Stem cell proliferation and differentiation during larval metamorphosis of the model tapeworm Hymenolepis microstoma.

Background: Tapeworm larvae cause important diseases in humans and domestic animals. During infection, the first larval stage undergoes a metamorphosis where tissues are formed de novo 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. Methods: In this work, we analyzed cell proliferation and differentiation during larval metamorphosis in the model tapeworm Hymenolepis microstoma, by in vivo 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. Results: 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. Discussion: 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.

Establishment and application of unbiased in vitro drug screening assays for the identification of compounds against Echinococcus granulosus sensu stricto.

Echinococcus multilocularis and E. granulosus s.l. are the causative agents of alveolar and cystic echinococcosis, respectively. Drug treatment options for these severe and neglected diseases are limited to benzimidazoles, which are not always efficacious, and adverse side effects are reported. Thus, novel and improved treatments are needed. In this study, the previously established platform for E. multilocularis in vitro drug assessment was adapted to E. granulosus s.s. In a first step, in vitro culture protocols for E. granulosus s.s. were established. This resulted in the generation of large amounts of E. granulosus s.s. metacestode vesicles as well as germinal layer (GL) cells. In vitro culture of these cells formed metacestode vesicles displaying structural characteristics of metacestode cysts generated in vivo. Next, drug susceptibilities of E. multilocularis and E. granulosus s.s. protoscoleces, metacestode vesicles and GL cells were comparatively assessed employing established assays including (i) metacestode vesicle damage marker release assay, (ii) metacestode vesicle viability assay, (iii) GL cell viability assay, and (iv) protoscolex motility assay. The standard drugs albendazole, buparvaquone, mefloquine, MMV665807, monepantel, niclosamide and nitazoxanide were included. MMV665807, niclosamide and nitazoxanide were active against the parasite in all four assays against both species. MMV665807 and monepantel were significantly more active against E. multilocularis metacestode vesicles, while albendazole and nitazoxanide were significantly more active against E. multilocularis GL cells. Albendazole displayed activity against E. multilocularis GL cells, but no effects were seen in albendazole-treated E. granulosus s.s. GL cells within five days. Treatment of protoscoleces with albendazole and monepantel had no impact on motility. Similar results were observed for both species with praziquantel and its enantiomers against protoscoleces. In conclusion, in vitro culture techniques and drug screening methods previously established for E. multilocularis were successfully implemented for E. granulosus s.s., allowing comparisons of drug efficacy between the two species. This study provides in vitro culture techniques for the reliable generation of E. granulosus s.s. metacestode vesicles and GL cell cultures and describes the validation of standardized in vitro drug screening methods for E. granulosus s.s.

Targeted and non-targeted proteomics to characterize the parasite proteins of Echinococcus multilocularis metacestodes.

The larval stage of the cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis. To investigate the biology of these stages and to test novel compounds, metacestode cultures represent a suitable in vitro model system. These metacestodes are vesicles surrounded by an envelope formed by the vesicle tissue (VT), which is formed by the laminated and germinal layer, and filled with vesicle fluid (VF). We analyzed the proteome of VF and VT by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified a total of 2,954 parasite proteins. The most abundant protein in VT was the expressed conserved protein encoded by EmuJ_000412500, followed by the antigen B subunit AgB8/3a encoded by EmuJ_000381500 and Endophilin B1 (protein p29). In VF, the pattern was different and dominated by AgB subunits. The most abundant protein was the AgB8/3a subunit followed by three other AgB subunits. In total, the AgB subunits detected in VF represented 62.1% of the parasite proteins. In culture media (CM), 63 E. multilocularis proteins were detected, of which AgB subunits made up 93.7% of the detected parasite proteins. All AgB subunits detected in VF (encoded by EmuJ_000381100-700, corresponding to AgB8/2, AgB8/1, AgB8/4, AgB8/3a, AgB8/3b, and AgB8/3c) were also found in CM, except the subunit encoded by EmuJ_000381800 (AgB8/5) that was very rare in VF and not detected in CM. The relative abundance of the AgB subunits in VF and CM followed the same pattern. In VT, only the subunits EmuJ_000381500 (AgB8/3a) and EmuJ_000381200 (AgB8/1) were detected among the 20 most abundant proteins. To see whether this pattern was specific to VF from in vitro cultured metacestodes, we analyzed the proteome of VF from metacestodes grown in a mouse model. Here, the AgB subunits encoded by EmuJ_000381100-700 constituted the most abundant proteins, namely, 81.9% of total protein, with the same order of abundance as in vitro. Immunofluorescence on metacestodes showed that AgB is co-localized to calcareous corpuscles of E. multilocularis. Using targeted proteomics with HA-tagged EmuJ_000381200 (AgB8/1) and EmuJ_000381100 (AgB8/2), we could show that uptake of AgB subunits from CM into VF occurs within hours.

