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Parasitic flatworms, such as cestodes and trematodes, are covered by a syncytial tissue known as the tegument. It consists of a superficial band of cytoplasm (the distal tegument) that is connected by cytoplasmic bridges to multiple cell bodies (cytons) that lay beneath the basal lamina and contain the nuclei. We characterized the cytoskeleton of the tegument of the model cestode Mesocestoides corti and determined the effects of albendazole and albendazole sulfoxide on its organization. These anthelmintics are known to target beta-tubulin in helminths, and their effects have been extensively studied in nematodes, but the specific cells and tissues that are affected are not well understood in parasitic flatworms. Using antibodies that detect different tubulin subunits and post-translational modifications, we show that microtubules in the distal tegument have a unique organization, with bouquets of microtubules radiating from the cytoplasmic bridges, suggesting a role in intracellular traffic. In contrast, actin filaments were largely absent from the distal tegument. The microtubules of the tegument were specifically sensitive to low, chemotherapeutically relevant concentrations of albendazole and albendazole sulfoxide. This was correlated with the accumulation of secretory material in the cytons, and low concentrations of albendazole strongly reduced the incorporation of newly synthesized proteins in the distal tegument, as determined by metabolic labeling. Unexpectedly, albendazole also induced a global decrease in protein synthesis, which was independent of the activation of the unfolded protein response. Our work identifies the tegument as a sensitive target of benzimidazoles in cestodes and indicates that translational inhibition may contribute to the anthelmintic effect of benzimidazoles.

Alveolar echinococcosis (AE) is a lethal zoonosis caused by infiltrative growth of the metacestode larva of the tapeworm Echinococcus multilocularis in host organs. We previously showed that the Echinococcus metacestode is an evolutionarily unique, broadly posteriorized tissue, leading us to hypothesize that canonical WNT (cWNT) signalling, which patterns the body axis across metazoans, might be critical for metacestode formation. Here, we report effective RNAi-mediated knockdown of the E. multilocularis β-catenin gene ( bcat-1 ), the central effector of cWNT signalling, in a primary parasite cell culture system that produces metacestode vesicles. bcat-1 (RNAi) cultures were markedly impaired in vesicle formation, exhibited stem-cell hyperproliferation, and displayed changes in muscle-fibre organisation. Genome-wide transcriptomics revealed a general anteriorization of gene expression, and in situ hybridization showed an overproduction of cells expressing head-inducing factors such as sfrp upon bcat-1 knockdown. Conversely, metacestode-specific genes including the tegumental factors muc-1 , TNFR , and antigen B as well as the posterior marker post2b were significantly downregulated, consistent with the observed vesicle-formation defects. In situ analyses further identified anterior markers frizzled-10 , nou-darake , notum , and follistatin that were overexpressed in bcat-1 (RNAi) cultures and localized to the future anterior pole at the earliest stages of protoscolex formation. Together, these findings establish a central role for cWNT signalling in directing Echinococcus body-axis formation and the posteriorization events driving metacestode growth within the host, providing insight into asexual parasite proliferation mediated by this biologically unique larval stage and pointing to potential targets for chemotherapy against AE.

Tapeworm larvae cause important diseases in humans and domestic animals. During infection, the first larval stage undergoes a metamorphosis where tissues are formed from a population of stem cells called germinative cells. This process is difficult to study for human pathogens, as these larvae are infectious and difficult to obtain in the laboratory. In this work, we analyzed cell proliferation and differentiation during larval metamorphosis in the model tapeworm , by labelling of proliferating cells with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), tracing their differentiation with a suite of specific molecular markers for different cell types. Proliferating cells are very abundant and fast-cycling during early metamorphosis: the total number of cells duplicates every ten hours, and the length of G2 is only 75 minutes. New tegumental, muscle and nerve cells differentiate from this pool of proliferating germinative cells, and these processes are very fast, as differentiation markers for neurons and muscle cells appear within 24 hours after exiting the cell cycle, and fusion of new cells to the tegumental syncytium can be detected after only 4 hours. Tegumental and muscle cells appear from early stages of metamorphosis (24 to 48 hours post-infection); in contrast, most markers for differentiating neurons appear later, and the detection of synapsin and neuropeptides correlates with scolex retraction. Finally, we identified populations of proliferating cells that express conserved genes associated with neuronal progenitors and precursors, suggesting the existence of tissue-specific lineages among germinative cells. These results provide for the first time a comprehensive view of the development of new tissues during tapeworm larval metamorphosis, providing a framework for similar studies in human and veterinary pathogens.

