Title: The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis*
Abstract: Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular “machinery” required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack. Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular “machinery” required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack. The trematode parasite, Fasciola hepatica is the causative agent of liver fluke disease (fasciolosis) in domestic animals. F. hepatica infects more than 300 million cattle and 250 million sheep worldwide resulting in losses of over $3 billion to global agriculture through lost productivity (1.Mas-Coma S. Bargues M.D. Valero M.A. Fascioliasis and other plant-borne trematode zoonoses.Int. J. Parasitol. 2005; 35: 1255-1278Crossref PubMed Scopus (658) Google Scholar). Fasciolosis is also an important zoonotic disease with an estimated 2.6 million people infected worldwide; human infection is particularly highly prevalent in Egypt and the Andes of South America, while outbreaks regularly occur in Northern Iran (2.Fürst T. Duthaler U. Sripa B. Utzinger J. Keiser J. Trematode infections: liver and lung flukes.Infect. Dis. Clin. North. Am. 2012; 26: 399-419Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Infection in animals and humans can last many years while adult parasites reside in the bile ducts. F. hepatica are obligate blood feeders and obtain blood by puncturing the bile duct wall. The regular regurgitation of parasite gut contents is thought to inject molecules into the bloodstream where they can exert an immunosuppressive activity on the host immune system (3.Molina-Hernández V. Mulcahy G. Pérez J. Martínez-Moreno Á. Donnelly S. O'Neill S.M. Dalton J.P. Cwiklinski K. Fasciola hepatica vaccine: we may not be there yet but we're on the right road.Vet. Parasitol. 2015; 208: 101-111Crossref PubMed Scopus (127) Google Scholar). Other molecules are secreted from the gut, excretory pores and surface tegument into the bile where they may be carried to the exterior via the host intestine (4.Fairweather I Threadgold L.T. Hanna R.E. B. Development of Fasciola hepatica in the mammalian host. In Fasciolosis (Dalton, J.P., ed).CABI, Oxford, United Kingdom. 1999; : 1-29Google Scholar, 5.Collins P.R. Stack C.M. O'Neill S.M. Doyle S. Ryan T. Brennan G.P. Mousley A. Stewart M. Maule A.G. Dalton J.P. Donnelly S. Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence: propeptide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells.J. Biol. Chem. 2004; 279: 17038-17046Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 6.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: 1347-1359Crossref PubMed Scopus (99) Google Scholar). We, and others, have previously used proteomic techniques to profile the secretome of adult F. hepatica (7.Jefferies J.R. Campbell A.M. van Rossum A.J. Barrett J. Brophy P.M. Proteomic analysis of Fasciola hepatica excretory-secretory products.Proteomics. 2001; 1: 1128-1132Crossref PubMed Scopus (0) Google Scholar, 8.Morphew R.M. Wright H.A. La Course E.J. Woods D.J. Brophy P.M. Comparative proteomics of excretory-secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex host.Mol. Cell. Proteomics. 2007; 6: 963-972Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 9.Robinson M.W. Tort J.F. Wong E. Donnelly S.M. Lowther J. Xu W. Stack C.M. Padula M. Herbert B. Dalton J.P. Proteomic 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: 1111-1123Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 10.Robinson M.W. Menon R. Donnelly S.M. Dalton J.P. Ranganathan S. An integrated transcriptomic and proteomic 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: 1891-1907Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) but these studies had a number of limitations: (1) they relied on very limited transcriptome data sets with considerable redundancy; (2) because genome sequence data was lacking it was not possible to determine precisely the identity and/or the number of the genes contributing to various secreted protein families; (3) the contribution of extracellular vesicles to fluke secretions was not investigated. However, our