727Triose-phosphate isomerase--Ex-+++
      2363637Cytidine deaminase--Cy/Ex----
      2295423Heat shock protein 70--Nu/Cy--+-
      889853Basement membrane-specific heparan sulfate proteoglycan core protein--Nu----
      14949429Hemoglobin F2--Cy-+--
      10852420Cubilin--Cy/Nu----
      990232GPI-anchored surface glycoprotein+3Pl++++--
      1152137LAMA-like protein 2 precursor--Er/Ex-+--
      21652251LAMA-like protein 2 precursor--Cy/Ex-+--
      11828327SH3-containing protein SH3GLB1-like--Cy----
      11926210Ornithine aminotransferase--Cy/Ex----
      181026Carbonic anhydrase+-Pl++--
      2270214Niemann-Pick C2 protein+-Ly+++--
      22436264Acid sphingomyelinase-like phosphodiesterase 3a+-Ex/Ly+---
      12409210Proteophosphoglycan ppg4--Cy/Ex----
      4722218Translation initiation inhibitor--Cy----
      Unknown
      11997415Hypothetical protein CLF_104189 [Clonorchis sinensis]--Mi/Cy----
      2247345Unknown--Cy----
      24853211Unknown--Cy----
      21118232Unknown--Cy/Ex----
      SC, number of unique peptides assigned to the protein; CO, percent cover; LO, putative subcellular localization of the protein; TM, number of transmembrane domains. The presence of a signal peptide (SP) or an N-linked glycosylation site (N) is denoted by “+”; the presence of the protein in the ES of adult Fasciola hepatica (Fh), Clonorchis sinensis (Cs), or Opisthorchis viverrini (Ov) is also denoted by “+.”
      The vast majority of F. magna ES products represented a complex mixture of proteins, predominantly of extracellular or cytoplasmic origin. In particular, a significant proportion of nonclassically secreted ES proteins, including a number of proteins predicted to be membrane bound, such as a tetraspanin and a glucose transporter, were identified. Unlike ES products from gastrointestinal parasitic nematodes, such as Ancylostoma caninum (
      • Mulvenna J.
      • Hamilton B.
      • Nagaraj S.H.
      • Smyth D.
      • Loukas A.
      • Gorman J.J.
      Proteomics analysis of the excretory/secretory component of the blood-feeding stage of the hookworm, Ancylostoma caninum.
      ), in which classically secreted proteins are very abundant, nonclassically secreted proteins have been consistently shown to represent a significant proportion of ES products from trematode parasites, including S. japonicum (
      • Liu F.
      • Cui S.J.
      • Hu W.
      • Feng Z.
      • Wang Z.Q.
      • Han Z.G.
      Excretory/secretory proteome of the adult developmental stage of human blood fluke, Schistosoma japonicum.
      ), C. sinensis (
      • Zheng M.
      • Hu K.
      • Liu W.
      • Hu X.
      • Hu F.
      • Huang L.
      • Wang P.
      • Hu Y.
      • Huang Y.
      • Li W.
      • Liang C.
      • Yin X.
      • He Q.
      • Yu X.
      Proteomic analysis of excretory secretory products from Clonorchis sinensis adult worms: molecular characterization and serological reactivity of excretory-secretory antigen-fructose-1,6-bisphosphatase.
      ), O. viverrini (
      • Mulvenna J.
      • Sripa B.
      • Brindley P.J.
      • Gorman J.
      • Jones M.K.
      • Colgrave M.L.
      • Jones A.
      • Nawaratna S.
      • Laha T.
      • Suttiprapa S.
      • Smout M.J.
      • Loukas A.
      The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini.
      ), and Fa. hepatica (
      • Wilson R.A.
      • Wright J.M.
      • de Castro-Borges W.
      • Parker-Manuel S.J.
      • Dowle A.A.
      • Ashton P.D.
      • Young N.D.
      • Gasser R.B.
      • Spithill T.W.
      Exploring the Fasciola hepatica tegument proteome.
      ). For the latter trematode, it has been proposed that the presence of these proteins in the ES products is a result of stress-induced shedding of the tegument during culture (
      • Morphew R.M.
      • Wright H.A.
      • LaCourse E.J.
      • Porter J.
      • Barrett J.
      • Woods D.J.
      • Brophy P.M.
      Towards delineating functions within the Fasciola secreted cathepsin I protease family by integrating in vivo based sub-proteomics and phylogenetics.
      ). However, morphological studies of this parasite have noted rapid turnover of its tegument, facilitated by subtegumental cells, leading to the hypothesis that the shedding of the tegument and associated proteins occurring in vivo might represent an immuno-defensive strategy (
      • Robinson M.W.
      • Menon R.
      • Donnelly S.M.
      • Dalton J.P.
      • Ranganathan S.
      An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.
      ,
      • Dalton J.P.
      • Skelly P.
      • Halton D.W.
      Role of the tegument and gut in nutrient uptake by parasitic platyhelminths.
      ). Two phospholipases and an ABC transporter were also identified among the ES products of F. magna. The presence of ABC transporters in the tegumental membrane of Fa. hepatica has prompted comparisons to the ER/Golgi-independent secretion of IL-1β and caspase-1 in mammalian cells (
      • Robinson M.W.
      • Menon R.
      • Donnelly S.M.
      • Dalton J.P.
      • Ranganathan S.
      An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.
      ). This process involves the formation of plasma membrane “blebs,” mediated by ABC transporters, which are subsequently released as microvesicles after phospholipase-mediated fusion with the plasma membrane (
      • MacKenzie A.