Alveolar echinococcosis (AE) is a lethal zoonosis caused by the metacestode larva of the tapeworm Echinococcus multilocularis. The infection is characterized by tumour-like growth of the metacestode within the host liver, leading to extensive fibrosis and organ-failure. The molecular mechanisms of parasite organ tropism towards the liver and influences of liver cytokines and hormones on parasite development are little studied to date. We show that the E. multilocularis larval stage expresses three members of the fibroblast growth factor (FGF) receptor family with homology to human FGF receptors. Using the Xenopus expression system we demonstrate that all three Echinococcus FGF receptors are activated in response to human acidic and basic FGF, which are present in the liver. In all three cases, activation could be prevented by addition of the tyrosine kinase (TK) inhibitor BIBF 1120, which is used to treat human cancer. At physiological concentrations, acidic and basic FGF significantly stimulated the formation of metacestode vesicles from parasite stem cells in vitro and supported metacestode growth. Furthermore, the parasite's mitogen activated protein kinase signalling system was stimulated upon addition of human FGF. The survival of metacestode vesicles and parasite stem cells were drastically affected in vitro in the presence of BIBF 1120. Our data indicate that mammalian FGF, which is present in the liver and upregulated during fibrosis, supports the establishment of the Echinococcus metacestode during AE by acting on an evolutionarily conserved parasite FGF signalling system. These data are valuable for understanding molecular mechanisms of organ tropism and host-parasite interaction in AE. Furthermore, our data indicate that the parasite's FGF signalling systems are promising targets for the development of novel drugs against AE.

The life-threatening diseases alveolar and cystic echinococcoses are caused by larvae of the tapeworms Echinococcus multilocularis and E. granulosus, respectively. In both cases, intermediate hosts, such as humans, are infected by oral uptake of oncosphere larvae, followed by asexual multiplication and almost unrestricted growth of the metacestode within host organs. Besides surgery, echinococcosis treatment relies on benzimidazole-based chemotherapy, directed against parasite beta-tubulin. However, since beta-tubulins are highly similar between cestodes and humans, benzimidazoles can only be applied at parasitostatic doses and are associated with adverse side effects. Mostly aiming at identifying alternative drug targets, the nuclear genome sequences of E. multilocularis and E. granulosus have recently been characterized, revealing a large number of druggable targets that are expressed by the metacestode. Furthermore, recent cell biological investigations have demonstrated that E. multilocularis employs pluripotent stem cells, called germinative cells, which are the only parasite cells capable of proliferation and which give rise to all differentiated cells. Hence, the germinative cells are the crucial cell type mediating proliferation of E. multilocularis, and most likely also E. granulosus, within host organs and should also be responsible for parasite recurrence upon discontinuation of chemotherapy. Interestingly, recent investigations have also indicated that germinative cells might be less sensitive to chemotherapy because they express a beta-tubulin isoform with limited affinity to benzimidazoles. In this article, we briefly review the recent findings concerning Echinococcus genomics and stem cell research and propose that future research into anti-echinococcosis drugs should also focus on the parasite’s stem cell population. Les échinococcoses alvéolaire et kystique, deux maladies potentiellement mortelles, sont respectivement causées par les larves des vers plats Echinococcus multilocularis et E. granulosus. Dans les deux cas, les hôtes intermédiaires, comme l’homme, s’infectent par l’ingestion des oncosphères, suivie de la multiplication asexuée et la croissance presque illimitée du métacestode dans les organes de l’hôte. À côté de la chirurgie, le traitement des échinococcoses repose sur une chimiothérapie par les benzimidazoles, dont l’action est dirigée contre la bêta-tubuline du parasite. Cependant, comme les bêta-tubulines sont extrêmement similaires chez les cestodes et les humains, les benzimidazoles ne peuvent être utilisés qu’à des posologies parasitostatiques et sont associés à des effets secondaires indésirables. Avec l’objectif principal d’identifier des cibles pour des médicaments alternatifs, le génome nucléaire d’E. multilocularis et d’E. granulosus a été récemment séquencé, et de nombreuses cibles potentielles pour des médicaments sont exprimées par le métacestode. De plus, des études récentes de biologie cellulaire ont montré qu’E. multilocularis dispose de cellules souches multipotentes, appelées cellules germinales, qui sont les seules cellules parasitaires capables de prolifération et à l’origine de toutes les cellules différenciées. Ces cellules germinales représentent donc un type cellulaire crucial pour la prolifération d’E. multilocularis, et très vraisemblablement aussi d’E. granulosus, dans les organes de l’hôte, et vraisemblablement responsables des récurrences parasitaires à l’arrêt de la chimiothérapie. Des études récentes ont aussi indiqué que les cellules germinales pourraient être moins sensibles à la chimiothérapie car elles expriment un isoforme de la bêta-tubuline à affinité limitée vis-à-vis des benzimidazoles. Dans cet article, nous faisons une courte revue des découvertes récentes concernant la génomique d’Echinococcus et la recherche sur les cellules souches. Nous proposons que les recherches futures sur de nouveaux médicaments contre les échinococcoses se focalisent sur la population des cellules souches du parasite.

