Transcriptomic and proteomic profiling of peptidase expression in Fasciola hepatica eggs developing at host's body temperature.

Fasciola hepatica is a global parasite of livestock which also causes a neglected zoonosis in humans. The parasite's communication with the host during its complicated lifecycle is based on an ingenious enzymatic apparatus which includes a variety of peptidases. These enzymes are implicated in parasite migration, pathogenesis of the disease, and modification of host immune response. Although the dynamics of proteolytic machinery produced by intra-mammalian F. hepatica life stages has been previously investigated in great detail, peptidases of the eggs so far received little scientific attention. In this study, we performed a comparative RNA-seq analysis aimed at identification of peptidases expressed in F. hepatica eggs, cultured at 37 °C to represent gall bladder retained eggs, for different time periods and employed mass spectrometry in order to identify and quantify peptidases translated in F. hepatica egg lysates. We demonstrated that F. hepatica eggs undergo significant molecular changes when cultured at the physiological temperature of the definitive host. Egg transcriptome is subject to numerous subtle changes while their proteome is even more variable. The peptidase profile is considerably modified on both transcriptome and proteome level. Finally, we measured and classified proteolytic activities in extracts from F. hepatica eggs using a library of fluorogenic substrates and peptidase class-selective inhibitors. Activities of threonine peptidases were detected constantly, while the cysteine peptidases prevailing in freshly laid eggs are substituted by aspartic peptidase and metallopeptidase activities in the later stages of egg development.

Molecular characterization of a novel GSTO2 of Fasciola hepatica and its roles in modulating murine macrophages.

Fascioliasis is an important zoonotic helminthic disease caused by Fasciola hepatica and poses a serious threat to global public health. To evade the immune response of its host (humans or animals), F. hepatica secretes various antioxidant enzymes such as glutathione transferase (GST) to facilitate its invasion, migration and parasitism in vivo. To investigate the biological functions of a novel omega-class GST (GSTO), the molecular features of GSTO2 of F. hepatica were analyzed by online software, and the biochemical properties in vitro of recombinant GSTO2 (rGSTO2) were dissected. Then, the regulatory roles of rGSTO2 protein in murine macrophages in vitro were further explored. The results revealed that the GSTO2 gene encodes 254 amino acids, which harbor the characteristic N-terminal domain (ßαßαßßα) and C-terminal domain (α-helical) of the cytoplasmic GST superfamily. GSTO2 was mainly expressed in F. hepatica vitelline follicles, intestinal tract, excretory pores and vitelline cells, with thioltransferase and dehydroascorbate reductase activities. Moreover, rGSTO2 protein could be taken up by murine macrophages and significantly inhibit the viability of macrophages. In addition, rGSTO2 protein could significantly promote apoptosis and modulate the expression of cytokines in macrophages. These findings suggested that F. hepatica GSTO2 plays an important role in modulating the physiological functions of macrophages, whereby this protein might be involved in immunomodulatory and anti-inflammatory roles during infection. This study provided new insights into the immune-evasion mechanism of F. hepatica and may contribute to the development of a potential anti-inflammatory agent.

Title: Caractérisation moléculaire d'une nouvelle GSTO2 de Fasciola hepatica et ses rôles dans la modulation des macrophages murins. Abstract: La fasciolase est une importante maladie helminthique zoonotique causée par Fasciola hepatica, qui constitue une menace sérieuse pour la santé publique mondiale. Pour échapper à la réponse immunitaire de son hôte (humain ou animal), F. hepatica sécrète diverses enzymes antioxydantes telles que la glutathion transférase (GST) pour faciliter son invasion, sa migration et son parasitisme in vivo. Pour étudier les fonctions biologiques d'une nouvelle GST de classe oméga (GSTO), les caractéristiques moléculaires de la GSTO2 de F. hepatica ont été analysées par un logiciel en ligne et les propriétés biochimiques in vitro de sa protéine recombinante (rGSTO2) ont été disséquées. Ensuite, les rôles régulateurs de la protéine rGSTO2 sur les macrophages murins in vitro ont été explorés plus avant. Les résultats ont révélé que le gène GSTO2 code pour 254 acides aminés, qui abritent le domaine N-terminal caractéristique (ßαßαßßα) et le domaine C-terminal (α-hélicoïdal) de la superfamille GST cytoplasmique. Chez F. hepatica, GSTO2 était principalement exprimée dans les follicules vitellins, le tractus intestinal, les pores excréteurs et les cellules vitellines, avec des activités de thioltransférase et de déhydroascorbate réductase. De plus, la protéine rGSTO2 a pu être absorbée par les macrophages murins et inhiber de manière significative la viabilité des macrophages. Enfin, la protéine rGSTO2 a pu favoriser de manière significative l'apoptose et moduler l'expression des cytokines dans les macrophages. Ces résultats suggèrent que la GSTO2 de F. hepatica joue un rôle important dans la modulation des fonctions physiologiques des macrophages, cette protéine pouvant être impliquée dans des rôles immunomodulateurs et anti-inflammatoires au cours de l'infection. Cette étude a fourni de nouvelles informations sur le mécanisme d'évasion immunitaire de F. hepatica et pourrait contribuer au développement d'un agent anti-inflammatoire potentiel.

