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.

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.

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.

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.

Metabolism of drugs and other xenobiotics in giant liver fluke (Fascioloides magna).

1. Giant liver fluke Fascioloides magna is a dangerous parasite, which infects herbivores. It was imported to Europe from North America and started to spread. Benzimidazoles like albendazole, mebendazole, triclabendazole and salicylanilides closantel and rafoxanide are the most used anthelmintics to control fascioloidosis. However their effect might be altered via drug-metabolizing enzymes of this parasite. 2. The aim of our study was to determine the activities of drug-metabolizing enzymes in F. magna and the metabolism of above mentioned anthelmintics. 3. Activities of several oxidative, reductive and conjugative enzymes towards various model xenobiotic substrates were found in F. magna subcellular fractions. 4. Subcellular fractions from F. magna oxidized albendazole to its sulphoxide metabolite and reduced mebendazole to hydroxyl-mebendazole. Under ex vivo conditions, only very-low concentrations of these compounds were detected using high-performance liquid chromatography/mass spectrometry. 5. The results indicate that the giant liver fluke possesses the active xenobiotic-metabolizing system. The overexpression of this system may play an important role in parasite resistance against these anthelmintics.

Biochemical characterisation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the liver fluke, Fasciola hepatica.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses one of the two steps in glycolysis which generate the reduced coenzyme NADH. This reaction precedes the two ATP generating steps. Thus, inhibition of GAPDH will lead to substantially reduced energy generation. Consequently, there has been considerable interest in developing GAPDH inhibitors as anti-cancer and anti-parasitic agents. Here, we describe the biochemical characterisation of GAPDH from the common liver fluke Fasciola hepatica (FhGAPDH). The primary sequence of FhGAPDH is similar to that from other trematodes and the predicted structure shows high similarity to those from other animals including the mammalian hosts. FhGAPDH lacks a binding pocket which has been exploited in the design of novel antitrypanosomal compounds. The protein can be expressed in, and purified from Escherichia coli; the recombinant protein was active and showed no cooperativity towards glyceraldehyde 3-phosphate as a substrate. In the absence of ligands, FhGAPDH was a mixture of homodimers and tetramers, as judged by protein-protein crosslinking and analytical gel filtration. The addition of either NAD⁺ or glyceraldehyde 3-phosphate shifted this equilibrium towards a compact dimer. Thermal scanning fluorimetry demonstrated that this form was considerably more stable than the unliganded one. These responses to ligand binding differ from those seen in mammalian enzymes. These differences could be exploited in the discovery of reagents which selectively disrupt the function of FhGAPDH.

UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors.

Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 µM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.

A single amino acid substitution in isozyme GST mu in Triclabendazole resistant Fasciola hepatica (Sligo strain) can substantially influence the manifestation of anthelmintic resistance.

The helminth parasite Fasciola hepatica causes fascioliasis in human and domestic ruminants. Economic losses due to this infection are estimated in U$S 2000-3000 million yearly. The most common method of control is the use of anthelmintic drugs. However, there is an increased concern about the growing appearance of F. hepatica resistance to Triclabendazole (TCBZ), an anthelmintic with activity over adult and young flukes. F. hepatica has eight Glutathione S-Transferase (GST) isozymes, which are enzymes involved in the detoxification of a wide range of substrates through chemical conjugation with glutathione. In the present work we identified and characterized the GST mu gene isolated from the TCBZ-susceptible and TCBZ-resistant F. hepatica strains. Total RNA was transcribed into cDNA by reverse transcription and a 657 bp amplicon corresponding to the GST mu gene was obtained. The comparative genetic analysis of the GST mu gene of the TCBZ susceptible strain (Cullompton) and TCBZ resistant strain (Sligo) showed three nucleotide changes and one amino acid change at position 143 in the GST mu isozyme of the TCBZ-resistant strain. These results have potential relevance as they contribute better understand the mechanisms that generate resistance to anthelmintics.

Isolation and characterization of Cu/Zn-superoxide dismutase in Fasciola gigantica.