recent report of the Fasciola genome and associated transcriptome data sets (11.Cwiklinski K. Dalton J.P. Dufresne P.J. La Course J. Williams D.J. Hodgkinson J. Paterson S. The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution.Genome Biol. 2015; 16: 71Crossref PubMed Scopus (191) Google Scholar) has now allowed us to address these limitations and perform a definitive characterization of the total secretome of adult F. hepatica. This is timely, given the report of exosome-like EVs released by adult F. hepatica (12.Marcilla A. Trelis M. Cortés A. Sotillo J. Cantalapiedra F. Minguez M.T. Valero M.L. Sánchez del Pino M.M. Muñoz-Antoli C. Toledo R. Bernal D. Extracellular vesicles from parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host cells.PLoS ONE. 2012; 7: e45974Crossref PubMed Scopus (246) Google Scholar). Extracellular vesicles (EVs) 1The abbreviations used are:EVextracellular vesicleNEJnewly excysted juvenileHDMhelminth defence moleculeCBcathepsin BCLcathepsin LLAPleucine aminopeptidasePrxperoxiredoxinFaBPfatty acid-binding proteinGSTglutathione transferaseMVBmultivesicular bodyESCRTendosomal sorting complex required for transportDAMPdamage-associated molecular pattern. 1The abbreviations used are:EVextracellular vesicleNEJnewly excysted juvenileHDMhelminth defence moleculeCBcathepsin BCLcathepsin LLAPleucine aminopeptidasePrxperoxiredoxinFaBPfatty acid-binding proteinGSTglutathione transferaseMVBmultivesicular bodyESCRTendosomal sorting complex required for transportDAMPdamage-associated molecular pattern. are small membrane bound organelles that are shed by most cell types. Although once considered to be “cellular garbage cans” with the sole purpose of discarding unwanted cellular material (13.Thébaud B. Stewart D.J. Exosomes: cell garbage can, therapeutic carrier, or trojan horse?.Circulation. 2012; 126: 2553-2555Crossref PubMed Scopus (36) Google Scholar), EVs are now recognized as important mediators of intercellular communication by transferring molecular signals, including proteins, lipids, mRNA, microRNA and other non-coding RNA species (14.EL Andaloussi S. Mäger I. Breakefield X.O. Wood M.J. Extracellular vesicles: biology and emerging therapeutic opportunities.Nat. Rev. Drug. Discov. 2013; 12: 347-357Crossref PubMed Scopus (2142) Google Scholar, 15.Record M. Carayon K. Poirot M. Silvente-Poirot S. Exosomes as new vesicular lipid transporters involved in cell-cell communication and various pathophysiologies.Biochim. Biophys. Acta. 2014; 1841: 108-120Crossref PubMed Scopus (592) Google Scholar). Variously described as exosomes or microvesicles depending on their cellular origin and mode of biogenesis, EVs perform a variety of roles in the maintenance of normal physiology such as blood coagulation, immune regulation and tissue repair (16.Raposo G. Nijman H.W. Stoorvogel W. Liejendekker R. Harding C.V. Melief C.J. Geuze. H.J. B lymphocytes secrete antigen-presenting vesicles.J. Exp. Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2461) Google Scholar, 17.Gatti S. Bruno S. Deregibus M.C. Sordi A. Cantaluppi V. Tetta C. Camussi G. Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury.Nephrol. Dial. Transplant. 2011; 26: 1474-1483Crossref PubMed Scopus (630) Google Scholar, 18.Del Conde I. Shrimpton C.N. Thiagarajan P. López J.A. Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation.Blood. 2005; 106: 1604-1611Crossref PubMed Scopus (802) Google Scholar), but also participate in pathological settings, notably in tumor progression (19.Rak J. Guha A. Extracellular vesicles - vehicles that spread cancer genes.Bioessays. 2012; 34: 489-497Crossref PubMed Scopus (132) Google Scholar). A number of recent studies have shown that parasite-derived EVs play an important role during infection. For instance, EVs released by the helminth parasites Heligmosomoides polygyrus and Schistosoma japonicum are capable of modulating the response of host innate immune cells (20.Buck A.H. Coakley G. Simbari F. McSorley H.J. Quintana J.F. Le Bihan T. Kumar S. Abreu-Goodger C. Lear M. Harcus Y. Ceroni A. Babayan S.A. Blaxter M. Ivens A. Maizels R.M. Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity.Nat. Commun. 2014; 5: 5488Crossref PubMed Scopus (473) Google Scholar, 21.Wang L. Li Z. Shen J. Liu Z. Liang J. Wu X. Sun X. Wu Z. Exosome-like vesicles derived by Schistosoma japonicum adult worms mediates M1 type immune- activity of macrophage.Parasitol. Res. 2015; 114: 1865-1873Crossref PubMed Scopus (86) Google Scholar). extracellular vesicle newly excysted juvenile helminth defence molecule cathepsin B cathepsin L leucine aminopeptidase peroxiredoxin fatty acid-binding protein glutathione transferase multivesicular body endosomal sorting complex required for transport damage-associated molecular pattern. extracellular vesicle newly excysted juvenile helminth defence molecule cathepsin B cathepsin L leucine aminopeptidase peroxiredoxin fatty acid-binding protein glutathione transferase multivesicular body endosomal sorting complex required for transport damage-associated molecular pattern. Here, we report the deep analysis of the soluble and vesicular components of the adult F. hepatica secretome. We found that F. hepatica secretes at least two subpopulations of EVs that differ according to size, cargo molecules and potential site of release from the parasite. Using a proteomic approach, integrated with newly available F. hepatica genome and transcriptome resources (11.Cwiklinski K. Dalton J.P. Dufresne P.J. La Course J. Williams D.J. Hodgkinson J. Paterson S. The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution.Genome Biol. 2015; 16: 71Crossref PubMed Scopus (191) Google Scholar), we have defined the protein composition of these two classes of vesicle and non-vesicle associated proteins. The larger 15K EVs are released from the specialized cells that line the parasite gastrodermus and contain, as a marker, the 37 kDa inactive zymogen of the cathepsin L1 digestive peptidase. In contrast, our analysis of the smaller 120K exosome-like vesicle population suggest that they are shed from the tegument of adult fluke. Although the 120K EV biogenesis “machinery” is constitutively expressed across the intramammalian developmental stages of the parasite, the cargo molecules packaged within the EVs are highly regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack. Western blots and fluorogenic assays were performed with at least two biological replicates and three technical triplicates. For the proteomics study, whole Fasciola EVs were initially analyzed by LC-MS/MS (two technical replicates). These identifications were subsequently mapped to the surface/membrane/lumen of the EVs following analysis of the EV fractions, which were run without replicates because of the paucity of parasite material. The aim of this experiment was to profile the molecules associated with fluke EVs and no quantitation was performed. Adult F. hepatica parasites were obtained from bovine livers from local abattoirs. To prepare secretions, adult flukes were thoroughly washed with PBS to void their gut contents and then maintained in RPMI 1640 culture medium containing 0.1% glucose, 100 U penicillin and 100 mg/ml streptomycin (Sigma), at 2 worms/ml for 5 h at 37 °C. EVs were purified according to the differential centrifugation protocol described by Marcilla et al. (12.Marcilla A. Trelis M. Cortés A. Sotillo J. Cantalapiedra F. Minguez M.T. Valero M.L. Sánchez del Pino M.M. Muñoz-Antoli C. Toledo R. Bernal D. Extracellular vesicles from parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host cells.PLoS ONE. 2012; 7: e45974Crossref PubMed Scopus (246) Google Scholar). Briefly, after the incubation period, the parasite culture media was collected and centrifuged at low speed (first at 300 × g/10 min, and then at 700 × g/30 min) to remove large debris. The resulting supernatant was centrifuged at 15,000 × g for 45 min at 4 °C to obtain large vesicles. Culture supernatants were then filtered using a 0.2 μm ultrafiltration membrane, and centrifuged at 120,000 × g/1 h at 4 °C to recover smaller vesicles that were subsequently washed with PBS. The protein content of adult fluke soluble secretions and EVs was determined using the BCA protein assay kit (Pierce, Loughborough, UK). Equal