Molecular characterization and phylogenetic analysis of Fasciola hepatica from high- plateau and steppe areas in Algeria.

A previous study based on mitochondrial DNA markers reported the presence of Fasciola hepatica in Algeria. However, a precise species identification is still required. In this report, a total of 68 Fasciola isolates, collected from high-plateau (Bordj-Bou-Arreridj) and steppe (Djelfa) areas of Algeria, were identified at the species level by multiplex PCR and PCR-restriction fragment length polymorphism (RFLP) for nuclear phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold), respectively. The result of the multiplex PCR conflicted with that of the PCR-RFLP; however, subsequent nucleotide sequencing of pepck clearly showed that all isolates should be classified as F. hepatica. The two mitochondrial markers, NADH dehydrogenase subunit I (nad1) and cytochrome c oxidase subunit 1 (cox1), revealed a close relationship between the parasite populations from the plateau and those from the steppe. A dispersal direction from the high plateau to the steppe was indicated because the former population was more diversified than the latter. Moreover, these populations were more closely related to populations from Spain than those from Egypt or Afghanistan. Given the population characteristic of F. hepatica in Spain and the history of cattle trade, it seems likely that the parasite was introduced to Algeria from Europe through a route across the Mediterranean Sea.

Regulation of the Fasciola hepatica newly excysted juvenile cathepsin L3 (FhCL3) by its propeptide: a proposed 'clamp-like' mechanism of binding and inhibition.

BACKGROUND: The zoonotic worm parasite Fasciola hepatica secretes an abundance of cathepsin L peptidases that are associated with virulence, invasiveness, feeding and migration. The peptidases are produced as inactive zymogens that activate at low pH by autocatalytic removal of their N-terminal pro-domain or propeptide. Propeptides bind to their cognate enzyme with high specificity. Little is known, however, about the mechanism by which the propeptide of FhCL3, a cathepsin L peptidase secreted by the infective newly excysted juveniles (NEJs), regulates the inhibition and activation of the mature enzyme before it is secreted into host tissues. RESULTS: Immunolocalisation/immunoblotting studies show that the FhCL3 zymogen is produced and secreted by gastrodermal cells of the NEJs gut. A recombinant propeptide of FhCL3 (ppFhCL3) was shown to be a highly potent and selective inhibitor of native and recombinant F. hepatica FhCL3 peptidase, and other members of the cathepsin L family; inhibition constant (Ki) values obtained for FhCL1, FhCL2 and FhCL3 were 0.04 nM, 0.004 nM and < 0.002 nM, respectively. These values are at least 1000-fold lower than those Ki obtained for human cathepsin L (HsCL) and human cathepsin K (HsCK) demonstrating the selectivity of the ppFhCL3 for parasite cathepsins L. By exploiting 3-D structural data we identified key molecular interactions in the specific binding between the ppFhCL3 and FhCL3 mature domain. Using recombinant variants of ppFhCL3 we demonstrated the critical importance of a pair of propeptide residues (Tyr46Lys47) for the interaction with the propeptide binding loop (PBL) of the mature enzyme and other residues (Leu66 and Glu68) that allow the propeptide to block the active site. CONCLUSIONS: The FhCL3 peptidase involved in host invasion by F. hepatica is produced as a zymogen in the NEJs gut. Regulation of its activation involves specific binding sites within the propeptide that are interdependent and act as a "clamp-like" mechanism of inhibition. These interactions are disrupted by the low pH of the NEJs gut to initiate autocatalytic activation. Our enzyme kinetics data demonstrates high potency and selectivity of the ppFhCL3 for its cognate FhCL3 enzyme, information that could be utilised to design inhibitors of parasite cathepsin L peptidases.

