Evolutionary analysis of species-specific duplications in flatworm genomes.

Platyhelminthes, also known as flatworms, is a phylum of bilaterian invertebrates infamous for their parasitic representatives. The classes Cestoda, Monogenea, and Trematoda comprise parasitic helminths inhabiting multiple hosts, including fishes, humans, and livestock, and are responsible for considerable economic damage and burden on human health. As in other animals, the genomes of flatworms have a wide variety of paralogs, genes related via duplication, whose origins could be mapped throughout the evolution of the phylum. Through in-silico analysis, we studied inparalogs, i.e., species-specific duplications, focusing on their biological functions, expression changes, and evolutionary rate. These genes are thought to be key players in the adaptation process of species to each particular niche. Our results showed that genes related with specific functional terms, such as response to stress, transferase activity, oxidoreductase activity, and peptidases, are overrepresented among inparalogs. This trend is conserved among species from different classes, including free-living species. Available expression data from Schistosoma mansoni, a parasite from the trematode class, demonstrated high conservation of expression patterns between inparalogs, but with notable exceptions, which also display evidence of rapid evolution. We discuss how natural selection may operate to maintain these genes and the particular duplication models that fit better to the observations. Our work supports the critical role of gene duplication in the evolution of flatworms, representing the first study of inparalogs evolution at the genome-wide level in this group.

Muscular remodeling and anteroposterior patterning during tapeworm segmentation.

BACKGROUND: Tapeworms are parasitic flatworms that independently evolved a segmented body plan, historically confounding comparisons with other animals. Anteroposterior (AP) patterning in free-living flatworms and in tapeworm larvae is associated with canonical Wnt signaling and positional control genes (PCGs) are expressed by their musculature in regionalized domains along the AP axis. Here, we extend investigations of PCG expression to the adult of the mouse bile-duct tapeworm Hymenolepis microstoma, focusing on the growth zone of the neck region and the initial establishment of segmental patterning. RESULTS: We show that the adult musculature includes new, segmental elements that first appear in the neck and that the spatial patterns of Wnt factors are consistent with expression by muscle cells. Wnt factor expression is highly regionalized and becomes AP-polarized in segments, marking them with axes in agreement with the polarity of the main body axis, while the transition between the neck and strobila is specifically demarcated by the expression domain of a Wnt11 paralog. CONCLUSION: We suggest that segmentation could originate in the muscular system and participate in patterning the AP axis through regional and polarized expression of PCGs, akin to the gene regulatory networks employed by free-living flatworms and other animals.

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.

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.

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.

Trans-splicing in the cestode Hymenolepis microstoma is constitutive across the life cycle and depends on gene structure and composition.

Spliced leader (SL) trans-splicing is a key process during mRNA maturation of many eukaryotes, in which a short sequence (SL) is transferred from a precursor SL-RNA into the 5' region of an immature mRNA. This mechanism is present in flatworms, in which it is known to participate in the resolution of polycistronic transcripts. However, most trans-spliced transcripts are not part of operons, and it is not clear if this process may participate in additional regulatory mechanisms in this group. In this work, we present a comprehensive analysis of SL trans-splicing in the model cestode Hymenolepis microstoma. We identified four different SL-RNAs which are indiscriminately trans-spliced to 622 gene models. SL trans-splicing is enriched in constitutively expressed genes and does not appear to be regulated throughout the life cycle. Operons represented at least 20% of all detected trans-spliced gene models, showed conservation to those of the cestode Echinococcus multilocularis, and included complex loci such as an alternative operon (processed as either a single gene through cis-splicing or as two genes of a polycistron). Most insertion sites were identified in the 5' untranslated region (UTR) of monocistronic genes. These genes frequently contained introns in the 5' UTR, in which trans-splicing used the same acceptor sites as cis-splicing. These results suggest that, unlike other eukaryotes, trans-splicing is associated with internal intronic promoters in the 5' UTR, resulting in transcripts with strong splicing acceptor sites without competing cis-donor sites, pointing towards a simple mechanism driving the evolution of novel SL insertion sites.

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.

Molecular characterization of the serotonergic transporter from the cestode Echinococcus granulosus: pharmacology and potential role in the nervous system.

Echinococcus granulosus, the etiological agent of human cystic echinococcosis (formerly known as hydatid disease), represents a serious worldwide public health problem with limited treatment options. The essential role played by the neuromuscular system in parasite survival and the relevance of serotonin (5-HT) in parasite movement and development make the serotonergic system an attractive source of drug targets. In this study, we cloned and sequenced a cDNA coding for the serotonin transporter from E. granulosus (EgSERT). Bioinformatic analyses suggest that EgSERT has twelve transmembrane domains with highly conserved ligand and ionic binding sites but a less conserved allosteric site compared with the human orthologue (HsSERT). Modeling studies also suggest a good degree of conservation of the overall structure compared with HsSERT. Functional and pharmacological studies performed on the cloned EgSERT confirm that this protein is indeed a serotonin transporter. EgSERT is specific for 5-HT and does not transport other neurotransmitters. Typical monoamine transport inhibitors also displayed inhibitory activities towards EgSERT, but with lower affinity than for the human SERT (HsSERT), suggesting a high divergence of the cestode transporter compared with HsSERT. In situ hybridization studies performed in the larval protoscolex stage suggest that EgSERT is located in discrete regions that are compatible with the major ganglia of the serotonergic nervous system. The pharmacological properties, the amino acidic substitutions at important functional regions compared with the HsSERT, and the putative role of EgSERT in the nervous system suggest that it could be an important target for pharmacological intervention.

