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

To infect their mammalian hosts, Fasciola hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum, and penetrate the liver. After migrating through and feeding on the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and produce eggs. Here we integrated a transcriptomics and proteomics approach to profile Fasciola secretory proteins that are involved in host-pathogen interactions and to correlate changes in their expression with the migration of the parasite. Prediction of F. hepatica secretory proteins from 14,031 expressed sequence tags (ESTs) available from the Wellcome Trust Sanger Centre using the semiautomated EST2Secretome pipeline showed that the major components of adult parasite secretions are proteolytic enzymes including cathepsin L, cathepsin B, and asparaginyl endopeptidase cysteine proteases as well as novel trypsin-like serine proteases and carboxypeptidases. Proteomics analysis of proteins secreted by infective larvae, immature flukes, and adult F. hepatica showed that these proteases are developmentally regulated and correlate with the passage of the parasite through host tissues and its encounters with different host macromolecules. Proteases such as FhCL3 and cathepsin B have specific functions in larvae activation and intestinal wall penetration, whereas FhCL1, FhCL2, and FhCL5 are required for liver penetration and tissue and blood feeding. Besides proteases, the parasites secrete an array of antioxidants that are also highly regulated according to their migration through host tissues. However, whereas the proteases of F. hepatica are secreted into the parasite gut via a classical endoplasmic reticulum/Golgi pathway, we speculate that the antioxidants, which all lack a signal sequence, are released via a non-classical trans-tegumental pathway.


Summary
In order to infect their mammalian hosts, F. hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum and penetrate the liver.After migrating through and feeding on the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and produce eggs.Here, we have integrated a transcriptomic and proteomic approach to profile Fasciola secretory proteins that are involved in hostpathogen interactions and to correlate changes in their expression with the migration of the parasite.Prediction of F. hepatica secretory proteins from 14,031 expressed sequence tags (ESTs) available from the Wellcome Trust Sanger Centre using the semi-automated EST2Secretome pipeline showed that the major components of adult parasite secretions are proteolytic enzymes including cathepsin L, cathepsin B and asparaginyl endopeptidase cysteine proteases as well as novel trypsin-like serine proteases and carboxypeptidases.Proteomic analysis of proteins secreted by infective larvae, immature flukes and adult F. hepatica showed that these proteases are developmentally regulated and correlate with the passage of the parasite through host tissues and its encounters with different host macromolecules.Proteases such as FhCL3 and cathepsin B have specific functions in larvae activation and intestinal wall penetration, while FhCL1, FhCL2 and FhCL5 are required for liver penetration, and tissue and blood feeding.Besides proteases, the parasites secrete an array of antioxidants that are also highly regulated according to their migration through host tissues.However, whereas the proteases of F. hepatica are secreted into the parasite gut via a classical ER/Golgi pathway, we speculate that the antioxidants, which all lack a signal sequence, are released via a non-classical trans-tegumental pathway.

Introduction
Fasciola hepatica is a helminth (worm) parasite with a world-wide distribution.
Although traditionally regarded as a parasite of livestock, particularly sheep and cattle, that results in a large economic loss to the agricultural community it has recently emerged as an important human infection in many regions of the world, including South America, Iran, Egypt and mainland South-East Asia (1).Dormant larvae contained within cysts adhere to vegetation and emerge as infective juveniles (newly excysted juveniles, NEJs) in the duodenum following ingestion by animals or humans.They infect their hosts by rapidly penetrating the intestinal wall and entering the peritoneal cavity where they break through the liver capsule.After 8-12 weeks of consistent burrowing, feeding and growth within the liver parenchyma they move to their final destination within the bile ducts where they mature and produce enormous numbers of eggs (2).The two distinct clinical phases of fasciolosis are directly related to the parasites' migration: acute fasciolosis, which manifests as fever, abdominal pain, weight loss and hepatomegaly, is associated with liver tissue damage and inflammation caused by the migrating immature parasites, whereas chronic fasciolosis (usually sub-clinical) is coupled with the presence of the mature adult flukes in the bile ducts (1,3).These invasive helminth parasites undergo complex changes as they migrate within their definitive mammalian hosts.The developing parasites encounter different host tissues and macromolecules and have to contend with a continually changing physiological microenvironment (such as pH and oxygen availability), and a mounting humoral and cellular host immune response.Morphological and ultrastructural studies clearly show major alterations of the parasite surface and gastrodermis, the two host-parasite interfaces, as they migrate and grow (4).However, we are only beginning to understand the molecular and biochemical interactions that occur between host and parasite and how these adjust as the parasite's development progresses.A deeper knowledge of such host-pathogen interplay should provide data on novel targets for anthelmintic compounds and potential anti-parasite vaccine candidates.
Increasingly, proteomic analysis is being employed as a means to investigate the interaction of helminth parasites and their hosts, particularly in cases where obtaining pathogen material is difficult (5)(6)(7)(8)(9).For some helminths these studies have been facilitated by the availability of large transcriptomic datasets (10)(11)(12).
Unfortunately, the Fasciola nucleotide sequences available in GenBank are relatively few and highly redundant (298 for F. hepatica and 142 for F. gigantica as of 15/01/2009) and the adult F. hepatica ESTs currently available from the Wellcome Trust Sanger Centre (14,031 reads) are unannotated (therefore the current identification of peptides of interest requires manual BLAST analysis using specific query sequences).Accordingly, in this study we employed the semi-automated EST2Secretome pipeline to analyse all available F. hepatica ESTs for secretory proteins potentially involved in host-pathogen interactions (13).EST2Secretome was developed in our laboratory by optimizing signal-peptide-mediated secreted protein prediction from our earlier predictions of parasitic nematode ESTs (14,15).We have integrated this transcriptomic data with a proteomic analysis of the molecules secreted by adult F. hepatica, with a particular emphasis on proteolytic enzymes.Furthermore, we have also analysed the somatic and secreted molecules of the infective NEJ parasites and compared these to the secretome of immature and adult parasites taken from liver tissues.In doing so we have produced a comprehensive view of how F. hepatica differentially and developmentally express and secrete proteolytic enzymes and other molecules according to the specific challenges faced in the intestine, liver and bile ducts.Fasciola hepatica cathepsins B and cathepsin L (FhCL3) are stored as zymogens within the infective larvae ready to be trans-activated by specific asparaginyl endopeptidases, and released to perform the highly-specific function of host tissue invasion (intestinal epithelium and liver capsule).By contrast, cysteine proteases belonging to the phylogenetic clades FhCL1, FhCL2 and FhCL5 are expressed during the later stages within the liver and bile duct and function in tissue degradation and feeding alongside the cell-lytic protein saposin and a newly-described prolylcarboxypeptidase.Several novel developmentally-regulated cathepsin L and cathepsin B cysteine proteases, and members of two serine protease families, namely carboxypeptidase and trypsin-like serine proteases, which may also have important roles in host-parasite interplay, have also been identified.Finally, our observations have led us to propose that whereas the major proteases of F. hepatica are secreted into the parasite gut via a classical ER/Golgi pathway, an array of abundant and highly-regulated antioxidants are released via a non-classical trans-tegumental pathway.as determined for the large-scale secretory protein prediction from helminth parasite ESTs (13).