Serpins in Fasciola hepatica: insights into host-parasite interactions.

Protease inhibitors play crucial roles in parasite development and survival, modulating the immune responses of their vertebrate hosts. Members of the serpin family are irreversible inhibitors of serine proteases and regulate systems related to defence against parasites. Limited information is currently available on protease inhibitors from the liver fluke Fasciola hepatica. In this study, we characterised four serpins from F. hepatica (FhS-1-FhS-4). Biochemical characterisation revealed that recombinant FhS-2 (rFhS) inhibits the activity of human neutrophil cathepsin G, while rFhS-4 inhibits the activity of bovine pancreatic chymotrypsin and cathepsin G. Consistent with inhibitor function profiling data, rFhS-4 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner.Similar to other serpins, rFhS2 and rFhS-4 bind to heparin with high affinity. Tissue localisation demonstrated that these serpins have different spatial distributions. FhS-2 is localised in the ovary, while FhS-4 was found in gut cells. Both of them co-localised in the spines within the tegument. These findings provide the basis for study of functional roles of these proteins as part of an immune evasion mechanism in the adult fluke, and in protection of eggs to ensure parasite life cycle continuity. Further understanding of serpins from the liver fluke may lead to the discovery of novel anti-parasitic interventions.

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.

Novel and selective inactivators of Triosephosphate isomerase with anti-trematode activity.

Trematode infections such as schistosomiasis and fascioliasis cause significant morbidity in an estimated 250 million people worldwide and the associated agricultural losses are estimated at more than US$ 6 billion per year. Current chemotherapy is limited. Triosephosphate isomerase (TIM), an enzyme of the glycolytic pathway, has emerged as a useful drug target in many parasites, including Fasciola hepatica TIM (FhTIM). We identified 21 novel compounds that selectively inhibit this enzyme. Using microscale thermophoresis we explored the interaction between target and compounds and identified a potent interaction between the sulfonyl-1,2,4-thiadiazole (compound 187) and FhTIM, which showed an IC50 of 5 µM and a Kd of 66 nM. In only 4 hours, this compound killed the juvenile form of F. hepatica with an IC50 of 3 µM, better than the reference drug triclabendazole (TCZ). Interestingly, we discovered in vitro inhibition of FhTIM by TCZ, with an IC50 of 7 µM suggesting a previously uncharacterized role of FhTIM in the mechanism of action of this drug. Compound 187 was also active against various developmental stages of Schistosoma mansoni. The low toxicity in vitro in different cell types and lack of acute toxicity in mice was demonstrated for this compound, as was demonstrated the efficacy of 187 in vivo in F. hepatica infected mice. Finally, we obtained the first crystal structure of FhTIM at 1.9 Å resolution which allows us using docking to suggest a mechanism of interaction between compound 187 and TIM. In conclusion, we describe a promising drug candidate to control neglected trematode infections in human and animal health.

Reference gene analysis and its use for kinase expression profiling in Fasciola hepatica.

The liver fluke Fasciola hepatica causes fasciolosis, a foodborne zoonosis affecting humans and livestock worldwide. A reliable quantification of gene expression in all parasite life stages relevant for targeting by anthelmintics in the mammalian host is fundamental. The aim of this study was to define a set of stably expressed reference genes for qRT-PCR in Fasciola studies. We determined the expression stabilities of eight candidate reference genes by the algorithms NormFinder, geNorm, BestKeeper, and comparative ΔCT method. The most stably expressed reference genes for the comparison of intra-mammalian life stages were glutamyl-prolyl-tRNA synthetase (Fheprs) and tubulin-specific chaperone D (Fhtbcd). The two best reference genes for analysis of in vitro-cultured juveniles were Fhtbcd and proteasome subunit beta type-7 (Fhpsmb7). These genes should replace the housekeeping gene gapdh which is used in most Fasciola studies to date, but in fact was differentially expressed in our analysis. Based on the new reference genes, we quantified expression of five kinases (Abl1, Abl2, PKC, Akt1, Plk1) discussed as targets in other parasitic flatworms. Distinct expression patterns throughout development were revealed and point to interesting biological functions. We like to motivate using this set of validated reference genes for future F. hepatica research, such as studies on drug targets or parasite development.

