RESUMO
Echinococcus granulosus sensu lato is a platyhelminth parasite and the etiological cause of cystic echinococcosis (CE), a zoonotic and neglected disease that infects animals and humans worldwide. As a part of the biological arsenal of the parasite, cathepsin L proteases are a group of proteins that are believed to be essential for parasite penetration, immune evasion, and establishment in the tissues of the host. In this work, we have cloned and sequenced a new putative cathepsin L protease from Echinococcus canadensis (EcCLP1). The bioinformatic analysis suggests that EcCLP1 could be synthesized as a zymogen and activated after proteolytic cleavage. The multiple sequence alignment with other cathepsin proteases reveals important functional conserved features like a conserved active site, an N-linked glycosylation residue, a catalytic triad, an oxyanion hole, and three putative disulfide bonds. The phylogenetic analysis suggests that EcCLP1 could indeed be a cathepsin L cysteine protease from clade 1 as it grouped with cathepsins from other species in this clade. Modeling studies suggest that EcCLP1 has two domains forming a cleft where the active site is located and an occluding role for the propeptide. The transcriptomic analysis reveals different levels of cathepsin transcript expression along the different stages of the parasite life cycle. The whole-mount immunohistochemistry shows an interesting superficial punctate pattern of staining which suggests a secretory pattern of expression. The putative cathepsin L protease characterized here may represent an interesting tool for diagnostic purposes, vaccine design, or a new pharmacological target for antiparasitic intervention.
Title: Caractérisation moléculaire d'EcCLP1, une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis. Abstract: Echinococcus granulosus sensu lato est un Plathelminthe parasite et la cause étiologique de l'échinococcose kystique (EK), une maladie zoonotique et négligée qui infecte les animaux et les humains dans le monde entier. En tant que partie de l'arsenal biologique du parasite, les protéases de type cathepsine L sont un groupe de protéines considérées comme essentielles à la pénétration du parasite, l'évasion immunitaire et son établissement dans les tissus de l'hôte. Dans ce travail, nous avons cloné et séquencé une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis (EcCLP1). L'analyse bioinformatique suggère qu'EcCLP1 pourrait être synthétisée sous forme de zymogène et activée après clivage protéolytique. L'alignement de séquences multiples avec d'autres protéases de type cathepsine révèle d'importantes caractéristiques fonctionnelles conservées telles qu'un site actif conservé, un résidu de glycosylation lié à N, une triade catalytique, un trou oxyanion et trois liaisons disulfure putatives. L'analyse phylogénétique suggère qu'EcCLP1 pourrait en effet être une protéase de type cathepsine L du clade 1 car elle se regroupe avec les cathepsines d'autres espèces de ce clade. Les études de modélisation suggèrent qu'EcCLP1 possède deux domaines formant une fente où se trouve le site actif et un rôle d'occlusion pour le propeptide. L'analyse transcriptomique révèle différents niveaux d'expression du transcrit de la cathepsine au cours des différentes étapes du cycle de vie du parasite. L'immunohistochimie de montages entiers montre un intéressant motif de coloration ponctuée superficielle qui suggère un modèle d'expression sécrétoire. La protéase putative de type cathepsine L caractérisée ici peut représenter un outil intéressant à des fins de diagnostic, de conception de vaccins ou une nouvelle cible pharmacologique pour une intervention antiparasitaire.
Assuntos
Sequência de Aminoácidos , Catepsina L , Echinococcus , Filogenia , Animais , Catepsina L/genética , Echinococcus/enzimologia , Echinococcus/genética , Echinococcus/classificação , Alinhamento de Sequência , Clonagem Molecular , Proteínas de Helminto/genética , Proteínas de Helminto/química , Estágios do Ciclo de Vida , Equinococose/parasitologia , Domínio Catalítico , Perfilação da Expressão GênicaRESUMO
Echinococcus granulosus hydatid cysts were examined in 41 patients from Neuquén and Tucumán provinces in Argentina. Sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) revealed in 19 patients common sheep strain (G1), in 6 patients Tasmania sheep strain (G2), in 1 patient cattle strain (G5), and in 15 patients camel strain (G6). In Argentina the only known is the domestic cycle that affects dogs and herbivorous, including ovine, swine, cattle and goats. These strains produced a total of 58.6% of primary liver infections, 29.2% primary in lung, 2.4% primary in spleen and 9.8% were multiorgan abdominal infections. The metacestode was classified using the evolutive stages proposed by WHO-IWGE (from CE1 to CE5). We estimated that CE1 cyst has a duration of about 22 years, CE2 of 14 years, CE3 of 10 years, CE4 of 19 years and CE5 was not determined. The active types CE1 and CE2 reached 75% of all cases from all strains. In 36 patients with cysts from G1, G5 and G6 strain, there were only two asymptomatic cases. The strains of the E. granulosus complex do not present important clinical differences; only G6 seems to have higher growth rate.
