Unique pharmacological properties of serotoninergic G-protein coupled receptors from cestodes.

BACKGROUND: Cestodes are a diverse group of parasites, some of them being agents of neglected diseases. In cestodes, little is known about the functional properties of G protein coupled receptors (GPCRs) which have proved to be highly druggable targets in other organisms. Notably, serotoninergic G-protein coupled receptors (5-HT GPCRs) play major roles in key functions like movement, development and reproduction in parasites. METHODOLOGY/PRINCIPAL FINDINGS: Three 5-HT GPCRs from Echinococcus granulosus and Mesocestoides corti were cloned, sequenced, bioinformatically analyzed and functionally characterized. Multiple sequence alignment with other GPCRs showed the presence of seven transmembrane segments and conserved motifs but interesting differences were also observed. Phylogenetic analysis grouped these new sequences within the 5-HT7 clade of GPCRs. Molecular modeling showed a striking resemblance in the spatial localization of key residues with their mammalian counterparts. Expression analysis using available RNAseq data showed that both E. granulosus sequences are expressed in larval and adult stages. Localization studies performed in E. granulosus larvae with a fluorescent probe produced a punctiform pattern concentrated in suckers. E. granulosus and M. corti larvae showed an increase in motility in response to serotonin. Heterologous expression revealed elevated levels of cAMP production in response to 5-HT and two of the GPCRs showed extremely high sensitivity to 5-HT (picomolar range). While each of these GPCRs was activated by 5-HT, they exhibit distinct pharmacological properties (5-HT sensitivity, differential responsiveness to ligands). CONCLUSIONS/SIGNIFICANCE: These data provide the first functional report of GPCRs in parasitic cestodes. The serotoninergic GPCRs characterized here may represent novel druggable targets for antiparasitic intervention.

A new approach for the characterization of proliferative cells in cestodes.

Cestodes show a remarkable proliferative capability that sustains the constant growth and differentiation of proglottids essential for their lifestyle. It is believed that a separate population of undifferentiated stem cells (the so-called germinative cells) are the only cells capable of proliferation during growth and development. The study of this particular cell subpopulation is hampered by the current lack of methods to isolate it. In this work, we developed a reproducible flow cytometry and cell sorting method to quantify and isolate the proliferating cells in the tetrathyridia larvae of the model cestode Mesocestoides corti, based on the DNA content of the cells. The isolated cells display the typical germinative cell morphology, and can be used for RNA isolation with a yield in the ng to µg range. We expect that this approach may facilitate the characterization of the germinative cells in M. corti and other model tapeworms.

Proteomic survey of the cestode Mesocestoides corti during the first 24 hours of strobilar development.

Despite the fact that cestodes represent major etiological agents of both human and domestic animal diseases, little is known about the molecular aspects of cestode development. In this work, Mesocestoides corti, a model cestode species, was studied from the early development of its larval form (tetrathyridium) into adult worms (strobilation) using different proteomic approaches. The protein profiles of M. corti tetrathyridia induced or not induced to undergo strobilation were compared. Proteomic mapping by two-dimensional gel electrophoresis showed the resolution of 248 and 154 spots from tetrathyridia that were subjected or not subjected to strobilation induction, respectively, allowing for the detection of at least nine spots exclusive to each group. Spot analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) or MALDI-TOF MS/MS identified four reference proteins (six spots). LC-MS/MS analyses of protein extracts identified 66 proteins, eight of which were found exclusively in non-induced tetrathyridia, while 13 were found exclusively in strobilation-induced tetrathyridia. Among the proteins exclusively identified in strobilation-induced worms, there was a predominance of proteins with functions relating to chaperone activity and protein synthesis and turnover. Quantitative differential expression analysis between M. corti tetrathyridia prior to and after strobilation induction revealed six proteins upregulated in strobilation-induced worms; these proteins were involved in metabolic pathways, cell proliferation, and cytoskeletal rearrangement. Overall, despite the absence of a sequenced M. corti genome, using sequences from other platyhelminthes, we were able to establish comprehensive protein profiles for tetrathyridia prior to and after strobilation induction and identify several proteins potentially involved in the early events leading to strobilation.

Developmental expression of high molecular weight tropomyosin isoforms in Mesocestoides corti.