The failure of a DNA prime/protein boost regime and CTLA-4 mediated targeting to improve the potency of a DNA vaccine encoding Fasciola hepatica phosphoglycerate kinase in sheep.

DNA vaccination in large animals has often been associated with poor immunogenicity, consequently several approaches have been evaluated to enhance its efficacy. Here, we tested a cDNA encoding a phosphoglycerate kinase from Fasciola hepatica (cDNA-FhPGK/pCMV) as a vaccine against ovine fasciolosis and investigated whether a DNA prime/protein boost regime or CTLA-4 (cytotoxic lymphocyte antigen 4) mediated targeting improved DNA vaccine efficacy. No statistically significant differences in the cellular responses were seen in either vaccine trial when compared with the respective control groups. However, specific antibody responses were considerably enhanced in DNA primed/protein boosted sheep, but not among CTLA-4 targeted cDNA-FhPGK/pCMV vaccinated animals. Nevertheless, increased titers of specific IgG1 did not contribute to protection against infection, with no differences in liver fluke recoveries reported. If DNA vaccines against fasciolosis in target species are to reach the market one day, more research in this area is needed.

Cathepsin L Inhibitors with Activity against the Liver Fluke Identified From a Focus Library of Quinoxaline 1,4-di-N-Oxide Derivatives.

Infections caused by Fasciola species are widely distributed in cattle and sheep causing significant economic losses, and are emerging as human zoonosis with increasing reports of human cases, especially in children in endemic areas. The current treatment is chemotherapeutic, triclabendazole being the drug of preference since it is active against all parasite stages. Due to the emergence of resistance in several countries, the discovery of new chemical entities with fasciolicidal activity is urgently needed. In our continuous search for new fasciolicide compounds, we identified and characterized six quinoxaline 1,4-di-N-oxide derivatives from our in-house library. We selected them from a screening of novel inhibitors against FhCL1 and FhCL3 proteases, two essential enzymes secreted by juvenile and adult flukes. We report compounds C7, C17, C18, C19, C23, and C24 with an IC50 of less than 10 µM in at least one cathepsin. We studied their binding kinetics in vitro and their enzyme-ligand interactions in silico by molecular docking and molecular dynamic (MD) simulations. These compounds readily kill newly excysted juveniles in vitro and have low cytotoxicity in a Hep-G2 cell line and bovine spermatozoa. Our findings are valuable for the development of new chemotherapeutic approaches against fascioliasis, and other pathologies involving cysteine proteases.

X-ray structure of Fasciola hepatica Sigma class glutathione transferase 1 reveals a disulfide bond to support stability in gastro-intestinal environment.

Sigma class GST (Prostaglandin D synthase), FhGST-S1, is present in the excretory-secretory products (ES) of the liver fluke parasite Fasciola hepatica as cargo of extracellular vesicles (EVs) released by the parasite. FhGST-S1 has a well characterised role in the modulation of the immune response; a key fluke intercession that allows for establishment and development within their hosts. We have resolved the three-dimensional structure of FhGST-S1 in complex with its co-factor glutathione, in complex with a glutathione-cysteine adduct, and in a glutathione disulfide complex in order to initiate a research pipeline to mechanistically understand how FhGST-S1 functions within the host environment and to rationally design selective inhibitors. The overall fold of FhGST-S1 shows high structural similarity to other Sigma class GSTs. However, a unique interdomain disulfide bond was found in the FhGST-S1 which could stabilise the structure within the host gastro-intestinal environment. The position of the two domains of the protein with respect to each other is seen to be crucial in the formation of the active site cleft of the enzyme. The interdomain disulfide bond raises the possibility of oxidative regulation of the active site of this GST protein.

Different SNPs in Fasciola hepatica P-glycoprotein from diverse Latin American populations are not associated with Triclabendazole resistance.