Analysis of the F. hepatica ESTs using EST2Secretome
Independently, un-annotated Fasciola ESTs that matched MS/MS data were used as queries for BLASTn (18) searches of all nucleotide sequences in the Genbank and EMBL databases.Searches were performed using the NCBI server (http://blast.ncbi.nlm.nih.gov/Blast.cgi).Open reading frames were constructed from the Fasciola EST sequences (guided by their best BLASTn hits) and hypothetical proteins derived from their conceptual translation were submitted to the InterProScan algorithm (19) to detect conserved domains and motifs to help assign putative protein identifications.

Proteomic analysis of parasite somatic and secreted proteins: gel electrophoresis and mass spectrometry
The profile of proteins secreted by adult F. hepatica has been recently studied using two-dimensional electrophoresis (2-DE) by us and others (8,20,21).However, because of the paucity of material that can be obtained for the infective juvenile NEJ parasites and 21 day-old liver stage parasites we employed 1-DE in the present study.
Nevertheless, we validated this approach by subjecting adult parasite secretory proteins to 1-DE and comparing the data with those reported by Robinson et al. (8).
Protein samples were analysed by 1-DE using NuPage

Quantitation of Fasciola proteins by emPAI
We analysed the exponentially modified protein abundance index (emPAI) provided in the output of the MASCOT MS/MS ion search to estimate the relative expression of proteases and antioxidants identified in the developmental stages of F. hepatica.
The emPAI used by MASCOT is a modification of the formula developed by Ishihama et al. (22) and gives a label-free relative quantification of the proteins in a mixture.The raw emPAI values obtained represent the transformed ratio of the number of experimentally observed peptides (composed of unique precursor ions, including different charge states of the same peptide, which match or exceed the threshold level for homology or identity) to the total number of peptides that can theoretically be detected within the operating mass range and retention time range of the mass spectrometer (calculated by MASCOT based on the mass of the protein, the average amino acid composition of the database searched and the enzyme specificity).
For this analysis, the raw emPAI values (averaged from 3 separate gels) for all Fasciola proteases or antioxidants identified were added to give a figure representing total expression for each within a particular developmental stage.The raw emPAI values for each individual protease or antioxidant were then converted to a percentage of this total to estimate their relative expression levels (Figures 2A-3A).As the method used by MASCOT to calculate the number of observable peptides differs from that originally described by Ishihama et al. (22) and is not freely available, it was decided not to calculate emPAI values manually for those proteins identified using the PEAKS software in order to avoid introducing errors into the subsequent analysis.In order to account for redundancy, molecules potentially containing shared sequences were grouped together (e.g.all cathepsin B variants were classed as FhCB, all cathepsin L1 variants were classed as FhCL1 etc).

Excystment of F. hepatica metacercariae and preparation of somatic larvae proteins
The dormant cysts of F. hepatica metacercariae contain two layers, the outer of which can be contaminated with plant or other extraneous material.Here we describe a method for removing the outer cyst and adhered material so that somatic proteins of the dormant infective larvae can be analysed.In addition, we describe a rapid method for activating the larvae and inducing them to emerge from the cysts so that in vitro-secreted proteins can be isolated.Thus, F. hepatica metacercariae (Baldwin Aquatics Inc., Monmouth, Oregon) were vortexed for 10 seconds in 0.5 % sodium hypochlorite and then incubated at room temperature for 20 min (this procedure dissolves the outer cyst layer).They were then washed three times in distilled water by centrifugation at 2000 x g for 2 min.The juvenile larvae, which were now only contained within a clear inner cyst layer, were used to prepare somatic protein extracts and secretory proteins.To prepare the somatic extracts the parasites were homogenised in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1 % Triton X-100, 1 % sodium deoxycholate (Sigma), 0.1% SDS, 1x Complete mini protease inhibitor cocktail (Roche)) and placed on ice for 30 min.The protein extract was centrifuged at 13,000 rpm for 10 min to remove insoluble debris and the supernatant stored at -20°C until use.
To determine whether cysteine proteases were essential to cyst rupture, F. hepatica metacercariae (fifty per treatment in triplicate) were either excysted with medium as described above or in medium lacking the 0.006 % (w/v) L-cysteine, or supplemented with the cysteine protease inhibitors trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64, Sigma) or 1 mM carbobenzoxy-phenylalanylalanine-diazomethyl ketone (Z-Phe-Ala-CHN 2 , Bachem St. Helens, UK) to a final concentration of 1 mM.The numbers of excysted parasites were counted after 3 h incubation at 37°C / 5 % CO 2 and the data was analysed using Student's t-test.The experiments were repeated twice.
Preparation of immature and mature adult F. hepatica secretory proteins Immature F. hepatica flukes (21 day-old) were recovered from the livers of female BALB/c mice (experimentally infected with 20 metacercariae) while adult parasites were recovered from the bile ducts of Merino sheep 16 weeks after an experimental infection with 200 metacercariae.Immature and adult parasites were washed in prewarmed (37°C), phosphate-buffered saline (PBS), pH 7.3, before transfer to culture medium (as described above) for 24 and 8 h, respectively.Secretory proteins were concentrated from the culture supernatants by precipitation with methanol / chloroform as previously described (6).Pellets were resuspended in 10 µl RIPA buffer and stored at -20°C prior to separation by electrophoresis.
All rESTs were then subjected to analysis using our recently reported semiautomated bioinformatics platform (EST2Secretome, 13) to predict secretory proteins from the F. hepatica EST database.Using this approach, we have identified a sub-set of parasite proteins likely to participate in the most significant interactions that occur between the adult stage of this parasite and its mammalian host.Thus, 173 Fasciola secretory proteins were predicted by the EST2Secretome pipeline, with 160 true positives based on the EST2Secretome annotations mapping homology to proteins in a non-redundant secreted protein database (SecProtSearch) derived from the literature, the secreted protein database, SPD (26) and the manually curated signal peptide database, SPdb (27); as well the GO annotations of subcellular localization of the top homologues identified by BLAST (92.5 % accuracy) (Table 1).Since ESTs are usually not full-length and often truncated, manual inspection of the final dataset is required as it is possible that transmembrane sequences are erroneously identified as secreted proteins and thus elude the filtration step by TMHMM.
From the detailed annotations of the 160 adult secreted proteins the predominance of cathepsin L cysteine proteases is clearly evident as these are represented by a total of 66 (41.2 %) proteins (Table 2, and Supplementary Table 1).that is found in eggs produced by the adult parasite (31) (Table 2).
A Fasciola protein disulphide isomerase was also predicted that has previously been identified in the secretions of adult flukes (32).Protein disulphide isomerases have roles in protein folding and a Fasciola recombinant enzyme was shown to mediate the oxidative refolding of reduced RNase (32).A putative peptide with a number of cubulin domains was also predicted in the current analysis.Cubulin domains occur predominantly in extracellular proteins or plasma membraneassociated proteins with a range of functions including complement activation, tissue repair, cell signalling and inflammation (33).Although the Fasciola peptide contains a predicted N-terminal transmembrane region, its molecular function remains unknown.We note that while orthologues are available for 6 proteins, including an uncharacterised secretory protein from Clonorchis sinensis, their function remains elusive.A total of 36 secreted proteins (21.9 %) are novel but no database matches exist at the present time (Table 2).
Proteomic profiling of adult F.hepatica secreted proteins. We