Tropomyosins are a family of actin-binding proteins with diverse roles in actin filament function. One of the best characterized roles is the regulation of muscle contraction. Tropomyosin isoforms can be generated from different genes, and from alternative promoters and alternative splicing from the same gene. In this work, we have isolated sequences for tropomyosin isoforms from the cestode Mesocestoides corti, and searched for tropomyosin genes and isoforms in other flatworms. Two genes are conserved in the cestodes M. corti and Echinococcus multilocularis, and in the trematode Schistosoma mansoni. Both genes have the same structure, and each gene gives rise to at least two different isoforms, a high molecular weight (HMW) and a low molecular weight (LMW) one. Because most exons are duplicated and spliced in a mutually exclusive fashion, isoforms from one gene only share one exon and are highly divergent. The gene duplication preceded the divergence of neodermatans and the planarian Schmidtea mediterranea. Further duplications occurred in Schmidtea, coupled to the selective loss of duplicated exons, resulting in genes that only code for HMW or LMW isoforms. A polyclonal antibody raised against a HMW tropomyosin from Echinococcus granulosus was demonstrated to specifically recognize HMW tropomyosin isoforms of M. corti, and used to study their expression during segmentation. HMW tropomyosins are expressed in muscle layers, with very low or absent levels in other tissues. No expression of HMW tropomyosins is present in early or late genital primordia, and expression only begins once muscle fibers develop in the genital ducts. Therefore, HMW tropomyosins are markers for the development of muscles during the final differentiation of genital primordia.

Mesocestoides corti: morphological features and glycogen mobilization during in vitro differentiation from larva to adult worm.

Mesocestodes corti has the capacity to develop from the tetrathyridium (larva) stage to adult worm in vitro by trypsin and serum stimulation. Consequently, it has been used as an experimental model system for studying cestode development, host-parasite relationships and anthelmintic drugs. We describe morphological features in 5 different developmental stages of M. corti obtained in vitro, including larvae from the peritoneal cavity of infected mice, trypsin- and serum-stimulated larvae, elongated parasites as well as segmented and mature worms. It is unambiguously confirmed that sexually mature worms are obtained as a result of this in vitro process of differentiation. Defined cellular regions are present in all stages of development studied, some of them surrounded by a basal lamina. Glycogen is present in the larvae obtained from the mouse peritoneal cavity and in parasites encapsulated in the mouse host liver. Glycogen distribution in the parasite changes on trypsin and serum stimulation to differentiate. We propose that changes in the distribution of neutral polysaccharides in the parenchyma of the parasite at different stages of development and degradation of polysaccharides in the transition from segmented to adult worm are related to energy needs necessary for the cellular processes leading to the mature specimen.

In vivo response of Mesocestoides vogae to human insulin.

SUMMARY: Successful host invasion by parasitic helminths involves detection and appropriate response to a range of host-derived signals. Insulin signal response pathways are ancient and highly-conserved throughout the metazoans. However, very little is known about helminth insulin signalling and the potential role it may play in host-parasite interactions. The response of Mesocestoides vogae (Cestoda: Cyclophyllidea) larvae to human insulin was investigated, focusing on tyrosine-phosphorylation status, glucose content, survival and asexual reproduction rate. Parasite larvae were challenged with different levels of insulin for variable periods. The parameters tested were influenced by human insulin, and suggested a host-parasite molecular dialogue.

Preliminary analysis of cold stress responsive proteins in Mesocestoides corti larvae.

Many parasites undergo sudden changes in environmental conditions at some stage during their life cycle. The molecular response to this variation is characterised by a rapid transcriptional activation of a specific set of genes coding for proteins generically known as stress proteins. They appear to be also involved in various biological processes including cell proliferation and differentiation. The platyhelminth parasite, Mesocestoides corti (Cestoda) presents important properties as a model organism. Under stress conditions, key molecules involved in metabolic pathways as well as in the growth and differentiation of the parasite can be identified. 2D protein expression profile of tetrathyridia of M. corti, submitted to nutritional starvation and cold stress is described, as well as the recovery pattern. A set of specifically expressed proteins was observed in each experimental condition. Quantitative and qualitative differences and stress recovery pattern are also reported. This work makes evident the high plasticity and resistance to extreme environmental conditions of these parasites at the molecular level.

Developmental expression pattern of histone H4 gene associated to DNA synthesis in the endoparasitic platyhelminth Mesocestoides corti.

