Evidence for Integrin - Venus Kinase Receptor 1 Alliance in the Ovary of Schistosoma mansoni Females Controlling Cell Survival.

In metazoan integrin signaling is an important process of mediating extracellular and intracellular communication processes. This can be achieved by cooperation of integrins with growth factor receptors (GFRs). Schistosoma mansoni is a helminth parasite inducing schistosomiasis, an infectious disease of worldwide significance for humans and animals. First studies on schistosome integrins revealed their role in reproductive processes, being involved in spermatogenesis and oogenesis. With respect to the roles of eggs for maintaining the parasite´s life cycle and for inducing the pathology of schistosomiasis, elucidating reproductive processes is of high importance. Here we studied the interaction of the integrin receptor Smß-Int1 with the venus kinase receptor SmVKR1 in S. mansoni. To this end we cloned and characterized SmILK, SmPINCH, and SmNck2, three putative bridging molecules for their role in mediating Smß-Int1/SmVKR1 cooperation. Phylogenetic analyses showed that these molecules form clusters that are specific for parasitic platyhelminths as it was shown for integrins before. Transcripts of all genes colocalized in the ovary. In Xenopus oocytes germinal vesicle breakdown (GVBD) was only induced if all members were simultaneously expressed. Coimmunoprecipitation results suggest that a Smß-Int1-SmILK-SmPINCH-SmNck2-SmVKR1 complex can be formed leading to the phosphorylation and activation of SmVKR1. These results indicate that SmVKR1 can be activated in a ligand-independent manner by receptor-complex interaction. RNAi and inhibitor studies to knock-down SmILK as a representative complex member concurrently revealed effects on the extracellular matrix surrounding the ovary and oocyte localization within the ovary, oocyte survival, and egg production. By TUNEL assays, confocal laser scanning microscopy (CLSM), Caspase-3 assay, and transcript profiling of the pro-apoptotic BCL-2 family members BAK/BAX we obtained first evidence for roles of this signaling complex in mediating cell death in immature and primary oocytes. These results suggest that the Smß-Int1/SmVKR1 signaling complex is important for differentiation and survival in oocytes of paired schistosomes.

Expression profile of heat shock response factors during hookworm larval activation and parasitic development.

When organisms are exposed to an increase in temperature, they undergo a heat shock response (HSR) regulated by the transcription factor heat shock factor 1 (HSF-1). The heat shock response includes the rapid changes in gene expression initiated by binding of HSF-1 to response elements in the promoters of heat shock genes. Heat shock proteins function as molecular chaperones to protect proteins during periods of elevated temperature and other stress. During infection, hookworm infective third stage larvae (L3) undergo a temperature shift from ambient to host temperature. This increased temperature is required for the resumption of feeding and activation of L3, but whether this increase initiates a heat shock response is unknown. To investigate the role of the heat shock in hookworm L3 activation and parasitic development, we identified and characterized the expression profile of several components of the heat shock response in the hookworm Ancylostoma caninum. We cloned DNAs encoding an hsp70 family member (Aca-hsp-1) and an hsp90 family member (Aca-daf-21). Exposure to a heat shock of 42°C for one hour caused significant up-regulation of both genes, which slowly returned to near baseline levels following one hour attenuation at 22°C. Neither gene was up-regulated in response to host temperature (37°C). Conversely, levels of hsf-1 remained unchanged during heat shock, but increased in response to incubation at 37°C. During activation, both hsp-1 and daf-21 are down regulated early, although daf-21 levels increase significantly in non-activated control larvae after 12h, and slightly in activated larvae by 24h incubation. The heat shock response modulators celastrol and KNK437 were tested for their effects on gene expression during heat shock and activation. Pre-incubation with celastrol, an HSP90 inhibitor that promotes heat shock gene expression, slightly up-regulated expression of both hsp-1 and daf-21 during heat shock. KNK437, an inhibitor of heat shock protein expression, slightly down regulated both genes under similar conditions. Both modulators inhibited activation-associated feeding, but neither had an effect on hsp-1 levels in activated L3 at 16h. Both celastrol and KNK437 prevent the up-regulation of daf-21 and hsf-1 seen in non-activated control larvae during activation, and significantly down regulated expression of the HSF-1 negative regulator Aca-hsb-1 in activated larvae. Expression levels of heat shock response factors were examined in developing Ancylostoma ceylanicum larvae recovered from infected hosts and found to differ significantly from the expression profile of activated L3, suggesting that feeding during in vitro activation is regulated differently than parasitic development. Our results indicate that a classical heat shock response is not induced at host temperature and is suppressed during larval recovery and parasitic development in the host, but a partial heat shock response is induced after extended incubation at host temperature in the absence of a developmental signal, possibly to protect against heat stress.