      • Wilson H.L.
      • Kiss-Toth E.
      • Dower S.K.
      • North R.A.
      • Surprenant A.
      Rapid secretion of interleukin-1β by microvescicle shedding.
      ); the presence of both phospholipases and ABC transporters in the ES products of F. magna suggests that a similar process might occur in this trematode. Although some ES products identified in the present study might relate to gut content regurgitated by the parasite (
      • Wilson R.A.
      • Wright J.M.
      • de Castro-Borges W.
      • Parker-Manuel S.J.
      • Dowle A.A.
      • Ashton P.D.
      • Young N.D.
      • Gasser R.B.
      • Spithill T.W.
      Exploring the Fasciola hepatica tegument proteome.
      ), 15 antioxidant proteins were identified, none of which possessed a signal peptide indicative of secretion. A similar profile of ES antioxidant proteins, including fatty-acid-binding proteins, glutathione S-transferase, peroxiredoxin, and thioredoxin, has been described for Fa. hepatica (
      • Robinson M.W.
      • Menon R.
      • Donnelly S.M.
      • Dalton J.P.
      • Ranganathan S.
      An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.
      ). In the latter trematode, these proteins are proposed to play an important role in the evasion of host immune responses, which likely relates to the protection of the parasite from reactive oxygen species released by host immune cells (
      • McGonigle L.
      • Mousley A.
      • Marks N.J.
      • Brennan G.P.
      • Dalton J.P.
      • Spithill T.W.
      • Day T.A.
      • Maule A.G.
      The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration.
      ,
      • Salazar-Calderón M.
      • Martin-Alonso J.M.
      • Ruiz de Eguino A.D.
      • Parra F.
      Heterologous expression and functional characterization of thioredoxin from Fasciola hepatica.
      ,
      • Salazar-Calderón M.
      • Martin Alonso J.M.
      • Castro A.M.
      • Parra F.
      Cloning, heterologous expression in Escherichia coli and characterization of a protein disulfide isomerase from Fasciola hepatica.
      ,
      • Sekiya M.
      • Mulcahy G.
      • Irwin J.A.
      • Stack C.M.
      • Donnelly S.M.
      • Xu W.
      • Collins P.
      • Dalton J.P.
      Biochemical characterisation of the recombinant peroxiredoxin (FhePrx) of the liver fluke, Fasciola hepatica.
      ), an inhibition of the proliferative potential of spleen cells (e.g. in rats) (
      • Cervi L.
      • Rossi G.
      • Masih D.T.
      Potential role for excretory-secretory forms of glutathione-S-transferase (GST) in Fasciola hepatica.
      ), and/or the recruitment and alternative activation of macrophages (
      • Cervi L.
      • Rossi G.
      • Masih D.T.
      Potential role for excretory-secretory forms of glutathione-S-transferase (GST) in Fasciola hepatica.
      ,
      • Donnelly S.
      • O'Neill S.M.
      • Sekiya M.
      • Mulcahy G.
      • Dalton J.P.
      Thioredoxin peroxidase secreted by Fasciola hepatica induces the alternative activation of macrophages.
      ,
      • Donnelly S.
      • Stack C.M.
      • O'Neill S.M.
      • Sayed A.A.
      • Williams D.L.
      • Dalton J.P.
      Helminth 2-Cys peroxiredoxin drives Th2 responses through a mechanism involving alternatively activated macrophages.
      ). All antioxidant proteins identified here, with the exception of dihydrolipoamide dehydrogenase, were also detected in the ES products from adult Fa. hepatica, suggesting that these proteins play a role in immune evasion (
      • McGonigle L.
      • Mousley A.
      • Marks N.J.
      • Brennan G.P.
      • Dalton J.P.
      • Spithill T.W.
      • Day T.A.
      • Maule A.G.
      The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration.
      ,
      • Salazar-Calderón M.
      • Martin-Alonso J.M.
      • Ruiz de Eguino A.D.
      • Parra F.
      Heterologous expression and functional characterization of thioredoxin from Fasciola hepatica.
      ,
      • Salazar-Calderón M.
      • Martin Alonso J.M.
      • Castro A.M.
      • Parra F.
      Cloning, heterologous expression in Escherichia coli and characterization of a protein disulfide isomerase from Fasciola hepatica.
      ,
      • Cervi L.
      • Rossi G.
      • Masih D.T.
      Potential role for excretory-secretory forms of glutathione-S-transferase (GST) in Fasciola hepatica.
      ,
      • Donnelly S.
      • O'Neill S.M.
      • Sekiya M.
      • Mulcahy G.
      • Dalton J.P.
      Thioredoxin peroxidase secreted by Fasciola hepatica induces the alternative activation of macrophages.
      ,
      • Donnelly S.
      • Stack C.M.
      • O'Neill S.M.
      • Sayed A.A.
      • Williams D.L.
      • Dalton J.P.
      Helminth 2-Cys peroxiredoxin drives Th2 responses through a mechanism involving alternatively activated macrophages.
      ). In Fa. hepatica, a variable antioxidant profile was observed in ES products from different developmental stages (
      • Robinson M.W.
      • Menon R.
      • Donnelly S.M.
      • Dalton J.P.
      • Ranganathan S.
      An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.
      ), which led to the hypothesis that these nonclassically secreted antioxidant proteins were transported through an alternative, transtegumental, secretory pathway (
      • Robinson M.W.
      • Menon R.
      • Donnelly S.M.