Discussion
The database of F. hepatica ESTs available from the Wellcome Trust Sanger Centre is now sufficiently large as to allow a significant transcriptomic analysis of this helminth pathogen, which until now has been lacking in this field.We have employed our newly developed EST2Secretome pipeline to analyse these data sets with the view to identifying molecules secreted by the adult trematode, F. hepatica, unlike earlier applications to nematode parasites which focused on the complete transcriptome.
Furthermore, we have integrated these results with data generated from proteomic analysis performed here and in previous reports (8,20,21) to build a picture of how the developing parasite sustains itself within the mammalian host, with particular emphasis on proteases as virulence and tissue-damaging factors.
EST2Secretome analysis of the adult F. hepatica ESTs identified 160 cDNAs encoding secreted proteins, 41% of which encoded cathepsin L cysteine proteases.
The abundance of adult cathepsin L sequences was noted in a previous analysis of entries in the public databases (8,35).When these sequences were subjected to a phylogenetic investigation it was demonstrated that they could be separated into five The F. hepatica cathepsin L proteases, the largest family of proteases known in any helminth pathogen, arose by a series of gene duplications.Members underwent selective functional diversity brought about by specific alterations within the active site that ultimately produced a repertoire of proteases with overlapping but distinct substrate specificity (8).Our proteomic investigation was consistent with the EST2Secretome analysis by demonstrating the predominance of these proteases in the adult F. hepatica secretome (approximately 80 % of total protein secreted) and indicated a major role for the enzymes in supporting the parasites' existence within the bile duct.Here, the adult parasites are obligate blood feeders and, therefore, the most obvious function for the cathepsin L enzymes is in the digestion of blood macromolecules.Recently, Lowther et al. (36) showed that members of the most predominant cathepsin L clade, FhCL1, have evolved an active site with a strong preference for hydrophobic amino acids, such as Leu, Ala, Phe and Val, that are particularly abundant in haemoglobin (42 %) indicating a specific adaptation for digestion of this protein.Haemoglobin is the prime supplier of amino acids which the parasites use in the anabolism of egg proteins, a major task since they produce 30-50,000 eggs/day/worm (37).Members of the other major clade, FhCL2, have specific amino acid substitutions in the active site cleft that gives these proteases the ability to cleave substrates with Pro residues and facilitates the degradation of proline-rich interstitial collagen during the parasites' migration through the tissues (38).
Consistent with the importance of assimilating nutrient from host red blood cells was the identification of ESTs encoding a saposin-like molecule as the next most abundant in the dataset.This protein, which was termed FhSAP-2 because of its similarity to a saponin-like protein family and the amoebapore precursors of Entamoeba histolytica, has been implicated in the penetration and lysis of ingested host red blood cells, not only in Fasciola (29,30) but also in other helminths (39).
Saposin-like molecules are likely secreted from granules within cells lining the parasite oesophagus so that ingested red blood cells are lysed as soon as they enter the digestive track.Hydrolysis of released haemoglobin by the cathepsin L proteases then takes place in the acidic caecum (37).
Several Fasciola proteins predicted as components of the secretome by our EST2Secretome analysis were not identified by mass spectrometry in secretions of adult parasites maintained in culture (8,20) or within the bile ducts (21).The most highly represented of these was the major eggshell protein, vitelline protein B1 (vpB1), which is produced by mature vitelline cells to form the hard protective trematode eggshell (31).However, since this process occurs within the ootype and uterus of the adult fluke it is likely that vpB1 is retained within the eggshell and not secreted outside the parasite.Other examples are the transcripts encoding six novel cathepsin B cysteine proteases and five new asparaginyl endopeptidases.While some isotypes of these proteases are secreted by the infective larvae and immature liverstage flukes (see below), they are absent from the secretome of adult F. hepatica or F. gigantica as previously reported using biochemical, proteomic and immunoblotting methods (8,40,41).Considering these observations, and the fact that cathepsin B and asparaginyl endopeptidases also function internally within cells (42,43), it is likely that these newly discovered proteases have functions within the internal tissues of this complex multicellular parasite, and are involved in generalised protein turnover, remodelling and/or catabolism.Alternatively or additionally, since immunocytochemistry and in situ hybridization has localised cysteine proteases and transcripts to the reproductive structures of other trematodes (44,45) these proteases may be involved in the process of egg production.Other proteins identified by EST2Secretome analysis but not by proteomics, including the previously uncharacterised proteins such as deoxyribonucleases, carboxypeptidase and trypsinlike peptidases that have homologs in S. japonicum and C. sinensis, may also be confined to internal tissues of the parasite or be expressed and secreted at levels below the detection capacity of the proteomic methods so far employed (cDNAs of these were poorly represented in the transcriptome of the adult parasite).
Conversely, a number of F. hepatica proteins that have previously been described as major components of the adult parasite secretions by standard protein and proteomics methods were not predicted by the EST2Secretome pipeline.These included the fatty acid-binding proteins, FaBP1, FaBP2, FaBP3 and Fh15 (20,21), and two redox enzymes, thioredoxin and peroxiredoxin (20,(46)(47)(48).Primary sequence analysis using SignalP (49) confirmed that these proteins lacked predicted N-terminal signal peptides for secretion via the classical ER/Golgi pathway and, hence, they must be released by alternate mechanisms.Morphew et al. (21) suggested that release of Fasciola FaBPs was due to shedding of the tegument as part of a stress response during in vitro culture of parasites since they were not detected in vivo.However, morphological studies have shown that blebbing or shedding of the F. hepatica surface tegument and renewal by proteins synthesised by highly metabolic subtegumental cells is a continuous property that may be an immuno-defensive strategy (4,50).This non-classical mechanism for protein secretion may be compared to those reported for some leaderless eukaryotic proteins, for example the ER/Golgiindependent secretion of leaderless peptides including interleukin (IL)-1α, IL-1β and fibroblast growth factor (FGF)-2 (51).In the case of IL-1β, which is processed and activated by caspase-1, it has been proposed that both molecules are packaged together into plasma membrane blebs (by ATP-binding cassette (ABC) transporters) and are rapidly released as microvesicles following phospholipase-mediated fusion with the plasma membrane (52)(53)(54).The identification of a phospholipase in the transcriptome of adult flukes (Table 2) together with the presence of ABC transporters within the tegument (55) supports the possibility of a similar mechanism for ER/Golgi-independent secretion pathway in Fasciola.
In summary, our EST2Secretome pipeline was successful in identifying the major secreted proteins of adult F. hepatica.Integration of this analysis with proteomic data is important for the study of helminth host-pathogen relationships to distinguish proteins that are secreted extra-corporeally from those secreted within the internal tissues of the parasites.Additionally, this integrated approach identified major helminth secreted proteins that may reach the exterior by novel or non-classical secretory pathways.

