RESUMO
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.
Assuntos
Cestoides/metabolismo , Proteínas de Helminto/metabolismo , Sistema Nervoso/metabolismo , Receptores 5-HT1 de Serotonina/metabolismo , Sequência de Aminoácidos , Animais , Echinococcus/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Helminto/química , Proteínas de Helminto/genética , Humanos , Hymenolepis/metabolismo , Receptores 5-HT1 de Serotonina/química , Receptores 5-HT1 de Serotonina/genética , Alinhamento de Sequência , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologiaRESUMO
Neglected tropical diseases caused by helminth infections currently affect millions of people worldwide. Among them, there are three tapeworm species of outstanding importance: Echinococcus granulosus, E. multilocularis, and Taenia solium, which are responsible for cystic echinococcosis, alveolar echinococcosis, and cysticercosis, respectively. Despite several attempts, there is still a need for an effective and low-cost serological diagnostic test that can be used in endemic countries. In the present work, we described an innovative bioinformatic workflow for a rational prediction of putative peptide candidates for one-step serological diagnosis of any of these infections. First, we predicted the theoretical secretome shared by the three tapeworms starting from their full reported proteomes. Then, through immunoinformatics, we identified proteins within the shared secretome displaying high antigenicity scores and bearing T cell epitopes able to bind most human MHC-II alleles. Secondly, in such proteins, we identified linear B cell epitopes without post-translational modifications, and mapped them on 3D modelled structures to visualize their antibody accessibilities. As a result, we finally suggested two antigenic peptides shared between the secretomes of the three parasite species, which could be further tested for their immunodiagnostic potential.
Assuntos
Biologia Computacional/métodos , Echinococcus/isolamento & purificação , Helmintíase/diagnóstico , Peptídeos/metabolismo , Taenia/isolamento & purificação , Animais , Anticorpos Anti-Helmínticos/metabolismo , Antígenos de Helmintos/imunologia , Echinococcus/metabolismo , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Genoma Helmíntico , Proteínas de Helminto/imunologia , Proteínas de Helminto/metabolismo , Helmintíase/imunologia , Helmintíase/parasitologia , Humanos , Doenças Negligenciadas/diagnóstico , Doenças Negligenciadas/imunologia , Doenças Negligenciadas/parasitologia , Processamento de Proteína Pós-Traducional , Proteoma/imunologia , Proteoma/metabolismo , Taenia/metabolismoRESUMO
BACKGROUND: The parasite Echinococcus canadensis (G7) (phylum Platyhelminthes, class Cestoda) is one of the causative agents of echinococcosis. Echinococcosis is a worldwide chronic zoonosis affecting humans as well as domestic and wild mammals, which has been reported as a prioritized neglected disease by the World Health Organisation. No genomic data, comparative genomic analyses or efficient therapeutic and diagnostic tools are available for this severe disease. The information presented in this study will help to understand the peculiar biological characters and to design species-specific control tools. RESULTS: We sequenced, assembled and annotated the 115-Mb genome of E. canadensis (G7). Comparative genomic analyses using whole genome data of three Echinococcus species not only confirmed the status of E. canadensis (G7) as a separate species but also demonstrated a high nucleotide sequences divergence in relation to E. granulosus (G1). The E. canadensis (G7) genome contains 11,449 genes with a core set of 881 orthologs shared among five cestode species. Comparative genomics revealed that there are more single nucleotide polymorphisms (SNPs) between E. canadensis (G7) and E. granulosus (G1) than between E. canadensis (G7) and E. multilocularis. This result was unexpected since E. canadensis (G7) and E. granulosus (G1) were considered to belong to the species complex E. granulosus sensu lato. We described SNPs in known drug targets and metabolism genes in the E. canadensis (G7) genome. Regarding gene regulation, we analysed three particular features: CpG island distribution along the three Echinococcus genomes, DNA methylation system and small RNA pathway. The results suggest the occurrence of yet unknown gene regulation mechanisms in Echinococcus. CONCLUSIONS: This is the first work that addresses Echinococcus comparative genomics. The resources presented here will promote the study of mechanisms of parasite development as well as new tools for drug discovery. The availability of a high-quality genome assembly is critical for fully exploring the biology of a pathogenic organism. The E. canadensis (G7) genome presented in this study provides a unique opportunity to address the genetic diversity among the genus Echinococcus and its particular developmental features. At present, there is no unequivocal taxonomic classification of Echinococcus species; however, the genome-wide SNPs analysis performed here revealed the phylogenetic distance among these three Echinococcus species. Additional cestode genomes need to be sequenced to be able to resolve their phylogeny.
