RESUMO
Cystic echinococcosis is caused by the larval stages (hydatids) of cestode parasites belonging to the species cluster Echinococcus granulosus sensu lato, with E. granulosus sensu stricto being the main infecting species. Hydatids are bladderlike structures that attain large sizes within various internal organs of livestock ungulates and humans. Hydatids are protected by the massive acellular laminated layer (LL), composed mainly of mucins. Parasite growth requires LL turnover, and abundant LL-derived particles are found at infection sites in infected humans, raising the question of how LL materials are dealt with by the hosts. In this article, we show that E. granulosus sensu stricto LL mucins injected into mice are taken up by Kupffer cells, the liver macrophages exposed to the vascular space. This uptake is largely dependent on the intact mucin glycans and on Clec4F, a C-type lectin receptor which, in rodents, is selectively expressed in Kupffer cells. This uptake mechanism operates on mucins injected both in soluble form intravenously (i.v.) and in particulate form intraperitoneally (i.p.). In mice harboring intraperitoneal infections by the same species, LL mucins were found essentially only at the infection site and in the liver, where they were taken up by Kupffer cells via Clec4F. Therefore, shed LL materials circulate in the host, and Kupffer cells can act as a sink for these materials, even when the parasite grows in sites other than the liver.
Assuntos
Equinococose , Echinococcus granulosus , Animais , Humanos , Camundongos , Equinococose/parasitologia , Echinococcus granulosus/química , Genótipo , Células de Kupffer , Lectinas , MucinasRESUMO
The larval stages of the cestode parasites belonging to the genus Echinococcus grow within internal organs of humans and a range of animal species. The resulting diseases, collectively termed echinococcoses, include major neglected tropical diseases of humans and livestock. Echinococcus larvae are outwardly protected by the laminated layer (LL), an acellular structure that is unique to this genus. The LL is based on a fibrillar meshwork made up of mucins, which are decorated by galactose-rich O-glycans. In addition, in the species cluster termed E. granulosus sensu lato, the LL features nano-deposits of the calcium salt of myo-inositol hexakisphosphate (Insp6). The main purpose of our article is to update the immunobiology of the LL. Major recent advances in this area are (i) the demonstration of LL "debris" at the infection site and draining lymph nodes, (ii) the characterization of the decoy activity of calcium Insp6 with respect to complement, (iii) the evidence that the LL mucin carbohydrates interact specifically with a lectin receptor expressed in Kupffer cells (Clec4F), and (iv) the characterization of what appear to be receptor-independent effects of LL particles on dendritic cells and macrophages. Much information is missing on the immunology of this intriguing structure: we discuss gaps in knowledge and propose possible avenues for research.
Assuntos
Equinococose , Echinococcus granulosus , Echinococcus , Animais , Cálcio , Equinococose/parasitologia , Echinococcus/imunologia , Echinococcus granulosus/química , Echinococcus granulosus/imunologia , MucinasRESUMO
Cystic echinococcosis is a zoonotic disease caused by the larval stage of Echinococcus granulosus. This affliction is an endemic worldwide condition that represents a neglected parasitic disease with important socioeconomic repercussions. Proteomic characterization of larval and adult stages of E. granulosus, as well as the association between expression profiles and host interactions, is relevant for a better understanding of parasite biology, and eventually for drug design and vaccine development. This study aimed to develop a synthesis of the evidence available related to proteomics of E. granulosus. A systematic review was carried out to collect data concerning the proteomics of E. granulosus, without language or host restriction, published between 1980 and 2019. A systematic search was carried out in the Trip Database, BIREME-BVS, SciELO, Web of Science, PubMed, EMBASE, SCOPUS, EBSCO host, and LILACS, using MeSH terms, free words, and Boolean connectors, and adapting strategies to each source of information. Additionally, a manual cross-reference search was performed. Variables studied were the year of publication, geographic origin of the study, number of samples, hosts, parasitic organs, proteomic techniques, and parasite proteins verified. Nine-hundred and thirty-six related articles were identified: 17 fulfilled selection criteria, including slightly more than 188 samples. Most articles were published between 2014 and 2019 (64.7%) and were from Brazil and China (35.3% each). In reference to confirmed hosts in the primary articles, cattle (41.2%) and humans (23.5%) were the most frequently reported. Concerning proteomic techniques applied in the primary articles, LC-MS/MS was the most used (41.1%), and 890 proteins were reported by the primary articles. As the results of our search suggest, the information related to E. granulosus proteomics is scarce, heterogeneous, and scattered throughout several articles that include a diversity of tissues, samples, intermediate hosts, and proteomic techniques. Consequently, the level of evidence generated by our search is type 4.
