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.     

Localisation of actin in the liver fluke,Fasciola hepatica

Adult and 3-week-old juvenileFasciola hepatica were examined for the presence of the cytoskeletal protein actin. Techniques of direct fluorescence using fluorescein isothiocyanate (FITC)-phalloidin and of indirect immunofluorescence using a monoclonal anti-actin antibody (MAA) demonstrated actin in the testes, sub-tegumental and gut musculature, tegumental cell bodies and tegumental spines. In contrast, polyclonal anti-actin antibody (PAA) revealed immunostaining only in the vitellaria. Effective removal of the tegument with 1% (w/v) sodium dodecyl sulphate (SDS) was confirmed by scanning electron microscopy (SEM), and this enabled immunoblotting of whole fluke and tegumental fractions with and without spines. Whole fluke fractions produced three bands corresponding to molecules exhibiting relative molecular weights of 43, 28 and 15 kDa, respectively, whereas the tegumental fraction with spines revealed a single band corresponding to 15 kDa in size. The fraction without spines displayed no bands. The present study localised actin in a number of different tissue types within the liver fluke. Using MAA, three forms of actin have been identified in the whole fluke and a single one in the tegumental spines.     

Characterization and differential expression of a ferritin protein from Fasciola hepatica

Ferritins are proteins that play a central role in maintaining intracellular iron balance. A cDNA clone of Fasciola hepatica (687 bp long) encoding a putative 228-amino acid polypeptide (FhFtn-1) homologous with ferritins of vertebrates and invertebrates was identified. FhFtn-1 contains a conserved motif of the ferroxidase center typical of vertebrate ferritins. Phylogenetic tree analysis showed that FhFtn-1 clusters with two ferritins of Paragonimus westermani, which suggests a common ancestry for the ferritins of these two trematodes. Recombinant FhFtn-1 protein expressed and purified from an Escherichia coli system showed iron-uptake ability. Moreover, FhFtn-1 showed strong reactivity with sera from rabbits infected with F. hepatica for 2-12 weeks, which suggests that this protein could be a potential antigen for immunodiagnosis of fascioliasis. qPCR analysis demonstrated that FhFtn-1-mRNA is expressed at significantly higher levels in adults and unembryonated eggs than in juveniles or miracidia. These results represent the first characterization of a ferritin protein from the liver fluke F. hepatica.     

Cloning,heterologous expression in Escherichia coli and characterization of a protein disulfide isomerase from Fasciola hepatica

A Fasciola hepatica cDNA clone of 1752 bp was isolated from an adult worm cDNA expression library by immunological screening using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 489 codons which encoded a 55 kDa polypeptide, showing a high degree of homology to protein disulfide isomerases. This putative antioxidant protein cDNA was expressed in Escherichia coli as a GST fusion protein. The cleaved recombinant protein was shown to be biologically active in vitro by mediating the oxidative refolding of reduced RNase. Immunoblotting studies using a specific antiserum raised against the recombinant protein showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The extracellular location of this protein was also supported by the specific immune responses found against this protein in F. hepatica experimentally infected rabbits.     

Evidence for multiple mitochondrial lineages of Fasciola hepatica (liver fluke) within infrapopulations from cattle and sheep

The economic, veterinary, and medical impact of the parasite Fasciola hepatica, liver fluke, is difficult to alleviate due to increasing incidences of resistance to the principal anthelmintic drugs. These have occurred in widely separated regions. The rate of response to selection imposed by such drugs will be dependent on the genetic variation present in the F. hepatica gene pool, but this is at present unknown. We have assessed the genetic diversity of mitochondrial haplotypes found in the infrapopulation of flukes recovered from a calf of known provenance and from six other cattle and sheep hosts located in Ireland and four from elsewhere. Our results revealed that at least ten different mitochondrial composite PCR–restriction fragment length polymorphism haplotypes had been acquired by a single animal in 1 year, and there was comparable diversity in six other definitive hosts carrying field-acquired infections. The extent of divergence between these fluke lineages suggests that they predate the last ice age and, thus, cannot have developed in Northern Europe. A consequence of this high level of diversity is that there will be frequent selection for anthelmintic resistance and rapid responses to climatic changes. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Developmental differences in the uterus of Fasciola hepatica between livestock liver fluke populations from Bolivian highlands and European lowlands