The use of Triclabendazole for controlling fasciolosis is compromised by increased drug resistance affecting livestock and humans. Although the mode of action of TCBZ is still unknown, putative candidates and markers of resistance have been advanced. A single nucleotide polymorphism (T687 G) in F. hepatica PGP was proposed as marker of resistance in a small scale study of European susceptible and resistant flukes, but the association was not found in Australian samples. The T687 G SNP was absent in more than 40 samples from 2 TCBZ-resistant and 3 susceptible isolates across Latin America here analyzed. While the American samples showed more variable SNPs than the previous ones, none of the SNPs detected showed a marked association with resistance. Analyzing the 42 kb of the FhPGP gene based on RNAseq data highlights that the variation has been underestimated, suggesting that more detailed efforts are needed in order to identify markers of resistance.

A galactokinase-like protein from the liver fluke Fasciola hepatica.

Galactokinase catalyses the ATP-dependent phosphorylation of galactose. A galactokinase-like sequence was identified in a Fasciola hepatica EST library. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein of approximately 50 kDa. The protein is monomeric, like galactokinases from higher animals, yeasts and some bacteria. The protein has no detectable enzymatic activity with galactose or N-acetylgalactosamine as a substrate. However, it does bind to ATP. Molecular modelling predicted that the protein adopts a similar fold to galactokinase and other GHMP kinases. However, a key loop in the active site was identified which may influence the lack of activity. Sequence analysis strongly suggested that this protein (and other proteins annotated as "galactokinase" in the trematodes Schistosoma mansoni and Clonorchis sinensis) are closer to N-acetylgalactosamine kinases. No other galactokinase-like sequences appear to be present in the genomes of these three species. This raises the intriguing possibility that these (and possibly other) trematodes are unable to catabolise galactose through the Leloir pathway due to the lack of a functional galactokinase.

Sex and vaccination: Insights from female rats vaccinated with juvenile-specific proteases from Fasciola hepatica.

Most animal research is less evidence-based for females, with the majority of studies conducted on males. Since immune responses vary between males and females, sexual dimorphism in immunity contributes, among other things, to sex-based differences post-vaccination. However, the issue of sex effects in animal vaccine research is rarely considered in vaccine study design. Previously, we have evaluated the efficacy of cathepsin L3 (FhCL3-1 and FhCL3-2) and B3 proteases (FhCB3) from juvenile Fasciola hepatica as vaccines against fasciolosis in male rats. Their administration resulted in reductions in liver fluke recovery in the range of 47-63% when compared with an infection control group. Here, we investigated if the protective effect of vaccination with these proteins can also be observed for female rats. The data indicates females were not protected from F. hepatica infection when vaccinated with juvenile cathepsins. Only in the FhCL3-2 vaccinated group was a low, non-significant, reduction in worm burden observed (21%). Although liver fluke mean body lengths and wet weights were reduced in vaccinated animals when compared with the infection controls, these effects were adjuvant- not vaccine-induced, while for males changes in these parameters were related primarily to vaccination. Specific humoral responses throughout the study were evident; however, trends in antibody responses in females replicated trends observed previously for male humoral responses. Formerly, elevated levels of FhCL3-1 and FhCL3-2 specific IgG1 and IgG2a were suggested to be correlated with protection. Here, despite increased and clear responses of these antibodies, protection was not observed. Hence, in the present study the roles of IgG1 and IgG2 in liver fluke reduction are questionable. Results demonstrated in our study show that observations obtained in one sex are not always applicable to the other sex. Hopefully, the findings of the study will stimulate discussion of the issue of sex impacts on post-vaccination outcomes and will encourage researchers to consider sex in their future vaccine studies.

Purification of native Sigma class glutathione transferase from Fasciola hepatica.

Fascioliasis is a parasitic disease of grazing livestock and a threat to global food security by significantly reducing the production value of sheep, goats and cattle. Moreover, the zoonotic parasite is also a re-emerging food borne threat to human populations. Driven by climate change, the prevalence of fascioliasis is set to increase. Efforts to control the main causative agent, Fasciola hepatica, are hampered by short lived chemotherapy approaches that are becoming increasingly obsolete due to therapeutic failure and resistance. A protective vaccine is urgently needed. A recombinant form of Sigma class glutathione transferase (Hematopoietic Prostaglandin D synthase) from F. hepatica (FhGSTS1) with confirmed prostaglandin synthase activity shows immune-modulation activity via suppression of Th17 responses in host dendritic cells. In vaccine trials recombinant FhGSTS1 reduces liver pathology but not worm burden. Native FhGSTS1 is yet to be tested for immune-modulation activities or for vaccine potential, primarily due to the technical difficulty in purifying FhGST-S1 away from the other more abundant GST members in F. hepatica cytosol. This paper reports a pipeline for the purification of native FhGSTS1 using a two-step process consisting of glutathione-agarose affinity and cationic exchange chromatography. The methodology allows for the isolation of purified and active FhGSTS1 or Sigma GSTs from other sources for analytical biochemical and immunological studies.