amounts (5 μg) of each sample were run on reducing NuPage Novex 4–12% Bis-Tris gels (Life Technologies), and transferred to nitrocellulose membranes (GE Healthcare, Paisley, UK) at 120 mA for 45 min. Following transfer, the membranes were incubated in blocking solution (TBST: 20 mm Tris-HCl, 150 mm NaCl, 1% Tween-20, pH 7.6) containing 5% skimmed milk for 2 h at room temperature (18–21 °C). Blots were probed with a 1:15,000 dilution of sheep antiserum raised against recombinant F. hepatica peroxiredoxin (FhPrx; 22) or recombinant cathepsin L1 (FhCL1; Dalton, unpublished); a 1:1000 dilution of rabbit antiserum raised against recombinant helminth defense molecule (FhHDM-1; 23) or a 1:5000 dilution of rabbit antiserum raised against recombinant Schistosoma mansoni leucine aminopeptidase which is reactive to F. hepatica leucine aminopeptidase (SmLAP; 24, Dalton unpublished) for 2 h at room temperature (18–21 °C). After washing in TBST (3 × 10 min), an appropriate alkaline phosphatase-conjugated IgG secondary antibody was applied to the membranes for 1 h at room temperature (18–21 °C) before detection using the BCIP/NBT substrate (Sigma). Peptides from surface-accessible proteins were released by trypsin hydrolysis of EVs isolated from adult F. hepatica using our previously described protocol (25.Robinson M.W. Buchtmann K.A. Jenkins C. Tacchi J.L. Raymond B.A. To J. Chowdhury P.R. Woolley L.K. Labbate M. Turnbull L. Whitchurch C.B. Padula M. Djordjevic S. MHJ_0125 is a M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of. Mycoplasma hyopneumoniae.Open Biol. 2013; 3: 130017Crossref PubMed Scopus (52) Google Scholar). Briefly, sequencing grade trypsin (Promega, Southampton, UK) was added to purified F. hepatica EVs at a final concentration of 50 μg/ml for 5 min at 37 °C. The treated EVs were then centrifuged at 100,000 × g for 1 h at 4 °C, and the released peptides recovered from the supernatant. To investigate subvesicular protein localization, purified F. hepatica EVs were sequentially extracted to identify proteins associated with the EV membrane or those contained within the EV lumen. First, the EVs were resuspended in water; the resulting osmotic shock causes the EVs to burst and release the soluble contents held within the EV lumen. The EV membranes were collected by centrifugation at 100,000 × g for 1 h at 4 °C, while the soluble contents of the EV lumen were recovered in the supernatant. The membrane pellet was sequentially extracted with 0.1 m Na2CO3 (pH 11) on ice for 30 min to extract peripheral proteins and the sample centrifuged at 100,000 × g for 1 h at 4 °C. The final pellet was then solubilized in 1% Triton X-100/2% SDS for 15 min at 37 °C to produce integral membrane protein fractions (26.Robinson M.W. Alvarado R. To J. Hutchinson A.T. Dowdell S.N. Lund M. Turnbull L. Whitchurch C.B. O'Brien B.A. Dalton J.P. Donnelly S. A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase.FASEB J. 2012; 26: 4614-4627Crossref PubMed Scopus (66) Google Scholar). Proteins in the various EV fractions were reduced with 2 mm DTT in 50 mm NH4HCO3 (60 °C/20 min) and alkylated with 5 mm iodoacetamide (room temperature (18–21 °C) in the dark/30 min). Samples were then incubated with 100 ng/μl sequencing grade trypsin (Promega) (37 °C/overnight). The digestions were stopped by the addition of trifluoroacetic acid (TFA) to a final concentration of 0.1% and dried in a vacuum centrifuge. The final mixtures were reconstituted with 10 μl of 0.1% TFA before analysis by LC-MS/MS. Five microliters of the resulting suspension were delivered to an analytical column (Eksigen C18-CL NanoLC Column, 3 μm; 75 μm × 15 cm) equilibrated in 5% acetonitrile/0.1% formic acid (FA). Elution was carried out with a linear gradient of 5–35% buffer B in buffer A for 30min (buffer A: 0.1% FA; buffer B: acetonitrile, 0.1% FA) at a flow rate of 300 nl/min. Peptides were analyzed in a nanoESI qQTOF mass spectrometer (5600 TripleTOF, ABSCIEX) operating in information-dependent acquisition mode, in which a 0.25-s TOF MS scan from 350–1250 m/z, was performed, followed by 0.05-s product ion scans from 100–1500 m/z on the 50 most