Fasciola hepatica Gastrodermal Cells Selectively Release Extracellular Vesicles via a Novel Atypical Secretory Mechanism.

The liver fluke, Fasciola hepatica, is an obligate blood-feeder, and the gastrodermal cells of the parasite form the interface with the host's blood. Despite their importance in the host-parasite interaction, in-depth proteomic analysis of the gastrodermal cells is lacking. Here, we used laser microdissection of F. hepatica tissue sections to generate unique and biologically exclusive tissue fractions of the gastrodermal cells and tegument for analysis by mass spectrometry. A total of 226 gastrodermal cell proteins were identified, with proteases that degrade haemoglobin being the most abundant. Other detected proteins included those such as proton pumps and anticoagulants which maintain a microenvironment that facilitates digestion. By comparing the gastrodermal cell proteome and the 102 proteins identified in the laser microdissected tegument with previously published tegument proteomic datasets, we showed that one-quarter of proteins (removed by freeze-thaw extraction) or one-third of proteins (removed by detergent extraction) previously identified as tegumental were instead derived from the gastrodermal cells. Comparative analysis of the laser microdissected gastrodermal cells, tegument, and F. hepatica secretome revealed that the gastrodermal cells are the principal source of secreted proteins, as well as showed that both the gastrodermal cells and the tegument are likely to release subpopulations of extracellular vesicles (EVs). Microscopical examination of the gut caeca from flukes fixed immediately after their removal from the host bile ducts showed that selected gastrodermal cells underwent a progressive thinning of the apical plasma membrane which ruptured to release secretory vesicles en masse into the gut lumen. Our findings suggest that gut-derived EVs are released via a novel atypical secretory route and highlight the importance of the gastrodermal cells in nutrient acquisition and possible immunomodulation by the parasite.

Role of Fasciola hepatica Small RNAs in the Interaction With the Mammalian Host.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression being involved in many different biological processes and play a key role in developmental timing. Additionally, recent studies have shown that miRNAs released from parasites are capable of regulating the expression of host genes. In the present work, we studied the expression patterns of ncRNAs of various intra-mammalian life-cycle stages of the liver fluke, Fasciola hepatica, as well as those packaged into extracellular vesicles and shed by the adult fluke. The miRNA expression profile of the intra-mammalian stages shows important variations, despite a set of predominant miRNAs that are highly expressed across all stages. No substantial variations in miRNA expression between dormant and activated metacercariae were detected, suggesting that they might not be central players in regulating fluke gene expression during this crucial step in the invasion of the definitive host. We generated a curated pipeline for the prediction of putative target genes that reports only sites conserved between three different prediction approaches. This pipeline was tested against an iso-seq curated database of the 3' UTR regions of F. hepatica genes to detect miRNA regulation networks within liver fluke. Several functions related to the host immune response or modulation were enriched among the targets of the most highly expressed parasite miRNAs, stressing that they might be key players during the establishment and maintenance of infection. Additionally, we detected fragments derived from the processing of tRNAs, in all developmental stages analyzed, and documented the presence of novel long tRNA fragments enriched in vesicles. We confirmed the presence of at least 5 putative vault RNAs (vtRNAs), that are expressed across different stages and enriched in vesicles. The presence of tRNA fragments and vtRNAs in vesicles raise the possibility that they could be involved in the host-parasite interaction.

The cellular and molecular origins of extracellular vesicles released by the helminth pathogen, Fasciola hepatica.