Isolation, enrichment and primary characterisation of vitelline cells from Schistosoma mansoni obtained by the organ isolation method.

In the emerging era of post-genomic research on schistosomes, new methods are required to functionally analyse genes of interest in more detail. Among other tools, schistosome cell lines are needed to overcome present research constraints. Based on a recently established organ isolation protocol for adult Schistosoma mansoni, we report here on the successful enrichment of vitellarium tissue and isolation of vitelline cells. Morphological analyses performed by bright field, fluorescence, scanning and transmission electron microscopy showed typical features of S1 to S4 stage vitelline cells. In addition, molecular analyses using reverse transcription-PCR confirmed the identity of vitelline cells. Cytological and physiological studies included staining experiments with viability dyes and a neutral lipid stain, as well as calcium (Ca2+) imaging. Together they demonstrated cell viability, the possibility to define the differentiation stage of individual vitelline cells, and the suitability to investigate Ca(2+)-associated processes herein. Finally, fluorescence-activated cell sorting was shown to be a convenient way to separate and enrich S1 to S4 stage vitelline cells. In summary, these results demonstrate the expedience of the organ isolation protocol to obtain vitellarium tissue. Importantly, the protocol allows vitelline cells representing defined differentiation stages to be purified, which can be cultured in vitro and used to investigate diverse aspects of schistosome reproductive biology in the post-genomic era.

Re-positioning protein-kinase inhibitors against schistosomiasis.

For decades, Praziquantel (PZQ) is the drug of choice against one of the most afflicting helminthic diseases worldwide, schistosomiasis. With respect to the fear of upcoming PZQ resistance, efforts are needed to find new chemotherapeutic options. Protein kinases (PKs) are essential molecules in signaling processes and indispensable to life. Aberrant PK functions take distinctive roles in human diseases and represent targets in chemotherapies. In schistosomes, conserved PKs were found to possess similar pivotal roles contributing not only to reproduction processes, but also to the pathology of schistosomiasis, which is closely associated to egg production. Exploiting the similarity of PKs of humans and schistosomes, PK inhibitors designed to treat human diseases may serve as lead compounds for new drugs against schistosomiasis.

Cryptic 3' mRNA processing signals hinder the expression of Schistosoma mansoni integrins in yeast.

The expression of parasite genes has often proven difficult in heterologous systems such as yeast or E. coli. Most often, promoter choice and codon usage were hypothesised to be the main reason for expression failures. The trematode parasite Schistosoma mansoni has five integrin genes named Smα-Int1-4 and Smß-Int1, which we aimed to express in the yeast Saccharomyces cerevisiae. This has not been achieved, however, as only Smß-Int1 integrin could be expressed. When the four α integrins were driven by a stronger promoter, this enabled Smα-Int1 to be expressed as well, but the remaining integrins, Smα-Int2-4, still could not be expressed. Evidence from RT-PCR experiments suggested that this was due to premature transcription termination. Using detailed in silico sequence analyses we identified AT-rich stretches in these integrin genes, which have high similarity to yeast mRNA 3'-end processing signals. We hypothesised that these signals were causing the premature truncation. To test this, we designed an optimised version of Smα-Int3, in which the sequence was modified to replace the yeast 3' processing signals. This strategy allowed us to express Smα-Int3 integrin successfully in S. cerevisiae. These findings show that the misinterpretation of AT-rich sequences by yeast 3'-mRNA processing machinery can cause problems when attempting to express genes containing such sequences in this host.

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.

Structural conservation of ligand binding reveals a bile acid-like signaling pathway in nematodes.

Bile acid-like molecules named dafachronic acids (DAs) control the dauer formation program in Caenorhabditis elegans through the nuclear receptor DAF-12. This mechanism is conserved in parasitic nematodes to regulate their dauer-like infective larval stage, and as such, the DAF-12 ligand binding domain has been identified as an important therapeutic target in human parasitic hookworm species that infect more than 600 million people worldwide. Here, we report two x-ray crystal structures of the hookworm Ancylostoma ceylanicum DAF-12 ligand binding domain in complex with DA and cholestenoic acid (a bile acid-like metabolite), respectively. Structure analysis and functional studies reveal key residues responsible for species-specific ligand responses of DAF-12. Furthermore, DA binds to DAF-12 mechanistically and is structurally similar to bile acids binding to the mammalian bile acid receptor farnesoid X receptor. Activation of DAF-12 by cholestenoic acid and the cholestenoic acid complex structure suggest that bile acid-like signaling pathways have been conserved in nematodes and mammals. Together, these results reveal the molecular mechanism for the interplay between parasite and host, provide a structural framework for DAF-12 as a promising target in treating nematode parasitism, and provide insight into the evolution of gut parasite hormone-signaling pathways.

