RESUMO
Parasites belonging to the class Cestoda include zoonotic species such as Echinococcus spp. and Taenia spp. that cause morbidity and mortality in endemic areas, mainly affecting pastoral and rural communities in low income countries but also upper middle income countries. Cestodes show remarkable developmental plasticity, implying tight regulation of gene expression throughout their complex life cycles. Despite the recent availability of genomic data for cestodes, little progress was made on postgenomic functional studies. MicroRNAs (miRNAs) are key components of gene regulatory systems that guide diverse developmental processes in multicellular organisms. miR-71 is a highly expressed miRNA in cestodes, which is absent in vertebrates and targets essential parasite genes, representing a potential key player in understanding the role of miRNAs in cestodes biology. Here we used transfection with antisense oligonucleotides to perform whole worm miRNA knockdown in tetrathyridia of Mesocestoides vogae (syn. Mesocestoides corti), a laboratory model of cestodes. We believe this is the first report of miRNA knockdown at the organism level in these parasites. Our results showed that M. vogae miR-71 is involved in the control of strobilation in vitro and in the establishment of murine infection. In addition, we identified miR-71 targets in M. vogae, several of them being de-repressed upon miR-71 knockdown. This study provides new knowledge on gene expression regulation in cestodes and suggests that miRNAs could be evaluated as new selective therapeutic targets for treating Neglected Tropical Diseases prioritised by the World Health Organization.
Assuntos
Cestoides , Infecções por Cestoides , Mesocestoides , MicroRNAs , Camundongos , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Cestoides/genética , Infecções por Cestoides/veterinária , Infecções por Cestoides/parasitologia , Mesocestoides/metabolismo , Estágios do Ciclo de VidaRESUMO
The histone chaperone SET/TAF-Iß is implicated in processes of chromatin remodelling and gene expression regulation. It has been associated with the control of developmental processes, but little is known about its function in helminth parasites. In Mesocestoides corti, a partial cDNA sequence related to SET/TAF-Iß was isolated in a screening for genes differentially expressed in larvae (tetrathyridia) and adult worms. Here, the full-length coding sequence of the M. corti SET/TAF-Iß gene was analysed and the encoded protein (McSET/TAF) was compared with orthologous sequences, showing that McSET/TAF can be regarded as a SET/TAF-Iß family member, with a typical nucleosome-assembly protein (NAP) domain and an acidic tail. The expression patterns of the McSET/TAF gene and protein were investigated during the strobilation process by RT-qPCR, using a set of five reference genes, and by immunoblot and immunofluorescence, using monospecific polyclonal antibodies. A gradual increase in McSET/TAF transcripts and McSET/TAF protein was observed upon development induction by trypsin, demonstrating McSET/TAF differential expression during strobilation. These results provided the first evidence for the involvement of a protein from the NAP family of epigenetic effectors in the regulation of cestode development.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas de Helminto/metabolismo , Chaperonas de Histonas/metabolismo , Mesocestoides/metabolismo , Sequência de Aminoácidos , Animais , Infecções por Cestoides/parasitologia , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Feminino , Proteínas de Helminto/genética , Chaperonas de Histonas/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda). Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C16. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C16 incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate's counterparts.
Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas de Helminto/metabolismo , Mesocestoides/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Dados de Sequência Molecular , Transporte ProteicoRESUMO
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
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.
Assuntos
Infecções por Cestoides/parasitologia , Glicogênio/metabolismo , Mesocestoides/crescimento & desenvolvimento , Mesocestoides/metabolismo , Animais , Metabolismo Energético , Feminino , Larva/crescimento & desenvolvimento , Larva/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Polissacarídeos/metabolismoRESUMO
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.