Parasitic helminths secrete extracellular vesicles (EVs) which have potent immunomodulatory effects. Whilst the cargo of EVs has been characterised for many species, we know little about the mechanisms that govern their biogenesis and release. Using antibodies raised against a panel of Fasciola hepatica EV (FhEV) marker proteins, we have identified multiple sites of EV production in the parasite. Discrete immunofluorescence patterns were observed within the gastrodermal cells and tegumental syncytium for different marker proteins whilst the protonephridial (excretory) system and parenchymal-type 2 cells were identified as additional sites of production (or transit) of FhEVs. Ligation was used to mechanically block the oral sucker, excretory pore, or both, to determine the effect on FhEV release from live adult flukes in vitro. This revealed that FhEVs are predominately derived from the gut, whilst the tegument releases EVs to a lesser extent. The data also suggest that the protonephridial system contributes to the small (120 K) EV sub-population. Sphingomyelinase (SMase) activity is a key driver of EV biogenesis in mammalian cells and we have previously identified SMases in FhEVs by mass spectrometry. SMase activity associated with isolated FhEVs was susceptible to the chemical inhibitor GW4869 and treatment of adult flukes with GW4869 led to a significant reduction in 120 K EV release in vitro, suggesting that a ceramide-dependent mechanism could drive 120 K EV formation. In contrast, the release of the larger 15 K EVs was only moderately impacted, indicating that they form independently of SMase activity. Ultrastructural observation of GW4869-treated F. hepatica tissue showed severe disruption to the parenchyma and vacuolation of the tegument, gastrodermal cells and epithelial lining of the excretory ducts. This work establishes that targeted disruption of EV biogenesis and release in helminths is possible, and provides proof-of-concept for future studies investigating EV secretion as a target for parasite control.

Helminth genome analysis reveals conservation of extracellular vesicle biogenesis pathways but divergence of RNA loading machinery between phyla.

A comparative genomics approach was used to determine whether established extracellular vesicle biogenesis pathways are conserved in helminths. This revealed conservation of membrane and cytoskeletal organising proteins as well as the endosomal sorting complex required for transport previously described in mammalian cells. Domain level analysis of this complex in helminths, however, indicated that some species may rely on atypical proteins to support subunit interactions and cargo recruitment. Interestingly, helminths displayed phylum level divergence of proteins associated with loading RNA into extracellular vesicles. These findings provide a framework for functional studies of helminth extracellular vesicle biogenesis and cargo sorting.

Isolation of Secreted and Tegumental Surface Proteins from Fasciola hepatica.

Proteins secreted by, or displayed on the surface tegument of, trematodes have key functions in the host-parasite interaction. As such, they are often leading targets for diagnostic tests or vaccine candidates. Here we describe methods for the isolation and analysis of soluble secreted proteins (i.e., the secretome) released during in vitro culture of adult Fasciola hepatica. We also describe two methods for the enrichment of proteins displayed on the outer tegumental surface of F. hepatica. These approaches enable downstream identification of the isolated proteins by mass spectrometry-based proteomics.

Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells.

Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.

Schistosoma bovis-host interplay: Proteomics for knowing and acting.

Schistosoma bovis is a parasite of ruminants that causes significant economic losses to farmers throughout Africa, Southwestern Asia and the Mediterranean. Additionally, recent studies have reported its zoonotic potential through the formation of S. bovis×Schistosoma haematobium hybrids. As observed in the Schistosoma species infecting humans, it is assumed that S. bovis has also evolved host regulatory molecules that ensure its long-term survival in the bloodstream of its host. Since these molecules could be potential targets for the development of new drugs and anti-schistosome vaccines, their identification and functional characterization were undertaken. With this aim in mind, the molecular interface between S. bovis and its vertebrate host was subjected to a series of proteomic studies, which started with the analysis of the proteomes of the S. bovis moieties exposed to the host, namely, the excretory/secretory products and the tegument surface. Thus, a wealth of novel molecular information of S. bovis was obtained, which in turn allowed the identification of several parasite proteins with fibrinolytic and anticoagulant activities that could be used by S. bovis to regulate the host defensive systems. Following on, the host interface was investigated by studying the proteome of the host vascular endothelium surface at two points along the infection: in the lung vessels during the schistosomula migration and in the portal vein after the parasites have reached adulthood and sexual maturity. These studies have provided original data regarding the proteomes of the endothelial cell surface of pulmonary vasculature and portal vein in S. bovis-infected animals, and have shown significant changes in these proteomes associated with infection. This review compiles current information and the analyses of all the proteomic data from S. bovis and the S. bovis-host interface, including the molecular and functional characterization of S. bovis proteins that were found to participate in the regulation of the host coagulation and fibrinolysis systems.