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.

Insights into in vivo adipocyte differentiation through cell-specific labeling in zebrafish.

White adipose tissue hyperplasia has been shown to be crucial for handling excess energy in healthy ways. Though adipogenesis mechanisms have been underscored in vitro, we lack information on how tissue and systemic factors influence the differentiation of new adipocytes. While this could be studied in zebrafish, adipocyte identification currently relies on neutral lipid labeling, thus precluding access to cells in early stages of differentiation. Here we report the generation and analysis of a zebrafish line with the transgene fabp4a(-2.7):EGFPcaax. In vivo confocal microscopy of the pancreatic and abdominal visceral depots of transgenic larvae, revealed the presence of labeled mature adipocytes as well as immature cells in earlier stages of differentiation. Through co-labeling for blood vessels, we observed a close interaction of differentiating adipocytes with endothelial cells through cell protrusions. Finally, we implemented hyperspectral imaging and spectral phasor analysis in Nile Red-labeled transgenic larvae and revealed the lipid metabolic transition towards neutral lipid accumulation of differentiating adipocytes. Altogether our work presents the characterization of a novel adipocyte-specific label in zebrafish and uncovers previously unknown aspects of in vivo adipogenesis. This article has an associated First Person interview with the first author of the paper.

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.

Identification and culture of proliferative cells in abnormal Taenia solium larvae: Role in the development of racemose neurocysticercosis.

Racemose neurocysticercosis is an aggressive disease caused by the aberrant expansion of the cyst form of Taenia solium within the subarachnoid spaces of the human brain and spinal cord resulting in a mass effect and chronic inflammation. Although expansion is likely caused by the proliferation and growth of the parasite bladder wall, there is little direct evidence of the mechanisms that underlie these processes. Since the development and growth of cysts in related cestodes involves totipotential germinative cells, we hypothesized that the expansive growth of the racemose larvae is organized and maintained by germinative cells. Here, we identified proliferative cells expressing the serine/threonine-protein kinase plk1 by in situ hybridization. Proliferative cells were present within the bladder wall of racemose form and absent from the homologous tissue surrounding the vesicular form. Cyst proliferation in the related model species Taenia crassiceps (ORF strain) occurs normally by budding from the cyst bladder wall and proliferative cells were concentrated within the growth buds. Cells isolated from bladder wall of racemose larvae were established in primary cell culture and insulin stimulated their proliferation in a dose-dependent manner. These findings indicate that the growth of racemose larvae is likely due to abnormal cell proliferation. The different distribution of proliferative cells in the racemose larvae and their sensitivity to insulin may reflect significant changes at the cellular and molecular levels involved in their tumor-like growth. Parasite cell cultures offer a powerful tool to characterize the nature and formation of the racemose form, understand the developmental biology of T. solium, and to identify new effective drugs for treatment.

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.

SLFinder, a pipeline for the novel identification of splice-leader sequences: a good enough solution for a complex problem.

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.

Analysis of Gene Expression in Fasciola hepatica Juveniles and Adults by In Situ Hybridization.

In situ hybridization (ISH) is a technique used for the spatial localization of nucleic acids within tissues and cells. It is based on the ability of labeled nucleic acids (probes) to hybridize under the right conditions with the nucleic acids present in fixed biological specimens. In this chapter, we describe protocols for detection of RNA by ISH using digoxigenin (DIG)-labeled probes for Fasciola hepatica adults (in cryosections, given their large size) and for newly excysted juveniles (NEJs, which are ideally suited given their small size for whole-mount ISH). We describe fluorogenic and chromogenic protocols, respectively, but the detection methods can be easily interchanged by using the appropriate enzyme-conjugated antibodies and detection solutions.

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.

The role of fibroblast growth factor signalling in Echinococcus multilocularis development and host-parasite interaction.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Highly conserved nucleotide motifs present in the 5'UTR of the heme-receptor gene shmR are required for HmuP-dependent expression of shmR in Ensifer meliloti.

Heme may represent a major iron-source for bacteria. In the symbiotic nitrogen-fixing bacterium Ensifer meliloti 1021, iron acquisition from heme depends on the outer-membrane heme-receptor ShmR. Expression of shmR gene is repressed by iron in a RirA dependent manner while under iron-limitation its expression requires the small protein HmuP. In this work, we identified highly conserved nucleotide motifs present upstream the shmR gene. These motifs are widely distributed among Alpha and Beta Proteobacteria, and correlate with the presence of HmuP coding sequences in bacterial genomes. According to data presented in this work, we named these new motifs as HmuP-responsive elements (HPREs). In the analyzed genomes, the HPREs were always present upstream of genes encoding putative heme-receptors. Moreover, in those Alpha and Beta Proteobacteria where transcriptional start sites for shmR homologs are known, HPREs were located in the 5'UTR region. In this work we show that in E. meliloti 1021, HPREs are involved in HmuP-dependent shmR expression. Moreover, we show that changes in sequence composition of the HPREs correlate with changes in a predicted RNA secondary structure element and affect shmR gene expression.