RESUMO
The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in areas where E. canadensis circulates. Furthermore, the presence of anti-AgB8/2 antibodies in serum may represent a useful parameter to rule out E. canadensis infection when human CE is diagnosed.
Assuntos
Equinococose/veterinária , Echinococcus/química , Proteínas de Helminto/química , Lipoproteínas/química , Doenças dos Suínos/parasitologia , Animais , Equinococose/parasitologia , Echinococcus/genética , Echinococcus/imunologia , Echinococcus/isolamento & purificação , Eletroforese em Gel Bidimensional , Genótipo , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Espectrometria de Massas , Proteômica , SuínosRESUMO
Echinococcus larvae are protected by a massive carbohydrate-rich acellular structure, called the laminated layer. In spite of being widely considered the crucial element of these host-parasite interfaces, the laminated layer has been historically poorly understood. In fact, it is still often called 'chitinous', 'hyaline' or 'cuticular' layer, or said to be composed of polysaccharides. However, over the past few years the laminated layer was found to be comprised of mucins bearing defined galactose-rich carbohydrates, and accompanied, in the case of Echinococcus granulosus, by calcium inositol hexakisphosphate deposits. In this review, the architecture and biosynthesis of this unusual structure is discussed at depth in terms of what is known and what needs to be discovered.
Assuntos
Echinococcus , Mucinas/química , Polissacarídeos/química , Animais , Echinococcus/anatomia & histologia , Echinococcus/química , Echinococcus/ultraestrutura , Interações Hospedeiro-Parasita , LarvaRESUMO
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
The identification of lectin-binding structures in adult worms of Echinococcus granulosus was carried out by lectin fluorescence; the distribution of carbohydrates in parasite glycoconjugates was also studied by lectin blotting. The lectins with the most ample recognition pattern were ConA, WGA, and PNA. ConA showed widespread reactivity in tegument and parenchyma components, including the reproductive system, suggesting that mannose is a highly expressed component of the adult glycans. Although reproductive structures appeared to be rich in N-acetyl-D-glucosamine (GlcNAc)-N-acetyl neuraminic acid (NeuAc) and galactose (Gal) as demonstrated by their strong reactivity with WGA and PNA, respectively, some differences were observed in their labeling patterns. This was very clear in the case of the vagina, which only reacted with WGA. Furthermore, WGA and ConA both had reactivity with the excretory canals. RCA, the other Gal binding lectin used, only reacted with the tegument, suggesting that widespread PNA reactivity with the reproductive system is related to the presence of the D-Gal-beta-(1,3)D-GalNAc terminal structure. UEA I failed to bind to any parasite tissues as determined by lectin fluorescence, whereas DBA and SBA showed a very faint staining of the tegument. However, in transferred glycans, N-acetyl-D-galactosamine (GalNAc) and fucose (Fuc) containing glycoproteins were distinctly detected.
Assuntos
Carboidratos/química , Echinococcus/química , Lectinas/metabolismo , Animais , Cães , Microscopia de Fluorescência , Sensibilidade e EspecificidadeRESUMO
Thioredoxin and glutathione systems are the major thiol-dependent redox systems in animal cells. They transfer via the reversible oxidoreduction of thiols the reducing equivalents of NADPH to numerous substrates and substrate reductases and constitute major defenses against oxidative stress. In this study, we cloned from the helminth parasite Echinococcus granulosus two trans-spliced mRNA variants that encode thioredoxin glutathione reductases (TGR). These variants code for mitochondrial and cytosolic selenocysteine-containing isoforms that possess identical glutaredoxin (Grx) and thioredoxin reductase (TR) domains and differ exclusively in their N termini. Western blot analysis of subcellular fractions with specific anti-TGR antibodies showed that TGR is present in both compartments. The biochemical characterization of the native purified TGR suggests that the Grx and TR domains of the enzyme can function either coupled or independently of each other, because the Grx domain can accept electrons from either TR domains or the glutathione system and the TR domains can transfer electrons to either the fused Grx domain or to E. granulosus thioredoxin.