Proteomic analysis of the dormant and infective stage larvae
Having collated the secretome data for adult F. hepatica we performed a comparative analysis of this with the infective larvae that invade their host by penetrating the intestinal wall.The larvae of F. hepatica (<1 mm in length) are released by the intermediate snail host (Galba truncatula) and encyst on vegetation that is consumed by the host.For research purposes these are produced in an aquarium and the larvae are allowed to encyst on cellophane.However, because under these conditions the encysted metacercariae can become contaminated with extraneous material we used a method of washing in 2 % hypochlorite to remove this and the outer cyst wall so that a proteomic study of the dormant larvae could be performed.We also used a medium containing bile duct surfactants to activate the larvae and induce them to excyst so that their secretome could be analysed.We found that the level of Fasciola excystment was significantly reduced when the excystment medium excluded the reducing agent L-cysteine (91 % reduction, p = 0.0008, not shown) compared with medium containing L-cysteine.The requirement for reducing conditions implied a role for cysteine proteases in the excystment process since the activity of these thioldependent proteases are enhanced in the presence of reducing agents.We therefore proved this point by showing that the level of excystment was significantly reduced when the broad-range cysteine protease inhibitors E-64 (98 % reduction, p = 0.0433) or the cathepsin-specific inhibitor Z-Phe-Ala-CHN 2 (99 % reduction, p = 0.0205) were added to the excystment medium.Proteomic analysis of soluble extracts of dormant larvae and the secretions of the newly excysted juveniles (NEJ) identified 26 and 29 proteins, respectively (Figures 2B-3B; Supplementary tables 2-3).
Significantly, 23 % ( 6) of all the somatic larval proteins and 31 % of the secreted proteins were cysteine proteases consisting of cathepsin L (37 %), cathepsin Bs (45 %) and asparaginyl endopeptidases (18 %).Since the former two classes are potently inhibited by E-64 and Z-Phe-Ala-CHN 2 , but the later class not, the results implicate cathepsin Ls and/or cathepsin B in the process of cyst rupture.
Phylogenetic studies showed that cDNA clones generated from transcripts of Fasciola larvae, designated clade FhCL3, encoded a cathepsin L protease that was specific to this infective stage (8,35); the cDNA encoding this protease, FhCL3_nl22, was originally described by Harmsen et al. (56).Earlier studies by Tkalcevic et al.
(34) using N-terminal sequencing to identify proteins expressed by larvae reported a sequence of a cathepsin L cysteine protease which we now know matches exactly with the predicted N-terminal of FhCL3_nl22 (56).More recently, Cancela et al. (28) confirmed the restricted expression of FhCL3, and another protease FhCL4, to infective larvae by isolating cDNAs encoding these enzymes from a F. hepatica larvae-specific cDNA library and, subsequently, by employing RT-PCR expression analysis.No FhCL4 peptides were identified in the present analysis suggesting that this enzyme may be expressed at low levels, performs specific intracellular functions and is not secreted by F. hepatica.However, we confirmed the presence of FhCL3 in NEJ by identifying several FhCL3-specific peptides with matches to FhCL3_nl22.It is noteworthy that the mass of the dormant larvae somatic cathepsin L3, which was found in a protein band migrating at ~37 kDa, is consistent with that of an inactive cathepsin L precursor, or zymogen, which is supported by the presence of mass ion m/z 644.28; +2 that matched to a peptide (SNDVSWHEWK) found only within the N-terminal prosegment region of the protease.No peptides matching with cathepsin Ls were identified in protein bands in the region corresponding to the fully processed and active mature enzymes i.e. mass ~24 kDa (Figure 1, NEJ secretome gel section 3).By contrast, while a few cathepsin L3 peptides were present at ~37 kDa in the secretome of NEJ (including the FhCL3 prosegment peptides LGLNQFTDLTFEEFK, m/z 901.49; +2, and SNDVSWHEWK, m/z 644.28; +2), the most robust peptide matches were obtained from gel sections at the molecular mass of ~24 kDa corresponding to the size of a fully activated mature enzyme (Figure 1, NEJ secretome gel section 3).
Collectively, these observations show that the dormant larvae of F. hepatica express a specific cathepsin L protease, FhCL3, stored as an inactive zymogen that is rapidly secreted and activated following emergence from their cysts to become infective larvae.
Fasciola cathepsin B-like cysteine proteases were also identified in somatic which spans the juncture between the prosegment and the mature domain) indicate that cathepsins B1 and B3 are also stored as zymogens by Fasciola NEJs.
However, unlike the FhCL3, we also found some cathepsin B at ~20 -36 kDa (Figure 1, NEJ secretome gel section 3) which is consistent with the occurrence of processed active enzymes within the dormant larvae (which have a predicted molecular mass of 29.6 kDa, 57).While each of the three cathepsin B were also found as fully active enzymes in the secretions of larvae, in the region of ~20 -36 kDa, we also discovered a novel family member, encoded by a Fasciola EST HAN4015b05.q1kT3 (here designated cathepsin B4) (Figure 2B; Table 3).
Cysteine proteases are produced as inactive zymogens consisting of a prosegment and mature domain (58).The prosegment lies along the active site groove (in reverse to the direction of protein substrates) preventing unwanted hydrolysis during trafficking and storage of the protease within the cell.Removal of the prosegment from the mature domain exposes the active site of the hydrolase to entry of macromolecular protein substrates.Dalton et al. (59) proposed that this activation step is mediated by proteolyic clipping at a 'protease-susceptible' region between the prosegment and mature enzyme, and that this event may be performed by the same or another protease molecule.Dalton  mansoni.In the present study, we identified two asparaginyl endopeptidases in the somatic proteins and secretome of Fasciola NEJs.One of the identified enzymes (legumain-like precursor TrEMBL accession number Q711M2, termed legumain 1) matched exactly with an N-terminal sequence for asparaginyl endopeptidase obtained from NEJ somatic proteins by Tkalcevic et al (34).Although this enzyme retains the His 158 of the conserved catalytic dyad residues, the Cys 200 is replaced by a serine residue and, therefore, this enzyme may display an altered substrate-specificity compared to the normal asparaginyl endopeptidases.However, the enzyme does contain a conserved asparagine residue (Asn 323 ) that is required for C-terminal processing to an active mature enzyme (63).The second legumain-like protease identified in the NEJ somatic protein extracts is encoded by a Fasciola EST (identifier Fhep29h09.q1k,termed legumain 2) but as this EST is incomplete at the 5` end, it is not possible to determine whether the enzyme encoded by this sequence retains the conserved active site His 158 /Cys 200 dyad.Interestingly, the enzyme encoded by the legumain 2 EST lacks the conserved Asn 323 required for auto-activation.The overlapping primary sequence of the two enzymes exhibit 51 % identity and, therefore, individual enzymes were easily differentiated on the basis of their tryptic peptides.Notwithstanding the anomalies within the sequence of these two enzymes, the data suggest that the NEJ possess the enzymatic machinery to rapidly process and activate the stored zymogens of cathepsin L and B.Recently Morita et al. (64) reported that purified bovine asparaginyl endopeptidase can degrade fibronectin, a major component of the extracellular matrix.If they possess similar biochemical properties, a second tangible role for the asparaginyl endopeptidases secreted by the infective larvae could be to facilitate penetration of the host intestine.

