Extracellular Vesicles from Parasitic Helminths Contain Specific Excretory/Secretory Proteins and Are Internalized in Intestinal Host Cells

The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30–100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents.


Introduction
Helminth infections are among the most neglected tropical diseases [1]. Many species of helminths are parasitic multicellular organisms of medical and economic importance, as they infect humans and sometimes induce fatal diseases. Moreover, parasitic helminth infections in livestock are responsible for significant economic losses due to decreases in animal productivity and the cost of antihelminthic treatments of parasitized individuals [1]. Echinostomes are intestinal trematodes with no tissue phases in the definitive host. They are important parasitic flatworms invading domestic and wildlife animals and occasionally humans [2].
These flukes, particularly Echinostoma caproni (Trematoda: Echinostomatidae) have been used for years as experimental models in different areas of parasitology because they are able to parasitize a wide range of invertebrate and vertebrate hosts during their life cycle [3]. More specifically, our group has used E. caproni as a model for chronic and acute intestinal helminth infections depending on the rodent host used [3].
Fasciola hepatica is a parasite of the liver and bile ducts of human and non-human definitive hosts, such as livestock [2,4]. The definitive host, including humans, becomes infected after ingestion of contaminated vegetation. The parasite excysts in the small intestine and juvenile worms penetrate through the gut wall and enter the peritoneal cavity. After 10-12 weeks of tissue migration the parasites enter the bile ducts where they mature [5]. Although fascioliasis has been traditionally considered as a livestock disease, it is now recognized as an important emerging zoonotic human disease. It is estimated that between 2.4 and 17 million people are currently infected and 91 million are at risk of infection [2].
Although studies of host-parasite interactions have led to important discoveries related to the identification of potential new targets for diagnosis and treatment, as well as new vaccine targets for helminthiases [1,2], further studies are required to identify new and specific targets for effective control of these important diseases. In this context, interesting helminth target molecules consist mainly of those present at the external surface (cuticle in nematodes and tegument in trematodes) and the excretory/secretory products (ESP) [6]. We and others have studied the proteins present in ESP from different helminths, which exhibit a common pattern in all the species studied, where cytoskeletal proteins, nuclear proteins and glycolytic enzymes are the most abundant [2,6,7]. Recent studies have indicated that the secretomes of many helminth pathogens contain a variety of highly-abundant proteins that are homologs of damage-associated host molecules. Helminths could have evolved mechanisms similar to their host in order to prevent their elimination by humoral and cellular immune responses [8].
In multicellular organisms, cells communicate with each other via extracellular molecules, but also by releasing membrane vesicles into their extracellular environment that can affect the cells that encounter these structures in complex ways. When, in 1963, L.T. Threadgold first characterized the tegument of F. hepatica, he described a great number of small membrane-limited vesicles, possibly indicating the process whereby metabolites may be secreted [9]. Moreover, Andresen et al. (1989) also reported the existence of ''membrane bound vesicles'' in E. caproni [10]. Nevertheless, the research on secretion vesicles and their wider involvement in intra and extracellular signaling began to emerge in the last decade [11]. This interest is reflected by the increasing number of papers published in last years, and the increasing information provided in databases like Exocarta (http://exocarta. org) [12]. For recent reviews see [13][14][15][16].
Exosomes represent a specific subtype of secreted vesicles. They were originally identified as small (30-100 nm) membrane vesicles that are actively secreted by several types of cells. It has been demonstrated that exosomes contribute to functions such as tissue repair, neural communication, immunological response and the transfer of pathogenic proteins [11,14,16]. It has been shown that protozoan parasites also secrete exosomes, representing a broadbased mechanism of protein export which can contribute either to tolerance of an invader (by dampening immune response), or, conversely, to elicit pathologic inflammatory reactions in the host [17,18]. In contrast, although several approaches have been employed to study the biology of helminths and their interactions with the host, the secretion of these structures by parasitic helminths has been poorly studied. The demonstration of the existence of exosome-like vesicles in helminths may offer a new point of view for the study of helminth infections. In the present study, we demonstrate the secretion of exosome-like vesicles and their potential role in the communication between the parasites and the host in two trematodes, Echinostoma caproni and Fasciola hepatica. Parasites E. caproni adults were obtained from ICR mice (Mus musculus) after four weeks of experimental infections with 75 metacercariae/ each as previously described [19]. Animals were maintained under standard conditions with food and water ad libitum. The worm egg release was investigated daily in each infected animal, as described previously [20].

