A MEKK1 - JNK mitogen activated kinase (MAPK) cascade module is active in Echinococcus multilocularis stem cells.

BACKGROUND: The metacestode larval stage of the fox-tapeworm Echinococcus multilocularis causes alveolar echinococcosis by tumour-like growth within the liver of the intermediate host. Metacestode growth and development is stimulated by host-derived cytokines such as insulin, fibroblast growth factor, and epidermal growth factor via activation of cognate receptor tyrosine kinases expressed by the parasite. Little is known, however, concerning signal transmission to the parasite nucleus and cross-reaction with other parasite signalling systems. METHODOLOGY/PRINCIPAL FINDINGS: Using bioinformatic approaches, cloning, and yeast two-hybrid analyses we identified a novel mitogen-activated kinase (MAPK) cascade module that consists of E. multilocularis orthologs of the tyrosine kinase receptor interactor Growth factor receptor-bound 2, EmGrb2, the MAPK kinase kinase EmMEKK1, a novel MAPK kinase, EmMKK3, and a close homolog to c-Jun N-terminal kinase (JNK), EmMPK3. Whole mount in situ hybridization analyses indicated that EmMEKK1 and EmMPK3 are both expressed in E. multilocularis germinative (stem) cells but also in differentiated or differentiating cells. Treatment with the known JNK inhibitor SP600125 led to a significantly reduced formation of metacestode vesicles from stem cells and to a specific reduction of proliferating stem cells in mature metacestode vesicles. CONCLUSIONS/SIGNIFICANCE: We provide evidence for the expression of a MEKK1-JNK MAPK cascade module which, in mammals, is crucially involved in stress responses, cytoskeletal rearrangements, and apoptosis, in E. multilocularis stem cells. Inhibitor studies indicate an important role of JNK signalling in E. multilocularis stem cell survival and/or maintenance. Our data are relevant for molecular and cellular studies into crosstalk signalling mechanisms that govern Echinococcus stem cell function and introduce the JNK signalling cascade as a possible target of chemotherapeutics against echinococcosis.

Enzymatic characteristics and preventive effect of leucine aminopeptidase against Echinococcus multilocularis.

Alveolar echinococcosis, a parasitic zoonotic disease caused by the larval stage of Echinococcus multilocularis infection, is a global epidemic in Eurasia and North America. Leucine aminopeptidase (LAP) of the M17 peptidase family could act on an ideal target antigen in diagnosis and prevention of parasitic diseases (schistosomiasis, malaria, fascioliasis) because of its good immunogenicity. In this study, the bioinformatic and enzymatic characterizations of recombinant Echinococcus multilocularis LAP (rEm-LAP) were evaluated. A prokaryotic expression system for rEm-LAP protein was established and its immunogenicity and preventive efficacy were demonstrated in a BALB/c mice model. This is the first report about the LAP of Echinococcus multilocularis and with a 57.4 KD purified rEm-LAP protein successfully expressed by pCzn1-LAP in Escherichia coli BL-21 cells. Enzymatic analysis results showed optimal rEm-LAP activity at pH 9. Serum indirect ELISA demonstrated that rEm-LAP could induce a Th1 and Th2 mixed-type immunological response and produce high levels of IgG, IgG1, IgG2a, IgM, and IgA. Furthermore, serum IFN-γ and IL-4 secretion were increased compared with the control groups. Finally, vaccination with rEm-LAP significantly decreased both the number and size of the cysts in Echinococcus multilocularis metacestode infected mice model. The current study provides evidence that rEm-LAP could be a potential vaccine antigen of Echinococcus multilocularis.

Impairing the maintenance of germinative cells in Echinococcus multilocularis by targeting Aurora kinase.