Proteomic analysis of the migrating liver-stage parasite
The developmental stage of F. hepatica that migrates through the liver tissue is responsible for the clinical manifestations associated with acute animal and human fasciolosis.The migrating parasite causes extensive physical tissue destruction, tunnelling and haemorrhaging, and induces immunologically-related inflammatory damage (65).To gain insight on the variety of molecules produced by this parasite stage that may induce this pathology 21 day-old immature parasites were removed from the liver of infected mice and maintained in culture.A proteomics analysis of the medium revealed that these parasites secreted a greater range of proteins than the infective and adult stage flukes.In particular, they secreted the greatest range of cathepsin L cysteine proteases, including the FhCL3 that we identified in the NEJ (this was secreted solely as a mature active form of ~24 kDa).Members of cathepsin L clades FhCL1, FhCL2 and FhCL5 originally identified in the adult parasite were also secreted by the immature flukes (these could be distinguished on the basis of the clade-/sub-clade-specific peptide matches described above and reported previously by Three asparaginyl endopeptidases were identified within the secretory proteins of the immature 21 day-old flukes.These included the NEJ legumain-like precursor, legumain 1, with the altered Cys 200 /Ser 200 active site residue, but did not include the NEJ legumain 2. However, two asparaginyl endopeptidases (designated legumains 3 and 4) appeared to be specific to the liver migrating stage and, like their homologues that were originally identified in F. gigantica (41), retained the conserved catalytic His 158 /Cys 200 dyad and Asn 323 that is required for C-terminal processing (63).Unlike the asparaginyl endopeptidases of NEJs, those secreted by the immature flukes all migrated at ~36 kDa suggesting that they had undergone some processing events to lead to fully active enzymes.It is possible that besides functioning in the transprocessing of cathepsin L and B, the asparaginyl endopeptidases of the immature parasites participate in host tissue degradation.
Mass spectrometry data from the immature 21 day-old F. hepatica (and subsequently found in adult parasite secretome) matched with several ESTs encoding a putative prolylcarboxypeptidase (Figure 2B; Table 3).The exopeptidase contains a predicted N-terminal signal peptide and a conserved catalytic triad (Ser 161 , Asp 413 , His 438 ) that is characteristic of the s28 family of serine peptidases including prolylcarboxypeptidase.Although this is the first report of a putative prolylcarboxypeptidase from a trematode pathogen, orthologues have been identified in parasitic nematodes including the gastrointestinal worm Haemonchus contortus where the enzymes act as anti-coagulants to facilitate a blood-feeding lifestyle (66).It is likely that the Fasciola prolylcarboxypeptidase performs a similar role, acting alongside the saposin-like proteins to prevent blood coagulation to ensure effective lysis of ingested host red blood cells.A role for the putative prolylcarboxypeptidase in Fasciola nutrition is supported by the absence of the enzyme in the NEJ which does not possess a functional gut.
Relative expression levels of Fasciola proteases and relationship to virulence and tissue invasion.
For F. hepatica NEJs, the emPAI data indicated that cathepsin L3 and cathepsin B enzymes were expressed at similar high levels (accounting for 37 % and 45 % of total proteases, respectively) whereas asparaginyl endopeptidases represented Correlating with the parasites' migration into the liver tissue, and the development of the gut apparatus (beginning at ~10 days, 36) the expression pattern of proteases becomes more complex.For the migrating parasite, the secretion of FhCL3 and cathepsin B becomes less important as these proteases account for only 3 % and 2 % of total proteases, respectively.In contrast, members of the other cathepsin L clades become more highly represented in the secretome, FhCL1 (50 %), FhCL2 (27 %) and FhCL5 (7 %) (Figure 2A).This striking shift in protease expression indicates a requirement for a new set of proteases for liver migration and for the degradation of tissue and blood macromolecules.As discussed above FhCL1 proteases have a particular adaptation to haemoglobin digestion while FhCL2 proteases exhibit unique collagenase-like activities (FhCL5 appears to be more closely related to FhCL1, 72).Asparaginyl endopeptidase secretion remains relatively high, accounting for 11 % of total proteases secreted by the immature flukes, which may be required for activation the cathepsin L proteases and possibly for directly engaging in tissue degradation.The expression of the prolylcarboxypeptidase and saposin at this stage signals the beginning of blood feeding.Working together this profile of molecules is an effective tissue-destroying machinery essential to the parasites migration and growth but, at the same time, responsible for the pathogenesis associated with acute animal and human fasciolosis.
After the parasite has entered the bile duct it completes its maturation and becomes an obligate blood feeder.Blood provides all the necessary nutrients (macroand micro-molecules) needed for production of enormous numbers of eggs, the principle function of the adult parasite (37).The total protease secretion is assumed completely by the various cathepsin L proteases, clade FhCL1 (69 %), FhCL2 (22 %) and FhCL5 (9 %), which can degrade macromolecules such a haemoglobin to small peptides which are absorbed by the parasite for further digestion to free amino acids within its tissues (36,37).The emPAI values determined for adult parasite proteases are similar to expression data obtained using by 2-DE and densitometry (67 %, 27 % and 5 % respectively, 8) and validates the use of emPAI for estimating Fasciola secretory protein levels in the other developmental stages.Within the bile duct the parasite can exist for years (cattle) or even decades (sheep) causing relatively little pathology, apart from anaemia and bile duct hyperplasia.