Investigation of the mechanism of action of mefloquine and derivatives against the parasite Echinococcus multilocularis.

Alveolar echinococcosis (AE) is caused by infection with the fox tapeworm E. multilocularis. The disease affects humans, dogs, captive monkeys, and other mammals, and it is caused by the metacestode stage of the parasite growing invasively in the liver. The current drug treatment is based on non-parasiticidal benzimidazoles. Thus, they are only limitedly curative and can cause severe side effects. Therefore, novel and improved treatment options for AE are needed. Mefloquine (MEF), an antimalarial agent, was previously shown to be effective against E. multilocularis in vitro and in experimentally infected mice. However, MEF is not parasiticidal and needs improvement for successful treatment of patients, and it can induce strong neuropsychiatric side-effects. In this study, the structure-activity relationship and mode of action of MEF was investigated by comparative analysis of 14 MEF derivatives. None of them showed higher activity against E. multilocularis metacestodes compared to MEF, but four compounds caused limited damage. In order to identify molecular targets of MEF and effective derivatives, differential affinity chromatography combined with mass spectrometry was performed with two effective compounds (MEF, MEF-3) and two ineffective compounds (MEF-13, MEF-22). 1'681 proteins were identified that bound specifically to MEF or derivatives. 216 proteins were identified as binding only to MEF and MEF-3. GO term enrichment analysis of these proteins and functional grouping of the 25 most abundant MEF and MEF-3 specific binding proteins revealed the key processes energy metabolism and cellular transport and structure, as well as stress responses and nucleic acid binding to be involved. The previously described ferritin was confirmed as an exclusively MEF-binding protein that could be relevant for its efficacy against E. multilocularis. The here identified potential targets of MEF will be further investigated in the future for a clear understanding of the pleiotropic effects of MEF, and improved therapeutic options against AE.

Dual inhibition of the Echinococcus multilocularis energy metabolism.

Alveolar echinococcosis is caused by the metacestode stage of the zoonotic parasite Echinococcus multilocularis. Current chemotherapeutic treatment options rely on benzimidazoles, which have limited curative capabilities and can cause severe side effects. Thus, novel treatment options are urgently needed. In search for novel targetable pathways we focused on the mitochondrial energy metabolism of E. multilocularis. The parasite relies hereby on two pathways: The classical oxidative phosphorylation including the electron transfer chain (ETC), and the anaerobic malate dismutation (MD). We screened 13 endochin-like quinolones (ELQs) in vitro for their activities against two isolates of E. multilocularis metacestodes and isolated germinal layer cells by the phosphoglucose isomerase (PGI) assay and the CellTiter Glo assay. For the five most active ELQs (ELQ-121, ELQ-136, ELQ-271, ELQ-400, and ELQ-437), EC50 values against metacestodes were assessed by PGI assay, and IC50 values against mammalian cells were measured by Alamar Blue assay. Further, the gene sequence of the proposed target, the mitochondrial cytochrome b, was analyzed. This allowed for a limited structure activity relationship study of ELQs against E. multilocularis, including analyses of the inhibition of the two functional sites of the cytochrome b. By applying the Seahorse XFp Extracellular Flux Analyzer, oxygen consumption assays showed that ELQ-400 inhibits the E. multilocularis cytochrome bc 1 complex under normoxic conditions. When tested under anaerobic conditions, ELQ-400 was hardly active against E. multilocularis metacestodes. These results were confirmed by transmission electron microscopy. ELQ-400 treatment increased levels of parasite-released succinate, the final electron acceptor of the MD. This suggests that the parasite switched to MD for energy generation. Therefore, MD was inhibited with quinazoline, which did not induce damage to metacestodes under anaerobic conditions. However, it reduced the production of succinate compared to control treated parasites (i.e., inhibited the MD). The combination treatment with quinazoline strongly improved the activity of the bc 1 inhibitor ELQ-400 against E. multilocularis metacestodes under anaerobic conditions. We conclude that simultaneous targeting of the ETC and the MD of E. multilocularis is a possible novel treatment approach for alveolar echinococcosis, and possibly also other foodborne diseases inflicted by platyhelminths, which cause substantial economic losses in livestock industry.

Cell repertoire and proliferation of germinative cells of the model cestode Mesocestoides corti.