Assuntos
Equinococose/genética , Echinococcus/genética , Genoma de Protozoário , Animais , Proteínas Argonautas/antagonistas & inibidores , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Hibridização Genômica Comparativa , Mapeamento de Sequências Contíguas , Ilhas de CpG , Metilação de DNA , Equinococose/parasitologia , Equinococose/patologia , Echinococcus/classificação , Echinococcus/metabolismo , Humanos , Sequências Repetitivas Dispersas/genética , Filogenia , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
BACKGROUND: microRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. The particular developmental and metabolic characteristics of cestode parasites highlight the importance of studying miRNA gene regulation in these organisms. Here, we perform a comprehensive analysis of miRNAs in the parasitic cestode Echinococcus canadensis G7, one of the causative agents of the neglected zoonotic disease cystic echinococcosis. METHODS: Small RNA libraries from protoscoleces and cyst walls of E. canadensis G7 and protoscoleces of E. granulosus sensu stricto G1 were sequenced using Illumina technology. For miRNA prediction, miRDeep2 core algorithm was used. The output list of candidate precursors was manually curated to generate a high confidence set of miRNAs. Differential expression analysis of miRNAs between stages or species was estimated with DESeq. Expression levels of selected miRNAs were validated using poly-A RT-qPCR. RESULTS: In this study we used a high-throughput approach and found transcriptional evidence of 37 miRNAs thus expanding the miRNA repertoire of E. canadensis G7. Differential expression analysis showed highly regulated miRNAs between life cycle stages, suggesting a role in maintaining the features of each developmental stage or in the regulation of developmental timing. In this work we characterize conserved and novel Echinococcus miRNAs which represent 30 unique miRNA families. Here we confirmed the remarkable loss of conserved miRNA families in E. canadensis, reflecting their low morphological complexity and high adaptation to parasitism. CONCLUSIONS: We performed the first in-depth study profiling of small RNAs in the zoonotic parasite E. canadensis G7. We found that miRNAs are the preponderant small RNA silencing molecules, suggesting that these small RNAs could be an essential mechanism of gene regulation in this species. We also identified both parasite specific and divergent miRNAs which are potential biomarkers of infection. This study will provide valuable information for better understanding of the complex biology of this parasite and could help to find new potential targets for therapy and/or diagnosis.
Assuntos
Equinococose/veterinária , Echinococcus/genética , MicroRNAs/genética , RNA de Helmintos/genética , Doenças dos Ovinos/parasitologia , Doenças dos Suínos/parasitologia , Animais , Sequência de Bases , Equinococose/parasitologia , Echinococcus/isolamento & purificação , Echinococcus/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/metabolismo , Dados de Sequência Molecular , RNA de Helmintos/metabolismo , Análise de Sequência de RNA , Ovinos , SuínosRESUMO
Histones from the parasitic platyhelminthes, Echinococcus granulosus and Fasciola hepatica, were systematically characterized. Core histones H2A, H2B, H3 and H4, which were identified on the basis of amino acid sequencing and mass spectrometry data, showed conserved electrophoretic patterns. Histones H1, identified on the basis of physicochemical properties, amino acid composition and amino acid sequencing, showed divergence, both in their number and electrophoretic mobilities, between the two species and among other organisms. According to these data, core histones but not H1 histones, would be stabilized during evolution at the level of platyhelminthes.