      • Dalton J.P.
      • Ranganathan S.
      An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.
      ). However, the existence of such a pathway in fasciolid trematodes remains to be demonstrated.
      The availability of entire genome sequences of related species of liver flukes, such as C. sinensis (
      • Wang X.
      • Chen W.
      • Huang Y.
      • Sun J.
      • Men J.
      • Liu H.
      • Luo F.
      • Guo L.
      • Lv Z.
      • Deng C.
      • Zhou C.
      • Fan Y.
      • Li X.
      • Huang L.
      • Hu Y.
      • Liang C.
      • Hu X.
      • Xum J.
      • Yum X.
      The draft genome of the carcinogenic human liver fluke Clonorchis sinensis.
      ) and blood flukes (
      • Young N.D.
      • Jex A.R.
      • Li B.
      • Liu S.
      • Yang L.
      • Xiong Z.
      • Li Y.
      • Cantacessi C.
      • Hall R.S.
      • Xu X.
      • Chen F.
      • Wu X.
      • Zerlotini A.
      • Oliveira G.
      • Hofmann A.
      • Zhang G.
      • Fang X.
      • Kang Y.
      • Campbell B.E.
      • Loukas A.
      • Ranganathan S.
      • Rollinson D.
      • Rinaldi G.
      • Brindley P.J.
      • Yang H.
      • Wang J.
      • Wang J.
      • Gasser R.B.
      Whole-genome sequence of Schistosoma haematobium.
      ,
      • The Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium
      The Schistosoma japonicum genome reveals features of host-parasite interplay.
      ,
      • Berriman M.
      • Haas B.J.
      • LoVerde P.T.
      • Wilson R.A.
      • Dillon G.P.
      • Cerqueira G.C.
      • Mashiyama S.T.
      • Al-Lazikani B.
      • Andrade L.F.
      • Ashton P.D.
      • Aslett M.A.
      • Bartholomeu D.C.
      • Blandin G.
      • Caffrey C.R.
      • Coghlan A.
      • Coulson R.
      • Day T.A.
      • Delcher A.
      • DeMarco R.
      • Djikeng A.
      • Eyre T.
      • Gamble J.A.
      • Ghedin E.
      • Gu Y.
      • Hertz-Fowler C.
      • Hirai H.
      • Hirai Y.
      • Houston R.
      • Ivens A.
      • Johnston D.A.
      • Lacerda D.
      • Macedo C.D.
      • McVeigh P.
      • Ning Z.
      • Oliveira G.
      • Overington J.P.
      • Parkhill J.
      • Pertea M.
      • Pierce R.J.
      • Protasio A.V.
      • Quail M.A.
      • Rajandream M.A.
      • Rogers J.
      • Sajid M.
      • Salzberg S.L.
      • Stanke M.
      • Tivey A.R.
      • White O.
      • Williams D.L.
      • Wortman J.
      • Wu W.
      • Zamanian M.
      • Zerlotini A.
      • Fraser-Liggett C.M.
      • Barrell B.G.
      • El-Sayed N.M.
      The genome of the blood fluke Schistosoma mansoni.
      ), now provides unprecedented opportunities to (i) conduct comparative proteomic comparisons; (ii) elucidate the structures and functions of key molecules (e.g. “endogenous” and “exogenous” cathepsins); (iii) explore novel biological pathways (e.g. transtegumental, secretory pathways); and (iv) establish relationships among genes, transcripts, and proteins involved specifically in the parasite's invasion of, establishment in, and interactions with the host. Advancing these areas will provide a basis for studies aimed at exploring the potential of these molecules as targets for the development of novel strategies for the control of trematodiases.

      REFERENCES

        • Boray J.C.
        Chemotherapy of infections with Fasciolidae.
        in: Boray J.C. Immunology, Pathobiology and Control of Fasciolosis. MSD AGVET, Rahway, NJ1997: 83-97
        • Kaplan R.M.
        Fasciola hepatica: a review of the economic impact in cattle and considerations for control.
        Vet. Ther. 2001; 2: 40-50
        • Young N.D.
        • Hall R.S.
        • Jex A.R.
        • Cantacessi C.
        • Gasser R.B.
        Elucidating the transcriptome of Fasciola hepatica——a key to fundamental and biotechnological discoveries for a neglected parasite.
        Biotechnol. Adv. 2010; 28: 222-231
        • Pybus M.J.
        Liver flukes.
        in: Samuel W.M. Pybus M.J. Kocan A.A. Parasitic Diseases in Wild Mammals. Iowa State Press, Iowa City, IA2001: 121-149
        • Coil W.H.
        The penetration of Fascioloides magna miracidia into the snail host Fossaria bulimoides. A scanning electron microscope study.
        Zeitschrift für Parasitenkunde. 1977; 52: 53-59
        • Fairweather I.
        Triclabendazole: new skills to unravel an old(ish) enigma.
        J. Helminthol. 2005; 79: 227-234
        • Maes L.
        • Vanparijs O.
        • Lauwers H.
        • Deckers W.
        Comparative efficacy of closantel and triclabendazole against Fasciola hepatica in experimentally infected sheep.
        Vet. Rec. 1990; 127: 450-452
        • Keiser J.
        • Utzinger J.
        Chemotherapy for major food-borne trematodes: a review.
        Expert Opin. Pharmacother. 2004; 5: 1711-1726
        • Flynn R.J.
        • Mulcahy G.
        • Elsheikha H.M.