Ethics Statement
F. hepatica adults were obtained from cow livers in Mercavalencia S.A. slaughterhouse.

Excretory-secretory products (ESP)
To prepare ESP, adult worms from either E. caproni (collected from the intestines of experimentally-infected mice) or F. hepatica (collected from cow livers from local abattoirs) were thoroughly washed with PBS and maintained in RPMI-1640 culture medium containing 100 U penicillin and 100 mg/mL streptomycin (all from Sigma), at concentrations either of 10 worms/mL (E. caproni) or 2 worms/mL (F. hepatica) at 37uC for 5 h. In all experiments, adult parasites were alive after incubations.

Extracellular vesicle purification
Exosome-like vesicles were purified by differential centrifugation as suggested by Mathivanan et al., (2010) [13], using a protocol based on ultracentrifugation coupled to membrane filtration to eliminate large contaminating extracellular vesicles, and following validation by electron microscopy, as described for other systems [21,22]. 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 larger debris, and the resulting supernatant was centrifuged at 15,000 g for 45 min at 4uC. ESP supernatants were then filtered using an ultrafiltration membrane (0.2 mm; Schleicher & Schuell) and centrifuged at 120000 g/1 h at 4uC in an Optima TL100 tabletop ultracentrifuge (Beckman) using a TLA-55 rotor.

Electron microscopy: TEM, Immunochemistry-EM and SEM
For TEM, LR-white resin inclusion was performed fixing parasite adults and extracellular vesicles with glutaraldehyde 2.5%, washed with phosphate buffer 0.1 M pH 7.2, and post-fixed with 2% osmium tetroxide in phosphate buffer. After washes with water, they were sequentially dehydrated in 30% EtOH, 50% EtOH, 70% EtOH and 96% EtOH. Finally, samples were sequentially incubated for 2 h in 33% LR-white resin in 96% EtOH, 66% LR-white resin in 96% EtOH, 66% LR-white resin in 100% EtOH and 100% LR-white resin in 100 EtOH Samples were filtered in resin and polymerized at 60uC for 48 h. Ultrathin slides (60 nm) were finally stained with 2% uranil acetate prior to viewing by transmission EM (TEM) using a Jeol JEM1010 microscope at 60 kV. Images were acquired with a digital camera MegaView III with Olympus Image Analysis Software.
For the immuno-gold labeling assays with antibodies, purified exosome-like vesicles and E.caproni adults were fixed with Karnovsky's fixative and then processed in resin as previously described. Grids containing the samples were blocked with PBS/ 0.8% BSA/0.1% gelatin, and 2 mL of each antibody in PBS/0.5% BSA were added. Goat anti-enolase antibody (Santa Cruz Biotechnology); rabbit sera obtained against actin from Echinostoma caproni and rabbit sera obtained against Leucine aminopeptidase (LAP) from Fasciola hepatica [23] (kindly provided by Dr. C. Carmona, Universidad de la Republica, Montevideo, Uruguay) were used at dilution of 1/20. The grids were then washed with PBS/0.5% BSA, incubated with gold-labeled secondary antibodies (Donkey anti-goat coupled to 18 nm gold particles; donkey antirabbit coupled to 12 nm gold particles, all from Jackson Immunoresearch) in PBS/0.5% BSA for 30 min, and then washed in 100 mL drops of PBS/0.5% BSA. Control grids incubated with only secondary antibodies were also used. The grids were stained with 2% uranyl acetate and then viewed for TEM using a Jeol JEM1010 microscope at 60 kV and images were acquired with a digital camera MegaView III with Olympus Image Analysis Software.
For SEM adult worms were fixed in Karnovsky's fixative, washed in buffer and post-fixed for 1-2 h with 2% osmium tetroxide in 0.1 M sodium phosphate buffer pH 7.2, and dehydrated by critical point. The mounted specimens were coated with Gold/Paladium and examined in a Hitachi S4100 scanning electron microscope at 5 kV.
Exosome-like vesicles membrane staining Extracellular vesicles isolated as described before were labeled with FM4-64 stain (Molecular Probes Inc.) according to the manufacturer's protocol. Briefly, purified vesicles from 6 mL of ESP were resuspended in 400 mL of PBS and mixed gently with FM4-64 (added to a final concentration of 5 mM) for 10 min on ice. The samples were then washed by ultracentrifugation at 120,000 g to remove the excess dye and resuspended in 200 mL of PBS. Labeling of vesicles was confirmed by confocal light microscopy in an Olympus FV1000 microscope. Optical excitation was carried out with a 488 nm argon laser beam and fluorescence emission was detected in the range of 655-698 nm.