Extracellular Vesicle Biogenesis in Helminths: More than One Route to the Surface?

The recent discovery that parasites release extracellular vesicles (EVs) that can transfer a range of effector molecules to host cells has made us re-think our understanding of the host-parasite interface. In this opinion article we consider how recent proteomics and transcriptomics studies, together with ultrastructural observations, suggest that more than one mechanism of EV biogenesis can occur in helminths. We propose that distinct EV subtypes have roles in immune modulation and repair of drug-induced damage, and put forward the case for targeting EV biogenesis pathways to achieve parasite control. In doing so we raise a number of outstanding research questions that must be addressed before this can happen.

The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis.

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.

Schistosome infections induce significant changes in the host biliary proteome.

Schistosomiasis is a disease caused by blood trematodes affecting man and animals that represents an important human health and veterinary problem. Main damages caused by this infection are a consequence of the host inflammatory reaction against the parasite eggs trapped inside the liver. Despite that the hepatic pathology of schistosomiasis is very well known, there are no specific studies dealing with the schistosome infection effects on the biliary function. The purpose of this work was to analyse the changes induced by Schistosoma bovis infection in the biliary proteome. For this, whole gallbladders from S. bovis-infected and non-infected mice were dissected, homogenized and fractionated by differential centrifugation. The resulting protein fractions were resolved by SDS-PAGE, the gels were sliced, and the gel pieces analysed by LC-MS/MS. Altogether, we identified 1937 proteins, which were classified according to their "protein class" and "molecular function", and then subjected to an "Enrichment analysis". The differences found in gallbladder proteomes between S. bovis-infected and non-infected mice are analysed. We show that chronic schistosome infections cause significant changes in the biliary proteome that may produce physiological alterations and affect the therapeutic actions of drugs when administered to human patients and animals with schistosomiasis. BIOLOGICAL SIGNIFICANCE: To identify the changes induced by the schistosome infection in bile protein composition, and therefore in bile function, we compared the proteome of the gallbladders collected from non-infected healthy mice and from mice infected with S. bovis during 4months. For this, gallbladders from both groups of mice were homogenized and these homogenates were fractionated by serial centrifugation and acrylamide gel electrophoresis. The proteins were in gel digested and analysed by LC-MS/MS for identification. The present work reports the first data on the proteome of the mouse gallbladder and provides a comprehensive catalogue of biliary proteins that may be of great use in other studies addressing biliary physiology and pathology. We observed that there were significant differences in the biliary proteome of mice infected and non-infected with S. bovis and that chronic schistosome infections may produce important physiological alterations in the bile function.

Proteomic mapping of the lung vascular endothelial cell surface in Schistosoma bovis-infected hamsters.

Schistosomes are blood trematodes that are perfectly adapted to living in their intravascular habitat and to achieve this they have developed mechanisms enabling them to evade the immune and haemostatic responses of the host and to regulate endothelial cell function to favour their own survival. The objective of this work was to analyse the changes induced by Schistosoma bovis schistosomula in the proteome expressed by infected hamsters, over 10 and 20 days, on the endothelial surface of their pulmonary vasculature. To accomplish this, we subjected the lungs of non-infected and S. bovis-infected hamsters to vascular perfusion with a biotin ester reactive. Analysis by liquid chromatography and tandem mass spectrometry analysis (LC-MS/MS) of endothelial surface proteins resulted in the identification of a total of 459 non-redundant proteins in the lung vasculature of infected and non-infected hamsters. Here we report the proteins identified, classified according to their biological function and cellular location, further analysing the differences in lung vascular proteomes between non-infected and S. bovis-infected hamsters for ten and twenty days. This work provides the first data on the vascular surface proteome of the lung after S. bovis infection and identifies some of the changes induced in it during infection. BIOLOGICAL SIGNIFICANCE: To identify the changes induced by schistosomula larvae of Schistosoma bovis in the proteome of the pulmonary vasculature of the host, we compared the proteins expressed on the vascular endothelium of the lungs of non-infected and infected hamsters over 10 and 20 days. Mass spectrometry analysis (LC-MS/MS) of the proteins isolated from the vascular endothelium resulted in the identification of a total of 459 non-redundant proteins in the lung of infected and non-infected hamsters. The proteins identified are classified according to their biological function and cellular location, further analysing the differences in lung vascular proteomes between non-infected and S. bovis-infected hamsters. This work provides the first data on the vascular surface proteome of the lung after S. bovis infection and identifies some of the changes induced in it during infection suggesting the possible involvement of these proteins during parasite infection.