The phylum Platyhelminthes shares a unique population of undifferentiated cells responsible for the proliferation capacity needed for cell renewal, growth, tissue repair and regeneration. These cells have been extensively studied in free-living flatworms, whereas in cestodes the presence of a set of undifferentiated cells, known as germinative cells, has been demonstrated in classical morphology studies, but poorly characterized with molecular biology approaches. Furthermore, several genes have been identified as neoblast markers in free-living flatworms that deserve study in cestode models. Here, different cell types of the model cestode Mesocestoides corti were characterized, identifying differentiated and germinative cells. Muscle cells, tegumental cells, calcareous corpuscle precursor cells and excretory system cells were identified, all of which are non-proliferative, differentiated cell types. Besides those, germinative cells were identified as a population of small cells with proliferative capacity in vivo. Primary cell culture experiments in Dulbecco's Modified Eagle Medium (DMEM), Echinococcus hydatid fluid and hepatocyte conditioned media in non-reductive or reductive conditions confirmed that the germinative cells were the only ones with proliferative capacity. Since several genes have been identified as markers of undifferentiated neoblast cells in free-living flatworms, the expression of pumilio and pL10 genes was analysed by qPCR and in situ hybridization, showing that the expression of these genes was stronger in germinative cells but not restricted to this cell type. This study provides the first tools to analyse and further characterise undifferentiated cells in a model cestode.

Global analysis of neuropeptides in cestodes identifies Attachin, a SIFamide homolog, as a stimulant of parasite motility and attachment.

Many anthelmintics target the neuromuscular system, in particular by interfering with signaling mediated by classical neurotransmitters. Although peptidergic signaling has been proposed as a novel target for anthelmintics, current knowledge of the neuropeptide complement of many helminth groups is still limited, especially for parasitic flatworms (cestodes, trematodes, and monogeneans). In this work, we have characterized the neuropeptide complement of the model cestode Hymenolepis microstoma. Peptidomic characterization of adults of H. microstoma validated many of the neuropeptide precursor (npp) genes previously predicted in silico, and identified novel neuropeptides that are conserved in parasitic flatworms. Most neuropeptides from parasitic flatworms lack significant similarity to those from other animals, confirming the uniqueness of their peptidergic signaling. Analysis of gene expression of ten npp genes by in situ hybridization confirmed that all of them are expressed in the nervous system and identified cryptic features, including the first evidence of dorsoventral asymmetry, as well as a new population of peripheral peptidergic cells that appears to be conserved in the trematode Schistosoma mansoni. Finally, we characterized in greater detail Attachin, an SIFamide homolog. Although its expression is largely restricted to the longitudinal nerve cords and cerebral commissure in H. microstoma, it shows widespread localization in the larval nervous system of Echinococcus multilocularis and Mesocestoides corti. Exogenous addition of a peptide corresponding to the highly conserved C-terminus of Attachin stimulated motility and attachment of M. corti larvae. Altogether, this work provides a robust experimental foothold for the characterization of peptidergic signaling in parasitic flatworms. Cover Image for this issue: https://doi.org/10.1111/jnc.15418.

Characterization of a new type of neuronal 5-HT G- protein coupled receptor in the cestode nervous system.

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-HT1a. Functional heterologous expression studies showed that Eca-5-HT1a 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-HT1a. 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-HT1a is localized in branched fibers which correspond to the nervous system of the parasite. The patterns of immunoreactive fibers for Eca-5-HT1a 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.

Inhibitors of protein kinases A and C impair the motility of oncospheres of the model tapeworm Hymenolepis microstoma.

The oncosphere larvae of tapeworms cyclically extend and retract their hooks during the penetration of the intestine of their intermediate hosts. The mechanisms regulating these movements are essentially unknown, in part due to the biohazardous nature of oncospheres from human pathogens. In this work, we standardized a method for the analysis of motility of hatched oncospheres (hexacanths) of the model tapeworm Hymenolepis microstoma. We used this assay to explore the relevance of protein kinases C (PKC) and A (PKA) in these processes. Pharmacological inhibition of the PKC pathway resulted in impaired larval motility. On the other hand, the PKA inhibitor H-89 potently blocked larval motility, as well as the motility of other life stages, although other inhibitors of the PKA pathway were not effective. This work represents the first study of the mechanisms that regulate the motility of oncospheres, and provides a path for further exploration.

Expression profiling of Echinococcus multilocularis miRNAs throughout metacestode development in vitro.

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.

Stage-specific transcriptomic analysis of the model cestode Hymenolepis microstoma.

Most parasitic flatworms go through different life stages with important physiological and morphological changes. In this work, we used a transcriptomic approach to analyze the main life-stages of the model tapeworm Hymenolepis microstoma (eggs, cysticercoids, and adults). Our results showed massive transcriptomic changes in this life cycle, including key gene families that contribute substantially to the expression load in each stage. In particular, different members of the cestode-specific hydrophobic ligand-binding protein (HLBP) family are among the most highly expressed genes in each life stage. We also found the transcriptomic signature of major metabolic changes during the transition from cysticercoids to adult worms. Thus, this work contributes to uncovering the gene expression changes that accompany the development of this important cestode model species, and to the best of our knowledge represents the first transcriptomic study with robust replicates spanning all of the main life stages of a tapeworm.