A morphometric study of the uterus area (UA) of Fasciola hepatica adults was carried out with the aid of a computer linked to a stereomicroscopic 3CCD colour video camera using image analysis software. The UA of adult liver flukes found in naturally infected sheep, cattle and pig from the endemic human fascioliasis zone of the northern Bolivian Altiplano highlands was compared with that of flukes found infecting sheep and cattle from Valencia, Spain and cattle from Corsica, France (collectively, European lowlands). Liver fluke UA was examined using an allometric model. A comparison of the allometry of the liver fluke UA in different host species from Bolivia revealed no significant differences. Similarly, no statistically significant differences were found between UA of Valencian and Corsican populations. However, the Bolivian sheep and cattle liver fluke populations proved to have a UA smaller than that of the European populations. These results indicate differences between the liver fluke population of highlands and lowlands, regarding UA. This paper discusses the possible relationship between the characteristic of having a reduced uterine development and the liver fluke adult stage adapting to host populations living at very high altitudes. Received: 9 June 2000 / Accepted: 29 June 2000     

Ultrastructural observations on oral ingestion and trans-tegumental uptake of clorsulon by the liver fluke, Fasciola hepatica

Three experiments have been carried out in vitro to determine the effect of oral and trans-tegumental uptake of clorsulon on the fine structure of the tegument and gut of Fasciola hepatica. Changes were assessed by transmission electron microscopy. In the first experiment, the flukes were ligatured to prevent the oral ingestion of drug and treated for 24 h in clorsulon (10 g/ml). Limited swelling of the basal infolds was observed in the tegumental syncytium. Swollen mitochondria were present in the syncytium, the underlying tegumental cells and in the gastrodermal cells. Swelling and vesiculation of the cisternae of the granular endoplasmic reticulum (ger) was evident in the gastrodermal cells, together with a reduction in secretory activity. In the second experiment, flukes were fed for 24 h on red blood cells isolated from rats dosed with clorsulon at 12.5 mg/kg body weight; this experiment was designed to prevent the exposure of the tegumental surface to the drug. There was severe swelling of the basal infolds in the tegumental syncytium and swelling of mitochondria in the syncytium, tegumental cells and gastrodermal cells. In the tegumental cells there was a decrease in the number of Golgi complexes as well. A number of changes were evident in the gastrodermal cells: swelling of the ger cisternae, an increase in the number of autophagic vacuoles, a reduction in the number of secretory bodies and disruption of the lamellae projecting from the surface of the cells. In the third experiment, flukes were incubated for 24 h in clorsulon (10 g/ml), with both absorptive surfaces being available for drug uptake. There was severe swelling of the basal infolds in the tegumental syncytium and large autophagic vacuoles were present. Swollen mitochondria were a feature of the tegument, tegumental cells and gastrodermal cells, as were swollen cisternae of ger in the tegumental and gastrodermal cells. Fewer Golgi complexes were observed in the tegumental cells and in the gastrodermal cells there were fewer secretory bodies and an increased number of autophagic vacuoles. Overall, the gastrodermal cells were more severely affected than the tegument. Greater disruption of the tegument occurred when the oral route of uptake was available. The results support those of previous studies which point to oral uptake of clorsulon being the major route of entry into the fluke.     

Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein.

A Fasciola hepatica cDNA clone of 994 bp was isolated from an adult worm cDNA expression library using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA clone revealed the presence of an open reading frame of 572 bp which encoded a 22 kDa polypeptide (Fh22) showing putative EF-hand domains. This gene was expressed in Escherichia coli and the recombinant protein used for the production of specific antibodies. Immunoblotting studies using the anti-Fh22 serum showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The recombinant Fh22 polypeptide showed calcium-dependent electrophoretic mobility (decreased with Ca2(+)-ions and increased with EGTA). The observed behaviour of recombinant Fh22 in gel filtration experiments also suggested calcium-induced conformational changes.     

On the fine structure of the Mehlis gland cells in the liver fluke,Fasciola hepatica L.

Summary The cells of Mehlis's gland in the liver fluke (Fasciola hepatica L.) have been studied with light and electron microscopy. The gland contained large cells in the periphery and small cells in the centre. The large cells presented morphological features characteristic of secretory cells, viz. large nucleoli, cytoplasmic basophilia, a well developed endoplasmic reticulum, abundant ribosomes, and secretory granules in different stages of development. The mitochondria were moderately frequent. Several small groups of vesicles and membraneous lamellae reminiscent of Golgi complexes occurred randomly in the cytoplasm. These structures were associated with mitochondria and secretory granules. Evidence of alterations and conversion into a cell débris was observed, and a process of holocrine secretion was suggested.The small cells were strongly basophilic, contained numerous ribosomes and mitochondria, and a few dense bodies. The internal membranes were few. The small cells had a primitive appearance and showed no secretory properties.With 7 Figures in the TextSupported by a grant from the Swedish Agricultural Research Council.     