Background Spliced Leader trans-splicing is an important mechanism for the maturation of mRNAs in several lineages of eukaryotes, including several groups of parasites of great medical and economic importance. Nevertheless, its study across the tree of life is severely hindered by the problem of identifying the SL sequences that are being trans-spliced. Results In this paper we present SLFinder, a four-step pipeline meant to identify de novo candidate SL sequences making very few assumptions regarding the SL sequence properties. The pipeline takes transcriptomic de novo assemblies and a reference genome as input and allows the user intervention on several points to account for unexpected features of the dataset. The strategy and its implementation were tested on real RNAseq data from species with and without SL Trans-Splicing. Conclusions SLFinder is capable to identify SL candidates with good precision in a reasonable amount of time. It is especially suitable for species with unknown SL sequences, generating candidate sequences for further refining and experimental validation.

The neglected zoonotic disease alveolar echinococcosis (AE) is caused by the metacestode stage of the tapeworm parasite Echinococcus multilocularis . MicroRNAs (miRNAs) are small non-coding RNAs with a major role in regulating gene expression in key biological processes. We analyzed the expression profile of E . multilocularis miRNAs throughout metacestode development in vitro , determined the spatial expression of miR-71 in metacestodes cultured in vitro and predicted miRNA targets. Small cDNA libraries from different samples of E . multilocularis were sequenced. We confirmed the expression of 37 miRNAs in E . multilocularis being some of them absent in the host, such as miR-71. We found a few miRNAs highly expressed in all life cycle stages and conditions analyzed, whereas most miRNAs showed very low expression. The most expressed miRNAs were miR-71, miR-9, let-7, miR-10, miR-4989 and miR-1. The high expression of these miRNAs was conserved in other tapeworms, suggesting essential roles in development, survival, or host-parasite interaction. We found highly regulated miRNAs during the different transitions or cultured conditions analyzed, which might suggest a role in the regulation of developmental timing, host-parasite interaction, and/or in maintaining the unique developmental features of each developmental stage or condition. We determined that miR-71 is expressed in germinative cells and in other cell types of the germinal layer in E . multilocularis metacestodes cultured in vitro . MiRNA target prediction of the most highly expressed miRNAs and in silico functional analysis suggested conserved and essential roles for these miRNAs in parasite biology. We found relevant targets potentially involved in development, cell growth and death, lifespan regulation, transcription, signal transduction and cell motility. The evolutionary conservation and expression analyses of E . multilocularis miRNAs throughout metacestode development along with the in silico functional analyses of their predicted targets might help to identify selective therapeutic targets for treatment and control of AE.

