Transcriptional effects of electroporation on Echinococcus multilocularis primary cell culture.

Echinococcus multilocularis is the etiological agent of alveolar echinococcosis (AE), a serious parasitic disease in the Northern Hemisphere. The E. multilocularis primary cell cultivation system, together with E. multilocularis genome data and a range of pioneering molecular-based tools have advanced the research on this and other cestodes. RNA interference (RNAi) and microRNA knock-down have recently contributed to the study of the cellular and molecular basis of tapeworm development and host-parasite interaction. These, as well as other techniques, normally involve an electroporation step for the delivery of RNA, DNA, peptides, and small molecules into cells. Using transcriptome data and bioinformatic analyses, we herein report a genome-wide comparison between primary cells of E. multilocularis and primary cells under electroporated conditions after 48 h of culture. We observed that ~ 15% of genes showed a significant variation in expression level, including highly upregulated genes in electroporated cells, putatively involved in detoxification and membrane remodeling. Furthermore, we found genes related to carbohydrate metabolism, proteolysis, calcium ion binding and microtubule processing significantly altered, which could explain the cellular dispersion and the reduced formation of cellular aggregates observed during the first 48 h after electroporation.

Molecular characterization of Echinococcus granulosus isolates from Bulgarian human cystic echinococcosis patients.

Although cystic echinococcosis (CE) is highly endemic in Bulgaria, there is still scarce information about species and/or genotypes of the Echinococcus granulosus complex that infect humans. Our study tackled the genetic diversity of E. granulosus complex in a cohort of 30 Bulgarian CE patients. Ten animal E. granulosus isolates from neighboring Greece were additionally included. Specimens were comparatively analyzed for partial sequences of five mitochondrial (mt) (cox I, nad I, rrnS, rrnL, and atp6) and three nuclear (nc) genes (act II, hbx 2, and ef-1α) using a PCR-sequencing approach. All 30 Bulgarian isolates were identified as E. granulosus sensu stricto (s.s.) and were showing identical sequences for each of the three examined partial nc gene markers. Based upon concatenated sequences from partial mtDNA markers, we detected 10 haplotypes: 6 haplotypes (H1-H6) clustering with E. granulosus s.s. (G1) and 4 haplotypes (H9-H13) grouping with E. granulosus s.s. (G3), with H1 and H10 being the most frequent in Bulgarian patients. The haplotypes H1, H4, and H11 were also present in Greek hydatid cyst samples of animal origin. In conclusion, E. granulosus s.s. (G1 and G3 genotypes) is the only causative agent found so far to cause human CE in Bulgaria. However, further studies including larger sample sizes and other additional geographic regions in Bulgaria will have to be performed to confirm our results.

A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa.

Reliable and rapid molecular tools for the genetic identification and differentiation of Echinococcus species and/or genotypes are crucial for studying spatial and temporal transmission dynamics. Here, we describe a novel dual PCR targeting regions in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rRNA) genes, which enables (i) the specific identification of species and genotypes of Echinococcus (rrnS + L-PCR) and/or (ii) the identification of a range of taeniid cestodes, including different species of Echinococcus, Taenia and some others (17 species of diphyllidean helminths). This dual PCR approach was highly sensitive, with an analytical detection limit of 1 pg for genomic DNA of Echinococcus. Using concatenated sequence data derived from the two gene markers (1225 bp), we identified five unique and geographically informative single nucleotide polymorphisms (SNPs) that allowed genotypes (G1 and G3) of Echinococcus granulosus sensu stricto to be distinguished, and 25 SNPs that allowed differentiation within Echinococcus canadensis (G6/7/8/10). In conclusion, we propose that this dual PCR-based sequencing approach can be used for molecular epidemiological studies of Echinococcus and other taeniid cestodes.

Comparison of ex vivo harvested and in vitro cultured materials from Echinococcus granulosus by measuring expression levels of five genes putatively involved in the development and maturation of adult worms.