BACKGROUND: The tumor-like growth of the metacestode larvae of the tapeworm E. multilocularis causes human alveolar echinococcosis, a severe disease mainly affecting the liver. The germinative cells, a population of adult stem cells, are crucial for the larval growth and development of the parasite within the hosts. Maintenance of the germinative cell pools relies on their abilities of extensive proliferation and self-renewal, which requires accurate control of the cell division cycle. Targeting regulators of the cell division progression may impair germinative cell populations, leading to impeded parasite growth. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we describe the characterization of EmAURKA and EmAURKB, which display significant similarity to the members of Aurora kinases that are essential mitotic kinases and play key roles in cell division. Our data suggest that EmAURKA and EmAURKB are actively expressed in the germinative cells of E. multilocularis. Treatment with low concentrations of MLN8237, a dual inhibitor of Aurora A and B, resulted in chromosomal defects in the germinative cells during mitosis, while higher concentrations of MLN8237 caused a failure in cytokinesis of the germinative cells, leading to multinucleated cells. Inhibition of the activities of Aurora kinases eventually resulted in depletion of the germinative cell populations in E. multilocularis, which in turn caused larval growth inhibition of the parasite. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate the vital roles of Aurora kinases in the regulation of mitotic progression and maintenance of the germinative cells in E. multilocularis, and suggest Aurora kinases as promising druggable targets for the development of novel chemotherapeutics against human alveolar echinococcosis.

Molecular and functional characterization of glucose transporter genes of the fox tapeworm Echinococcus multilocularis.

Alveolar echinococcosis (AE) is a zoonotic parasitosis caused by larvae of the fox tapeworm, Echinococcus multilocularis. E. multilocularis is distributed widely in the Northern hemisphere, causing serious health problems in various animals and humans. E. multilocularis, like other cestodes, lacks a digestive tract and absorbs essential nutrients, including glucose, across the syncytial tegument on its external surface. Therefore, it is hypothesized that E. multilocularis uses glucose transporters on its surface similar to a closely-related species, Taenia solium. Based on this hypothesis, we cloned and characterized glucose transporter homologues from E. multilocularis. As a result, we obtained full-length sequences of 2 putative glucose transporter genes (EmGLUT1 and EmGLUT2) from E. multilocularis. In silico analysis predicted that these were classified in the solute carrier family 2 group. Functional expression analysis using Xenopus oocytes demonstrated clear uptake of 2-deoxy-D-glucose (2-DG) by EmGLUT1, but not by EmGLUT2 in this experimental system. EmGLUT1 was shown to have relatively high glucose transport activity. Further analyses using the Xenopus oocyte system revealed that 2-DG uptake of EmGLUT1 did not depend on the presence or concentration of Na+ nor H+, respectively. Immunoblot analyses using cultured metacestode, ex vivo protoscolex, and adult worm samples demonstrated that both EmGLUTs were stably expressed during each developmental stage of the parasite. Based on the above-mentioned findings, we conclude that EmGLUT1 is a simple facilitated glucose transporter and possibly plays an important role in glucose uptake by E. multilocularis throughout its life cycle.

Suppression of nemo-like kinase by miR-71 in Echinococcus multilocularis.

Echinococcus multilocularis metacestodes are a causative pathogen for alveolar echinococcosis in human beings, and have been found to express miRNAs including emu-miR-71. miR-71 is evolutionarily conserved and highly expressed across platyhelminths, but little is known about its role. Here it was shown that emu-miR-71 was differentially expressed in protoscoleces and was unlikely to be expressed in neoblasts. The results of the luciferase assay indicated that emu-miR-71 was able to bind in vitro to the 3'-UTR of emu-nlk, encoding a key regulator of cell division, causing significant downregulation of luciferase activity (p < 0.01) compared to the negative control and the construct with mutations in the binding site. Consistent with the decreased luciferase activity, transfection of emu-miR-71 mimics into protoscoleces notably repressed emu-NLK (p < 0.05). These results demonstrate the suppression of emu-nlk by emu-miR-71, potentially involved in the protoscolex development.

Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI) assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ), a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC50 of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors.