Culture assays with labeled exosome-like vesicles and confocal microscopy
Rat intestinal epithelial cancer (IEC-18) cells (ATCC nu CRL-1589, kindly provided by Dr. Duñ ach, Universitat Autonoma de Barcelona, Spain) were routinely grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum until 80% confluence. IEC-18 cells were grown in 800 mL of DMEM media in m-slide chambers (ibiTreat, Ibidi GmbH) at 37uC and 5% CO 2 . Prior to assays, cells were starved in DMEM without FBS for 4 h.
We performed duplicate assays with 10 and 100 mL of FM4-64 labeled vesicles per well, and keeping the cultured cells in a chamber at 37uC coupled to an Olympus FV1000 laser scanning confocal microscope for the times indicated.
As a control, IEC-18 cells were grown in several m-slide chambers, starved in DMEM without FBS for 4 h, cooled to 4uC and incubated with 10 and100 mL of FM4-64 labeled vesicles per well, keeping the cultured cells at 4uC. Images were recorded at 0, 30, 60, 90 and 120 minutes.
Images were obtained using the Olympus FV10-ASW software v 02.01.03.10 (Olympus Corporation) and Olympus UPLSAPO 606 oil objective. Optical excitation was carried out with a 488 nm argon laser beam and fluorescence emission was detected in the range of 655-698 nm.
Five microliters of the resulting suspension were delivered to a trap column (LC Packings Amsterdam, C18 PepMap100, 5 mm; 300 mm65 mm) using capillary HPLC (Switchos, LC Packings) via an isocratic flow of mobile phase (0.1% TFA in water) at a rate of 30 mL/min for 3 min. The flow rate was then switched to 300 nL/min, and the peptides were flushed into the analytical column (LC Packings C18 PepMap100, 3 mm, 100 Å , 75 mm615 cm) and eluted via a mobile phase gradient: 2-30% of B buffer in A buffer (A buffer: 0.1% formic acid in water, B buffer: 0.1% formic acid in 95% acetonitrile) over 120 min. The elution was directly applied to a nanospray source of a QSTAR XL instrument (Applied Biosystems). The QSTAR XL was operated in information-dependent acquisition mode, in which a 1-s TOF MS scan from 400-2000 m/z was performed, followed by 3-s product ion scans from 65-2000 m/z on the three most intense doubly or triply charged ions.

Database search
Database search on NCBInr databases was performed using both the MASCOT (Matrix-Science) search engine and the ProteinPilot software v3.0.1 (Applied Biosystems) for metazoan organisms, as previously described [24]. Basically, MASCOT searches allowed one missed cleavage and a tolerance on the mass measurement of 100 ppm in MS mode and 0.5 Da for MS/MS ions. We used carbamidomethylation of Cys as a fixed modification and oxidation of Met and deamidation of Asn and Gln as variable modifications [24]. With the ProteinPilot software, the default parameters for Paragon algorithm [25] were used.