In vivo intravascular biotinylation of Schistosoma bovis adult worms and proteomic analysis of tegumental surface proteins.

Schistosoma bovis is a blood-dwelling fluke of ruminants that lives for years inside the vasculature of their hosts. The parasite tegument covers the surface of the worms and plays a key role in the host-parasite relationship. The parasite molecules expressed at the tegument surface are potential targets for immune or drug intervention. The purpose of this work was the identification of the proteins expressed in vivo on the surface of the tegument of S. bovis adult worms. To accomplish this we used a method based on in vivo vascular perfusion of mice infected with S. bovis which allowed the labelling of the surface of the worms inside the blood vasculature. The biotinylation of parasite inside blood vessels prevents the handling of worms in vitro and hence possible damage to the tegument that could produce results that would be difficult to interpret. Trypsin digestion of biotinylated proteins and subsequent liquid chromatography and tandem mass spectrometry analysis (LC-MS/MS) resulted in the identification on the S. bovis tegument of 80 parasite proteins and 28 host proteins. The proteins identified were compared with the findings from other proteomic studies of the schistosome surface. The experimental approach used in this work is a reliable method for selective investigation of the surface of the worms and provides valuable information about the exposed protein repertoire of the tegument of S. bovis in the environmental conditions that the parasite faces inside the blood vessels. BIOLOGICAL SIGNIFICANCE: To identify the proteins expressed on the surface of the tegument of S. bovis adult worms we used a method based on in vivo vascular perfusion, with biotin, of mice infected with S. bovis which allowed the labelling of the surface of the worms inside the blood vasculature. This methodology prevents the handling of worms in vitro and hence possible damage to the tegument that could produce results that would be difficult to interpret. This work is the first in which vascular perfusion has been used to investigate, in vivo, the protein exposed by an intravascular pathogen on its surface to the host, and provides valuable information about the exposed protein repertoire of the tegument of S. bovis in the environmental conditions that the parasite faces inside the blood vessels.

Molecular cloning, characterization and diagnostic performance of the Schistosoma bovis 22.6 antigen.

Animal schistosomiasis caused by Schistosoma bovis is a veterinary problem in many areas of the world. It affects a large number of animals and causes important economic losses in livestock production. The 22.6 kDa antigen is a tegumental protein of unknown function, restricted to schistosomes. In S. bovis it has been identified in the tegument and in an excretion-secretion extract, consisting of several, non-glycosylated isoforms that are recognised by the sera of animals infected with S. bovis. The aims of the present work were to clone, sequence, express and characterize at molecular level the S. bovis 22.6 antigen (Sb22.6), as well as to assess the usefulness of the corresponding recombinant protein as a diagnostic antigen in ELISA tests for the detection of free-range cattle farms infested with S. bovis. Immunolocalization studies revealed that Sb22.6 is expressed in the tegument and some internal tissues of the adult worms, but it is not exposed on the surface of the adult worms and schistosomula. The reactivity of the recombinant Sb22.6 (rSb22.6) in ELISA against antibodies in sera from S. bovis experimentally infected hamsters and sera from free-range cattle from a S. bovis endemic area showed that the recombinant protein and the soluble extract of adult worms (SbC) exhibited a similar diagnostic performance. In addition, rSb22.6 did not show cross-reactions with antibodies against Fasciola hepatica, also a frequent trematode parasite in cattle. The rSb22.6 antigen can be readily produced in large amounts and in a highly reproducible fashion, avoiding the types of problem that arise upon using crude extracts such as the SbC. In conclusion, this protein represents a promising epidemiological tool for the surveillance of S. bovis and may help to implement control measures in the areas and farms were the parasite is present.