Fibrinogen and fibrin are novel substrates for Fasciola hepatica cathepsin L peptidases.

Cathepsin peptidases form a major component of the secreted proteins of the blood-feeding trematodes Fasciola hepatica and Schistosoma mansoni. These peptidases fulfill many functions, from facilitating infection to feeding and immune evasion. In this study, we examined the Fasciola cathepsin L peptidases FhCL1, FhCL2, and FhCL3 and the schistosomal cathepsin peptidases SmCB1 and SmCL3 for their anticoagulant properties. Although no direct anticoagulant effect of these peptidases was observed, we discovered that cathepsin peptidases from Fasciola, but not from Schistosoma, were able to degrade purified fibrinogen, with FhCL1 having the highest fibrinogenolytic activity. Additionally, FhCL1 and FhCL2 both efficiently degraded fibrin. The lack of a direct anticoagulant or fibrinolytic effect of these peptidases is explained by their inhibition by plasma components. However, within the parasite gut, high concentrations of these peptidases could induce an anticoagulant environment, facilitating blood-feeding for extended periods.

Vaccination against Fasciola hepatica using cathepsin L3 and B3 proteases delivered alone or in combination.

No licensed vaccine is currently available for prevention of Fasciola hepatica infections. However, considering the alarming increase in drug resistance, there is an urgent need for a safe and fully effective vaccine against fasciolosis. Here, we tested if cathepsins L (FhCL3-1, FhCL3-2) and B (FhCB3) secreted by juvenile liver flukes are viable vaccine targets when delivered alone or in combination in a rat model. Since control over the early immune response is crucial for parasite's establishment in its host, it was hypothesised that targeting fluke juvenile stages may prove beneficial. Moreover, it was assumed that selected antigens will act in a cumulative manner to interfere with liver fluke migration and thereby will reduce F. hepatica infection. Recombinant FhCL3-1 and FhCL3-2 delivered alone reduced liver fluke burdens by 47 % and 63 %, respectively. A trivalent vaccine containing rFhCL3-1/CL3-2/CB3 did not increase the protective vaccine efficacy compared to the rFhCL3-2 vaccinated group (53 %), although, reductions in liver fluke wet weight (statistically significant) and liver damage score were most pronounced. Further, the highest IgG1 and IgG2a levels were seen in rFhCL3-2 vaccinated rats, the group for which the highest reduction in worm burden was demonstrated. Moreover, IgG1 and IgG2a levels in vaccinated rats were significantly elevated compared to those reported for control groups up to 4 week post-infection. While the mechanism of protection remains unknown, it appears that it depends on vaccine-induced antibodies directed against cathepsins. The obtained results imply that F. hepatica juvenile-specific cathepsins are promising vaccine candidates that induce responses that successfully target early migratory liver fluke stages. Now, the challenge is to evaluate these juvenile-specific cathepsins for use in livestock.

Purification and biochemical characterization of a 22-kDa stable cysteine- like protease from the excretory-secretory product of the liver fluke Fasciola hepatica by using conventional techniques.

This study describes the purification and characterization of a stable protease activity isolated from Fasciola hepatica adult worms maintained in vitro by employing acetone precipitation (40-60%) followed by a gel filtration through Sephadex G-100 and DEAE- cellulose ion exchange column. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9U/mg and 31.5% recovery. After the final ultrafiltration step, the purification fold was increased up to 13.1 and the overall activity yield reached a rate of 18.8%. The MW of the purified protease was estimated by reducing SDS-PAGE to be 22kDa while the proteolytic activity detection was carried out by zymography on non-denaturing SDS-PAGE containing the casein as substrate. Using this substrate, the protease showed extreme proteolytic activity at pH 5.5 and temperature 35-40°C and was highly stable over a wide range of pH, from 5.0 to 10.0. In addition to its preference for the Z-Phe-Arg-AMC fluorogenic substrate resulting in maximum proteolytic activity (99.7%) at pH 7.0, the pure protease exhibited highest cleavage activity against hemoglobin and casein substrates at pH 5.5 (85.6% and 82.8%, respectively). The Km values obtained for this protease were 5.4, 13, 160 and approximately 1000µM using respectively the fluorogenic substrate Z-Phe-Arg-AMC, hemoglobin, casein and albumin. The protease activity was completely inhibited either by E-64 inhibitor (5mM) or iodoacetamide (10mM), indicating its cysteine nature. The usefulness of the purified protease as an antigen was studied by immunoblotting. Thus, sera from sheep experimentally infected with F. hepatica recognized the protease band at 2 weeks post-infection (WPI) and strongly at 7 WPI. The early detection of antibodies anti- F. hepatica suggests the application of this molecule as a specific epitope for the serodiagnosis of fascioliasis disease.