Characterization of the secreted cathepsin B cysteine proteases family of the carcinogenic liver fluke Clonorchis sinensis

Clonorchis sinensis excretory/secretory products (ESP) have gained high attentions because of their potential to be vaccine candidates and drug targets in C. sinensis prevention. In this study, we extensively profiled the characteristics of four C. sinensis cathepsin B cysteine proteases (CsCB1, CsCB2, CsCB3, and CsCB4). Bioinformatics analysis showed all CsCBs contained signal peptides at the N-terminal. Functional domains and residues were found in CsCB sequences. We expressed four CsCBs and profiled immune responses followed by vaccine trials. Recombinant CsCBs could induce high IgG titers, indicating high immunogenicity of CsCB family. Additionally, ELISA results showed that both IgG1 and IgG2a levels apparently increased post-immunization with all four CsCBs, showing that combined Th1/Th2 immune responses were triggered by CsCB family. Both Real-time polymerase chain reaction (RT-PCR) and Western blotting confirmed that four CsCBs have distinct expression patterns in C. sinensis life stages. More importantly, we validated our hypothesis that CsCBs were C. sinensis excretory/secretory products. CsCBs could be recognized by C. sinensis-infected sera throughout the infection period, indicating that secreted CsCBs are immune triggers during C. sinensis infection. The protective effect was assessed by comparing the worm burden and egg per gram (EPG) between CsCB group and control group, showing that worm burden (P?P?CsCB2 and CsCB3 groups were significantly lower than in control group. In conclusion, we profiled secreted cathepsin B cysteine proteases family for the first time and demonstrated that all CsCB family were C. sinensis excretory/secretory products that may regulate host immune responses.     

A scanning electron microscope study on the route of entry of clorsulon into the liver fluke, Fasciola hepatica

Three experiments were carried out in vitro to determine the roles of the tegument and gut of Fasciola hepatica in the uptake of the flukicidal drug, clorsulon. Changes to the two surfaces were assessed by scanning electron microscopy. In the first experiment, the flukes were ligatured to prevent the oral ingestion of drug and treated for 24 h in clorsulon (10 g/ml). The gastrodermal surface remained normal and few changes to the tegumental surface were observed. In the second experiment, flukes were fed for 24 h on red blood cells isolated from rats dosed with clorsulon at 12.5 mg/kg body weight; this experiment was designed to prevent the exposure of the tegumental surface to the drug. The gastrodermal surface was severely disrupted and the gut lamellae were disorganised and necrotic. Swelling of the tegument and blebbing on the tegumental surface were evident, but the changes were not severe. More severe swelling of the tegument was observed in the third experiment, in which flukes were incubated for 24 h in clorsulon (10 g/ml), with both absorptive surfaces being available for drug uptake. The gastrodermal surface was badly disrupted and the gut lamellae were disorganised and necrotic. Taking the results of the three experiments together, the gastrodermal surface was more affected than the tegument and the greatest disruption to the two surfaces was seen when both routes of entry were available to the fluke. The data support a previous study which indicated that entry of clorsulon into the fluke in vivo is principally by the oral ingestion of drug bound to the red blood cells.An erratum to this article can be found at     

Heterologous expression and functional characterization of thioredoxin from Fasciola hepatica

The full thioredoxin coding sequence from Fasciola hepatica has been cloned into the pGEX-2T expression vector and produced in Escherichia coli as a fusion protein. The recombinant protein proved to be biologically active, using an insulin reduction assay, and was also able to activate thioredoxin peroxidase from F. hepatica. These observations suggest that this protein could participate in a redox cascade involved in the maintenance of cell homeostasis as well as in parasite protection against reactive oxygen species produced by the host.     

Identification, expression and in situ hybridization of an eggshell protein gene from Fasciola hepatica

The molecular basis of egg formation in the parasitic liver fluke, Fasciola hepatica, was investigated by isolating and characterizing an abundant cDNA from a female genital complex cDNA library. It was expressed in Escherichia coli as a beta-galactosidase fusion protein, which was purified and used to produce polyclonal antibodies. Using immunoblots, the antiserum recognized two soluble constituents of isolated egg shells, both significantly larger than predicted from cDNA sequencing. Using in situ hybridization, the message was detected in cells in the adult vitelline follicles. Eggshell protein mRNA expressed in E. coli will provide a source of precursor protein for further studies of parasite eggshell formation.     