Parts of the natural life cycle of Echinococcus granulosus can be retraced in vitro such as the development of protoscoleces into semiadult worms with three or more proglottids, or the redifferentiation of in vitro cultured protoscoleces into metacestode-like cystic structures. Most in vitro generated samples share-at the microscopical level-high similarities with those naturally grown, but developmental differences have also been documented, such as missing egg production in in vitro grown adults or unusual bladder/vesicle formation in protoscoleces cultured into the metacestode direction. The aim of the present study was to explore how far different in vitro generated stage-specific materials/structures match the natural situation on the transcriptome level, based on testing five exemplarily chosen different genes: the frizzled receptor eg-fz4 (posterior marker), the FGF receptor-like factor eg-fgfrl (anterior association), the cell differentiation protein eg-rcd1 (part of the CCR4-NOT complex, a key regulator of eukaryotic gene expression), the rapidly accelerated fibrosarcoma serin/threonin kinase eg-braf (part of the MAPK pathway involved, e.g., in EGF signaling) and the co-smad eg-smadD (downstream factor of TGFß/BMP2/activin signaling). These genes-tested via qPCR-were selected such as to allow a discussion on their potential role in the development of E. granulosus into the adult stage. Thus, testing took place with three ex vivo isolated samples, namely (i) egg-containing adult worms, (ii) invaginated protoscoleces, and (iii) protoscolex-free germinal layer tissue. Respective data were compared (a) with in vitro generated metacestode-like microcysts developed from protoscolices, and (b) different development stages of protoscoleces in vitro cultured toward adult maturation. As a finding, only eg-smadD and partially eg-fz4 showed high expression similarities between ex vivo harvested and in vitro cultured E. granulosus, thus suggesting a putative role in adult maturation. Conclusively, the fact of using "only" five genes did not allow answering the question if ex vivo and in vitro materials are similar on the transcriptome level. Another experimental restriction arises from different growth conditions of the in vitro cultured materials, and comparing these to the ex vivo harvested ones. Future experiments may solve the problems by using fully standardized E. granulosus sample collection and fully standardized culture conditions.

Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development.

BACKGROUND: The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host's liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood. RESULTS: Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite's glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin. CONCLUSIONS: Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs.

Genome-wide transcriptome analysis of Echinococcus multilocularis larvae and germinative cell cultures reveals genes involved in parasite stem cell function.

The lethal zoonosis alveolar echinococcosis is caused by tumour-like growth of the metacestode stage of the tapeworm Echinococcus multilocularis within host organs. We previously demonstrated that metacestode proliferation is exclusively driven by somatic stem cells (germinative cells), which are the only mitotically active parasite cells that give rise to all differentiated cell types. The Echinococcus gene repertoire required for germinative cell maintenance and differentiation has not been characterised so far. We herein carried out Illumina sequencing on cDNA from Echinococcus metacestode vesicles, from metacestode tissue depleted of germinative cells, and from Echinococcus primary cell cultures. We identified a set of ~1,180 genes associated with germinative cells, which contained numerous known stem cell markers alongside genes involved in replication, cell cycle regulation, mitosis, meiosis, epigenetic modification, and nucleotide metabolism. Interestingly, we also identified 44 stem cell associated transcription factors that are likely involved in regulating germinative cell differentiation and/or pluripotency. By in situ hybridization and pulse-chase experiments, we also found a new general Echinococcus stem cell marker, EmCIP2Ah, and we provide evidence implying the presence of a slow cycling stem cell sub-population expressing the extracellular matrix factor Emkal1. RNA-Seq analyses on primary cell cultures revealed that metacestode-derived Echinococcus stem cells display an expanded differentiation capability and do not only form differentiated cell types of the metacestode, but also cells expressing genes specific for protoscoleces, adult worms, and oncospheres, including an ortholog of the schistosome praziquantel target, EmTRPMPZQ. Finally, we show that primary cell cultures contain a cell population expressing an ortholog of the tumour necrosis factor α receptor family and that mammalian TNFα accelerates the development of metacestode vesicles from germinative cells. Taken together, our analyses provide a robust and comprehensive characterization of the Echinococcus germinative cell transcriptome, demonstrate expanded differentiation capability of metacestode derived stem cells, and underscore the potential of primary germinative cell cultures to investigate developmental processes of the parasite. These data are relevant for studies into the role of Echinococcus stem cells in parasite development and will facilitate the design of anti-parasitic drugs that specifically act on the parasite germinative cell compartment.