Assuntos
Cromatina/química , Echinococcus/química , Fasciola hepatica/química , Histonas/química , Histonas/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Bovinos , Cromatina/metabolismo , Cromatografia Líquida de Alta Pressão , Echinococcus/metabolismo , Eletroforese em Gel de Poliacrilamida , Fasciola hepatica/metabolismo , Histonas/genética , Histonas/metabolismo , Ouriços-do-Mar/química , Ouriços-do-Mar/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Timo/química , Timo/metabolismo , Trypanosoma cruzi/química , Trypanosoma cruzi/metabolismoRESUMO
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.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Helmintos/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Animais , Antígenos Glicosídicos Associados a Tumores/química , Western Blotting , Bovinos , Cães , Echinococcus/enzimologia , Echinococcus/imunologia , Echinococcus/metabolismo , Eletroforese em Gel de Poliacrilamida , Fasciola hepatica/enzimologia , Fasciola hepatica/imunologia , Fasciola hepatica/metabolismo , Glicopeptídeos/metabolismo , Glicoproteínas/análise , Glicosilação , Helmintos/enzimologia , Helmintos/imunologia , Humanos , Mesocestoides/enzimologia , Mesocestoides/imunologia , Mesocestoides/metabolismo , Camundongos , Nippostrongylus/enzimologia , Nippostrongylus/imunologia , Nippostrongylus/metabolismo , Ratos , Ratos Wistar , Taenia/enzimologia , Taenia/imunologia , Taenia/metabolismo , Toxocara canis/enzimologia , Toxocara canis/imunologia , Toxocara canis/metabolismo , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
We describe the preparation of Echinococcus granulosus metacestode protein extracts for two-dimensional electrophoresis (2-DE). Protoscoleces and hydatid fluid were prepared by precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants. Compared to the untreated control, TCA precipitation improved the 2-DE gel profile of the protoscoleces proteins. Comparison of 2-DE gels from insoluble and soluble fractions of the protoscoleces protein extract showed that most proteins are insoluble after lysis by sonication. Host serum proteins, especially albumin and globulins, caused horizontal streaking problems on the hydatid fluid 2-DE gels due to their high content in this sample. Even after the preparation of a hydatid fluid parasite enriched fraction, the high amount of bovine serum albumin and globulins made parasite-specific proteins difficult to detect by 2-DE. Despite the absence of an E. granulosus genome sequencing or expressed sequence tag (EST) projects, it was possible to identify 15 prominent protein spots from a whole protein protoscoleces 2-DE gel by peptide mass fingerprinting. These include actins, tropomyosin, paramyosin, thioredoxin reductase, antigen P-29, cyclophilin, and the heat shock proteins hsp70 and hsp20. This work demonstrates that 2-DE and PMF are important tools to identify proteins from the hydatid fluid and protoscoleces and for the comparative analysis of cysts from different hosts or between active and resting cysts.
Assuntos
Equinococose/parasitologia , Echinococcus/metabolismo , Larva/metabolismo , Proteoma , Animais , Echinococcus/patogenicidade , Eletroforese em Gel Bidimensional , Etiquetas de Sequências ExpressasAssuntos
Echinococcus/crescimento & desenvolvimento , Echinococcus/metabolismo , Proteínas de Helminto/biossíntese , Tropomiosina/biossíntese , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Echinococcus/genética , Regulação da Expressão Gênica , Proteínas de Helminto/genética , Larva/genética , Larva/crescimento & desenvolvimento , Tropomiosina/genéticaRESUMO
This work describes a new gene coding for a fatty acid binding protein (FABP) in the parasite Echinococcus granulosus, named EgFABP2. The complete gene structure, including the promoter sequence, is reported. The genomic coding domain organisation of the previously reported E. granulosus FABP gene (EgFABP1) has been also determined. The corresponding polypeptide chains share 76% of identical residues and an overall 96% of similarity. The two EgFABPs present the highest amino acid homologies with the mammalian FABP subfamily containing heart-FABPs (H-FABPs). The coding sequences of both genes are interrupted by a single intron located in the position of the third intron reported for vertebrate FABP genes. Both genes are expressed in the protoscolex stage of the parasite. The promoter region of EgFABP2 presents several consensus putative cis-acting elements found in other members of the family, suggesting interesting possible mechanisms involved in the host-parasite adaptation.
Assuntos
Proteínas de Transporte/genética , Echinococcus/genética , Proteínas de Peixes , Proteínas de Neoplasias , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/biossíntese , Proteínas de Transporte/química , Echinococcus/metabolismo , Proteínas de Ligação a Ácido Graxo , Expressão Gênica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
EgFABP1 is a developmentally regulated intracellular fatty acid binding protein characterized in the larval stage of parasitic platyhelminth Echinococcus granulosus. It is structurally related to the heart group of fatty acid binding proteins (H-FABPs). Binding properties and ligand affinity of recombinant EgFABP1 were determined by fluorescence spectroscopy using cis- and trans-parinaric acid. Two binding sites for cis- and trans-parinaric acid were found (K(d(1)) 24+/-4 nM, K(d(2)) 510+/-60 nM for cis-parinaric acid and K(d(1)) 32+/-4 nM, K(d(2)) 364+/-75 nM for trans-parinaric). A putative third site for both fatty acids is discussed. Binding preferences were determined using displacement assays. Arachidonic and oleic acids presented the highest displacement percentages for EgFABP1. The Echinococcus FABP is the unique member of the H-FABP group able to bind two long chain fatty acid molecules with high affinity. Structure-function relationships and putative roles for EgFABP1 in E. granulosus metabolism are discussed.