Results
The parasitic trematodes Echinostoma caproni and Fasciola hepatica produce exosome-like vesicles Scanning electron microscopy (SEM) assays with Echinostoma caproni adults showed the presence of microvesicles on the surface of their tegument. The external surface of E. caproni adults is evaginated into vesicular-shaped bodies with a size ranging from 30-100 nm, which is in the range of exosome-like structures (Fig. 1A, B).
Transmission electron microscopy (TEM) assays confirmed the presence of different microvesicular structures on the E. caproni adult tegument (Fig. 2). They appeared as typical spherical structures released into the media ( Fig. 2A, B, C), as previously described in E. caproni and Fasciola hepatica [9,10]. These vesicles are apparently nipped off into the environment. We also detected these vesicles in membranous multivesicular bodies similar to exosome structures (Fig. 2 D).
To ascertain whether these exosome-like vesicles were also produced by other trematodes and to explore their nature, we next carried out classical purification assays of these structures from E. caproni and F. hepatica ESPs, and used TEM to visualize them. As shown in figure 3, abundant round-shaped material with the expected size of exosomes was obtained after ultracentrifugation from both E. caproni (Fig. 3A) and F. hepatica (Fig. 3B) ESPs, confirming their presence in the excreted/secreted material by both parasitic trematodes.
Helminth parasite exosome-like vesicles contain typical excretory/secretory proteins We next carried out two complementary approaches (proteomics and immune-TEM) to identify the proteins present in purified extracellular vesicles from E. caproni and F. hepatica.
Exosome-like vesicle preparations, which were previously confirmed by TEM, were subjected to proteomic analysis. Purified vesicles were digested with trypsin and no proteins were detected by MS/MS (data not shown). In order to release the protein content, purified vesicles were precipitated with CHCl 3 /MeOH and then digested with trypsin. Protein identification using MASCOT from three different experiments confirmed the presence of 45 different proteins in exosome-like vesicles from E. caproni ESP (Table S1). Application of the ProteinPilot software allowed for the identification of 6 additional proteins (Table S2). Most of the identified proteins had been found in ESP in previous studies, including cytoskeletal proteins (i.e. actin, tubulin, myosin, paramyosin, tropomyosin), glycolytic enzymes (i.e. enolase, aldolase, GAPDH, PEPCK), calcium-binding proteins (i.e. calmodulin, calponin), nuclear proteins (histones and elongation factors), as well as stress-related proteins (i.e. HSPs) and detoxifying enzymes like peroxiredoxins [2,19,26]. In addition to parasite proteins, the vesicles from E. caproni adults also contained host-derived proteins. In E. caproni exosome-like vesicles, MASCOT searches produced  the identification of 36 different host proteins in these vesicles (Table S1). The identified mice proteins mainly corresponded to immunoglobulins, histones, partial sequences of mucins and metabolic enzymes (Table S1).
The exosome proteome of E. caproni accounted for 54% (Table  S3) of the proteins currently identified in the secretome [19,27].
In the case of F. hepatica vesicles, we could identify 79 different proteins (Table S4). F. hepatica exosome-like vesicles exhibited qualitative differences in composition, when compared to E. caproni vesicles, including a higher number of proteases (like cathepsins and leucine aminopeptidase, LAP), as well as detoxifying enzymes (i.e. heat-shock proteins, and fatty-acid binding proteins), all of them also previously described as being present in F. hepatica ESP [6,[28][29][30]. The proteins identified in these vesicles accounted for the 52% of the secretome (Table S5).
The high protein overlap between the proteins in the secretome and the exosomes suggests that these vesicles are the primary mechanism of protein release from these trematodes similarly to what has been described for Leishmania [31].
In F. hepatica vesicles, we identified 19 host proteins including immunoglobulins, metabolic enzymes and typical exosomal molecules like CD19 (Table S4).
We further confirmed the presence of some of the most abundant proteins (actin, enolase, and LAP) in purified vesicles by immunohistochemistry and TEM using antibodies against some of the abundant molecules [26,28]. As shown in figure 4, goldlabeling was detected in E. caproni vesicles when using antibodies specific for enolase (Fig. 4B) and actin (Fig. 4C), and no label was detected when incubating with pre-immune antibodies, confirming the specific detection of these molecules in the exosome-like vesicles (Fig. 4A). Gold-labeled antibodies against F. hepatica LAP [23] clearly detected the presence of this protease in F. hepatica vesicles (Fig. 4D), confirming its identification by proteomics. No label was detected when F. hepatica exosome-like vesicles were incubated with mock antibodies (data not shown).
To investigate the origin of these vesicles, we next performed immunohistochemistry and TEM of E. caproni and F. hepatica adults with antibodies against actin and LAP, respectively (Fig. 5).These assays revealed gold labeled vesicles in E. caproni tegument suggesting the process whereby actin molecules may be secreted (Fig. 5A, B). Labeling was detected in vesicles that were detached from the surface (Fig. 5B). Similar results were obtained in F. hepatica tegument when using anti-LAP antibodies (data not shown). We conclude that exosome-like vesicles are already formed in the tegument of these trematodes.