Development of three PCR assays for the differentiation between Echinococcus shiquicus, E. granulosus (G1 genotype), and E. multilocularis DNA in the co-endemic region of Qinghai-Tibet plateau, China.

To investigate echinococcosis in co-endemic regions, three polymerase chain reaction (PCR) assays based on the amplification of a fragment within the NADH dehydrogenase subunit 1 (ND1) mitochondrial gene were optimized for the detection of Echinococcus shiquicus, Echinococcus granulosus G1, and Echinococcus multilocularis DNA derived from parasite tissue or canid fecal samples. Specificity using parasite tissue-derived DNA was found to be 100% except for E. shiquicus primers that faintly detected E. equinus DNA. Sensitivity of the three assays for DNA detection was between 2 and 10 pg. Ethanol precipitation of negative PCR fecal samples was used to eliminate false negatives and served to increase sensitivity as exemplified by an increase in detection from 0% to 89% of E. shiquicus coproDNA using necropsy-positive fox samples.

In vitro efficacy of the anticancer drug imatinib on Echinococcus multilocularis larvae.

The metacestode stage of Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), a lethal zoonosis with very limited treatment options. Chemotherapy of AE currently employs benzimidazoles (BZs); however, these exert only a parasitostatic action in vivo and have to be given life-long. In the search for novel drug targets, we have concentrated on parasite signalling pathways. Here we report significant antiparasitic effects of imatinib, an ABL kinase inhibitor that is in clinical use to treat certain cancers. At concentrations of 25 µM, imatinib was highly effective in killing Echinococcus stem cells, metacestode vesicles and protoscoleces in vitro. Moreover, already at concentrations as low as 10 µM, imatinib significantly inhibited the formation of metacestode vesicles from parasite stem cells, inactivated 50% of vesicles after 7 days, and induced morphological alterations in the metacestode upon short-term treatment. We also demonstrate that E. multilocularis larvae express enzymes with high homology to previously identified ABL-like kinases that act as imatinib targets in Schistosoma mansoni. In particular, amino acids known to mediate the binding of imatinib to target kinases are well conserved between human and Echinococcus ABL kinases. Taken together, these data demonstrate effective inactivation of Echinococcus larvae using imatinib concentrations that do not significantly affect cultivated human cells, indicating that imatinib might be a promising alternative to BZs in anti-AE chemotherapy. Furthermore, imatinib can also act as a lead substance for the identification of related compounds with higher antiparasitic activity, the identification of which will be facilitated by the Echinococcus ABL kinase sequences determined in this study.

Fructose-bisphosphate aldolase and enolase from Echinococcus granulosus: genes, expression patterns and protein interactions of two potential moonlighting proteins.

Glycolytic enzymes, such as fructose-bisphosphate aldolase (FBA) and enolase, have been described as complex multifunctional proteins that may perform non-glycolytic moonlighting functions, but little is known about such functions, especially in parasites. We have carried out in silico genomic searches in order to identify FBA and enolase coding sequences in Echinococcus granulosus, the causative agent of cystic hydatid disease. Four FBA genes and 3 enolase genes were found, and their sequences and exon-intron structures were characterized and compared to those of their orthologs in Echinococcus multilocularis, the causative agent of alveolar hydatid disease. To gather evidence of possible non-glycolytic functions, the expression profile of FBA and enolase isoforms detected in the E. granulosus pathogenic larval form (hydatid cyst) (EgFBA1 and EgEno1) was assessed. Using specific antibodies, EgFBA1 and EgEno1 were detected in protoscolex and germinal layer cells, as expected, but they were also found in the hydatid fluid, which contains parasite's excretory-secretory (ES) products. Besides, both proteins were found in protoscolex tegument and in vitro ES products, further suggesting possible non-glycolytic functions in the host-parasite interface. EgFBA1 modeled 3D structure predicted a F-actin binding site, and the ability of EgFBA1 to bind actin was confirmed experimentally, which was taken as an additional evidence of FBA multifunctionality in E. granulosus. Overall, our results represent the first experimental evidences of alternative functions performed by glycolytic enzymes in E. granulosus and provide relevant information for the understanding of their roles in host-parasite interplay.