Transforming growth factor-ß signalling regulates protoscolex formation in the Echinococcus multilocularis metacestode.

The lethal zoonosis alveolar echinococcosis (AE) is caused by tumor-like, infiltrative growth of the metacestode larval stage of the tapeworm Echinococcus multilocularis. We previously showed that the metacestode is composed of posteriorized tissue and that the production of the subsequent larval stage, the protoscolex, depends on re-establishment of anterior identities within the metacestode germinative layer. It is, however, unclear so far how protoscolex differentiation in Echinococcus is regulated. We herein characterized the full complement of E. multilocularis TGFß/BMP receptors, which is composed of one type II and three type I receptor serine/threonine kinases. Functional analyzes showed that all Echinococcus TGFß/BMP receptors are enzymatically active and respond to host derived TGFß/BMP ligands for activating downstream Smad transcription factors. In situ hybridization experiments demonstrated that the Echinococcus TGFß/BMP receptors are mainly expressed by nerve and muscle cells within the germinative layer and in developing brood capsules. Interestingly, the production of brood capsules, which later give rise to protoscoleces, was strongly suppressed in the presence of inhibitors directed against TGFß/BMP receptors, whereas protoscolex differentiation was accelerated in response to host BMP2 and TGFß. Apart from being responsive to host TGFß/BMP ligands, protoscolex production also correlated with the expression of a parasite-derived TGFß-like ligand, EmACT, which is expressed in early brood capsules and which is strongly expressed in anterior domains during protoscolex development. Taken together, these data indicate an important role of TGFß/BMP signalling in Echinococcus anterior pole formation and protoscolex development. Since TGFß is accumulating around metacestode lesions at later stages of the infection, the host immune response could thus serve as a signal by which the parasite senses the time point at which protoscoleces must be produced. Overall, our data shed new light on molecular mechanisms of host-parasite interaction during AE and are relevant for the development of novel treatment strategies.

Serological Assays for Alveolar and Cystic Echinococcosis-A Comparative Multi-Test Study in Switzerland and Kyrgyzstan.

Both alveolar (AE) and cystic echinococcosis (CE) are lacking pathognomonic clinical signs; consequently imaging technologies and serology remain the main pillars for diagnosis. The present study included 100 confirmed treatment-naïve AE and 64 CE patients that were diagnosed in Switzerland or Kyrgyzstan. Overall, 10 native Echinococcus spp. antigens, 3 recombinant antigens, and 4 commercial assays were comparatively evaluated. All native E. multilocularis antigens were produced in duplicates with a European and a Kyrgyz isolate and showed identical test values for the diagnosis of AE and CE. Native antigens and three commercial tests showed high diagnostic sensitivities (Se: 86-96%) and specificities (Sp: 96-99%) for the diagnosis of AE and CE in Swiss patients. In Kyrgyz patients, values of sensitivities and specificities were 10-20% lower as compared to the Swiss patients' findings. For the sero-diagnosis of AE in Kyrgyzstan, a test-combination of an E. multilocularis protoscolex antigen and the recombinant antigen Em95 appears to be the most suitable test strategy (Se: 98%, Sp: 87%). For the diagnosis of CE in both countries, test performances were hampered by major cross-reactions with AE patients and other parasitic diseases as well as by limited diagnostic sensitivities (93% in Switzerland and 76% in Kyrgyzstan, respectively).

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.

Regulation of hepatic microRNAs in response to early stage Echinococcus multilocularis egg infection in C57BL/6 mice.