Echinostoma caproni exosome-like vesicles are internalized by rat intestinal epithelial cells in culture
Once confirmed the existence and composition of E. caproni exosome-like vesicles, we next performed functional assays to ascertain their role in host-parasite interactions. We analyzed whether these structures could interact with host cells and be internalized in cultured cells. Intestinal rat cell (IEC-18) cultures were incubated with FM4-64-labeled E. caproni vesicles for 2 h. Images were taken at the indicated time points by time-lapse confocal fluorescence microscopy (Fig. 6). Upon addition of labeled vesicles to the culture media at 37uC, fluorescence was observed outside the cells (data not shown). As observed in figure 6, after only 30 min, fluorescence was observed at the cell surface and the intracellular fluorescence increased markedly during the time course. At 4uC, the fluorescence was observed outside the cells at the different times analyzed (data not shown). These results strongly suggest that the parasite extracellular vesicles are taken up by the rat cells in a time-dependent and metabolically-dependent manner, most likely by endocytosis, confirming interspecific communications mediated by exocytic vesicles.

Discussion
In this study, we report the active release of microvesicles exhibiting the morphological and biochemical characteristics of typical exosomes by the parasitic trematodes E. caproni and F. hepatica. The present study constitutes the first report of the existence and composition of exosome-like vesicles in parasitic helminths, since functional exosomes in helminths only had been previously described in the free-living nematode Caenorhabditis elegans [32]. Furthermore, our results indicate that these vesicles may play an essential role in host-parasite communication since they are actively taken up by intestinal rat cells. Our results confirm the ultrastructure of the tegument of F. hepatica described by Threadgold [9] in which describes ''a great number of small membrane-limited vacuoles and vesicles''. Interestingly, Andresen et al. [10] also reported the existence of ''membrane bound vesicles'' containing surface antigens in the adult worms of E. caproni that may well correspond to the structures described herein (Fig. 2). Remarkably, we found multivesicular bodies near the surface containing typical spherical exosome-like vesicles (Fig. 2D), exhibiting the typical morphology of exosome vesicles [13][14][15][16].
Most of the proteins we identified in the extracellular vesicles correspond to those described previously in the secretome of E. caproni, F. hepatica and other parasitic trematodes [2,19,29,30]. In fact, more than a half of the molecules identified in the secretome of E. caproni and F. hepatica are found in these exosome-like vesicles. This suggests that extracellular vesicles constitute an important mechanism for protein export in trematodes, as suggested for the parasitic protozoa Leishmania [31]. Furthermore, this mechanism may explain the presence of atypical proteins lacking classical secretion signal peptides, like enolase, in the helminth ESP [32][33][34][35][36][37][38]. A very recent study has confirmed the presence of enolase in the outer membrane vesicles from spirochaetae as responsible for fixing enymes involved in external proteolysis in the pericellular environment, having roles in nutrition and in enhancing dissemination [39]. Future studies should confirm the role of secreted helminth enolases in the host-parasite interaction.
Interestingly, all the molecules identified herein are among the most abundant exosome proteins identified in other organisms (see Exocarta.org; [12]). Among the proteins identified in exosomes, some are considered as damage-associated molecular pattern molecule (DAMP) homologs, which lack typical secretion signals and exit the cell by non-classical secretory pathways [8,40]. We now confirm that some of these DAMP homologues (i.e. annexins and heat shock proteins) are actively secreted in trematodes. An interesting DAMP homologue found in both E. caproni and F. hepatica parasites was thioredoxin peroxidase (peroxiredoxin). This molecule is also considered as a pathogen-associated molecular pattern molecule (PAMP) [40], able to modulate immune cells [8,41], thus constituting a promising target for pharmacological interventions [8].