Divergent Axin and GSK-3 paralogs in the beta-catenin destruction complexes of tapeworms.

The Wnt/beta-catenin pathway has many key roles in the development of animals, including a conserved and central role in the specification of the primary (antero-posterior) body axis. The posterior expression of Wnt ligands and the anterior expression of secreted Wnt inhibitors are known to be conserved during the larval metamorphosis of tapeworms. However, their downstream signaling components for Wnt/beta-catenin signaling have not been characterized. In this work, we have studied the core components of the beta-catenin destruction complex of the human pathogen Echinococcus multilocularis, the causative agent of alveolar echinococcosis. We focused on two Axin paralogs that are conserved in tapeworms and other flatworm parasites. Despite their divergent sequences, both Axins could robustly interact with one E. multilocularis beta-catenin paralog and limited its accumulation in a heterologous mammalian expression system. Similarly to what has been described in planarians (free-living flatworms), other beta-catenin paralogs showed limited or no interaction with either Axin and are unlikely to function as effectors in Wnt signaling. Additionally, both Axins interacted with three divergent GSK-3 paralogs that are conserved in free-living and parasitic flatworms. Axin paralogs have highly segregated expression patterns along the antero-posterior axis in the tapeworms E. multilocularis and Hymenolepis microstoma, indicating that different beta-catenin destruction complexes may operate in different regions during their larval metamorphosis.

Analysis of classical neurotransmitter markers in tapeworms: Evidence for extensive loss of neurotransmitter pathways.

Parasitic flatworms have complex neuromuscular systems that serve important functions in their life cycles. However, our understanding of neurotransmission in parasitic flatworms is limited. Pioneering studies have suggested the presence of several classical neurotransmitter systems, but their molecular components have not been characterized in most cases. Because these components are conserved in bilaterian animals, we searched the genomes of parasitic flatworms for orthologs of genes required for neurotransmitter synthesis, vesicular transport, reuptake, and reception. Our results indicate that tapeworms have lost the genes that are specifically required in other animals for synaptic signaling using the classical neurotransmitters dopamine, tyramine, octopamine, histamine and gamma-aminobutyric acid (GABA). These results imply that these signaling pathways are either absent in these parasites, or that they require completely different molecular components in comparison with other animals. The orthologs of genes related to histaminergic and GABA signaling are also missing in trematodes (although Schistosoma-specific histaminergic receptors have been previously described). In contrast, conserved genes required for glutamatergic, serotonergic and cholinergic signaling could be found in all analyzed flatworms. We analyzed the expression of selected markers of each pathway in the tapeworm Hymenolepis microstoma by whole-mount in situ hybridization. Each marker was specifically expressed in the nervous system, although with different patterns. In addition, we analyzed the expression of proprotein convertase 2 as a marker of peptidergic cells. This gene showed the widest expression in the nervous system, but was also expressed in other tissues, suggesting additional roles of peptidergic signaling in tapeworm development and reproduction.

De novo discovery of neuropeptides in the genomes of parasitic flatworms using a novel comparative approach.

Neuropeptide mediated signalling is an ancient mechanism found in almost all animals and has been proposed as a promising target for the development of novel drugs against helminths. However, identification of neuropeptides from genomic data is challenging, and knowledge of the neuropeptide complement of parasitic flatworms is still fragmentary. In this work, we have developed an evolution-based strategy for the de novo discovery of neuropeptide precursors, based on the detection of localised sequence conservation between possible prohormone convertase cleavage sites. The method detected known neuropeptide precursors with good precision and specificity in the models Drosophila melanogaster and Caenorhabditis elegans. Furthermore, it identified novel putative neuropeptide precursors in nematodes, including the first description of allatotropin homologues in this phylum. Our search for neuropeptide precursors in the genomes of parasitic flatworms resulted in the description of 34 conserved neuropeptide precursor families, including 13 new ones, and of hundreds of new homologues of known neuropeptide precursor families. Most neuropeptide precursor families show a wide phylogenetic distribution among parasitic flatworms and show little similarity to neuropeptide precursors of other bilaterian animals. However, we could also find orthologs of some conserved bilaterian neuropeptides including pyrokinin, crustacean cardioactive peptide, myomodulin, neuropeptide-Y, neuropeptide KY and SIF-amide. Finally, we determined the expression patterns of seven putative neuropeptide precursor genes in the protoscolex of Echinococcus multilocularis. All genes were expressed in the nervous system with different patterns, indicating a hidden complexity of peptidergic signalling in cestodes.