intense 2–5 charged ions. Peak list files were generated by Protein Pilot v4.5 (Applied Biosystems) using default parameters and exported to Mascot v2.4.1 (Matrix Science) for database searching. All MS/MS samples were analyzed using Mascot v2.4.1 (Matrix Science). Mascot was set up to search a database comprised of the gene models identified within the F. hepatica genome (version 1.0, 101,780 entries; 11) assuming trypsin digestion with 1 missed cleavage permitted. The F. hepatica gene model sequences can be accessed through WormBase ParaSite (http://parasite.wormbase.org/) under accession PRJEB6687 (genomic read data and gene model transcripts). Mascot was searched with a fragment ion mass tolerance of 0.1 Da and a parent ion tolerance of 0.1 Da. Iodoacetamide derivative of cysteine was specified in Mascot as a fixed modification. Glu->pyro-Glu of the N terminus, Gln->pyro-Glu of the N terminus, deamidation of Asn and Gln and oxidation of Met were specified in Mascot as variable modifications. Scaffold (version Scaffold_4.3.0, Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm (27.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: 5383-5392Crossref PubMed Scopus (3897) Google Scholar) with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (28.Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3631) Google Scholar). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. RNA sequencing and gene expression analysis was carried out for multiple lifecycle stages, including metacercariae, NEJ 1, 3 and 24 h postexcystment, juvenile 21-day-old and adult liver flukes, as described by Cwiklinski et al. (11.Cwiklinski K. Dalton J.P. Dufresne P.J. La Course J. Williams D.J. Hodgkinson J. Paterson S. The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution.Genome Biol. 2015; 16: 71Crossref PubMed Scopus (191) Google Scholar). Briefly, Illumina TruSeq RNA libraries were prepared and sequenced, with the resulting RNAseq data being mapped to F. hepatica genome scaffolds and gene models. Differential expression analysis in edgeR was carried out for all genes to which at least five Illumina reads across all libraries could be mapped. Discovery of F. hepatica genes associated with EV machinery was carried out using BLAST analysis against the F. hepatica genome. Briefly, human proteins, inferred from the literature to have roles in exosome biogenesis, cargo sorting or uptake, were used as BLAST queries to identify homologs in closely-related trematode species (NCBI; trematoda). Both the human proteins and trematode homologs were used as BLAST queries to interrogate the F. hepatica genome (NCBI v2.2.29). Each match was manually validated and confirmed using InterPro (http://www.ebi.ac.uk/interpro/) which detects the presence of conserved protein domains. Expression of these identified genes across the F. hepatica lifecycle was investigated using the differential expression analysis of all the gene models identified within the genome. Hierarchical clustering using Gene Cluster 3.0 (29.Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Cluster analysis and display of genome-wide expression patterns.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 14863-14868Crossref PubMed Scopus (13218) Google Scholar) was used to group differentially expressed genes by similarity of expression, graphically represented using heat-maps. The soluble contents of F. hepatica EVs recovered following the 15,000 × g and 120,000 × g centrifugation of fluke culture media were isolated by osmotic shock. Briefly, the vesicle pellets were resuspended in hypotonic buffer (20 mm HEPES-KOH; pH 7.3) and placed on ice for 30 min with intermittent vortexing followed by sonication (5 × 20s at 20% power). The vesicle membranes were then removed by centrifugation at 20,000 × g for 1 h at 4 °C. Auto-activation of the native procathepsin L was carried out by incubating the soluble extract from the 15,000 × g vesicle pellet (50 μg total protein) at 37 °C in 100 mm citrate phosphate buffer, pH 4.5, containing 1 mm DTT. Aliquots (15 μl) were removed at time intervals and added to tubes containing 1 μl of 1 mm E-64 to stop the