Assuntos
Equinococose/parasitologia , Echinococcus granulosus/química , Proteínas de Helminto/análise , Proteômica , Animais , Proteínas de Helminto/químicaRESUMO
The genus Echinococcus of cestode parasites includes important pathogens of humans and livestock animals. Transcriptomic and genomic studies on E. granulosus and E. multilocularis uncovered striking expansion of monodomain Kunitz proteins. This expansion is accompanied by the specialization of some family members away from the ancestral protease inhibition function to fulfill cation channel blockade functions. Since cation channels are involved in immune processes, we tested the effects on macrophage physiology of two E. granulosus Kunitz-type inhibitors of voltage-activated cation channels (Kv) that are close paralogs. Both inhibitors, EgKU-1 and EgKU-4, inhibited production of the Th1/Th17 cytokine subunit IL-12/23p40 by macrophages stimulated with the TLR4 agonist LPS. In addition, EgKU-4 but not EgKU-1 inhibited production of the inflammatory cytokine IL-6. These activities were not displayed by EgKU-3, a family member that is a protease inhibitor without known activity on cation channels. EgKU-4 potently inhibited macrophage proliferation in response to M-CSF, whereas EgKU-1 displayed similar activity but with much lower potency, similar to EgKU-3. We discuss structural differences, including a heavily cationic C-terminal extension present in EgKU-4 but not in EgKU-1, that may explain the differential activities of the two close paralogs.
Assuntos
Echinococcus granulosus/química , Proteínas de Helminto/farmacologia , Interleucina-12/antagonistas & inibidores , Interleucina-6/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica , Proteínas de Helminto/isolamento & purificação , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/imunologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
The interaction of dendritic cells and macrophages with a variety of rigid noncellular particles triggers activation of the NLRP3 inflammasome and consequent secretion of interleukin 1ß (IL-1ß). Noncellular particles can also be generated in the context of helminth infection, since these large pathogens often shed their outermost structures during growth and/or molting. One such structure is the massive, mucin-based, soft, flexible laminated layer (LL), which protects the larval stages of cestodes of the genus Echinococcus We show that particles from the Echinococcus granulosus LL (pLL) trigger NLRP3- and caspase-1-dependent IL-1ß in lipopolysaccharide (LPS)-primed mouse bone marrow-derived dendritic cells (BMDC). This response can be elicited by pLL too large for phagocytosis and nonetheless requires actin dynamics, Syk, and phosphatidylinositol 3-kinase (PI3K). These three requirements had already been observed in our previous study on the alteration by pLL of CD86, CD40, IL-10, and IL-12 responses to LPS in BMDC; however, we now show that these alterations are independent of NLRP3 and caspase-1. In other words, an initial interaction with particles requiring actin dynamics, Syk, and PI3K, but not phagocytosis, elicits both NLRP3-dependent and NLRP3-independent responses. Intraperitoneal injection of pLL induced IL-1ß, suggesting that contact with LL materials induces IL-1ß in the E. granulosus infection setting. Our results extend our understanding of NLRP3 inflammasome activation by noncellular particulate materials both to helminth-derived materials and to flexible/soft materials.