        Coordinating innate and adaptive immunity in Fasciola hepatica infection: implications for control.
        Vet. Parasitol. 2010; 169: 235-240
        • Overend D.J.
        • Bowen F.L.
        Resistance of Fasciola hepatica to triclabendazole.
        Aust. Vet. J. 1995; 72: 275-276
        • Coles G.C.
        • Rhodes A.C.
        • Stafford K.A.
        Activity of closantel against adult triclabendazole-resistant Fasciola hepatica.
        Vet. Rec. 2000; 146: 504
        • Moll L.
        • Gaasenbeek C.P.H.
        • Vellema P.
        • Borgsteede F.H.M.
        Resistance of Fasciola hepatica against triclabendazole in cattle and sheep in the Netherlands.
        Vet. Parasitol. 2000; 91: 153-158
        • Thomas I.
        • Coles G.C.
        • Duffus K.
        Triclabendazole-resistant Fasciola hepatica in southwest Wales.
        Vet. Rec. 2000; 146: 200
        • Fairweather I.
        Triclabendazole progress report, 2005–2009: an advancement of learning?.
        J. Helminthol. 2009; 83: 139-150
        • Gaasenbeek C.P.H.
        • Moll L.
        • Cornelissen J.
        • Vellema P.
        • Borgsteede F.H.M.
        An experimental study on triclabendazole resistance of Fasciola hepatica in sheep.
        Vet. Parasitol. 2001; 95: 37-43
        • Spithill T.W.
        • Dalton J.P.
        Progress in development of liver fluke vaccines.
        Parasitol. Today. 1998; 14: 224-228
        • Piacenza L.
        • Acosta D.
        • Basmadjian I.
        • Dalton J.P.
        • Carmona C.
        Vaccination with cathepsin L proteinases and with leucine aminopeptidase induces high levels of protection against fascioliasis in sheep.
        Infect. Immun. 1999; 67: 1954-1961
        • Dalton J.P.
        • Mulcahy G.
        Parasite vaccines—a reality?.
        Vet. Parasitol. 2001; 98: 149-167
        • Dalton J.P.
        • Brindley P.J.
        • Knox D.P.
        • Brady C.P.
        • Hotez P.J.
        • Donnelly S.
        • O'Neill S.M.
        • Mulcahy G.
        • Loukas A.
        Helminth vaccines: from mining genomic information for vaccine targets to systems used for protein expression.
        Int. J. Parasitol. 2003; 33: 621-640
        • Dalton J.P.
        • O'Neill S.
        • Stack C.
        • Collins P.
        • Walshe A.
        • Sekiya M.
        • Doyle S.
        • Mulcahy G.
        • Hoyle D.
        • Khaznadji E.
        • Moire N.
        • Brennan G.
        • Mousley A.
        • Kreshchenko N.
        • Maule A.G.
        • Donnelly S.M.
        Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines.
        Int. J. Parasitol. 2003; 33: 1173-1181
        • McManus D.P.
        • Dalton J.P.
        Vaccines against the zoonotic trematodes Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica.
        Parasitology. 2006; 133: S43-S61
        • Acosta D.
        • Cancela M.
        • Piacenza L.
        • Roche L.
        • Carmona C.
        • Tort J.F.
        Fasciola hepatica leucine aminopeptidase, a promising candidate for vaccination against ruminant fasciolosis.
        Mol. Biochem. Parasitol. 2008; 158: 52-64
        • Jayaraj R.
        • Piedrafita D.
        • Dynon K.
        • Grams R.
        • Spithill T.W.
        • Smooker P.M.
        Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens.
        Vet. Parasitol. 2009; 160: 230-236
        • Robinson M.W.
        • Menon R.
        • Donnelly S.M.
        • Dalton J.P.
        • Ranganathan S.
        An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host.
        Mol. Cell. Proteomics. 2009; 8: 1891-1907
        • Young N.D.
        • Jex A.R.
        • Cantacessi C.
        • Hall R.S.
        • Campbell B.E.
        • Spithill T.W.
        • Tangkawattana S.
        • Tangkawattana P.
        • Laha T.
        • Gasser R.B.
        A portrait of the transcriptome of the neglected trematode, Fasciola gigantica—biological and biotechnological implications.
        PLoS Negl. Trop. Dis. 2011; 5: e1004
        • Kasny M.
        • Mikes L.
        • Hampl V.
        • Dvorak J.
        • Caffrey C.R.
        • Dalton J.P.
        • Horak P.
        Chapter 4. Peptidases of trematodes.
        Adv. Parasitol. 2009; 69: 205-297
        • DeMarco R.
        • Verjovski-Almeida S.
        Schistosomes—proteomics studies for potential novel vaccines and drug targets.
        Drug Discov. Today. 2009; 14: 472-478
        • Chuan J.
        • Feng Z.
        • Brindley P.J.
        • McManus D.P.
        • Han Z.
        • Jianxin P.
        • Hu W.
        Our wormy world: genomics, proteomics and transcriptomics in East and southeast Asia.
        Adv. Parasitol. 2010; 73: 327-371
        • Cantacessi C.
        • Campbell B.E.
        • Gasser R.B.
        Key strongylid nematodes of animals—impact of next-generation transcriptomics on systems biology and biotechnology.
        Biotechnol. Adv. 2012; 30: 469-488
        • Verjovski-Almeida S.
        • DeMarco R.
        Current developments in Schistosoma proteomics.
        Acta Trop. 2008; 108: 183-185
        • Mulvenna J.
        • Hamilton B.
