RESUMO
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.
Assuntos
Cestoides , Infecções por Cestoides , Mesocestoides , Platelmintos , Animais , Proliferação de Células , Cestoides/genética , Infecções por Cestoides/veterinária , Meios de Cultivo Condicionados , Mesocestoides/genética , Platelmintos/genéticaRESUMO
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.
Assuntos
Cestoides/fisiologia , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Animais , Cestoides/genética , Cestoides/crescimento & desenvolvimento , Infecções por Cestoides/tratamento farmacológico , Clonagem Molecular , Biologia Computacional , Echinococcus granulosus/genética , Echinococcus granulosus/fisiologia , Larva/fisiologia , Mesocestoides/genética , Mesocestoides/crescimento & desenvolvimento , Mesocestoides/fisiologia , Modelos Moleculares , Filogenia , Conformação Proteica , Receptores Acoplados a Proteínas G/genética , Alinhamento de Sequência , Serotonina/farmacologiaRESUMO
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.
Assuntos
Mesocestoides/crescimento & desenvolvimento , Mesocestoides/genética , Tropomiosina/biossíntese , Animais , Sequência Conservada , DNA de Helmintos/química , DNA de Helmintos/genética , Echinococcus granulosus/genética , Echinococcus multilocularis/genética , Evolução Molecular , Duplicação Gênica , Camundongos , Dados de Sequência Molecular , Músculos/química , Filogenia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Schistosoma mansoni/genética , Análise de Sequência de DNA , Homologia de Sequência , Tropomiosina/genética , Turbelários/genéticaRESUMO
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.
Assuntos
Proteínas de Helminto/metabolismo , Mesocestoides/crescimento & desenvolvimento , Proteoma/análise , Animais , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Feminino , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mesocestoides/química , Mesocestoides/genética , Mesocestoides/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Análise Serial de Proteínas , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Temperatura Baixa , Proteínas de Choque Térmico/análise , Proteínas de Helminto/análise , Mesocestoides/metabolismo , Animais , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Helminto/biossíntese , Proteínas de Helminto/química , Proteínas de Helminto/genética , Ponto Isoelétrico , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Mesocestoides/genética , Mesocestoides/crescimento & desenvolvimento , Camundongos , Peso Molecular , Proteômica , Coloração pela PrataRESUMO
With the aim of identifying genes involved in development and parasite adaptation in cestodes, four coding sequences were isolated from the cyclophyllidean Mesocestoides corti larval stage (tetrathyridium). Genes showed significant similarity to the cysteine-rich secreted protein (CRISP) encoding genes, a large family that includes stage and tissue-specific genes from diverse organisms, many associated with crucial biological processes. The full-length McCrisp2 cDNA encodes a predicted protein of 202 residues in length, containing 10 cysteines and a putative signal peptide. The expression level of McCrisp2 was estimated by Real-time PCR, relative to GAPDH, showing an increase of 75% in segmented worms compared to tetrathyridia. By in situ hybridization, McCrisp2 expression was localized mainly at the larvae apical region of tetrathyridia and in the proglottids of segmented worms. Taken together our results suggest a possible role for M. corti CRISP proteins as ES products, potentially involved in differentiation processes as proposed for homologs in other organisms.
Assuntos
Glicoproteínas/genética , Proteínas de Helminto/genética , Mesocestoides/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , Expressão Gênica , Genes Homeobox , Glicoproteínas/química , Glicoproteínas/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Hibridização In Situ , Mesocestoides/química , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , RNA de Helmintos/genética , RNA Mensageiro/genética , Ratos , Alinhamento de SequênciaRESUMO
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.
Assuntos
Replicação do DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histonas/biossíntese , Histonas/genética , Mesocestoides/genética , Sequência de Aminoácidos , Animais , Feminino , Proteínas de Helminto/biossíntese , Proteínas de Helminto/genética , Masculino , Mesocestoides/embriologia , Mesocestoides/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
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.