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, EmTRPM PZQ . 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.

Cestodes are platyhelminth parasites with a wide range of hosts that cause neglected diseases. Neurotransmitter signaling is of critical importance for these parasites which lack circulatory, respiratory and digestive systems. For example, serotonin (5-HT) and serotonergic G-protein coupled receptors (5-HT GPCRs) play major roles in cestode motility, development and reproduction. In previous work, we deorphanized a group of 5-HT7 type GPCRs from cestodes. However, little is known about another type of 5-HT GPCR, the 5-HT1 clade, which has been studied in several invertebrate phyla but not in platyhelminthes. Three putative 5-HT GPCRs from Echinococcus canadensis , Mesocestoides vogae ( syn . M . corti ) and Hymenolepis microstoma were cloned, sequenced and bioinformatically analyzed. Evidence grouped these new sequences within the 5-HT1 clade of GPCRs but differences in highly conserved GPCR motifs were observed. Transcriptomic analysis, heterologous expression and immunolocalization studies were performed to characterize the E . canadensis receptor, called Eca-5-HT 1a . Functional heterologous expression studies showed that Eca-5-HT 1a is highly specific for serotonin. 5-Methoxytryptamine and α-methylserotonin, both known 5-HT GPCR agonists, give stimulatory responses whereas methysergide, a known 5-HT GPCR ligand, give an antagonist response in Eca-5-HT 1a . Mutants obtained by the substitution of key predicted residues resulted in severe impairment of receptor activity, confirming that indeed, these residues have important roles in receptor function. Immunolocalization studies on the protoscolex stage from E . canadensis , showed that Eca-5-HT 1a is localized in branched fibers which correspond to the nervous system of the parasite. The patterns of immunoreactive fibers for Eca-5-HT 1a and for serotonin were intimately intertwined but not identical, suggesting that they are two separate groups of fibers. These data provide the first functional, pharmacological and localization report of a serotonergic receptor that putatively belongs to the 5-HT1 type of GPCRs in cestodes. The serotonergic GPCR characterized here may represent a new target for antiparasitic intervention.

Alveolar echinococcosis (AE) is a life-threatening disease caused by larvae of the fox-tapeworm Echinococcus multilocularis. Crucial to AE pathology is continuous infiltrative growth of the parasite's metacestode stage, which is driven by a population of somatic stem cells, called germinative cells. Current anti-AE chemotherapy using benzimidazoles is ineffective in eliminating the germinative cell population, thus leading to remission of parasite growth upon therapy discontinuation. We herein describe the characterization of EmPlk1, encoded by the gene emplk1, which displays significant homologies to members of the Plk1 sub-family of Polo-like kinases that regulate mitosis in eukaryotic cells. We demonstrate germinative cell-specific expression of emplk1 by RT-PCR, transcriptomics, and in situ hybridization. We also show that EmPlk1 can induce germinal vesicle breakdown when heterologously expressed in Xenopus oocytes, indicating that it is an active kinase. This activity was significantly suppressed in presence of BI 2536, a Plk1 inhibitor that has been tested in clinical trials against cancer. Addition of BI 2536 at concentrations as low as 20 nM significantly blocked the formation of metacestode vesicles from cultivated Echinococcus germinative cells. Furthermore, low concentrations of BI 2536 eliminated the germinative cell population from mature metacestode vesicles in vitro, yielding parasite tissue that was no longer capable of proliferation. We conclude that BI 2536 effectively inactivates E. multilocularis germinative cells in parasite larvae in vitro by direct inhibition of EmPlk1, thus inducing mitotic arrest and germinative cell killing. Since germinative cells are decisive for parasite proliferation and metastasis formation within the host, BI 2536 and related compounds are very promising compounds to complement benzimidazoles in AE chemotherapy.