We present a comprehensive analysis of the hepatic miRNA transcriptome at one month post-infection of experimental primary alveolar echinococcosis (AE), a parasitic infection caused upon ingestion of E. multilocularis eggs. Liver tissues were collected from infected and non-infected C57BL/6 mice, then small RNA libraries were prepared for next-generation sequencing (NGS). We conducted a Stem-loop RT-qPCR for validation of most dysregulated miRNAs. In infected mice, the expression levels of 28 miRNAs were significantly altered. Of these, 9 were up-regulated (fold change (FC) ≥ 1.5) and 19 were down-regulated (FC ≤ 0.66) as compared to the non-infected controls. In infected livers, mmu-miR-148a-3p and mmu-miR-101b-3p were 8- and 6-fold down-regulated, respectively, and the expression of mmu-miR-22-3p was reduced by 50%, compared to non-infected liver tissue. Conversely, significantly higher hepatic levels were noted for Mus musculus (mmu)-miR-21a-5p (FC = 2.3) and mmu-miR-122-5p (FC = 1.8). In addition, the relative mRNA expression levels of five genes (vegfa, mtor, hif1-α, fasn and acsl1) that were identified as targets of down-regulated miRNAs were significantly enhanced. All the five genes exhibited a higher expression level in livers of E. multilocularis infected mice compared to non-infected mice. Finally, we studied the issue related to functionally mature arm selection preference (5p and/or 3p) from the miRNA precursor and showed that 9 pre-miRNAs exhibited different arm selection preferences in normal versus infected liver tissues. In conclusion, this study provides first evidence that miRNAs are regulated early in primary murine AE. Our findings raise intriguing questions such as (i) how E. multilocularis affects hepatic miRNA expression;(ii) what are the alterations in miRNA expression patterns in more advanced AE-stages; and (iii) which hepatic cellular, metabolic and/or immunologic processes are modulated through altered miRNAs in AE. Thus, further research on the regulation of miRNAs during AE is needed, since miRNAs constitute an attractive potential option for development of novel therapeutic approaches against AE.

Axenic in vitro cultivation of Echinococcus multilocularis metacestode vesicles and the generation of primary cell cultures.

Parasitic helminths are a major cause of disease worldwide, yet the molecular mechanisms of host-helminth interaction and parasite development are only rudimentarily studied. A main reasons for this lack of knowledge are the tremendous experimental difficulties in cultivating parasitic helminths under defined laboratory conditions and obtaining sufficient amounts of parasite material for molecular analyses. For one member of this neglected group of pathogens, the fox-tapeworm Echinococcus multilocularis, we have established and optimized in vitro cultivation systems by which the major part of the parasite's life cycle, leading from early metacestode vesicles to the production of protoscoleces, can be mimicked under laboratory conditions. The methodology comprises co-cultivation systems for host cells and parasite larvae by which large amounts of parasite vesicles can be generated. Furthermore, we have established an axenic (host cell-free) cultivation system that allows studies on the influence of defined host factors on parasite growth and development. On the basis of this system, the isolation and maintenance of primary Echinococcus cells that are devoid of overgrowing host cells is now possible. The availability of the primary cell culture system constitutes a first step toward the establishment of genetic manipulation methods for the parasite that will be of great interest for further research on infection strategies and development of Echinococcus and other cestodes.

Deciphering the role of miR-71 in Echinococcus multilocularis early development in vitro.