Assuntos
Processamento Alternativo , Echinococcus/genética , Mitocôndrias/enzimologia , Complexos Multienzimáticos/genética , NADH NADPH Oxirredutases/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Citosol/enzimologia , Primers do DNA , DNA Complementar/química , DNA Complementar/genética , Echinococcus/química , Echinococcus/enzimologia , Éxons , Variação Genética , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA de Helmintos/química , RNA de Helmintos/genética , RNA Mensageiro/química , Ratos , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
myo-Inositol hexakisphosphate (IP(6)) is an abundant intracellular component of animal cells. In this study we describe the presence of extracellular IP(6) in the hydatid cyst wall (HCW) of the larval stage of the cestode parasite Echinococcus granulosus. The HCW comprises an inner cellular layer and an outer, acellular (laminated) layer up to 2 mm in thickness that protects the parasite from host immune cells. A compound, subsequently identified as IP(6), was detected in and purified from an HCW extract on the basis of its capacity to inhibit complement activation. The identification of the isolated compound was carried out by a combination of NMR, MS and TLC. The majority of IP(6) in the HCW was found in the acellular layer, with only a small fraction of the compound being extracted from cells. In the laminated layer, IP(6) was present in association with calcium, and accounted for up to 15% of the total dry mass of the HCW. IP(6) was not detected in any other structures or stages of the parasite. Our results imply that IP(6) is secreted by the larval stage of the parasite in a polarized fashion towards the interface with the host. This is the first report of the secretion of IP(6), and the possible implications beyond the biology of E. granulosus are discussed.
Assuntos
Echinococcus/química , Ácido Fítico/análise , Animais , Antígenos de Helmintos/análise , Cálcio/análise , Parede Celular/química , Cromatografia em Camada Fina , Ciclofilina A/análise , Echinococcus/crescimento & desenvolvimento , Espaço Extracelular/química , Proteínas de Helminto/análise , Magnésio/análise , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Ácido Fítico/isolamento & purificaçãoRESUMO
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
Nuclei from Echinococcus granulosus protoscolices were isolated from infected sheep. Protein extracts were prepared for analysis of DNA-protein interactions involving specific transcriptional regulatory factors. Gel mobility-shift assays were done using a heterologous probe containing binding sites for widespread transcription factors. A fragment of the promoter of GATA-1 transcription factor from the chicken was selected. When nuclear extracts from E. granulosus protoscolices were assayed a specific band shift was observed. The methodologies developed in this study could provide an important contribution for the characterization of the DNA-protein interactions involved in transcriptional regulation within the context of recent developments in the molecular biology of this parasite.
Assuntos
Proteínas de Ligação a DNA/isolamento & purificação , Echinococcus/química , Proteínas de Helminto/isolamento & purificação , Proteínas Nucleares/isolamento & purificação , Animais , Autorradiografia , Fracionamento Celular , Núcleo Celular/química , Galinhas , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Fatores de Ligação de DNA Eritroide Específicos , Proteínas de Helminto/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ovinos , Fatores de Transcrição/genéticaRESUMO
A novel intracellular calcium-binding protein from Echinococcus granulosus is described in this work. A cDNA was isolated from a lambdagt11 protoscolex expression library and the deduced amino acid sequence has at least fifteen sequentially repeated twelve-residue repeats that resemble the calcium-binding loop of EF-hands; however, the dodecamer motif has no flanking helices. The cDNA was expressed in Escherichia coli using the pGEX vector, and a recombinant fusion protein (EgCaBP1-GST) was obtained. The recombinant fusion protein binds calcium when assayed with 45Ca. It is possible that the calcium-binding motifs present a secondary structure similar to the parallel beta roll structure described for an alkaline protease from Pseudomonas aeruginosa. A native protein of more than 300 kDa was recognized by an anti-EgCaBP1 monoclonal antibody by Western-blot analysis. Immunohistochemistry using a pool of anti-EgCaBP1-GST mouse sera demonstrated a strong association of the protein with calcareous corpuscles. The possible role of this protein and that of the calcareous corpuscles in the protoscolex are discussed.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Echinococcus/química , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , DNA Complementar , Escherichia coli/genética , Dados de Sequência MolecularRESUMO
A 107-pS (symmetrical 150 mM KCl), nonselective cation channel was reconstituted from a microsomal membrane fraction of the larval stage of the tapeworm Echinococcus granulosus. Most of the time, it displayed a high open probability (>>0.95) irrespective of either the applied voltage, Ca2+, Ba2+, or tetraethylammonium concentration. Nevertheless, in contrast with this "leaklike" behavior, less frequently this "all-the-time-open" channel reversibly entered two different kinetic modes. One of them was characterized by lower Po values and some voltage sensitivity (V(1/2) congruent with 129 mV, and an equilibrium constant for channel closing changing e-fold per 63-mV change) the kinetic analysis revealing that it resulted from the appearance of voltage-sensitivity in the mean closed times and a sixfold increase in the equilibrium constant for channel closing at 0 mV. The other mode was characterized by a very fast open-close activity leading to poorly resolved current levels and a Po around 0.6-0.7 which, occasionally and in a voltage-sensitive manner, entered a long-lived nonconducting state. However, the rare nature of these mode-shifting transitions precluded a more detailed analysis of their kinetics. The conductive properties of the channel were not affected by these switches. Model gating alone does not seem to ensure any physiological role of this channel and, instead, some other channel changes must occur if this phenomenon were to be of regulatory importance in vivo. Thus, mode-shifting might constitute an alternative target for channel activity modulation also in tapeworms.