Assuntos
Micropartículas Derivadas de Células/química , Células Dendríticas/efeitos dos fármacos , Echinococcus granulosus/química , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Parasita/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Caspase 1/genética , Caspase 1/imunologia , Micropartículas Derivadas de Células/imunologia , Células Dendríticas/imunologia , Echinococcus granulosus/imunologia , Feminino , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Parasita/genética , Indazóis/farmacologia , Inflamassomos/efeitos dos fármacos , Inflamassomos/genética , Inflamassomos/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/agonistas , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fagocitose/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais , Estilbenos/farmacologia , Sulfonamidas/farmacologia , Wortmanina/farmacologiaRESUMO
La información disponible referente a las características proteómicas del E. granulosus es escasa (no supera los 50 estudios publicados); y nos parece que la identificación proteómica, podría mejorar la comprensión de algunas características bioquímicas e inmunológicas de la Equinococosis Quística (EQ). De tal modo que el proteoma de E. granulosus aún no está bien descrito. Sólo existen reportes de algunas secuencias de proteínas. El objetivo de este manuscrito fue comentar algunos aspectos de la evidencia existente respecto de los estudios del perfil proteómico del E. granulosus. Se recomienda el estudio de al menos el quiste y su pared, el líquido hidatídico y la víscera del hospedero. Para ello, existen metodologías que han sido empleadas para estudiar las características proteómicas de la EQ. Entre ellas, destacan SDS-PAGE, electroforesis bidimensional combinada con Western Blot, inmunoanálisis, y espectrometría de masas mediante técnicas MALDI-TOF. Se han identificado una serie de proteínas en muestras de EQ. Algunas de ellas, asociadas a procesos inmunológicos, de gluconeogénesis, glucogenolisis y glucogénesis. Por otra parte, se ha documentado la liberación de exosomas al líquido hidatídico por parte de los protoescólex y la capa germinativa; estructuras en las que se han identificado factores de virulencia asociados con la supervivencia del quiste. No obstante lo anteriormente señalado, se requiere de múltiples estudios exhaustivos en la materia para comprender mejor la caracterización perfil proteómico del E. granulosus.
The information available regarding the proteomic characteristics of E. granulosus is scarce; and it seems that the proteomic identification could improve the understanding of some biochemical and immunological characteristics of cystic echinococcosis (CE). So, the proteome of E. granulosus is still not well described yet. There are only reports of some protein sequences. The objective of this manuscript was to comment on some aspects of the existing evidence regarding studies of the proteomic profile of E. granulosus. The study of at least the cyst and its wall, the hydatid fluid and the viscera of the host are recommended. There are a series of methodologies that have been used to study the proteomic characteristics of EQ. These include SDS-PAGE, bidimensional electrophoresis combined with Western Blot, immunoassay, and mass spectrometry using MALDI-TOF techniques. A series of proteins have been identified in CE samples. Some of them, associated with immune response, gluconeogenesis, glycogenolysis and glycogenesis. On the other hand, release of exosomes to the hydatid fluid by protoescolex and germinative layer has been documented (associated virulence factors have been identified in these structures). Notwithstanding the foregoing, it requires multiple exhaustive studies in the field to better understand the characterization of the proteomic profile of E. granulosus.
Assuntos
Proteínas/análise , Proteômica/métodos , Echinococcus granulosus/química , Echinococcus granulosus/genética , Echinococcus granulosus/metabolismo , Eletroforese em Gel de PoliacrilamidaRESUMO
Glutathione transferases (GSTs) comprise a major detoxification system in helminth parasites, displaying both catalytic and non-catalytic activities. The kinetic mechanism of these enzymes is complex and depends on the isoenzyme which is being analyzed. Here, we characterized the kinetic mechanism of rEgGST1, a recombinant form of a cytosolic GST from Echinococcus granulosus (EgGST1), which is related to the Mu-class of mammalian enzymes, using the canonical substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Initial rate and product inhibition studies were consistent with a steady-state random sequential mechanism, where both substrates are bound to the enzyme before the products are released. Kinetic constants were also determined (pH 6.5 and 30 °C). Moreover, rEgGST1 lowered the pKa of GSH from 8.71 ± 0.07 to 6.77 ± 0.08, and enzyme-bound GSH reacted with CDNB 1 × 105 times faster than free GSH at pH 7.4. Finally, the dissociation of the enzyme-GSH complex was studied by means of intrinsic fluorescence, as well as that of the complex with the anthelminth drug mebendazole. This is the first report on mechanistic issues related to a helminth parasitic GST.