Identification of novel glutathione transferases in Echinococcus granulosus. An evolutionary perspective.

Glutathione transferase enzymes (GSTs) constitute a major detoxification system in helminth parasites and have been related to the modulation of host immune response mechanisms. At least three different GSTs classes have been described in Platyhelminthes: Mu, Sigma and Omega. Mining the genome of Echinococcus multilocularis and the ESTs databases of Taenia solium and E. granulosus identified two new GSTs from the cestode E. granulosus, named EgGST2 and EgGST3. It also revealed that the Omega class of GSTs was absent from the Taenidae family. EgGST2 and EgGST3 are actively expressed in the parasite. In order to know the origin of these new GSTs, in silico analyses were performed. While EgGST2 is classified as belonging to the Sigma class, the data obtained for EgGST3 allowed a less clear interpretation. The study of the evolutionary relatedness based on the C-terminal domain sequence, gene structure conservation and three-dimensional structure predictions, suggests that EgGST3 is derived from the Platyhelminthes' Sigma-class cluster. Interestingly, the N-terminal domain displays some characteristic Omega-class residues, including a Cys residue that is likely to be involved in the catalytic mechanism. We discuss different evolutionary scenarios that could explain the observed patterns.

Echinococcus multilocularis: identification and functional characterization of cathepsin B-like peptidases from metacestode.

Cysteine peptidases have potent activities in the pathogenesis of various parasitic infections, and are considered as targets for chemotherapy and antigens for vaccine. In this study, two cathepsin B-like cysteine peptidases (EmCBP1 and EmCBP2) from Echinococcus multilocularis metacestodes were identified and characterized. Immunoblot analyses demonstrated that EmCBP1 and EmCBP2 were present in excretory/secretory products and extracts of E. multilocularis metacestodes. By immunohistochemistry, EmCBP1 and EmCBP2 were shown to localize to the germinal layer, the brood capsule and the protoscolex. Recombinant EmCBP1 and EmCBP2 expressed in Pichia pastoris, at optimum pH 5.5, exhibited substrate preferences for Z-Phe-Arg-MCA, Z-Val-Val-Arg-MCA, and Z-Leu-Arg-MCA, and low levels of hydrolysis of Z-Arg-Arg-MCA. Furthermore, recombinant enzymes degraded IgG, albumin, type I and IV collagens, and fibronectin. These results suggested that EmCBP1 and EmCBP2 may play key roles in protein digestion for parasites' nutrition and in parasite-host interactions.

Echinococcus multilocularis phosphoglucose isomerase (EmPGI): a glycolytic enzyme involved in metacestode growth and parasite-host cell interactions.

In Echinococcus multilocularis metacestodes, the surface-associated and highly glycosylated laminated layer, and molecules associated with this structure, is believed to be involved in modulating the host-parasite interface. We report on the molecular and functional characterisation of E. multilocularis phosphoglucose isomerase (EmPGI), which is a component of this laminated layer. The EmPGI amino acid sequence is virtually identical to that of its homologue in Echinococcus granulosus, and shares 64% identity and 86% similarity with human PGI. Mammalian PGI is a multi-functional protein which, besides its glycolytic function, can also act as a cytokine, growth factor and inducer of angiogenesis, and plays a role in tumour growth, development and metastasis formation. Recombinant EmPGI (recEmPGI) is also functionally active as a glycolytic enzyme and was found to be present, besides the laminated layer, in vesicle fluid and in germinal layer cell extracts. EmPGI is released from metacestodes and induces a humoral immune response in experimentally infected mice, and vaccination of mice with recEmPGI renders these mice more resistant towards secondary challenge infection, indicating that EmPGI plays an important role in parasite development and/or in modulating the host-parasite relationship. We show that recEmPGI stimulates the growth of isolated E. multilocularis germinal layer cells in vitro and selectively stimulates the proliferation of bovine adrenal cortex endothelial cells but not of human fibroblasts and rat hepatocytes. Thus, besides its role in glycolysis, EmPGI could also act as a factor that stimulates parasite growth and potentially induces the formation of novel blood vessels around the developing metacestode in vivo.