Proteomic identification of endothelial cell surface proteins isolated from the hepatic portal vein of mice infected with Schistosoma bovis.

Schistosomes are blood parasites adapted to their intravascular habitat that have evolved mechanisms to evade the immune and hemostatic responses of their hosts. It has been observed that the schistosome can regulate the endothelium function along the infection, which contributes to modulation of host defensive responses and parasite survival. The purpose of this work was the analysis of the changes induced by Schistosoma bovis adult worms in the proteome expressed by infected mice on the endothelial surface of their portal vein. With this aim, we have utilized a methodology that allows the purification, identification and relative quantification of endothelial cell surface proteins after their selective in vivo labeling with biotin. Trypsin digestion of the biotinylated proteins and subsequent liquid chromatography and tandem mass spectrometry analysis (LC-MS/MS) resulted in the identification of a total 127 non-redundant proteins. All these proteins have been classified according to their function and cellular location, and the differences between S. bovis-infected and non-infected mice in their endothelial surface proteomes have been analyzed. The present work provides the first data on the proteome of the endothelial surface of the portal vein, and identifies some of the changes induced in it after an infection by S. bovis.

Molecular and functional characterization of a Schistosoma bovis annexin: fibrinolytic and anticoagulant activity.

Annexins belong to an evolutionarily conserved multigene family of proteins expressed throughout the animal and plant kingdoms. Although they are soluble cytosolic proteins that lack signal sequences, they have also been detected in extracellular fluids and have been associated with cell surface membranes, where they could be involved in anti-haemostatic and anti-inflammatory functions. Schistosome annexins have been identified on the parasite's tegument surface and excretory/secretory products, but their functions are still unknown. Here we report the cloning, sequencing, in silico analysis, and functional characterization of a Schistosoma bovis annexin. The predicted protein has typical annexin secondary and tertiary structures. Bioassays with the recombinant protein revealed that the protein is biologically active in vitro, showing fibrinolytic and anticoagulant properties. Finally, the expression of the native protein on the tegument surface of S. bovis schistosomula and adult worms is demonstrated, revealing the possibility of exposure to the host's immune system and thus offering a potential vaccine target for the control of schistosomiasis in ruminants.

Cloning and characterization of a plasminogen-binding surface-associated enolase from Schistosoma bovis.

Schistosoma bovis is a ruminant parasite able to survive prolonged periods in the vasculature of its host without either being cleared by the host defensive systems or inducing thrombotic or coagulation disturbances. This suggests that the parasite modulates both the immune and haemostatic host responses. Previous studies have shown that host plasminogen binds to the surface of S. bovis adult worms, and that a tegument extract from S. bovis fixes and activates host plasminogen, generating plasmin, which in turn could both inhibit blood clotting and dissolve clots. Enolase has been identified among the tegumental proteins that bind plasminogen. The aim of the present study is to determine the physiological role of the enolase found in the tegument of S. bovis adult worms as regards plasminogen-binding and activation, and to confirm its surface exposure on the parasite. The study included the cloning and sequencing of S. bovis enolase cDNA, collection of the corresponding recombinant protein and evaluation of its plasminogen-binding and activation activity, and an exploration of the expression and localization of native enolase in adult worms and lung schistosomulae. Here we show that S. bovis male adult worms express enolase on their tegumental surface and that this protein binds host plasminogen and increases its activation in the presence of host tissue plasminogen activator (t-PA). This suggests that the surface-associated enolase found here is a physiological receptor of plasminogen that plays a role in the activation of the host fibrinolytic system, most probably to avoid blood clot formation on the worm's surface.