Understanding the main route of drug entry in adult Fasciola hepatica: Further insights into closantel pharmacological activity.

Closantel (CLS) is highly effective against adult liver flukes after its oral or subcutaneous (sc) administration in ruminants. Trans-tegumental diffusion and oral ingestion are the two potential routes available for the entry of drugs into Fasciola hepatica. The work reported here contributes to improve the understanding of CLS pharmacology. The main goals of were: I) to determine the pattern of in vivo CLS accumulation into adult F. hepatica and relevant tissues in CLS-treated sheep; II) to investigate the influence of the physicochemical composition of the incubation medium on the CLS diffusion process into adult F. hepatica; III) to assess the ovicidal activity of CLS against F. hepatica eggs; and IV) to investigate the in vivo effect of CLS treatment on glutathione S-transferases activity in adult liver flukes exposed to CLS. Fourteen healthy sheep were each orally infected with 75 F. hepatica metacercariae. Sixteen (16) weeks after infection, animals were treated with CLS by oral (n = 6, 10 mg/kg) or sub-cutaneous (sc) (n = 6, 5 mg/kg) route. At 12, 24 and 36 h post-treatment, animals were sacrificed (n = 2) and samples of blood, bile and adult F. hepatica were collected. In addition, flukes recovered from non-treated sheep (n = 2) were ex vivo incubated (60 min) in the presence of CLS in either RPMI or bile as incubation medium. CLS concentration was measured by HPLC. The ovicidal activity of CLS was investigated using eggs obtained from the bile of untreated sheep. Finally, glutathione S-transferase activity in F. hepatica recovered from untreated and CLS-treated sheep was assessed. In the in vivo studies, the highest CLS concentrations were measured in plasma and adult liver flukes. A positive correlation was observed between CLS concentration in plasma and in F. hepatica. Results obtained in the current work indicate that the in vivo accumulation of CLS into adult liver flukes occurs mainly by the oral route. After ex vivo incubation, the uptake of CLS by the parasite was markedly diminished in the presence of bile compared with that observed in the presence of RPMI as incubation medium. CLS lacks ovicidal activity at therapeutically relevant concentrations. Lastly, CLS significantly increased glutathione S-transferase activity in flukes recovered at 12 h (oral treatment) and 24 h (sc treatment), compared to the control liver flukes.

Functional characterization of single-domain cystatin-like cysteine proteinase inhibitors expressed by the trematode Fasciola hepatica.

Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F. hepatica. For this purpose, we first surveyed the available sequence databases, identifying three different F. hepatica single-domain cystatins. In agreement with the in silico predictions, at least three small proteins with cysteine proteinase binding activity were identified. Phylogenetic analyses showed that the three cystatins (named FhStf-1, -2 and -3) are members of the I25A subfamily (stefins). Whereas FhStf-1 grouped with classical stefins, FhStf-2 and 3 fell in a divergent stefin subgroup unusually featuring signal peptides. Recombinant rFhStf-1, -2 and -3 had potent inhibitory activity against F. hepatica cathepsin L cysteine proteinases but differed in their capacity to inhibit mammalian cathepsin B, L and C. FhStf-1 was localized in the F. hepatica reproductive organs (testes and ovary), and at the surface lamella of the adult gut, where it may regulate cysteine proteinases related with reproduction and digestion, respectively. FhStf-1 was also detected among F. hepatica excretion-secretion (E/S) products of adult flukes. This suggests that it is secreted by non-classical secretory pathway and that it may interact with host lysosomal cysteine proteinases.