Molecular characterisation of MEK1/2- and MKK3/6-like mitogen-activated protein kinase kinases (MAPKK) from the fox tapeworm Echinococcus multilocularis.

Mitogen-activated protein kinase kinases (MAPKKs) are essential components of evolutionary conserved signalling modules that regulate a variety of fundamental cellular processes in response to environmental stimuli. To date, no MAPKK ortholog has been characterised in free-living or parasitic flatworm species. Here, we report the identification and molecular characterisation of two such molecules in the human parasitic cestode Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Using degenerative PCR approaches as well as 3'- and 5'-rapid amplification of cDNA ends (RACE), the cDNAs encoding two different E. multilocularis MAPKKs, EmMKK1 and EmMKK2, have been identified and fully cloned. Structurally, EmMKK1 and EmMKK2 closely resemble members of the MKK3/6- and the MEK1/2-MAPKK sub-families, respectively, from a variety of vertebrate and invertebrate organisms, and contain all catalytically important residues of MAPKKs at the corresponding positions. By reverse transcriptase-PCR analyses, expression of the EmMKK2-encoding gene, emmkk2, was observed in the larval stages, metacestode and protoscolex while emmkk1 displayed a protoscolex-specific expression pattern. In yeast two-hybrid analyses, EmMKK1 strongly interacted with the previously identified Echinococcus MAPKK kinase EmRaf but not with the Erk-like MAP kinase EmMPK1 or the p38-like MAP kinase EmMPK2. EmMKK2, on the other hand, not only interacted with EmRaf and a member of the parasite's 14-3-3 protein family, but also with EmMPK1, which was confirmed by co-immunoprecipitation assays. Incubation of in vitro cultivated metacestode vesicles with small-molecule inhibitors of Raf- and MEK-kinases resulted in a marked de-phosphorylation of EmMPK1 and negatively affected parasite growth, but was ineffective in vesicle killing. Taken together, our results define EmRaf, EmMKK2 and EmMPK1 as the three components of the Erk-like E. multilocularis MAPK cascade module and provide a solid basis for further investigations into the role of Erk-like MAPK signalling in parasite development and stem cell function.

Echinococcus multilocularis metacestode metabolites contain a cysteine protease that digests eotaxin, a CC pro-inflammatory chemokine.

In many helminthic infections, eotaxin, a CC-chemokine, triggers the mobilization of eosinophils, thus, contributing to an elevated blood and periparasitic eosinophil level. Following an experimental intraperitoneal infection of C57BL6 mice with Echinococcus multilocularis metacestodes, however, we observed the absence of eosinophils in the peritoneal cavity and a low number of such cells in the blood of infected animals. Therefore, we carried out an explorative study to address the question why eosinophilia did not occur especially in the peritoneal cavity of such secondarily AE-infected mice. In an in vitro assay, we showed that metacestode antigens (in vitro generated vesicle fluid and E/S products) were able to proteolytically digest eotaxin. This effect was confirmed with semiquantitative Western blotting, which demonstrated a decreasing intensity of remaining eotaxin signals. Proteolysis of eotaxin was, thus, dose-dependent and proportional to the time of incubation with the metacestode antigens. Using appropriate inhibitors, the respective protease was identified as a cysteine protease, which required the presence of Ca(++) as co-enzyme. A chromatographic fractionation procedure by successive separation of VF molecules using a superpose column and subsequently a MonoQ column mounted on an FPLC system allowed to yield a fraction, referred to us as fraction 6; containing the enriched cysteine protease, this fraction will be used for further molecular studies. Eotaxin inactivation by VF and E/S products may contribute to explain the absence of eosinophils within the peritoneal cavity of AE-secondary infected mice. Absent eosinophils, thus, may be a part of a series of events that maintain a low level of inflammation displayed within the peritoneal cavity of experimentally infected mice.