Assuntos
Proteínas de Transporte/metabolismo , Echinococcus/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Peixes , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Proteínas de Transporte/biossíntese , Proteínas de Transporte/química , Echinococcus/química , Echinococcus/embriologia , Escherichia coli/metabolismo , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/química , Ácidos Graxos Insaturados/química , Larva/metabolismo , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
In the present work we demonstrate that the cancer-associated O-glycosylated Tn antigen (GalNAc-O-Ser/Thr) is expressed by the cestode Echinococcus granulosus. This antigen was detected in both larval and adult worm extracts, with the highest specific activity observed in the adult excretion/secretion preparation. Histochemical analysis showed that Tn is preferentially expressed in the parenchyma in both parasite stages and the external part of tegument in adult worms. A similar pattern was observed for sialyl-Tn, a related O-linked antigen. Tn glycoproteins from protoscoleces were resolved by SDS-PAGE in two main components of 43 and 49 kDa. After purification, this material was reactive with lectins which bind GlcNAc/sialic acid, GalNAc, and T antigen. In a preliminary evaluation, high levels of Tn antigen were detected in serum samples from patients with hydatid cyst, suggesting that the measure of Tn in serum could be a biomarker of this disease, although extensive work is necessary in order to determine the clinical usefulness of this assay. The results reported here, the first evidence of O-glycosylation pathways in E. granulosus and the presence of Tn antigen in cestodes, suggest that the evaluation of O-glycosylated antigens might give new insights in the host-parasite relationship.
Assuntos
Antígenos Glicosídicos Associados a Tumores/isolamento & purificação , Equinococose/imunologia , Echinococcus/imunologia , Adulto , Animais , Antígenos Glicosídicos Associados a Tumores/sangue , Antígenos Glicosídicos Associados a Tumores/metabolismo , Western Blotting , Neoplasias da Mama/sangue , Neoplasias da Mama/imunologia , Cromatografia de Afinidade , Cães , Equinococose/sangue , Echinococcus/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Glicosilação , Humanos , Lectinas/metabolismo , MasculinoRESUMO
Infection of Balb/c mice with Echinococcus granulosus protoscoleces constitutes the model for secondary hydatid infection. The immune response of Balb/c mice infected with E. granulosus is characterized by secretion of antibodies specific for carbohydrate epitopes and production of type-2 cytokines. A role for glycoconjugates in the induction of type-2 responses has been suggested in other host--parasite systems. Although glycoconjugates are immunogenic in E. granulosus infection, the role of these molecules in the establishment of the type-2 response has never been analysed. In this study, a carbohydrate rich fraction (E4+) from E. granulosus protoscoleces was obtained using the monoclonal antibody E492/G1 specific for the moiety Galalpha(1,4)Gal which is widely represented in protoscoleces and other E. granulosus antigenic preparations. The results showed that E4+ was immunogenic in Balb/c mice evoking an antibody response mainly directed against carbohydrate epitopes. In addition, splenocytes from E4+-immunized mice showed suppressed proliferative responses to Con A and E4+ induced IL-10 secretion by E4+-primed and naive splenocytes. The fraction E4+ also was immunogenic in infected mice during early infection. In this case also, splenocytes from infected mice as well as peritoneal cells from infected or naive mice, when stimulated in vitro with E4+, secreted IL-10. Collectively, these results suggest that E4+ may be involved in immunosuppression phenomena and, by stimulating IL-10 secretion, may contribute to the induction and sustaining of the type-2 cytokine response established in early experimental infection.
Assuntos
Antígenos de Helmintos/imunologia , Equinococose/imunologia , Echinococcus/imunologia , Glicoconjugados/imunologia , Células Th2/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Citocinas/biossíntese , Equinococose/parasitologia , Echinococcus/crescimento & desenvolvimento , Echinococcus/metabolismo , Imunização , Terapia de Imunossupressão , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The 14-3-3 protein, already described in the metacestode of Echinococcus multilocularis, has been characterized in the Echinococcus granulosus adult worm. Immunolocalization studies show the presence of the 14-3-3 protein in the periphery of testes and externally associated with the apical rostellum and adjacent worm tegument. The alcian blue staining in consecutive parasite sections gave similar reactivity patterns, suggesting that the 14-3-3 protein is produced and secreted by rostellar glands. Immunoblot analysis showed the presence of the 14-3-3 protein in somatic and excretory-secretory worm products with higher and smaller apparent molecular masses, respectively, than those detected in E. multilocularis or E. granulosus metacestode tissues. Conversely, the 14-3-3 protein was not detected in metacestode secretory products. Detection of anti-E. granulosus 14-3-3 reactivity in sera of experimentally infected dogs was achieved at early stages of infection. Specific antibody titres decreased during the course of infection. The possible origin and functions of the 14-3-3 protein produced by the adult worm are discussed.