        • Nagaraj S.H.
        • Smyth D.
        • Loukas A.
        • Gorman J.J.
        Proteomics analysis of the excretory/secretory component of the blood-feeding stage of the hookworm, Ancylostoma caninum.
        Mol. Cell. Proteomics. 2009; 8: 109-121
        • Young N.D.
        • Campbell B.E.
        • Hall R.S.
        • Jex A.R.
        • Cantacessi C.
        • Laha T.
        • Sohn W.M.
        • Sripa B.
        • Loukas A.
        • Brindley P.J.
        • Gasser R.B.
        Unlocking the transcriptomes of two carcinogenic parasites, Clonorchis sinensis and Opisthorchis viverrini.
        PLoS Negl. Trop. Dis. 2010; 4: e719
        • Bentley D.R.
        • Balasubramanian S.
        • Swerdlow H.P.
        • George D.
        • Gietzen K.J.
        • Goddard C.P.
        • Golda G.S.
        • Granieri P.A.
        • Green D.E.
        • Gustafson D.L.
        • Hansen N.F.
        • Harnish K.
        • Haudenschild C.D.
        • Heyer N.I.
        • Hims M.M.
        • Ho J.T.
        • Horgan A.M.
        • Hoschler K.
        • Hurwitz S.
        • Ivanov D.V.
        • Johnson M.Q.
        • James T.
        • Huw Jones T.A.
        • Kang G.D.
        • Kerelska T.H.
        • Kersey A.D.
        • Khrebtukova I.
        • Kindwall A.P.
        • Kingsbury Z.
        • Kokko-Gonzales P.I.
        • Kumar A.
        • Laurent M.A.
        • Lawley C.T.
        • Lee S.E.
        • Lee X.
        • Liao A.K.
        • Loch J.A.
        • Lok M.
        • Luo S.
        • Mammen R.M.
        • Martin J.W.
        • McCauley P.G.
        • McNitt P.
        • Mehta P.
        • Moon K.W.
        • Mullens J.W.
        • Newington T.
        • Ning Z.
        • Ling Ng B.
        • Novo S.M.
        • O'Neill M.J.
        • Osborne M.A.
        • Osnowski A.
        • Ostadan O.
        • Paraschos L.L.
        • Pickering L.
        • Pike A.C.
        • Pike A.C.
        • Chris Pinkard D.
        • Pliskin D.P.
        • Podhasky J.
        • Quijano V.J.
        • Raczy C.
        • Rae V.H.
        • Rawlings S.R.
        • Chiva Rodriguez A.
        • Roe P.M.
        • Rogers J.
        • Rogert Bacigalupo M.C.
        • Romanov N.
        • Romieu A.
        • Roth R.K.
        • Rourke N.J.
        • Ruediger S.T.
        • Rusman E.
        • Sanches-Kuiper R.M.
        • Schenker M.R.
        • Seoane J.M.
        • Shaw R.J.
        • Shiver M.K.
        • Short S.W.
        • Sizto N.L.
        • Sluis J.P.
        • Smith M.A.
        • Ernest Sohna Sohna J.
        • Spence E.J.
        • Stevens K.
        • Sutton N.
        • Szajkowski L.
        • Tregidgo C.L.
        • Turcatti G.
        • Vandevondele S.
        • Verhovsky Y.
        • Virk S.M.
        • Wakelin S.
        • Walcott G.C.
        • Wang J.
        • Worsley G.J.
        • Yan J.
        • Yau L.
        • Zuerlein M.
        • Rogers J.
        • Mullikin J.C.
        • Hurles M.E.
        • McCooke N.J.
        • West J.S.
        • Oaks F.L.
        • Lundberg P.L.
        • Klenerman D.
        • Durbin R.
        • Smith A.J.
        Accurate whole human genome sequencing using reversible terminator chemistry.
        Nature. 2008; 456: 53-59
        • Schulz M.H.
        • Zerbino D.R.
        • Vingron M.
        • Birney E.
        Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels.
        Bioinformatics. 2012; 28: 1086-1092
        • Li R.
        • Yu C.
        • Li Y.
        • Lam T.W.
        • Yiu S.M.
        • Kristiansen K.
        • Wang J.
        SOAP2: an improved ultrafast tool for short read alignment.
        Bioinformatics. 2009; 25: 1966-1967
        • Mortazavi A.
        • Williams B.A.
        • McCue K.
        • Schaeffer L.
        • Wold B.
        Mapping and quantifying mammalian transcriptomes by RNA-seq.
        Nat. Methods. 2008; 5: 621-628
        • Cantacessi C.
        • Jex A.R.
        • Hall R.S.
        • Young N.D.
        • Campbell B.E.
        • Joachim A.
        • Nolan M.J.
        • Abubucker S.
        • Sternberg P.W.
        • Ranganathan S.
        • Mitreva M.
        • Gasser R.B.
        A practical, bioinformatic workflow system for large data sets generated by next-generation sequencing.
        Nucleic Acids Res. 2010; 38: e171
        • Young N.D.
        • Jex A.R.
        • Li B.
        • Liu S.
        • Yang L.
        • Xiong Z.
        • Li Y.
        • Cantacessi C.
        • Hall R.S.
        • Xu X.
        • Chen F.
        • Wu X.
        • Zerlotini A.
        • Oliveira G.
        • Hofmann A.
        • Zhang G.
        • Fang X.
        • Kang Y.
        • Campbell B.E.
        • Loukas A.
        • Ranganathan S.
        • Rollinson D.