In higher eukaryotes, histone gene expression is coupled to DNA replication during the S-phase of the cell cycle. This coupling is primarily controlled at the transcriptional level. Considering the basal phylogenic position of platyhelminthes in the bilateria phylum, we have cloned a partial sequence of the histone H4 gene of Mesocestoides corti and studied its expression during the post larval development of this endoparasitic platyhelminth. In in vitro trypsin-induced tetrathyridia development to segmented adult worm, we found that histone H4 is expressed concomitantly with DNA synthesis throughout all stages of development. DNA synthesis and histone H4 mRNA levels were sharply increased at 24 h after inducing development. Afterwards, tetrathyridia grew in length from days 4 to 12 of development as proliferative cells gradually increased in number. Consequently, during this period of development histone H4 mRNA levels were upregulated. Taken together these results suggest that a replication-dependent expression pattern of histone H4 occurs in ancient bilateria, such as platyhelminthes, as previously observed in higher eukaryotes.

Localization, specific activity, and molecular forms of acetylcholinesterase in developmental stages of the cestode Mesocestoides corti.

The nervous system of flatworms is quite simple although there is increasing evidence indicating that it is chemically complex. Studies of the nervous system in these animals have only been performed in the larval stage or in the adult worms, which are easy to obtain in nature, while the description of the nervous system in developing stages of these organisms is missing. Mesocestoides corti is a parasitic platyhelminth whose larvae can be induced in vitro to develop to adult, sexually mature worms, opening the possibility of studying the nervous system of a flatworm in different stages of development. Here, we describe the presence, activity, location, and molecular forms of acetylcholinesterase (AChE) in different stages of development of M. corti, from the larvae to adult forms of this endoparasite, obtained in in vitro cultures after induction of the larval stage with trypsin. Our results point to AChE as a molecular marker of the nervous system in platyhelminthes. The change in molecular forms of this enzyme and the increase in its activity during development from larvae to adult worm may reflect the presence of a more complex nervous system, necessary to adjust and coordinate the movement of a much bigger structure. A relationship between the development of the reproductive apparatus in segmented and adult worms with a more complex nervous system in these stages is also apparent. Finally, our study opens the possibility of applying anti-AChE as more effective therapeutic strategies against cestode parasites.

Praziquantel and albendazole damaging action on in vitro developing Mesocestoides corti (Platyhelminthes: Cestoda).

Parasitic flatworms present several steps of body architecture rearrangement during their fast transition from one developmental stage to another, which are, at least in part, responsible for their evasion from host immune response. Besides, different developmental stages present different degrees of susceptibility to drug action, and the identification of more susceptible stages is of importance for the definition of therapeutical approaches. Mesocestoides corti (syn. Mesocestoides vogae) is considered a good model to study cestode biology because it can be easily manipulated both in vivo and in vitro and due to its relatively close relationship to cestodes of medical relevance, such as those from genera Echinococcus or Taenia. We have analyzed the damaging action of two broad spectrum anthelmintic drugs (praziquantel and albendazole) throughout the in vitro strobilization process of M. corti in order to identify developmental stages or body structures more susceptible to these drugs. Tetrathyridia (larval stage) and segmented-induced worms were cultivated and treated with praziquantel and albendazole. Whole mounted samples, taken from different developmental stages, were fixed and stained with fluorophore-labeled WGA lectin and phalloidin for the analysis of tegument and muscles, respectively. Confocal laser scanning microscopy was used to identify anatomical changes and lesions caused by each anthelmintic drug in a 3D view. We demonstrated that both praziquantel and albendazole cause extensive tissue damage, especially on tegument, and that adult forms were the most susceptible to drug exposure.

Early post-larval development of the endoparasitic platyhelminth Mesocestoides corti: trypsin provokes reversible tegumental damage leading to serum-induced cell proliferation and growth.

Mesocestoides corti is a suitable in vitro model for studying the development of human endoparasitic platyhelminthes. Treatment with trypsin, supplemented with fetal bovine serum (FBS), induces M. corti development from larvae (tetrathyridia) to segmented adult worm; however, the role of this protease and of FBS in post-larval development induction remains unknown. To characterize the participation of trypsin enzymatic activity and of FBS in the induction of tetrathyridia growth and development, both stimuli were added to the larvae either together or sequentially. Additionally, specific inhibition of trypsin activity was also monitored. Finally, the effect of the enzyme on the parasite tegument as well as the proliferative activity and location of proliferating cells after induction of tetrathyridia development were also studied. We conclude that trypsin-induced tetrathyridia development to adult worm is FBS-dependent and that the effect of serum factors is dependent upon a previous trypsin-induced reversible damage to the larva tegument. In dividing and non-dividing tetrathyridia, proliferative activity of cells is mainly located within the apical massif in the anterior region and nerve cords of larvae, respectively. In tetrathyridia stimulated to develop to adult worms, an intense proliferative activity is evident along the nerve cords. Our results suggest that in natural infections the tetrathyridia tegument is temporally made permeable to growth factors by proteolytic enzyme activity in the intestine juice of the definitive host, thus leading to development to adult worms.