A full-length complementary DNA (cDNA) encoding Cu/Zn-superoxide dismutase was isolated from Fasciola gigantica that on nucleotide sequencing showed a close homology (98.9%) with Cu/Zn-superoxide dismutase (SOD) of the temperate liver fluke, F. hepatica. Expression of the gene was found in all the three developmental stages of the parasite viz. adult, newly excysted juvenile and metacercaria at transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. F. gigantica Cu/Zn-SOD cDNA was cloned and expressed in Escherichia coli. Enzyme activity of the recombinant protein was determined by nitroblue tetrazolium (NBT)-polyacrylamide gel electrophoresis (PAGE) and this activity was inactivated by hydrogen peroxide but not by sodium azide, indicating that the recombinant protein is Cu/Zn-SOD. The enzyme activity was relatively stable at a broad pH range of pH 4.0-10.0. Native Cu/Zn-superoxide dismutase protein was detected in the somatic extract and excretory-secretory products of the adult F. gigantica by Western blotting. NBT-PAGE showed a single Cu/Zn-SOD present in the somatic extract while three SODs are released ex vivo by the adult parasite. The recombinant superoxide dismutase did not react with the serum from buffaloes infected with F. gigantica. The role of this enzyme in defense by the parasite against the host reactive oxygen species is discussed.

[Immunoreactivity and immunogenicity analysis of the recombinant cathepsin L-like protease of Fasciola hepatica in SD rats].

OBJECTIVE: To analyze the immunoreactivity of recombinant cathepsin L-like proteases (CatL) protein of Fasciola hepatica and its immunogenicity in SD rats. METHODS: The E. coli BL21(DE3) cells harbouring recombinant plasmid pET30a-FhCatL were inoculated in LB medium, and the protein expression was induced with IPTG. The recombinant protein FhCatL was analyzed by SDS-PAGE and the immunoreactivity was identified by Western blotting with sera from Fasciola hepatica-infected goat as the primary antibody. Twenty SD rats were randomly divided into immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 microg of purified FhCatL protein. All the rats received three immunizations at 3-week intervals. The adjuvant control group with 10 SD rats received only adjuvants emulsified with the same amount of PBS. Serum samples were collected at the day before the second and final immunization, 3, 6, and 9 weeks after the final immunization. The IgG antibody of rats' sera was examined by indirect ELISA and spleen lymphocyte proliferation (SLP) was tested by methyl thiazolyl tetrazolium (MTT). RESULTS: The molecular weight of purified FhCatL was about Mr 42,000. The recombinant FhCatL was recognized by pool sera of goats naturally infected with F. hepatica. The titer of specific antibody IgG in SD rats induced by the recombinant protein against CatL protein was significantly higher than that of the control, and the antibody titer reached the peak at three weeks after the final immunization (1 : 102,400). The stimulation index of splenocytes in immunized group was 2.176 +/- 0.047, which was significantly higher than that of the control (1.171 +/- 0.032) (P<0.05). CONCLUSION: The recombinant FhCatL protein bears stronger immnoreactivity and immunogenicity.

Microarray analysis of rat immune responses to liver fluke infection following vaccination with Fasciola hepatica phosphoglycerate kinase.

Fasciolosis is a considerable veterinary problem, causing significant economic losses to livestock production and the food industry. Research in the area of Fasciola hepatica infection immunology is necessary to improve our knowledge about immunological mechanism evoked by the parasite and to develop new control strategies against liver fluke. In this present paper we analyzed the expression levels of cytokines in rats infected with F. hepatica following immunization with F. hepatica phosphoglycerate kinase - a novel vaccine antigen. Immune response analysis using microarray was undertaken six weeks after infection. Expression levels of INF-γ and IL-4, which are characteristic cytokines secreted during Th1-like and Th2-like immune responses, respectively, were unchanged in vaccinated animals as compared to control animals. This indicates the vaccine did not influence the major modulation of immune responses typically observed during Fasciola infections, however, other subtle but significant variations were observed that indicated altered inflammatory and possibly T helper cell responses. A significant rise in IL-12α chain expression levels was observed. Expression levels of TNF-α and some related molecules, such as ADAM17, FasL, CD40 and TRAF3 were also elevated. Expression levels of molecules involved in IL-1 signaling pathways were reduced, although a rise in IL-1α expression was noted.