F. hepatica secretes a battery of antioxidant molecules
It is worth highlighting that for all three developmental stages of F. hepatica investigated in this study an array of antioxidant molecules were secreted in an abundance second only to the proteases.These included peroxiredoxin, thioredoxin, protein disulphide isomerase, four fatty acid-binding proteins (FaBP 1-3 and Fh15) and five glutathione S-transferases (GST sigma and mu class) (20,21,32,(46)(47)(48)73).
These molecules have been implicated in fluke immune avoidance mechanisms; for example, secreted isoforms of Fasciola GSTs were shown to decrease the proliferative response of rat spleen cells and diminished nitric oxide production by macrophages (73) whereas peroxiredoxin plays a key role driving host Th2 immune responses via the recruitment and alternative activation of peritoneal macrophages (74,75).Fasciola peroxiredoxin, thioredoxin and protein disulphide isomerase may also protect the parasites from harmful reactive oxygen species released by host immune cells (32,47,48,76).
Analysis of the emPAI values for these various anti-oxidants indicates that, like the proteases, their secretion is highly regulated during the migration of the parasite (Figure 3A).The profile of secretory antioxidants is similar for the NEJs and adult flukes; both secrete several FaBPs together with peroxiredoxin (although thioredoxin is also secreted by the adult worms).However, the diversity of the antioxidant molecules secreted by the immature liver-stage parasites is notably different to these two stages with the dramatic production of glutathione-Stransferases (GST), including sigma class GST and four isoforms of mu class GSTs (GST1, GST7, GST47 and GST 51); together these account for 50 % of total antioxidants expressed at this stage.GST may provide a particular defence for the fluke against a mounting host cellular immune response as the immature parasite is in direct contact with the immune systems as it migrates through the liver parenchyma.
At this stage also the parasite has moved from an aerobic to an anaerobic environment and therefore significant changes take place in its metabolism, particularly in the tegument which is the primary host-parasite interface (77).Clearly, the combined action of FaBPs, peroxiredoxin and thioredoxin offer sufficient protection for the adult flukes residing within the immunologically-privileged bile ducts.
It was also noted that each of the aforementioned antioxidant classes found in the secretome of F. hepatica do not possess a signal sequence for secretion (with the exception of protein disulphide isomerase that also has a putative ER-retention signal and may act as a chaperone to ensure proper folding of classically secreted Fasciola peptides).This suggests that they are secreted via non-classical mechanisms, possibly shedding or blebbing of the tegument (see above).Accordingly, we propose that two distinct mechanisms for protein export operate for the most abundant proteins in

Conclusion
Our integrated transcriptomics and proteomics analysis has provided an in-depth overview of protein secretion by the animal and human pathogen, F. hepatica that represents a significant step towards a comprehensive understanding of the hostparasite interactions in fasciolosis.Of vital importance now is the completion of transcriptome datasets for both the infective NEJ larvae and immature liver-stage parasites so that a comparative analysis of secretory proteins can be performed.
Nevertheless, we have described the secretion of the major molecules of this parasite, and correlated their expression with critical steps in its migration and development within the mammalian host.This will allow a more strategic and rational approach to     an approximate quantification of the protein (22).These values were converted to a percentage of total antioxidant expression within each Fasciola developmental stage (see experimental procedures section) and were used to generate the pie charts shown in A; 3 section of the 1-D gel from which the peptides were identified.Putative peptides 2960 (68.9 % rESTs) Secreted proteins (SignalP cut-off: 0.5) 160 (5.4 % peptides) yielded 4236 representative ESTs (rESTs; 2749 contig and 1487 singleton sequences), of which 2960 (68.9 %) had open reading frames.These pre-processed ESTs ranged from 60-2093 bp, with a mean of 569 bp and a standard deviation (S.D.) of 268 bp.After clustering, the mean length of the contigs increased to 788 bp and standard deviation (S.D) of 358 bp.The G+C content of the coding sequences was 44.5 % which is similar to the figure of 43.5 % reported for the adult bovine lungworm, Dictyocaulus viviparus from EST analysis (