        • Rinaldi G.
        • Brindley P.J.
        • Yang H.
        • Wang J.
        • Wang J.
        • Gasser R.B.
        Whole-genome sequence of Schistosoma haematobium.
        Nat. Genet. 2012; 44: 221-225
        • The Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium
        The Schistosoma japonicum genome reveals features of host-parasite interplay.
        Nature. 2009; 460: 345-351
        • Berriman M.
        • Haas B.J.
        • LoVerde P.T.
        • Wilson R.A.
        • Dillon G.P.
        • Cerqueira G.C.
        • Mashiyama S.T.
        • Al-Lazikani B.
        • Andrade L.F.
        • Ashton P.D.
        • Aslett M.A.
        • Bartholomeu D.C.
        • Blandin G.
        • Caffrey C.R.
        • Coghlan A.
        • Coulson R.
        • Day T.A.
        • Delcher A.
        • DeMarco R.
        • Djikeng A.
        • Eyre T.
        • Gamble J.A.
        • Ghedin E.
        • Gu Y.
        • Hertz-Fowler C.
        • Hirai H.
        • Hirai Y.
        • Houston R.
        • Ivens A.
        • Johnston D.A.
        • Lacerda D.
        • Macedo C.D.
        • McVeigh P.
        • Ning Z.
        • Oliveira G.
        • Overington J.P.
        • Parkhill J.
        • Pertea M.
        • Pierce R.J.
        • Protasio A.V.
        • Quail M.A.
        • Rajandream M.A.
        • Rogers J.
        • Sajid M.
        • Salzberg S.L.
        • Stanke M.
        • Tivey A.R.
        • White O.
        • Williams D.L.
        • Wortman J.
        • Wu W.
        • Zamanian M.
        • Zerlotini A.
        • Fraser-Liggett C.M.
        • Barrell B.G.
        • El-Sayed N.M.
        The genome of the blood fluke Schistosoma mansoni.
        Nature. 2009; 460: 352-358
        • Hunter S.
        • Apweiler R.
        • Attwood T.K.
        • Bairoch A.
        • Bateman A.
        • Binns D.
        • Bork P.
        • Das U.
        • Daugherty L.
        • Duquenne L.
        • Finn R.D.
        • Gough J.
        • Haft D.
        • Hulo N.
        • Kahn D.
        • Kelly E.
        • Laugraud A.
        • Letunic I.
        • Lonsdale D.
        • Lopez R.
        • Madera M.
        • Maslen J.
        • McAnulla C.
        • McDowall J.
        • Mistry J.
        • Mitchell A.
        • Mulder N.
        • Natale D.
        • Orengo C.
        • Quinn A.F.
        • Selengut J.D.
        • Sigrist C.J.
        • Thimma M.
        • Thomas P.D.
        • Valentin F.
        • Wilson D.
        • Wu C.H.
        • Yeats C.
        InterPro: the integrative protein signature database.
        Nucleic Acids Res. 2009; 37: D211-D215
        • Ashburner M.
        • Ball C.A.
        • Blake J.A.
        • Botstein D.
        • Butler H.
        • Cherry J.M.
        • Davis A.P.
        • Dolinski K.
        • Dwight S.S.
        • Eppig J.T.
        • Harris M.A.
        • Hill D.P.
        • Issel-Tarver L.
        • Kasarskis A.
        • Lewis S.
        • Matese J.C.
        • Richardson J.E.
        • Ringwald M.
        • Rubin G.M.
        • Sherlock G.
        Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.
        Nat. Genet. 2000; 25: 25-29
        • Ye J.
        • Fang L.
        • Zheng H.
        • Zhang Y.
        • Chen J.
        • Zhang Z.
        • Wang J.
        • Li S.
        • Li R.
        • Bolund L.
        • Wang J.
        WEGO: a web tool for plotting GO annotations.
        Nucleic Acids Res. 2006; 34: W293-W297
        • Xie C.
        • Mao X.
        • Huang J.
        • Ding Y.
        • Wu J.
        • Dong S.
        • Kong L.
        • Gao G.
        • Li C.Y.
        • Wei L.
        KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases.
        Nucleic Acids Res. 2011; 39: W316-W322
        • Bendtsen J.D.
        • Nielsen H.
        • von Heijne G.
        • Brunak S.
        Improved prediction of signal peptides: SignalP 3.0.
        J. Mol. Biol. 2004; 340: 783-795
        • Sonnhammer E.L.L.
        • von Heijne G.
        • Krogh A.
        A hidden Markov model for predicting transmembrane helices in protein sequences.
        Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology. AAAI, Menlo Park, CA1998: 175-182
        • Krogh A.
        • Larsson B.
        • von Heijne G.
        • Sonnhammer E.L.
        Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.
        J. Mol. Biol. 2001; 305: 567-580
        • Moller S.
        • Croning M.D.R.
        • Apweiler R.
        Evaluation of methods for the prediction of membrane spanning regions.
        Bioinformatics. 2001; 17: 646-653
        • Chen Y.
        • Zhang Y.
        • Yin Y.
        • Gao G.
        • Li S.
        • Jiang Y.
        • Gu X.
        • Luo J.
        SPD—a web based secreted protein database.
        Nucleic Acids Res. 2005; 33: D169-D173
        • Choo K.H.
        • Tan T.W.
        • Ranganathan S.
        SPdb—a signal peptide database.
        BMC Bioinformatics. 2005; 6: 249
        • Laemmli U.K.
        Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
        Nature. 1970; 227: 680-907
        • Searle B.C.
        Scaffold: a bioinformatics tool for validating MS/MS-based proteomics studies.
        Proteomics. 2010; 10: 1265-1269
        • Keller A.
        • Nesvizhskii A.I.
        • Kolker E.
        • Aebersold R.
        Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.
        Anal. Chem. 2002; 74: 5383-5392
        • Nesvizhsii A.I.
        • Keller A.
        • Kolker E.
        • Aebersold R.
        A statistical model for identifying proteins by tandem mass spectrometry.
        Anal. Chem. 2003; 75: 4646-4658
        • Emanuelsson O.
        • Brunak S.
        • von Heijne G.
        • Nielsen H.
        Locating proteins in the cell using TargetP, SignalP and related tools.
        Nat. Protoc. 2007; 2: 953-971
        • Blom N.
        • Sicheritz-Pontén T.
        • Gupta R.
        • Gammeltoft S.
        • Brunak S.
        Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence.
        Proteomics. 2004; 4: 1633-1649
        • Cho P.Y.
        • Lee M.J.
        • Kim T.I.
        • Kang S.Y.
        • Hong S.J.
        Expressed sequence tag analysis of adult Clonorchis sinensis, the Chinese liver fluke.
        Parasitol. Res. 2006; 99: 602-608
        • Cho P.Y.
        • Kim T.I.
        • Whang S.M.
        • Hong S.J.
        Gene expression profile of Clonorchis sinensis metacercariae.
        Parasitol. Res. 2008; 102: 277-282
        • Kang J.M.
        • Bahk Y.Y.
        • Cho P.Y.
        • Hong S.J.
        • Kim T.S.
        • Sohn W.M.
        • Na B.K.
        A family of cathepsin F cysteine proteases of Clonorchis sinensis is the major secreted proteins that are expressed in the intestine of the parasite.
        Mol. Biochem. Parasitol. 2010; 170: 7-16
        • Yoo W.G.
        • Kim D.W.
        • Ju J.W.
        • Cho P.Y.
        • Kim T.I.
        • Cho S.H.
        • Choi S.H.
        • Park H.S.
        • Kim T.S.
        • Hong S.J.
        Developmental transcriptomic features of the carcinogenic liver fluke, Clonorchis sinensis.
        PLoS Negl. Trop. Dis. 2011; 5: e1208
        • Kim T.S.
        • de Guzman J.V.
        • Kong H.H.
        • Chung D.I.
        Comparison of gene representation between diploid and triploid Paragonimus westermani by expressed sequence tags analyses.
        J. Parasitol. 2006; 92: 803-816
        • Horton P.
        • Park K.
        • Obayashi T.
        • Fujita N.
        • Harada H.
        • Adams-Collier C.J.
        • Nakai K.
        WoLF PSORT: protein localization predictor.
        Nucleic Acids Res. 2007; 35: W585-W587
        • Jefferies J.R.
        • Campbell A.M.
        • vanRossum A.J.
        • Barrett J.
        • Brophy P.M.
        Proteomic analysis of Fasciola hepatica excretory-secretory products.
        Proteomics. 2001; 1: 1128-1132
        • Morphew R.M.
        • Wright H.A.
        • LaCourse E.J.
        • Porter J.
        • Barrett J.
        • Woods D.J.
        • Brophy P.M.
        Towards delineating functions within the Fasciola secreted cathepsin I protease family by integrating in vivo based sub-proteomics and phylogenetics.
        PLoS Negl. Trop. Dis. 2011; 5: e937
        • Robinson M.W.
        • Tort J.F.
        • Lowther J.
        • Donnelly S.M.
        • Wong E.
        • Xu W.
        • Stack C.M.
        • Padula M.
        • Herbert B.
        • Dalton J.P.
        Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulence-associated factors.
        Mol. Cell. Proteomics. 2008; 7: 1111-1123
        • Wilson R.A.
        • Wright J.M.
        • de Castro-Borges W.
        • Parker-Manuel S.J.
        • Dowle A.A.
        • Ashton P.D.
        • Young N.D.
        • Gasser R.B.
        • Spithill T.W.
        Exploring the Fasciola hepatica tegument proteome.
        Int. J. Parasitol. 2011; 41: 1347-1359
        • Zheng M.
        • Hu K.
        • Liu W.
        • Hu X.
        • Hu F.
        • Huang L.
        • Wang P.
        • Hu Y.
        • Huang Y.
        • Li W.
        • Liang C.
        • Yin X.
        • He Q.
        • Yu X.
        Proteomic analysis of excretory secretory products from Clonorchis sinensis adult worms: molecular characterization and serological reactivity of excretory-secretory antigen-fructose-1,6-bisphosphatase.
        Parasitol. Res. 2011; 1: 1-8
        • Mulvenna J.
        • Sripa B.
        • Brindley P.J.
        • Gorman J.
        • Jones M.K.
        • Colgrave M.L.
        • Jones A.
        • Nawaratna S.
        • Laha T.
        • Suttiprapa S.
        • Smout M.J.
        • Loukas A.
        The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini.
        Proteomics. 2010; 10: 1063-1078
        • Robinson M.W.
        • Corvo I.
        • Jones P.M.
        • George A.M.
        • Padula M.P.
        • To J.
        • Cancela M.
        • Rinaldi G.
        • Tort J.F.
        • Roche L.
        • Dalton J.P.
        Collagenolytic activities of the major secreted cathepsin L peptidases involved in the virulence of the helminth pathogen, Fasciola hepatica.