Partial purification and characterization of cAMP-dependent protein kinase from Fasciola hepatica

Three cyclic AMP (cAMP)-dependent protein kinases, designated A1, A2, and B, were isolated from the liver fluke Fasciola hepatica using Phenyl-Sepharose and DEAE-cellulose chromatography. These enzymes differed with respect to activation by cAMP and their molecular weights. The half-maximal activation constant for cAMP-dependent protein kinases A1 and B was 20 nM, while that of A2 was about five-fold higher (110 nM). The estimated molecular weights for cAMP-dependent protein kinases A1 and A2 (both 98,000) suggest a dimeric form for these enzymes; whereas, the higher molecular weight for cAMP-dependent protein kinase B (187,000) indicates that this enzyme is a tetramer. The physical and kinetic properties of the catalytic subunit of fluke cAMP-dependent protein kinase were similar to those reported for the mammalian enzyme. The molecular weight of the catalytic subunit was estimated to be 41,000. The pH optimum for the enzyme was 6.0, 6.5, or 7.0 when casein, histone, or protamine were used as substrates. The protein substrate specificity was in the order histone greater than arginine-rich histone greater than casein greater than protamine greater than lysine-rich histone. Free Mg2+ 'stimulated' enzyme activity at low concentrations (0.5 to 5 mM), whereas at higher concentrations (greater than 5 mM) it became inhibitory. Of the divalent cations tested, only Co2+ and Mn2+ could substitute for Mg2+. Kinetic studies indicated that the reaction mechanism of this enzyme is sequential and that MgATP and MgADP are competitive ligands. Reconstitution experiments using the subunits of fluke and bovine heart cAMP-dependent protein kinase showed that there is sufficient structural homology between these enzymes such that the catalytic subunit from one species can combine with the regulatory subunit of the other species to form inactive holoenzyme. Thus, the present results indicate that cAMP-dependent protein kinase from F. hepatica is similar but not identical to the mammalian enzyme.     

Distribution and fate of 2-C14-glucose in the liver fluke,Fasciola hepatica L., after short in vitro incubation

Summary Adult stages of Fasciola hepatica L. were incubated in 2-C¹⁴-glucose for 10 minutes. The distribution and fate of C¹⁴ was followed by microautoradiography and paper electrophoresis and paper chromatography in combination with autoradiography. It was found that C¹⁴ accumulated predominately in the parenchyme and in the suckers. Within the fluke C¹⁴ was found in high molecular weight compounds like glycogen and in acids belonging to the citric acid cycle. These acids were not observed in the incubation medium. Labelled compounds behaving like amino acids were found both in the fluke and in the incubation medium.

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Zusammenfassung Erwachsene Stadien von Fasciola hepatica L. wurden in 2-C¹⁴-Glucose 10 min lang inkubiert. Verteilung und Schicksal des C¹⁴ wurden mittels Microautoradiographie und Papierelektrophorese und Papierchromatographie kombiniert mit Autoradiographie studiert. C¹⁴ wurde insbesondere im Parenchym und in den Saugnäpfen angereichert. C¹⁴ fand sich in hoch molekularen Verbindungen wie Glykogen und in Säuren des Citronensäurezyklus. Diese Säuren wurden nicht in dem Inkubationsmedium gefunden. Markierte Verbindungen mit Eigenschaften wie Aminosäuren wurden sowohl in dem Leberegel wie in dem Inkubationsmedium nachgewiesen.

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Supported by a grant A 1460/B 1110 from Jordbrukets Forskningsr»d.     

Molecular cloning and expression of Cu/Zn-containing superoxide dismutase from Fasciola hepatica

The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.     

Ultrastructural localisation of FMRFamide- and pancreatic polypeptide-immunoreactivities within the central nervous system of the liver fluke,Fasciola hepatica (Trematoda,Digenea)

A post-embedding immunogold technique was used to examine the subcellular distribution of immunoreactivities to the invertebrate peptide, FMR-Famide, and to vertebrate pancreatic polypeptide (PP) within the central nervous system of the trematode,Fasciola hepatica. Gold labeling of peptide was localised exclusively over both dense-cored and ellipsoidal electron-dense vesicles (with a homogeneous matrix) present within nerve cell bodies, small and giant nerve processes of the neuropile in the cerebral ganglia and transverse commissure, as well as in the main longitudinal nerve cords. Double labeling demonstrated an apparent co-localisation of FMRFamide and PP immunoreactivities in the same dense-cored vesicles, although populations of ellipsoidal electron-dense vesicles that labeled solely for FMRFamide were also evident. Antigen pre-absorption studies indicated little, if any, cross-reactivity of the two antisera.