Finally, molecules with
roles in protein turnover such as ubiquitin and a putative serpin were also identified.Identification of immature F. hepatica secretory proteinsParasites were removed from the livers of infected mice and maintained in vitro for collection of secreted proteins.The overall complexity of the secretory proteins of these 21 day-old immature F. hepatica was greater compared with that of the NEJs or adult parasites (Figure1) and yielded a total of 45 different protein identifications (Figures2B-3B; Supplementary tables 2-3).Of these, 34 were matched to previously identified Fasciola cDNAs and 11 corresponded to putative proteins encoded by novel ESTs.Mass spectrometry data also matched to peptides encoded by a further two F. hepatica EST sequences that lacked conserved protein domains and could not be assigned putative functions based on BLAST searches.Of the 45 positively-matched proteins 22 were proteases and included 14 cathepsin Ls, four cathepsin Bs, three asparaginyl endopeptidases (legumains) and a newly discovered prolylcarboxypeptidase.The remaining 23 proteins secreted by the immature liver stage parasites included a glutathione S-transferase (GST) sigma class enzyme and four isoforms of mu class GSTs (GST1, GST7, GST47 and GST 51), four fatty acid-binding proteins (FaBP1, FaBP2, FaBP3 and Fh15), two saposin-like proteins (SAP1 and SAP3), two enzymes of glycolysis (enolase and triosephosphate isomerase) and two enzymes involved in cell redox homeostasis (peroxiredoxin and protein disulphide isomerase).Other significant peptide matches included annexin, ferritin, ubiquitin, a 14-3-3 protein, a multi-cystatin and a putative ABC transporter protein.
clusters or clades, FhCL1, FhCL2, FhCL3, FhCL4 and FhCL5.Due to the high level of conservation between the clades it was not possible to study temporal patterns of specific cathepsin gene expression by conventional means (such as RT-PCR).Analysis of the >14,000 adult parasite EST sequences in the present study confirmed the expression of three of these distinct clades (FhCL1, FhCL2 and FhCL5) in this fully mature stage of the parasite.It also supported the lack of expression of clades FhCL3 and FhCL4 in adult parasites, although proteases encoding these genes are expressed by NEJs (see below and 28).We also identified five novel cathepsin L proteases that although encoded by partial nucleotide sequences (contigs 1553, 2626, 1886 and singletons Fhep55b05.q1kand Fhep18b10.q1k)may represent a new phylogenetic cluster.
extracts of larvae, with peptides matching to proteins encoded by three different cDNAs that were previously designated cathepsin B1 (accession number A7UNB2, Ljunggren et al. unpublished), cathepsin B2 (Q8I7B2, Khaznadji et al. unpublished) and cathepsin B3 (A5X494, 28).All three cathepsin B proteases were identified in intense protein bands that migrated in reducing SDS-PAGE at molecular mass of ~36 -50 kDa and ~50 -70 kDa (Figure 1, dormant larvae gel sections 1 and 2) suggesting that like FhCL3 these may represent stored zymogens.The presence of peptides FINIEHFK (m/z 524.28, +2) and QHLGLLEETPEER (m/z 775.89, +2) confirmed the existence of cathepsin B2 as an unprocessed zymogen whereas peptides QNLGVLEETPEDR (m/z 750.36, +2) and YSVSENDLPESFDAR (m/z 864.90, +2; and Brindley(60) and Dalton et al.(59) proposed that in F. hepatica and S. mansoni an asparaginyl endopeptidase (otherwise known as legumain), which cleaves peptide bonds C-terminal to Asn residues, are involved in the trans-processing and activation of cathepsin L and B cysteine proteases.In support of this argument all members of these classes of cysteine protease in both helminth parasites possess an Asn residue in the vicinity of the prosegment/mature juncture(8,60).Furthermore, Sajid et al.(62) and Beckham et al.(57) provided experimental evidence by showing that a recombinant asparaginyl endopeptidase could trans-process and activate recombinant cathepsin B of F. hepatica and S.
18 % of total proteases detected.By using inhibitors of cathepsin-like proteases we have shown that excystment of the infective larvae are dependant on the cathepsin L3 and cathepsin B proteases.Asparaginyl endopeptidases are likely to be essential for the rapid trans-processing and activation of the stored zymogens of cathepsin L and B and, hence, it is probable that one of the first steps in activation of the dormant larvae is the switching on of the genes encoding these enzymes.A role for cathepsin L and B proteases in the penetration of the host by NEJ was recently demonstrated using RNA interference methods by McGonigle et al.(67).Knockdown of both cathepsin L and cathepsin B transcripts in NEJ parasites blocked the ability of the larvae to penetrate and traverse the intestinal wall of a rodent host.Therefore, the two types of cysteine proteases, working together, must be responsible for degrading the intestinal tissue macromolecules to enable the rapid penetration of the host intestine.Cysteine proteases are also essential for infectivity of the closely-related schistosome parasites (Schistosoma mansoni and Trichobilharzia regentii) which enter their hosts via the skin(68), and for the cyst emergence and infectivity of larvae of the trematode Paragonimus westermani(69)(70)(71).

Fasciola:
(a) a classical secretion of proteases and other molecules via the ER/Golgi from specialised gastrodermal cells of the gut as previously reported (78), and (b) a non-classical secretion of leaderless Fasciola antioxidants via a trans-tegumental route.This has clear implications for the development of novel anti-F.hepatica chemotherapy and could explain the additive effects of two of the most potent antiflukicides triclabendazole and clorsulon which act upon the tegument and gastrodermal cells, respectively (79-81).
future anthelmintic-or vaccine-development programs.It is interesting that several of the major components of Fasciola secretions identified here are currently leading candidates for development as first generation anti-fluke vaccines (including cathepsins, peroxiredoxin, glutathione S-transferase and fatty acid-binding proteins; 82) or are promising targets for novel flukicidal drugs (cathepsins; 83) demonstrating the value of our integrated approach for the future identification of new targets for therapeutic intervention.Project, Grant 352912.JPD is a recipient of a NSW Government BioFirst Award 2004.The bioinformatics analysis was partially supported by a grant from the Australian Research Council (ARC) (LP0667795).