Assuntos
Equinococose/parasitologia , Echinococcus/classificação , Adolescente , Adulto , Idoso , Animais , Argentina , Criança , DNA de Protozoário/química , DNA de Protozoário/genética , Equinococose/cirurgia , Echinococcus/enzimologia , Echinococcus/genética , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Pessoa de Meia-Idade , Análise de Sequência de DNARESUMO
A sample of 114 isolates of Echinococcus granulosus (Cestoda: Taeniidae) collected from different host species and sites in Argentina has been sequenced for 391 bp from the mitochondrial cytochrome c oxidase subunit I gene to analyze genetic variability and population structure. Nine different haplotypes were identified, 5 of which correspond to already characterized strains. Analysis of molecular variance and nested clade analysis of the distribution of haplotypes among localities within 3 main geographic regions indicate that geographic differentiation accounts for the overall pattern of genetic variability in E. granulosus populations. Significant geographic differentiation is also present when the sheep strain alone is considered. Our results suggest that geographic patterns are not due to actual restricted gene flow between regions but are rather a consequence of past history, probably related to the time and origin of livestock introduction in Argentina.
Assuntos
Animais Domésticos/parasitologia , Equinococose/veterinária , Echinococcus/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Variação Genética , Análise de Variância , Animais , Argentina/epidemiologia , Sequência de Bases , Bovinos , DNA de Helmintos/química , Cães , Equinococose/epidemiologia , Equinococose/parasitologia , Echinococcus/classificação , Echinococcus/enzimologia , Cabras , Haplótipos , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/veterinária , Polimorfismo Genético , Alinhamento de Sequência/veterinária , Ovinos , SuínosRESUMO
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
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 AminoácidosAssuntos
Echinococcus/enzimologia , Superóxido Dismutase/metabolismo , Animais , Cromatografia por Troca Iônica , Echinococcus/genética , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/isolamento & purificaçãoAssuntos
Echinococcus/enzimologia , Proteínas de Neoplasias , Peroxidases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA de Helmintos/química , DNA de Helmintos/genética , Echinococcus/genética , Peróxido de Hidrogênio/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Peroxidases/química , Peroxidases/metabolismo , Peroxirredoxinas , Reação em Cadeia da Polimerase , Alinhamento de SequênciaAssuntos
Citosol/enzimologia , Echinococcus/enzimologia , Proteínas de Helminto/isolamento & purificação , Malato Desidrogenase/isolamento & purificação , Sequência de Aminoácidos , Animais , Equinococose Hepática/parasitologia , Echinococcus/crescimento & desenvolvimento , Echinococcus/isolamento & purificação , Proteínas de Helminto/metabolismo , Cinética , Malato Desidrogenase/metabolismo , Dados de Sequência Molecular , Ovinos , Doenças dos Ovinos/parasitologia , Especificidade por SubstratoRESUMO
Deoxyribonuclease activity was detected in E. granulosus protoscoleces from sheep hydatid cysts by electrophoresis in agarose gels of DNA fragments obtained after incubation of integral DNA with a protoscoleces preparation. Preliminary characterization showed that deoxyribonuclease activity was optimal at neutral-alkaline pH, magnesium ions were required, and it was able to digest different types of DNA, making random cuts. Electrophoresis in DNA-containing sodium dodecylsulfate (SDS)-polyacrylamide gels indicated a relative molecular mass, under non-reducing conditions, of 32 kDa. Deoxyribonuclease activity was also found in sheep hydatid fluid. It shared optimal pH, ionic and substrate requirements with the enzyme from protoscoleces but had a higher relative molecular mass (40 kDa), the same as that of normal sheep serum deoxyribonuclease.
Assuntos
Desoxirribonucleases/metabolismo , Echinococcus/enzimologia , Animais , DNA/metabolismo , Equinococose Pulmonar/parasitologia , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Ovinos , Doenças dos Ovinos/parasitologiaRESUMO
Proteolytic activity in hydatid cyst fluid, cyst membranes and protoscoleces of E. granulosus was analyzed by electrophoresis in gelatin-containing polyacrylamide gels, including characterization with a set of protease inhibitors. All contained metalloproteinases in the range 60-120 kDa, with neutral/alkaline pH optima. Major activity was observed in hydatid fluid and the membranes (five bands) with both exhibiting similar electrophoretic patterns. The samples prepared from protoscoleces shared only some of these bands.