Assuntos
Echinococcus/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas 14-3-3 , Animais , Cães , Equinococose/parasitologia , Ensaio de Imunoadsorção Enzimática/métodos , Immunoblotting/métodos , Imuno-Histoquímica/métodos , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/imunologiaRESUMO
The insert of a clone from a lambdagt11 Echinococcus granulosus (Platyhelminth, Cestoda) protoscolex cDNA library, showed an open reading frame whose deduced protein sequence presents a high homology with all described thioredoxins (TRX). The TRX active site (Trp-Cys-Gly-Pro-Cys) is completely conserved. With a monospecific antibody, selected from a total anti-protoscolex sera by the isolated clone, a 12 kDa polypeptide was immunoprecipitated from a protoscolex total protein extract. Furthermore, an antiserum raised against a recombinant EgTRX also recognizes a 12 kDa band in these extracts. The recombinant protein presents TRX activity, using the insulin reduction assay. Finally, a TRX activity was characterized in protoscolex extracts. In all organisms where TRXs were studied, they participate in a cascade of redox exchanges, contributing to the maintaining of cell homeostasis. Considering that the parasitic flatworm E. granulosus is probably submitted to an important oxidative stress due to host defences, EgTRX protein could be involved in the survival strategies of this parasite.
Assuntos
Echinococcus/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Southern Blotting , Western Blotting , Clonagem Molecular , Echinococcus/crescimento & desenvolvimento , Echinococcus/metabolismo , Expressão Gênica , Proteínas de Helminto/química , Proteínas de Helminto/classificação , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Soros Imunes , Insulina/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Oxirredução , Estrutura Secundária de Proteína , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tiorredoxinas/química , Tiorredoxinas/classificaçãoRESUMO
Two cationic channels present in the microsomal fraction from Echinococcus granulosus protoscoleces of the sheep strain were studied in planar bilayer reconstitution experiments. A whole-worm homogenate was subjected to differential centrifugation and the postmitochondrial supernatant was laid on the top of a discontinuous sucrose gradient. The 15-30% (w/v) membrane fraction, enriched in NADPH-cytochrome c reductase, exhibited the highest fusion rate, two cationic channels being most frequently reconstituted. Both of them were highly cation-selective and had high conductances (244 and 107 pS in symmetrical 150 mM KCl). In most experiments, none of them displayed voltage dependence. The 244-pS channel was activated by Ca2+ and blocked by Ba2+, both in the micromolar range, thus partially resembling the Ca(2+)-activated K+ channel from more highly evolved animals. The 107-pS channel exhibited a Cs+ approximately equal to K+ > Na+ > Li+ > Ca2+ selectivity sequence (as measured by permeability ratios) and, most frequently, a high open probability (> 0.9) irrespective of the experimental conditions used, therefore sharing many properties of Schistosoma mansoni outer tegumental membrane cation channels.
Assuntos
Echinococcus/metabolismo , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Microssomos/metabolismo , Animais , Bário/farmacologia , Cálcio/metabolismo , Cátions , Fracionamento Celular , Condutividade Elétrica , Ativação do Canal Iônico , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/isolamento & purificação , Lítio/metabolismo , Potássio/metabolismo , Ovinos , Sódio/metabolismoRESUMO
A stage-specific expressed gene has been isolated from a cDNA expression library of Echinococcus granulosus protoscolices. The isolated clone contains the complete coding sequence. The corresponding protein (EgDf1) has a molecular weight of 15.5 kDa and is expressed at the tegumental level in the protoscolices, being undetectable in the germinal layer of the metacestode. This protein shares an important homology with a family of low-molecular weight proteins involved in the binding of hydrophobic ligands. This family includes a protein of Schistosoma mansoni (Sm 14) that has immunoprotective activity in rodents. Histochemical and metabolic data already reported for E. granulosus suggest that EgDf1 could be a molecular marker for early events in the process of protoscolex differentiation.