Characterization and inhibition of a p38-like mitogen-activated protein kinase (MAPK) from Echinococcus multilocularis: antiparasitic activities of p38 MAPK inhibitors.

Alveolar echinococcosis (AE), caused by the metacestode larval stage of the fox-tapeworm Echinococcus multilocularis, is a life-threatening disease with very limited treatment options. In search for novel drug targets, we concentrate on factors of the cellular signaling machinery and report herein the characterization of a novel gene, Emmpk2, which is expressed in the parasite's larval stage and which codes for a member of the mitogen-activated protein kinase (MAPK) family. On the amino acid sequence level, the encoded protein, EmMPK2, shares considerable homologies with p38 MAPKs from a wide variety of animal organisms but also displays several distinct differences, particularly in amino acid residues known to be involved in the regulation of enzyme activity. Upon heterologous expression in Escherichia coli, purified EmMPK2 showed prominent autophosphorylation activity and strongly elevated basal activity towards a MAPK substrate, when compared to the closest human orthologue, p38-alpha. EmMPK2 activity could be effectively inhibited in the presence of ML3403 and SB202190, two ATP-competitive pyridinyl imidazole inhibitors of p38 MAPKs, in a concentration-dependent manner. When added to in vitro cultivated metacestode vesicles, SB202190 and particularly ML3403 led to dephosphorylation of EmMPK2 in the parasite and effectively killed parasite vesicles at concentrations that did not affect cultivated mammalian cells. Taken together, these results identify pyridinyl imidazoles as a novel class of anti-Echinococcus compounds and EmMPK2 as a promising target for the development of drugs against alveolar echinococcosis.

Anaerobic NADH-fumarate reductase system is predominant in the respiratory chain of Echinococcus multilocularis, providing a novel target for the chemotherapy of alveolar echinococcosis.

Alveolar echinococcosis, which is due to the massive growth of larval Echinococcus multilocularis, is a life-threatening parasitic zoonosis distributed widely across the northern hemisphere. Commercially available chemotherapeutic compounds have parasitostatic but not parasitocidal effects. Parasitic organisms use various energy metabolic pathways that differ greatly from those of their hosts and therefore could be promising targets for chemotherapy. The aim of this study was to characterize the mitochondrial respiratory chain of E. multilocularis, with the eventual goal of developing novel antiechinococcal compounds. Enzymatic analyses using enriched mitochondrial fractions from E. multilocularis protoscoleces revealed that the mitochondria exhibited NADH-fumarate reductase activity as the predominant enzyme activity, suggesting that the mitochondrial respiratory system of the parasite is highly adapted to anaerobic environments. High-performance liquid chromatography-mass spectrometry revealed that the primary quinone of the parasite mitochondria was rhodoquinone-10, which is commonly used as an electron mediator in anaerobic respiration by the NADH-fumarate reductase system of other eukaryotes. This also suggests that the mitochondria of E. multilocularis protoscoleces possess an anaerobic respiratory chain in which complex II of the parasite functions as a rhodoquinol-fumarate reductase. Furthermore, in vitro treatment assays using respiratory chain inhibitors against the NADH-quinone reductase activity of mitochondrial complex I demonstrated that they had a potent ability to kill protoscoleces. These results suggest that the mitochondrial respiratory chain of the parasite is a promising target for chemotherapy of alveolar echinococcosis.

Biochemical properties of an intracellular serpin from Echinococcus multilocularis.

A serpin of the intracellular type from the tapeworm Echinococcus multilocularis was expressed in Escherichia coli, purified by ion exchange chromatography and tested for inhibitory activity against several proteinases. The recombinant protein, which after transcriptional induction, represents about 20 % of total cellular protein, is biochemically active and inhibits trypsin and the trypsin-like plasmin as well as pig pancreatic and human neutrophil elastase. Implications regarding its biochemistry and biological function are discussed.