        PLoS Negl. Trop. Dis. 2011; 4: e1012
        • Nickel W.
        The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes.
        Eur. J. Biochem. 2003; 270: 2109-2119
        • Robinson M.W.
        • Colhoun L.M.
        • Fairweather I.
        • Brennan G.P.
        • Waite J.H.
        Development of the vitellaria of the liver fluke, Fasciola hepatica, in the rat host.
        Parasitology. 2001; 123: 509-518
        • Chen J.M.
        • Dando P.M.
        • Stevens R.A.
        • Fortunato M.
        • Barrett A.J.
        Cloning and expression of mouse legumain, a lysosomal endopeptidase.
        Biochem. J. 1998; 335: 111-117
        • Turk D.
        • Guncar G.
        Lysosomal cysteine proteases (cathepsins): promising drug targets.
        Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 203-213
        • Robinson M.W.
        • Dalton J.P.
        • Donnelly S.
        Helminth pathogen cathepsin proteases: it's a family affair.
        Trends Biochem. 2008; 33: 601-608
        • Irving J.A.
        • Spithill T.W.
        • Pike R.N.
        • Whisstock J.C.
        • Smooker P.M.
        The evolution of enzyme specificity in Fasciola spp.
        J. Mol. Evol. 2003; 57: 1-15
        • Tort J.
        • Brindley P.J.
        • Knox D.
        • Wolfe K.H.
        • Dalton J.P.
        Proteinases and associated genes of parasitic helminths.
        Adv. Parasitol. 1999; 43: 161-266
        • Dalton J.P.
        • Brindley P.J.
        • Donnelly S.
        • Robinson M.W.
        The enigmatic asparaginyl endopeptidase of helminth parasites.
        Trends Parasitol. 2009; 25: 59-61
        • Hall S.L.
        • Braschi S.
        • Truscott M.
        • Mathieson W.
        • Cesari I.M.
        • Wilson R.A.
        Insights into blood feeding by schistosomes from a proteomic analysis of worm vomitus.
        Mol. Biochem. Parasitol. 2011; 179: 18-29
        • Liu F.
        • Cui S.J.
        • Hu W.
        • Feng Z.
        • Wang Z.Q.
        • Han Z.G.
        Excretory/secretory proteome of the adult developmental stage of human blood fluke, Schistosoma japonicum.
        Mol. Cell. Proteomics. 2008; 8: 1236-1251
        • Dalton J.P.
        • Skelly P.
        • Halton D.W.
        Role of the tegument and gut in nutrient uptake by parasitic platyhelminths.
        Can. J. Zool. 2004; 24: 211-232
        • MacKenzie A.
        • Wilson H.L.
        • Kiss-Toth E.
        • Dower S.K.
        • North R.A.
        • Surprenant A.
        Rapid secretion of interleukin-1β by microvescicle shedding.
        Immunity. 2001; 15: 825-835
        • McGonigle L.
        • Mousley A.
        • Marks N.J.
        • Brennan G.P.
        • Dalton J.P.
        • Spithill T.W.
        • Day T.A.
        • Maule A.G.
        The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration.
        Int. J. Parasitol. 2008; 38: 149-155
        • Salazar-Calderón M.
        • Martin-Alonso J.M.
        • Ruiz de Eguino A.D.
        • Parra F.
        Heterologous expression and functional characterization of thioredoxin from Fasciola hepatica.
        Parasitol. Res. 2001; 87: 390-395
        • Salazar-Calderón M.
        • Martin Alonso J.M.
        • Castro A.M.
        • Parra F.
        Cloning, heterologous expression in Escherichia coli and characterization of a protein disulfide isomerase from Fasciola hepatica.
        Mol. Biochem. Parasitol. 2003; 126: 15-23
        • Sekiya M.
        • Mulcahy G.
        • Irwin J.A.
        • Stack C.M.
        • Donnelly S.M.
        • Xu W.
        • Collins P.
        • Dalton J.P.
        Biochemical characterisation of the recombinant peroxiredoxin (FhePrx) of the liver fluke, Fasciola hepatica.
        FEBS Lett. 2006; 580: 5016-5022
        • Cervi L.
        • Rossi G.
        • Masih D.T.
        Potential role for excretory-secretory forms of glutathione-S-transferase (GST) in Fasciola hepatica.
        Parasitology. 1999; 119: 627-633
        • Donnelly S.
        • O'Neill S.M.
        • Sekiya M.
        • Mulcahy G.
        • Dalton J.P.
        Thioredoxin peroxidase secreted by Fasciola hepatica induces the alternative activation of macrophages.
        Infect. Immun. 2005; 73: 166-173
        • Donnelly S.
        • Stack C.M.
        • O'Neill S.M.
        • Sayed A.A.
        • Williams D.L.
        • Dalton J.P.
        Helminth 2-Cys peroxiredoxin drives Th2 responses through a mechanism involving alternatively activated macrophages.
        FASEB J. 2008; 22: 4022-4032
        • Wang X.
        • Chen W.
        • Huang Y.
        • Sun J.
        • Men J.
        • Liu H.
        • Luo F.
        • Guo L.
        • Lv Z.
        • Deng C.
        • Zhou C.
        • Fan Y.
        • Li X.
        • Huang L.
        • Hu Y.
        • Liang C.
        • Hu X.
        • Xum J.
        • Yum X.
        The draft genome of the carcinogenic human liver fluke Clonorchis sinensis.
        Genome Biol. 2011; 12: R107