Mesocestoides corti: a LIM-homeobox gene upregulated during strobilar development.

To understand the molecular processes regulating morphological changes during cestode life histories we focused on homeodomain (HD) proteins, a family of transcription factors essential for pattern formation during development. In this study we report the isolation of the partial sequence of MvLim, a LIM-HD gene of Mesocestoides corti. Other members of this gene family, characterized in Drosophila melanogaster, Caenorhabditis elegans and vertebrates contribute to cell fate determination of various neuronal subtypes. Phylogenetic analyses showed that MvLim clusters with members of the LIN-11 group and that platyhelminths have at least two different LIM-HD genes. By real time PCR we determined that MvLim expression is 20-fold greater in segmented worms than in tetrathyridia. The enhancement of MvLim expression during strobilation could be associated to changes in the innervation pattern occurring in proglottids development.

The toxicity of praziquantel against Mesocestoides vogae (syn. corti) tetrathyridia can be assessed using a novel in vitro system.

We recently standardised Mesocestoides vogae (syn. corti) tetrathyridia cultures in the presence of sodium taurocholate. Parasite clustering and segmentation were observed as taurocholate-dependent effects in biphasic and monophasic media, respectively, and both were inhibited by a specific minimum inhibitory concentration (m.i.c.) of the cestocidal drugs albendazol and praziquantel. In the present study, we analysed the relationship between clustering inhibition and drug toxicity using praziquantel and a mouse experimental infection. In an "in vitro-in vivo" trial, a significant (ANOVA, P<0.05) reduction was observed in the infectivity of tetrathyridia previously cultured with praziquantel m.i.c. (0.06 micro g/ml) for 10 days. In an "in vivo-in vitro" trial, the clustering of tetrathyridia recovered from mice treated with praziquantel was found to be markedly reduced: 22%, compared with 83% cluster-containing wells of parasites from control mice. These results show that the outcome of infection and the suppression of taurocholate-induced clustering provide consistent indications of praziquantel toxicity against M. vogae, an observation confirmed by histological studies. The easily recorded clustering inhibition of M. vogae tetrathyridia in biphasic medium is a potentially useful system for the assessment of drug toxicity against cestode larvae.

In vitro taurocholate-induced segmentation and clustering of Mesocestoides vogae (syn. corti) tetrathyridia (Cestoda)--inhibition byh cestocidal drugs.

Mesocestoides vogae (syn. M. corti) tetrathyridia were cultured in the presence of sodium taurocholate, for the purpose of exploring the suitability of this organism for the in vitro assay of cestocidal drugs. Parasite clustering and segmentation were observed as taurocholate-dependent effects in biphasic and monophasic media, respectively. Interestingly, representative members of two major classes of known cestocidal agents (namely, albendazole and praziquantel) blocked these effects. Furthermore, it was possible to determine a specific concentration of the drugs that inhibited clustering and segmentation (minimum inhibitory concentration). In contrast, no inhibition was obtained in the presence of anthelmintics without cestocidal activity. These observations open the way for further studies focused at understanding how the activity of the drugs is involved in the suppression of the taurocholate-induced effects.

Effect of praziquantel on the strobilar development of Mesocestoides corti in vitro.

The effect of praziquantel (PZQ) on the strobilar development of the cyclophyllidean cestode Mesocestoides corti was explored. Mesocestoides corti larvae were cultivated under conditions reported to favour their differentiation to the adult stage. Parasites were exposed to 0.1 microg ml(-1) PZQ for 16 h and subsequently transferred to drug-free medium. The ocurrence of segmentation--an early event of the larval somatic differentiation to the adult worm-- was considered as quantitative data. This phenomenon was evidenced earlier in worms transiently exposed to PZQ with respect to control cultures. Moreover, the rate of segmentation of drug-treated worms at the end of the experiment almost doubled that of control worms. To date, no similar effect on any cestode developmental process has been reported for an anthelmintic drug. In the light of the existing knowledge and understanding of PZQ mechanisms of action, the proposed experimental approach could contribute to the elucidation of pathways and mechanisms involved in cestode strobilar development.