Transgenic C. elegans dauer larvae expressing hookworm phospho null DAF-16/FoxO exit dauer.

Parasitic hookworms and the free-living model nematode Caenorhabtidis elegans share a developmental arrested stage, called the dauer stage in C. elegans and the infective third-stage larva (L3) in hookworms. One of the key transcription factors that regulate entrance to and exit from developmental arrest is the forkhead transcription factor DAF-16/FoxO. During the dauer stage, DAF-16 is activated and localized in the nucleus. DAF-16 is negatively regulated by phosphorylation by the upstream kinase AKT, which causes DAF-16 to localize out of the nucleus and the worm to exit from dauer. DAF-16 is conserved in hookworms, and hypothesized to control recovery from L3 arrest during infection. Lacking reverse genetic techniques for use in hookworms, we used C. elegans complementation assays to investigate the function of Ancylostoma caninum DAF-16 during entrance and exit from L3 developmental arrest. We performed dauer switching assays and observed the restoration of the dauer phenotype when Ac-DAF-16 was expressed in temperature-sensitive dauer defective C. elegans daf-2(e1370);daf-16(mu86) mutants. AKT phosphorylation site mutants of Ac-DAF-16 were also able to restore the dauer phenotype, but surprisingly allowed dauer exit when temperatures were lowered. We used fluorescence microscopy to localize DAF-16 during dauer and exit from dauer in C. elegans DAF-16 mutant worms expressing Ac-DAF-16, and found that Ac-DAF-16 exited the nucleus during dauer exit. Surprisingly, Ac-DAF-16 with mutated AKT phosphorylation sites also exited the nucleus during dauer exit. Our results suggest that another mechanism may be involved in the regulation DAF-16 nuclear localization during recovery from developmental arrest.

RNA and protein synthesis is required for Ancylostoma caninum larval activation.

The developmentally arrested infective larva of hookworms encounters a host-specific signal during invasion that initiates the resumption of suspended developmental pathways. The resumption of development during infection is analogous to recovery from the facultative arrested dauer stage in the free-living nematode Caenorhabditis elegans. Infective larvae of the canine hookworm Ancylostoma caninum resume feeding and secrete molecules important for infection when exposed to a host mimicking signal in vitro. This activation process is a model for the initial steps of the infective process. Dauer recovery requires protein synthesis, but not RNA synthesis in C. elegans. To determine the role of RNA and protein synthesis in hookworm infection, inhibitors of RNA and protein synthesis were tested for their effect on feeding and secretion by A. caninum infective larvae. The RNA synthesis inhibitors α-amanitin and actinomycin D inhibit feeding dose-dependently, with IC(50) values of 30 and 8 µM, respectively. The protein synthesis inhibitors puromycin (IC(50)=110 µM), cycloheximide (IC(50)=50 µM), and anisomycin (IC(50)=200 µM) also displayed dose-dependent inhibition of larval feeding. Significant inhibition of feeding by α-amanitin and anisomycin occurred when the inhibitors were added before 12h of the activation process, but not if the inhibitors were added after 12h. None of the RNA or protein synthesis inhibitors prevented secretion of the activation-associated protein ASP-1, despite nearly complete inhibition of feeding. The results indicate that unlike dauer recovery in C. elegans, de novo gene expression is required for hookworm larval activation, and the critical genes are expressed within 12h of exposure to activating stimuli. However, secretion of infection-associated proteins is independent of gene expression, indicating that the proteins are pre-synthesized and stored for rapid release during the initial stages of infection. The genes that are inhibited represent a subset of those required for the transition to parasitism, and therefore represent interesting targets for further investigation. Furthermore, while dauer recovery provides a useful model for hookworm infection, the differences identified here highlight the importance of exercising caution before making generalizations about parasitic nematodes based on C. elegans biology.

Characterisation of hookworm heat shock factor binding protein (HSB-1) during heat shock and larval activation.