reaction. Proteolytic cleavage of the prosegment was visualized by immunoblotting as described above. Cathepsin L activity was also monitored in the presence of the fluorogenic peptide substrate Z-Leu-Arg-NHMec (20 μm) by measuring the release of fluorescence over time using a Polarstar Omega micro-plate reader (BMG Labtech) in 96-well fluorescent plates as previously described (30.Lowther J. Robinson M.W. Donnelly S.M. Xu W. Stack C.M. Matthews J.M. Dalton J.P. The importance of pH in regulating the function of Fasciola hepatica cathepsin L1 cysteine protease.PLoS. Negl. Trop. Dis. 2009; 3: e369Crossref PubMed Scopus (66) Google Scholar). Cathepsin B activity was measured by incubating the soluble vesicular extracts with Z-Arg-Arg-NHMec (10 μm) in 100 mm citrate-phosphate buffer (pH 6.5) containing 1 mm DTT and 0.01% Triton X-100. E-64 (10 μm) was used to inhibit cathepsin L and B activity. Leucine aminopeptidase activity was measured by incubating the vesicular extracts with the substrate H-Leu-NHMec (10 μm) in 100 mm Tris-HCl buffer (pH 7.5) containing 1 mm MnCl2 and 0.01% Triton X-100. Bestatin (50 μm), a broad-spectrum aminopeptidase inhibitor (31.Rich D.H. Moon B.J. Harbeson S. Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes.J. Med. Chem. 1984; 27: 417-422Crossref PubMed Scopus (218) Google Scholar) was used to demonstrate inhibition of the reaction. Transmission electron microscopy (TEM) was performed on adult flukes as previously described (32.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: 509-518Crossref PubMed Scopus (19) Google Scholar). Briefly, adult flukes were fixed for 1 h in 2% double-distilled glutaraldehyde (DDG; Agar Scientific) in 0.1 m sodium cacodylate buffer (pH 7.2) containing 3% sucrose at 4 °C. After washing in buffer, specimens were dehydrated in ethanol and embedded in Agar 100 resin (Agar Scientific, Stansted, UK). Ultrathin sections (60–70 nm) were cut on a Reichert Ultracut E ultramicrotome and collected on bare 200-mesh nickel grids. For immunogold labeling, sections were etched with 10% hydrogen peroxide for 5 min then washed with 20 mm Tris-HCl buffer (pH 8.2) containing 0.1% bovine serum albumin and 2.5% Tween 20. Grids were incubated in normal goat serum (1:20 dilution in Tris-HCl buffer) for 30 min and then transferred to primary antibody diluted to 1:20,000 with 0.1% bovine serum albumin/Tris-HCl buffer for 12–18 h. Grids were washed in bovine serum albumin/Tris-HCl and then incubated with 10 nm gold-conjugated goat anti-sheep IgG (Bio Cell International) for 2 h at room temperature (18–21 °C). Grids were then lightly fixed with 2% DDG for 3 min, double stained with uranyl acetate and lead citrate and examined in a FEI (Philips) CM100 transmission electron microscope, operating at 100 keV. Controls included omission of primary antibody and incubation of grids with pre-immune serum followed by the secondary antiserum. The exosome-like 120K vesicle pellet was analyzed by TEM as previously described (12.Marcilla A. Trelis M. Cortés A. Sotillo J. Cantalapiedra F. Minguez M.T. Valero M.L. Sánchez del Pino M.M. Muñoz-Antoli C. Toledo R. Bernal D. Extracellular vesicles from parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host cells.PLoS ONE. 2012; 7: e45974Crossref PubMed Scopus (246) Google Scholar). The 15K vesicle pellet was resuspended in PBS and aliquots placed on formvar-coated grids and examined in a FEI (Philips) CM100 transmission electron microscope, operating at 100 keV. Taking advantage of our recently reported F. hepatica genome and transcriptome data sets for the major intramammalian life-cycle stages (newly excysted juveniles, NEJs; 21-day old immature liver stage juveniles, and adult flukes) (11.Cwiklinski K. Dalton J.P. Dufresne P.J. La Course J. Williams D.J. Hodgkinson J. Paterson S. The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution.Genome Biol. 2015; 16: 71Crossref PubMed Scopus (191) Google Scholar), we were able to perform a definitive characterization of the secretions of adult F. hepatica. Sixty-nine proteins secreted by adult F. hepatica were identified in this analysis (supplemental Tables S1 and S2)