Cloning and characterization of cathepsin L-like peptidases of Echinococcus multilocularis metacestodes.

Cysteine peptidases have potent activities in the pathogenesis of various parasitic infections. Two cDNA clones encoding cysteine peptidases were isolated from Echinococcus multilocularis metacestode (EmCLP1 and EmCLP2). EmCLP1 and EmCLP2 shared high similarity to the cathepsin L-like peptidases. Immunoblot analyses demonstrated that native EmCLP1 and EmCLP2 were present in excretory/secretory products and extracts of E. multilocularis metacestodes. By immunohistochemistry, native EmCLP1 and EmCLP2 were shown to localize to the germinal layer, the brood capsule and the protoscolex. Recombinant EmCLP1 and EmCLP2 expressed in Saccharomyces cerevisiae exhibited substrate specificity against synthetic peptidyl substrates, Z-Leu-Arg-MCA and Z-Phe-Arg-MCA. Furthermore, recombinant enzymes degraded IgG, albumin, type I and IV collagens, and fibronectin, which suggested those key roles in parasite-host interactions. This is the first report of cysteine peptidases from E. multilocularis, and would contribute to control E. multilocularis infections by chemotherapeutic drugs and/or immunoprophylaxis.

Genetic uniformity of Echinococcus multilocularis collected from different intermediate host species in Hokkaido, Japan.

DNA from several isolates of Taenia taeniaeformis and Echinococcus multilocularis were digested with restriction enzymes and hybridized with digoxigenated oligonucleotide probe (CAC)5. Within the six wild isolates of Taenia taeniaeformis from Norway rats in Hokkaido, although several bands were common among isolates, fingerprinting patterns were specific to each isolate. In the case of E. multilocularis, regardless of hosts from which each isolate has been isolated, the five isolates collected from Hokkaido, showed the same fingerprinting pattern. These results indicate that there was very little genetic difference among these isolates. Although the fingerprinting pattern of E. multilocularis from St. Lawrence Is. was similar to that of the Hokkaido isolates, some bands were different from those in the Hokkaido isolates. Echinococcus multilocularis in Hokkaido seems to be closely-related genetically to that from St. Lawrence Is.

In vitro metacestodicidal activities of genistein and other isoflavones against Echinococcus multilocularis and Echinococcus granulosus.

Echinococcus multilocularis and Echinococcus granulosus metacestode infections in humans cause alveolar echinococcosis and cystic echinococcosis, respectively, in which metacestode development in visceral organs often results in particular organ failure. Further, cystic hydatidosis in farm animals causes severe economic losses. Although benzimidazole derivatives such as mebendazole and albendazole are being used as therapeutic agents, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of a number of isoflavones against Echinococcus metacestodes and protoscoleces. The most prominent isoflavone, genistein, exhibits significant metacestodicidal activity in vitro. However, genistein binds to the estrogen receptor and can thus induce estrogenic effects, which is a major concern during long-term chemotherapy. We have therefore investigated the activities of a number of synthetic genistein derivatives carrying a modified estrogen receptor binding site. One of these, Rm6423, induced dramatic breakdown of the structural integrity of the metacestode germinal layer of both species within 5 to 7 days of in vitro treatment. Further, examination of the culture medium revealed increased leakage of parasite proteins into the medium during treatment, but zymography demonstrated a decrease in the activity of metalloproteases. Moreover, two of the genistein derivatives, Rm6423 and Rm6426, induced considerable damage in E. granulosus protoscoleces, rendering them nonviable. These findings demonstrate that synthetic isoflavones exhibit distinct in vitro effects on Echinococcus metacestodes and protoscoleces, which could potentially be exploited further for the development of novel chemotherapeutical tools against larval-stage Echinococcus infection.