Echinococcosis represents a major public health problem worldwide and is considered a neglected disease by the World Health Organization. The etiological agents are Echinococcus tapeworms, which display elaborate developmental traits that imply a complex control of gene expression. MicroRNAs (miRNAs), a class of small regulatory RNAs, are involved in the regulation of many biological processes such as development and metabolism. They act through the repression of messenger RNAs (mRNAs) usually by binding to the 3' untranslated region (3'UTR). Previously, we described the miRNome of several Echinococcus species and found that miRNAs are highly expressed in all life cycle stages, suggesting an important role in gene expression regulation. However, studying the role of miRNAs in helminth biology remains a challenge. To develop methodology for functional analysis of miRNAs in tapeworms, we performed miRNA knockdown experiments in primary cell cultures of Echinococcus multilocularis, which mimic the development of metacestode vesicles from parasite stem cells in vitro. First, we analysed the miRNA repertoire of E. multilocularis primary cells by small RNA-seq and found that miR-71, a bilaterian miRNA absent in vertebrate hosts, is one of the top five most expressed miRNAs. Using genomic information and bioinformatic algorithms for miRNA binding prediction, we found a high number of potential miR-71 targets in E. multilocularis. Inhibition of miRNAs can be achieved by transfection of antisense oligonucleotides (anti-miRs) that block miRNA function. To this end, we evaluated a variety of chemically modified anti-miRs for miR-71 knockdown. Electroporation of primary cells with 2'-O-methyl modified anti-miR-71 led to significantly reduced miR-71 levels. Transcriptomic analyses showed that several predicted miR-71 targets were up-regulated in anti-miR-treated primary cells, including genes potentially involved in parasite development, host parasite interaction, and several genes of as yet unknown function. Notably, miR-71-silenced primary cell cultures showed a strikingly different phenotype from control cells and did not develop into fully mature metacestodes. These findings indicate an important function of miR-71 in Echinococcus development and provide, for the first time, methodology to functionally study miRNAs in a tapeworm.

Transient transfection of Echinococcus multilocularis primary cells and complete in vitro regeneration of metacestode vesicles.

A major limitation in studying molecular interactions between parasitic helminths and their hosts is the lack of suitable in vitro cultivation systems for helminth cells and larvae. Here we present a method for long-term in vitro cultivation of larval cells of the tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Primary cells isolated from cultivated metacestode vesicles in vitro showed a morphology typical of Echinococcus germinal cells, displayed an Echinococcus-specific gene expression profile and a cestode-like DNA content of approximately 300Mbp. When kept under reducing conditions in the presence of Echinococcus vesicle fluid, the primary cells could be maintained in vitro for several months and proliferated. Most interestingly, upon co-cultivation with host hepatocytes in a trans-well system, mitotically active Echinococcus cells formed cell aggregates that subsequently developed central cavities, surrounded by germinal cells. After 4 weeks, the cell aggregates gave rise to young metacestode vesicles lacking an outer laminated layer. This layer was formed after 6 weeks of cultivation indicating the complete in vitro regeneration of metacestode larvae. As an initial step toward the creation of a fully transgenic strain, we carried out transient transfection of Echinococcus primary cells using plasmids and obtained heterologous expression of a reporter gene. Furthermore, we successfully carried out targeted infection of Echinococcus cells with the facultatively intracellular bacterium Listeria monocytogenes, a DNA delivery system for genetic manipulation of mammalian cells. Taken together, the methods presented herein constitute important new tools for molecular investigations on host-parasite interactions in alveolar echinococcosis and on the roles of totipotent germinal cells in parasite regeneration and metastasis formation. Moreover, they enable the development of fully transgenic techniques in this group of helminth parasites for the first time.

Recent advances in the in vitro cultivation and genetic manipulation of Echinococcus multilocularis metacestodes and germinal cells.

In order to elucidate host-parasite interactions and infection strategies of helminths at the molecular level, the availability of suitable in vitro cultivation systems for this group of parasites is of vital importance. One of the few helminth systems for which in vitro cultivation has been relatively successfully carried out in the past is the larval stage of the fox-tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Respective 'first generation' cultivation systems relied on the co-incubation of larval tissue, isolated from laboratory rodents, with host feeder cells. Although these techniques have been very successful in producing metacestode material for drug screening assays or the establishment of cDNA libraries, the continuous presence of host cells prevented detailed studies on the influence of defined host factors on larval growth. To facilitate such investigations, we have recently introduced the first truly axenic system for long-term in vitro maintenance of metacestode vesicles and used it to establish a technique for parasite cell cultivation. The resulting culture system, which allows the complete in vitro regeneration of metacestode vesicles from germinal cells, is a highly useful tool to study the cellular and molecular basis of a variety of developmental processes that occur during the infection of the mammalian host. Furthermore, it provides a solid basis for establishing transgenic techniques in cestodes for the first time. We consider it an appropriate time point to discuss the characteristics of these 'second generation' cultivation systems in comparison with former techniques, to present our first successful attempts to introduce foreign DNA into Echinococcus cells, and to share our ideas on how a fully transgenic Echinococcus strain can be generated in the near future.