Figure 1 .
Figure 1.Analysis of F. hepatica somatic and secretory proteins by 1-DE.

( 19 )
to detect conserved domains and motifs.1 MASCOT scores are given for matches to F. hepatica cDNAs; 2 number of unique peptide matches; 3 raw emPAI values provided by the MASCOT search engine give an approximate quantification of the protein(22).These values were converted to a percentage of total protease expression within each Fasciola developmental stage (see experimental procedures section) and were used to generate the pie charts shown in A; 4 section of the 1-D gel from which the peptides were identified;5 PEAKS scores (expressed as a percentage) are given for matches to peptides encoded by F. hepatica ESTs; 6 single peptide-based identification -see supplementary figure 4.
Transcriptome analysis for secretory proteins from 14031 Fasciola EST sequences, (16)EST2Secretome pipeline integrates a number of high quality programs for EST cleaning (SeqClean and RepeatMasker), assembly into contigs and singletons (CAP3), conceptual translation (ESTSCAN) and prediction of secreted proteins, based on the presence of N-terminal secretory signal sequences (SignalP), and follows with the elimination of membrane proteins using TMHMM.This approach has proven very reliable in identifying secretory proteins from a number of helminths including the bovine lungworm, Dictyocaulus viviparus(16), gastrointestinal worm, protein-protein interactions (comparison to IntAct), mapping to Caenorhabditis elegans proteins (from WormPep) and subsequent correlation to RNAi phenotype data.The SignalP threshold value for secretory signal peptide prediction was set at 0.5 (34)lar to the banding pattern of Fasciola larval somatic proteins reported by Tkalcevic et al.(34).Proteins in these bands were in-gel digested with trypsin and analysed by nanoLc-ESI-MS/MS and the resulting CID data was used for database searching.A total of 26 different Fasciola dormant larvae proteins were identified: 12 matched to previously identified Fasciola cDNAs (or cDNAs from related trematode species) and 14 corresponded to proteins encoded by novel ESTs (Figures2B-3B; Supplementary tables 2-3).A further five peptides encoded by Fasciola ESTs were also identified but these lacked conserved protein domains and could not be assigned putative functions based on BLAST searches.themedium,separated by 1-DE and analysed by nano-Lc-ESI-MS/MS (Figure1).Matches to 29 different proteins, of which ten were proteases including seven cathepsin L3 proteases, one cathepsin B, and two asparaginyl endopeptidaselike proteases were obtained from MS/MS data from Fasciola NEJ secreted proteins.
(8,20,21)s have previously characterised the major secretory proteins expressed by adult F. hepatica using 2-DE(8,20,21).To complement these earlier studies, and to validate the use of 1-DE for proteomic analysis, we analysed tryptic digests extracted from gel sections of adult F. hepatica secreted proteins (see Figure1) by mass spectrometry.Twenty-two different proteins secreted by adult F. hepatica were identified in this analysis: 19 matched to previously identified Fasciola cDNAs and 3 corresponded to putative proteins encoded by novel ESTs identified by our present EST2Secretome analysis but were unidentified by Robinson et al. (8) (Figures 2B-3B; Supplementary tables 2-3).enzymes, and the egg-shell vitelline protein B1.On the other hand, proteomic analysis detected four fatty acid-binding proteins (FaBP1, FaBP2, FaBP3 and Fh15) and two redox enzymes (peroxiredoxin and thioredoxin) that were not predicted by EST2Secretome analysis.A putative novel prolylcarboxypeptidase (also observed in 21-day old immature parasites, see below) was also identified by mass spectrometry and yet was not predicted to be a secretory peptide.This is surprising since at least one adult fluke EST (Fhep06a01.q1k)encoding the N-terminal end of the enzyme contains a putative signal peptide.However, this EST was not retained as a singleton, after repeat-masking and truncation, in the early stages of EST processing in EST2Secretome.Identification of F. hepatica dormant larval somatic proteins and NEJ secretory proteinsBy removing the outer cyst layer of the dormant metacercarial infective stage of the parasite we could extract somatic protein without contamination with extraneous proteins and analyse these by 1-DE.Approximately 14 protein bands could be visualised following Coomassie blue-stained SDS-PAGE (Figure1) which on inspection appeared very Of the 26 positively-matched Fasciola proteins six were proteases including, two cathepsin L3 proteases, three cathepsin B endopeptidases and an asparaginyl endopeptidase-like precursor (discussed in detail below).Others included structural proteins related to muscle function such as actin, myosin-regulatory light chain and a troponin C homologue (Supplementary tables 2-3).Four metabolic enzymes were also identified; pyruvate carboxylase (gluconeogenesis), malate dehydrogenase (TCA cycle) as well as aldolase and enolase (glycolysis).Mass spectrometry data also matched to a putative cullin protein (roles in protein degradation and ubiquitination), a cyclophilin-like peptidyl-prolyl cis-trans isomerase and a tetraspanin membrane protein.Other notable peptide matches included the Fasciola antioxidant enzymes peroxiredoxin, two histone proteins, a heat shock protein 70, dynein light chain, a conditions in the duodenum we could activate the dormant infective larvae and induce them to excyst.Proteins secreted by F. hepatica NEJs during in vitro culture were isolated from fructose-bisphosphate aldolase, phosphoenolpyruvate carboxykinase, glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase and an ATPase.Other matches included putative structural proteins such as calponin, spermadhesin and histones (H2A, H2B and H4), redox enzymes (thioredoxin and peptidyl-prolyl cis-trans isomerase) and an uncharacterised protein with predicted transmembrane regions.

Table 1 .
Summary analysis of 14031 adult F. hepatica ESTs using EST2Secretome.The contigs and singletons generated by pre-processing, overall representative ESTs (rESTs), peptides from conceptual translation and putative secretory proteins identified are shown.