One of the most striking features in our study is the evidence that extracellular vesicles may play an important role in the communication between the parasite and the host. This is based on: (i) we have shown that parasite vesicle proteins are species-specific; (ii) we have identified some host proteins in the parasite exosome-like vesicles; and (iii) parasite vesicles are incorporated into live intestinal cells. Although some common proteins were identified in the exosome-like vesicles released from E. caproni and F. hepatica, particularly structural and metabolic enzymes, our results clearly indicate that they are species-specific, since major differences were detected between both trematodes. For example, vesicles released from F. hepatica were enriched in stress-related proteins and proteases. The presence of proteases, such as cathepsins and aminopeptidases, in the ESP of F. hepatica has been reported previously and implicated in tissue migration of the juvenile worms from the intestine to the definitive microhabitat in the bile ducts [42,43]. Similarly, in the migrating parasitic protozoa Trypanosoma brucei, the presence of cathepsin L-like in 50-to 100 nm vesicles budding from the coated plasma membrane has also been demonstrated [44]. In contrast, in extracellular vesicles of E. caproni, which is an intestinal trematode with no tissue phases within the host, no proteases were identified. This suggests that biological characteristics of the parasite species determine the protein composition of the vesicles and their involvement in the success of the infection.
The identification of host proteins in purified parasite exosomelike vesicles clearly implicates these structures in host-parasite communication processes. The host proteins identified also appear to be species-specific, corresponding to those highly produced by hosts in response to helminth infection in each case (i.e. mucin-2 was only identified in E. caproni vesicles, whereas CD19 was only found in F. hepatica ones). Recent studies have also identified host proteins like IgA heavy chain constant region in secretion preparations of Fasciola hepatica [30]. The role of these host proteins in parasitic vesicles warrants further investigation. Furthermore, we show that labeled parasite extracellular vesicles are incorporated into intestinal cells in culture, suggesting a role for these structures in host-parasite communication. Other examples of the involvement of extracellular vesicles in hostpathogen communication include HIV [45,46], spirochaetes like Borrelia burgdorferi [39], fungi like Cryptococcus neoformans [47,48] or Malasezia sympodialis [49], and protozoans like Trypanosoma brucei [50] or Leishmania spp. [31]. In some cases, exosomes have been implicated in modulating the host response to those pathogens [31,47,49]. Studies are needed to confirm this role for exosomes in parasitic helminths, but would support previous studies suggesting an important role for helminth ESP in modulating the host immune response [6].
Recent studies have demonstrated that exosomes can be internalized by endocytosis [51]. We believe it could be also the case for helminth vesicles, which could be internalized either by an unspecific endocytic pathway, with no ligand-receptor involved, or by specific mechanisms requiring unidentified proteins that would act as a ligand for receptor recognition. Currently, we have not identified proteins at the exosome-like vesicle surface, and more studies are required to address this question.
In summary, although the secretion of exosome-like vesicles has been demonstrated in several organisms, we have shown the production of these structures by parasitic helminths for the first time. These structures have been isolated and purified following specific and validated protocols used for a wide range of organisms and systems [13,17,21,22,[44][45][46][47][48][49].
Moreover, we reveal the involvement of these structures in the communication with the host, suggesting an important role for exosome-like vesicles in the establishment of the infection. In this context, further studies will address whether these vesicles constitute good targets for new control strategies in parasitic diseases, implementing new vaccines, diagnostic and treatment tools.