Characterisation of EmMPK1, an ERK-like MAP kinase from Echinococcus multilocularis which is activated in response to human epidermal growth factor.

Mitogen-activated protein (MAP) kinases are key regulators of cellular signalling systems that mediate responses to a wide variety of extracellular stimuli and should also play a central role in developmental mechanisms of parasitic helminths. Until now, however, no MAP kinase orthologue has been characterised in a member of this parasite group. Here, we report the identification and characterisation of such a molecule, EmMPK1, from the human parasitic cestode Echinococcus multilocularis. Using a degenerative PCR approach, we isolated and completely sequenced the 1.2kb cDNA for EmMPK1 which displays significant homologies to known MAP kinases of different phylogenetic origin. EmMPK1 contains all amino acid residues which are characteristic for MAP kinases, including a conserved TEY motif which identifies the protein as a member of the ERK subfamily of MAP kinases. The corresponding gene, emmpk1 (6.9 kb), was characterised and contained 10 introns. Southern blot hybridisation studies showed that emmpk1 is present as single copy locus in E. multilocularis. Using RT-PCR analyses we demonstrated that emmpk1 is expressed in form of three different transcripts which derive from alternative splice acceptor site utilisation at intron 9. Using EmMPK1-specific antibodies in Western blot studies and immunohistochemistry, we detected the Echinococcus protein and its phosphorylated form in the larval stages metacestode and protoscolex during in vitro cultivation and during an infection of the intermediate host. EmMPK1, immunoprecipitated from Echinococcus lysate, was able to phosphorylate myelin basic protein in activity assays, indicating that it is a functionally active MAP kinase. Finally, we also show that phosphorylation of EmMPK1 is specifically induced in vitro-cultivated E. multilocularis metacestode vesicles in response to exogenous host serum and upon addition of human epidermal growth factor. These data indicate that the E. multilocularis metacestode is able to sense epidermal growth factor from the host which results in an activation of the parasite's MAP kinase cascade.

The molecular mechanisms of larval cestode development: first steps into an unknown world.

Several hundred million years ago, the free-living ancestors of all extant helminth parasites decided to colonize entirely new habitats, the bodies of other metazoan animals. As a consequence of the resulting adaptation processes, they evolved highly complex life-cycles in which many developmental transitions were initiated and controlled by host-derived signals. Understanding the molecular basis of the original developmental mechanisms, and the modifications that occurred during co-evolution with the host, is not only fundamental to our understanding of parasitism but also highly relevant for the design of anti-parasitic drugs and vaccines. In the past several years, molecular investigations on parasitic nematode and trematode development have made considerable progress and, supported by respective genome sequencing projects and emerging methods of genetic manipulation, will be a flourishing field in the years to come. We consider it time that corresponding studies are also pushed for the third large group of parasitic helminths, the cestodes. Here, we review the first experimental steps into that area, which have been undertaken recently. We report on cestode genomics, the identification of signaling factors associated with larval development, and the establishment as well as improvement of in vitro cultivation systems by which cestode life-cycles can be studied in the laboratory.

Repeated Long-Term DT Application in the DEREG Mouse Induces a Neutralizing Anti-DT Antibody Response.

Regulatory T (Tregs) cells play an important role in mediating tolerance to self-antigens but can also mediate detrimental tolerance to tumours and pathogens in a Foxp3-dependent manner. Genetic tools exploiting the foxp3 locus including bacterial artificial chromosome- (BAC-) transgenic DEpletion of REGulatory T cells (DEREG) mice have provided essential information on Treg biology and the potential therapeutic modulation of tolerance. In DEREG mice, Foxp3+ Tregs selectively express enhanced green fluorescent protein (eGFP) and diphtheria toxin (DT) receptor, allowing for the specific depletion of Tregs through DT administration. We here provide a detailed overview about an important consideration that long-term administration of DT induces a humoral immune response with an appropriate production of anti-DT antibodies that can inactivate DT and thus abrogate its effect in the DEREG mouse. Additionally, we showed that anti-DT mouse serum partially neutralized DT-induced Foxp3 inhibition.

Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis.

The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 µM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 µM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 µM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load.

Molecular cloning and characterization of Ras- and Raf-homologues from the fox-tapeworm Echinococcus multilocularis.

To better understand growth regulation in the human parasitic cestode Echinococcus multilocularis, we have cloned and characterized the parasite's orthologues of the key regulatory factors Ras and Raf. Using a degenerative PCR approach a gene, emras, was identified whose gene product, EmRas, showed high homology (79% identical residues) to human Ras and contained all amino acid residues which are characteristic for this subfamily of small GTPases at the corresponding positions. Recombinantly expressed EmRas bound GTP and was farnesylated, but not geranyl-geranylated, by Echinococcus lysate in an in vitro prenylation assay. Furthermore, upon expression in yeast, emras was able to functionally complement the Saccharomyces orthologue RAS2 in an invasive growth assay. In Western blot analyses using an anti-EmRas antibody, the Echinococcus factor could be detected in lysates of the larval stages metacestode and protoscolex. By immune-histochemistry, EmRas was shown to localize to the germinal layer of the metacestode and to tegumental structures of the protoscolex, particularly around the rostellum and the sucker regions. In addition, we fully characterized the gene emraf whose product, EmRaf, displayed considerable homology to mammalian Raf-kinases and orthologous factors from Drosophila and Caenorhabditis elegans. emraf was co-expressed with emras in the larval stages metacestode and protoscolex during in vitro cultivation and during an infection of the intermediate host as assessed by RT-PCR experiments. The emraf gene was composed of nine exons and eight introns and shared four highly conserved exon-intron boundaries with the human gene encoding Raf-1, suggesting that both genes derived from a common evolutionary ancestor. Southern blot hybridizations demonstrated that emraf is a single copy gene. Using the yeast two-hybrid system, EmRaf was shown to interact with EmRas, but not with EmRal, a previously characterized orthologue of mammalian Ral GTPases. This is the first characterization of a Ras orthologue from a cestode and the first report on a Raf-like kinase from a platyhelminth. The data presented herein will form a solid basis for further investigations on Echinococcus signaling systems that are involved in growth control and development of the parasite.

Susceptibility versus resistance in alveolar echinococcosis (larval infection with Echinococcus multilocularis).

Epidemiological studies have demonstrated that the majority of human individuals exposed to infection with Echinococcus spp. eggs exhibit resistance to disease as shown by either seroconversion to parasite--specific antigens, and/or the presence of 'dying out' or 'aborted' metacestodes, not including hereby those individuals who putatively got infected but did not seroconvert and who subsequently allowed no development of the pathogen. For those individuals where infection leads to disease, the developing parasite is partially controlled by host immunity. In infected humans, the type of immune response developed by the host accounts for the subsequent trichotomy concerning the parasite development: (i) seroconversion proving infection, but lack of any hepatic lesion indicating the failure of the parasite to establish and further develop within the liver; or resistance as shown by the presence of fully calcified lesions; (ii) controlled susceptibility as found in the "conventional" alveolar echinococcosis (AE) patients who experience clinical signs and symptoms approximately 5-15 years after infection, and (iii) uncontrolled hyperproliferation of the metacestode due to an impaired immune response (AIDS or other immunodeficiencies). Immunomodulation of host immunity toward anergy seems to be triggered by parasite metabolites. Beside immunomodulating IL-10, TGFß-driven regulatory T cells have been shown to play a crucial role in the parasite-modulated progressive course of AE. A novel CD4+CD25+ Treg effector molecule FGL2 recently yielded new insight into the tolerance process in Echinococcus multilocularis infection.