Assuntos
Insulina/metabolismo , Mesocestoides/fisiologia , Sequência de Aminoácidos , Animais , Glucose/metabolismo , Humanos , Insulina/farmacologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mesocestoides/crescimento & desenvolvimento , Mesocestoides/metabolismo , Dados de Sequência Molecular , Fosforilação , Reprodução Assexuada , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de Proteína , Transdução de Sinais , Tirosina/metabolismoRESUMO
Protein glycosylation is an important post-translational modification underlying host-parasite interactions, which may determine the outcome of infection. Although Mesocestoides vogae represents an important model for investigating the various aspects of cestode biology, virtually no information is available about the structure and synthesis of glycans in this parasite. In this work, focused on the initiation pathway of mucin-type O-glycosylation in M. vogae, we characterized O-glycoproteins bearing the simple mucin-type cancer-associated Tn and sialyl-Tn antigens, and the expression and activity of ppGalNAc-T, the key enzyme responsible for the first step of mucin-type O-glycosylation. Using immunohistochemistry, Tn and sialyl-Tn antigens were detected mainly in the tegument (microtriches) and in parenchymal cells. Tn expression was also observed in lateral nerve cords. Both Tn and sialyl-Tn antigens were detected in in vitro cultured parasites. Based on their electrophoretic mobility, Tn- and sialyl-Tn-bearing glycoproteins from M. vogae were separated into several components of 22 to 60 kDa. The observation that Tn and sialyl-Tn glycoproteins remained in the 0.6N perchloric acid-soluble fraction suggested that they could be good candidates for characterizing mucin-type glycosylation in this parasite. O-glycoproteins were purified and initially characterized using a proteomic approach. Immunohistochemical analysis of the tissue distribution of ppGalNAc-T revealed that this enzyme is expressed in the sub-tegumental region and in the parenchyma of the parasite. In M. vogae cultured in vitro, ppGalNAc-T was mainly detected in the suckers. Using a panel of 8 acceptor substrate synthetic peptides, we found that M. vogae ppGalNAc-T preferentially glycosylate threonine residues, the best substrates being peptides derived from human mucin MUC1 and from Trypanosoma cruzi mucin. These results suggest that M. vogae might represent a useful model to study O-glycosylation, and provide new research avenues for future studies on the glycopathobiology of helminth parasites.
Assuntos
Antígenos de Helmintos/metabolismo , Mesocestoides/metabolismo , Animais , Antígenos de Helmintos/análise , Antígenos Glicosídicos Associados a Tumores/metabolismo , Western Blotting , Sequência de Carboidratos , Infecções por Cestoides/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicosilação , Interações Hospedeiro-Parasita , Imuno-Histoquímica , Mesocestoides/química , Camundongos , Camundongos Endogâmicos , Mucinas/metabolismo , N-Acetilgalactosaminiltransferases/análise , Parasitologia/métodos , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
This work describes two new fatty acid binding proteins (FABPs) identified in the parasite platyhelminth Mesocestoides vogae (syn. corti). The corresponding polypeptide chains share 62% identical residues and overall 90% similarity according to CLUSTALX default conditions. Compared with Cestoda FABPs, these proteins share the highest similarity score with the Taenia solium protein. M. vogae FABPs are also phylogenetically related to the FABP3/FABP4 mammalian FABP subfamilies. The native proteins were purified by chromatographical procedures, and apparent molecular mass and isoelectric point were determined. Immunolocalization studies determined the localization of the expression of these proteins in the larval form of the parasite. The genomic exon-intron organization of both genes is also reported, and supports new insights on intron evolution. Consensus motifs involved in splicing were identified.
Assuntos
Evolução Molecular , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Mesocestoides/metabolismo , Sequência de Aminoácidos , Animais , Éxons , Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Proteínas de Helminto/isolamento & purificação , Íntrons , Microscopia Confocal , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
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
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.
Assuntos
Acetilcolinesterase/metabolismo , Estágios do Ciclo de Vida/fisiologia , Mesocestoides/crescimento & desenvolvimento , Mesocestoides/metabolismo , Acetilcolinesterase/química , Animais , Feminino , Estágios do Ciclo de Vida/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Tripsina/farmacologiaRESUMO
This article focuses on the initiation pathway of mucin-type O-glycosylation in helminth parasites. The presence of the GalNAc-O-Ser/Thr structure, also known as Tn antigen, a truncated determinant related to aberrant glycosylation in mammal cells, and the activity of the UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferase (ppGaNTase), the enzyme responsible for its synthesis, were studied in species from major taxonomic groups. Tn reactivity was determined in extracts from Taenia hydatigena, Mesocestoides corti, Fasciola hepatica, Nippostrongylus brasiliensis, and Toxocara canis using the monoclonal antibody 83D4. The Tn determinant was revealed in all preparations, and multiple patterns of Tn-bearing glycoproteins were observed by immunoblotting. Additionally, the first evidence that helminth parasites express ppGaNTase activity was obtained. This enzyme was studied in extracts from Echinococcus granulosus, F. hepatica, and T. canis by measuring the incorporation of UDP-(3H)GalNAc to both deglycosylated ovine syalomucin (dOSM) and synthetic peptide sequences derived from tandem repeats of human mucins. Whereas significant levels of ppGaNTase activity were detected in all the extracts when dOSM was used as a multisite acceptor, it was only observed in F. hepatica and E. granulosus extracts when mucin-derived peptides were used, suggesting that T. canis ppGaNTase enzyme(s) may represent a member of the gene family with a more restricted specificity for worm O-glycosylation motifs. The widespread expression of Tn antigen, capable of evoking both humoral and cellular immunity, strongly suggests that simple mucin-type O-glycosylation does not constitute an aberrant phenomenon in helminth parasites.