Citrate synthase from the liver fluke Fasciola hepatica.

Citrate synthase catalyses the first step of the Krebs' tricarboxylic acid cycle. A sequence encoding citrate synthase from the common liver fluke, Fasciola hepatica, has been cloned. The encoded protein sequence is predicted to fold into a largely α-helical protein with high structural similarity to mammalian citrate synthases. Although a hexahistidine-tagged version of the protein could be expressed in Escherichia coli, it was not possible to purify it by nickel-affinity chromatography. Similar results were obtained with a version of the protein which lacks the putative mitochondrial targeting sequence (residues 1 to 29). However, extracts from bacterial cells expressing this version had additional citrate synthase activity after correcting for the endogenous, bacterial activity. The apparent K m for oxaloacetate was found to be 0.22 mM, which is higher than that observed in mammalian citrate synthases. Overall, the sequence and structure of F. hepatica citrate synthase are similar to ones from other eukaryotes, but there are enzymological differences which merit further investigation.

[Immunogenicity analysis of recombinant GST protein of Fasciola hepatica in SD rats].

OBJECTIVE: To analyze the immunogenicity of recombinant glutathione S-transferase protein of Fasciola hepatica (FhGST) in SD rats. METHODS: The recombinant expression plasmid pET30a-FhGST was transformed into E. coli BL21 (DE3) cells and induced with IPTG for protein expression. The recombinant protein FhGST was analyzed by SDS-PAGE and identified by Western blotting. Twenty SD rats were randomly divided into two groups: immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 microg of purified FhGST protein. The adjuvant control group with 10 SD rats received only adjuvants emulsified with PBS. All the rats received three immunizations at 3-week intervals. Serum samples were collected at pre-immunization, the day after each immunization, 3 weeks and 6 weeks after the final immunization. The IgG antibody of rats' sera was examined by indirect ELISA and spleen lymphocyte proliferation (SLP) was tested by MTT. RESULTS: The molecular weight of purified FhGST was about M(r) 31 300. The recombinant FhGST was recognized by pool sera of goats naturally infected with F. hepatica. The recombinant protein induced specific antibody IgG against GST protein in SD rats significantly higher than that of the control, and the antibody titer reached the peak at 9 weeks after the first immunization (GMT 1:89 144). FhGST protein significantly enhanced the growth and proliferation of rat splenocytes. CONCLUSION: The recombinant FhGST protein induces specific immune response in SD rats.

Helminth pathogen cathepsin proteases: it's a family affair.

Helminth pathogens express papain-like cysteine peptidases, termed cathepsins, which have important roles in virulence, including host entry, tissue migration and the suppression of host immune responses. The liver fluke Fasciola hepatica, an emerging human pathogen, expresses the largest cathepsin L cysteine protease family yet described. Recent phylogenetic, biochemical and structural studies indicate that this family contains five separate clades, which exhibit overlapping but distinct substrate specificities created by a process of gene duplication followed by subtle residue divergence within the protease active site. The developmentally regulated expression of these proteases correlates with the passage of the parasite through host tissues and its encounters with different host macromolecules.

Molecular cloning and characterization of leucine aminopeptidase from Fasciola gigantica.

M17 leucine aminopeptidase (LAP) is one of a family of metalloexopeptidases, of which short peptide fragments are cleaved from the N-terminals. In this study, the full length of cDNA encoding Fasciola gigantica LAP (FgLAP) was cloned from adult parasites. The amino acid sequences of FgLAP showed a high degree of identity (98%) with that from Fasciola hepatica and a low degree of identities (11% and 9%) with those from cattle and human. Phylogenetic analysis revealed that the FgLAP was closely related and grouped with F. hepatica LAP (FhLAP). Northern analysis showed that FgLAP transcriptional products have 1800 base pairs. Analysis by RNA in situ hybridization indicated that LAP gene was expressed in the cecal epithelial cells of adult parasites. A polyclonal antibody to a recombinant FgLAP (rFgLAP) detected the native LAP protein in various developmental stages of the parasite. In a functional test, this rFgLAP displayed aminolytic activity using a fluorogenic Leu-MCA substrate, and was significantly inhibited by bestatin. Its maximum activity was at pH 8.0 and enhanced by Mn(2+) ions. Localization of LAP proteins by immunohistochemistry and immunofluorescence techniques indicated that the enzyme was distributed in the apical cytoplasm of cecal epithelial cells. Because of its important metabolic role and fairly exposed position, FgLAP is a potential drug target and a possible vaccine candidate against fasciolosis.