When hookworm infective L3s infect their mammalian host, they undergo a temperature shift from that of the ambient environment to that of their endothermic host. Additionally, L3s living in the environment can be exposed to temperature extremes associated with weather fluctuations. The heat shock response (HSR) is a conserved response to heat shock and other stress that involves the expression of protective heat shock proteins (HSPs). The HSR is controlled by heat shock factor-1 (HSF-1), a conserved transcription factor that binds to a heat shock element in the promoter of HSPs, causing their expression. HSF-1 is negatively regulated in part by a HSF binding protein (HSB-1) that binds to and removes HSF-1 trimers bound to HSP gene promoters, resulting in attenuation of the HSR. Herein we describe an HSB-1 orthologue, Ac-HSB-1, from the hookworm Ancylostoma caninum. The Ac-hsb-1 cDNA encodes a 79 amino acid protein that is 71% identical to the Caenorhabditis elegans HSB-1, and is predicted to share the characteristic coiled-coil structural motif comprised of two interacting alpha helices. Recombinant Ac-HSB-1 immunoprecipitated Ce-HSF-1 expressed in mammalian cells that had been heat shocked for 1h at 42°C, but not from cells incubated at 37°C, indicating that HSB-1 only bound to the active DNA binding form of HSF-1. Expression of Ac-hsb-1 transcripts decreased following 1h of heat shock, but increased when L3s were incubated at 37°C for 1h. Activation of hookworm L3s induces a five-sixfold increase in Ac-hsb-1 expression that peaks at 12h, coincident with L3 feeding, but that subsequently decreases to two-threefold above control at 24h. Recombinant Ac-HSB-1 immunoprecipitates greater amounts of 70 and 40kDa proteins from extracts of activated L3s than from non-activated L3s. We propose that an increase in Ac-hsb-1 levels early in activation allows feeding to resume, but that a subsequent decrease in expression permits a HSR that protects non-developing L3s at host-like temperatures. Further investigations of the HSR will clarify the role of HSB-1 and HSF-1 in hookworm infection.

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.

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.

Molecular characterisation of a second structurally unusual AR-Smad without an MH1 domain and a Smad4 orthologue from Echinococcus multilocularis.

Members of the transforming growth factor-beta/bone morphogenetic protein (TGF-beta/BMP) family of cytokines play crucial roles in animal development and are candidate molecules for host-parasite cross-communication in helminth diseases. TGF-beta/BMP-signalling involves binding of the cytokines to receptor kinases which subsequently activate intracellular transcription factors of the Smad family. We have previously characterized two members of the receptor-regulated Smad (R-Smad) family, EmSmadA and EmSmadB, from the human parasitic cestode Echinococcus multilocularis and now present evidence for two additional Smads that are expressed by the larval stages of the parasite. The full-length cDNAs coding for a third R-Smad, EmSmadC, and a common mediator Smad (Co-Smad), EmSmadD, were characterized. While EmSmadD displayed a typical Co-Smad structure, EmSmadC lacked the N-terminal MH1 domain which is typically found in Smads. In yeast two-hybrid analyses, EmSmadC and EmSmadD were capable of homo- and heterodimer formation with other Echinococcus Smads. Furthermore, EmSmadC displayed autonomous transcription activation activity and interacted with EmSkip, a member of the SNW/SKIP family of transcriptional regulators. In a heterologous expression system, EmSmadC was specifically phosphorylated by mammalian TGF-beta receptors, indicating that it is a member of the AR-Smad sub-family. Finally, in activity assays, the parasite's Erk-like kinase EmMPK1 phosphorylated EmSmadD, indicating cross-regulation between mitogen-activated protein kinase cascade- and TGF-beta/BMP-signalling in Echinococcus. The data presented herein significantly broaden our knowledge of Smad-signalling factors in E. multilocularis and will facilitate studies on TGF-beta/BMP-regulated genes in the parasite as well as TGF-beta/BMP mediated host-parasite cross-interaction during alveolar echinococcosis.

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.

Echinococcus multilocularis: cloning and characterization of a member of the SNW/SKIP family of transcriptional coregulators.

We have isolated a cDNA from the fox-tapeworm Echinococcus multilocularis that encodes EmSkip, a novel member of the SNW/SKIP family of transcriptional coregulators. EmSkip displays significant amino acid sequence homologies to already known members of the protein family and contains all the characteristic amino acid residues at their corresponding positions. RT-PCR experiments showed that the EmSkip encoding gene, emskip, is expressed in the Echinococcus larval stages metacestode and protoscolex during an infection of the intermediate host. By yeast two-hybrid analyses, EmSkip was found to be capable of forming homodimers in vivo. Furthermore, EmSkip was found to interact with EmSmadA and EmSmadB, two previously identified TGF-beta/BMP signal transducers of E. multilocularis, indicating a role of this protein in TGF-beta signaling processes in the parasite. In view of the role played by SNW/SKIP proteins in splicing mechanisms and intracellular signaling, the data presented herein should facilitate the identification of Echinococcus factors involved in such processes.