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.

Piperonyl butoxide enhances triclabendazole action against triclabendazole-resistant Fasciola hepatica.

A study has been carried out to determine whether the action of triclabendazole (TCBZ) against the liver fluke, Fasciola hepatica is altered by inhibition of the cytochrome P450 (CYP 450)-mediated drug metabolism pathway. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments, the basic design of which is given in the paper by Devine et al. (2010a). Piperonyl butoxide (PB) was the CYP P450 inhibitor used. Morphological changes resulting from drug treatment and following metabolic inhibition were assessed by means of transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with PB+TCBZ, but more particularly PB+TCBZ.SO, led to greater changes to the TCBZ-resistant isolate than with each drug on its own, with blebbing of the apical plasma membrane, severe swelling of the basal infolds and their associated mucopolysaccharide masses in the syncytium and flooding in the internal tissues. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis and production of secretory bodies were badly disrupted. The mitochondria were swollen throughout the tegumental system and the somatic muscle blocks were disrupted. With the TCBZ-susceptible Cullompton isolate, there was a limited increase in drug action following co-incubation with PB. The results provide evidence that the condition of a TCBZ-resistant fluke can be altered by inhibition of drug metabolism. Moreover, they support the concept that altered drug metabolism contributes to the mechanism of resistance to TCBZ.

A recombinant thioredoxin-glutathione reductase from Fasciola hepatica induces a protective response in rabbits.

Antioxidant systems are fundamental components of host-parasite interactions, and often play a key role in parasite survival. Here, we report the cloning, heterologous expression, and characterization of a thioredoxin glutathione reductase (TGR) from Fasciola hepatica. The deduced polypeptide sequence of the cloned open reading frame (ORF) confirmed the experimental N-terminus previously determined for a native F. hepatica TGR showing thioredoxin reductase (TR) activity. The sequence revealed the presence of a fusion between a glutaredoxin (Grx) and a TR domain, similar to that previously reported in Schistosoma mansoni and Echinococcus granulosus. The F. hepatica TGR sequence included an additional redox active center (ACUG; U being selenocysteine) located at the C-terminus. The addition of a recombinant selenocysteine insertion sequence (SECIS) element in the Escherichia coli expression vector, or the substitution of the native selenocysteine by a cysteine, indicated the relevance of this unusual amino acid residue for the activity of F. hepatica TGR. Rabbit vaccination with recombinant F. hepatica TGR reduced the worm burden by 96.7% following experimental infection, further supporting the relevance of TGR as a promising target for anti Fasciola treatments.

The phylogeny, structure and function of trematode cysteine proteases, with particular emphasis on the Fasciola hepatica cathepsin L family.

Helminth parasites (nematodes, flatworms and cestodes) infect over 1 billion of the world's population causing high morbidity and mortality. The large tissue-dwelling worms express papain-like cysteine peptidases, termed cathepsins that play important roles in virulence including host entry, tissue migration and the suppression of host immune responses. Much of our knowledge of helminth cathepsins comes from studies using flatworms or trematode (fluke) parasites. The developmentally-regulated expression of these proteases correlates with the passage of parasites through host tissues and their encounters with different host macromolecules. Recent phylogenetic, biochemical and structural studies indicate that trematode cathepsins exhibit overlapping but distinct substrate specificities due to divergence within the protease active site. Here we provide an overview of the evolution, biochemistry and structure of these important enzymes and highlight how recent advances in proteomics and gene silencing techniques are allowing researchers to probe their biological functions. We focus mainly on members of the cathepsin L gene family of the animal and human pathogen, Fasciola hepatica, because of our deep understanding of their function, biochemistry and structure.