Assuntos
Echinococcus granulosus/química , Glutationa Transferase/metabolismo , Glutationa/metabolismo , Proteínas de Helminto/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anti-Helmínticos/farmacologia , Clonagem Molecular , Dinitroclorobenzeno/metabolismo , Echinococcus granulosus/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/genética , Proteínas de Helminto/antagonistas & inibidores , Proteínas de Helminto/genética , Concentração de Íons de Hidrogênio , Inativação Metabólica/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Mebendazol/farmacologia , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Especificidade por SubstratoRESUMO
AIMS: New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosus TGR, free and complexed with AuI-MPO, a novel gold inhibitor, together with inhibition assays were performed. RESULTS: AuI-MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: (i) unique monomer-monomer interactions, (ii) distinct binding sites for thioredoxin and the glutaredoxin (Grx) domain, (iii) a single glutathione disulfide reduction site in the Grx domain, (iv) rotation of the Grx domain toward the Sec-containing redox active site, and (v) a single gold atom bound to Cys519 and Cys573 in the AuI-TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, CysâSer mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site. INNOVATION: The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments. CONCLUSIONS: The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center but also by rotation of the Grx domain and distinct binding sites for Grx domain and thioredoxin. The conserved Cys519 and Cys573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. Antioxid. Redox Signal. 27, 1491-1504.
Assuntos
Echinococcus granulosus/enzimologia , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Compostos Organoáuricos/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Cisteína/metabolismo , Echinococcus granulosus/química , Echinococcus granulosus/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Glutarredoxinas/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Modelos Moleculares , Complexos Multienzimáticos/genética , Mutação , NADH NADPH Oxirredutases/genética , Compostos Organoáuricos/química , Ligação Proteica , Conformação ProteicaRESUMO
The larval stages of the cestodes Echinococcus multilocularis and Echinococcus granulosus cause the important zoonoses known as larval echinococcoses. These larvae are protected by a unique, massive, mucin-based structure known as the laminated layer. The mucin glycans of the E. granulosus laminated layer are core 1- or core 2-based O-glycans in which the core Galpß1-3 residue can initiate a chain comprising one to three additional Galpß1-3 residues, a motif not known in mammalian carbohydrates. This chain can be capped with a Galpα1-4 residue, and can be ramified with GlcNAcpß1-6 residues. These, as well as the GlcNAcpß1-6 residue in core 2, can be decorated with the Galpα1-4Galpß1-4 disaccharide. Here we extend our analysis to the laminated layer of E. multilocularis, showing that the non-decorated cores, together with Galpß1-3(Galpα1-4Galpß1-4GlcNAcpß1-6)GalNAc, comprise over 96% of the glycans in molar terms. This simple laminated layer glycome is exhibited by E. multilocularis grown either in vitro or in vivo. Interestingly, all the differences with the complex laminated layer glycome found in E. granulosus may be explained in terms of strongly reduced activity in E. multilocularis of a putative glycosyltransferase catalysing the elongation with Galpß1-3. Comparative inter-species analysis of available genomic and transcriptomic data suggested a candidate for this enzyme, amongst more than 20 putative (non-core 1) Gal/GlcNAc ß1-3 transferases present in each species as a result of a taeniid-specific gene expansion. The candidate gene was experimentally verified to be transcribed at much higher levels in the larva of E. granulosus than that of E. multilocularis.
Assuntos
Echinococcus granulosus/química , Echinococcus multilocularis/química , Mucinas/química , Polissacarídeos/química , Animais , Cromatografia em Gel , Mineração de Dados , Echinococcus granulosus/classificação , Echinococcus granulosus/genética , Echinococcus multilocularis/classificação , Echinococcus multilocularis/genética , Regulação Enzimológica da Expressão Gênica , Genoma , Gerbillinae , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Larva/química , Larva/classificação , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Mucinas/metabolismo , Cavidade Peritoneal/parasitologia , Polissacarídeos/biossíntese , Polissacarídeos/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , TranscriptomaRESUMO
Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes.
Assuntos
Echinococcus granulosus/química , Proteínas de Ligação a Ácido Graxo/análise , Proteínas de Helminto/análise , Animais , Western Blotting , Bovinos , Doenças dos Bovinos/parasitologia , Equinococose/parasitologia , Equinococose/veterinária , Echinococcus granulosus/metabolismo , Echinococcus granulosus/ultraestrutura , Eletroforese em Gel Bidimensional , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita , Larva/química , Larva/metabolismo , Larva/ultraestrutura , Metabolismo dos Lipídeos , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , UruguaiRESUMO
INTRODUCTION: The aim of this study was to explore the environment of Echinococcus granulosus (E. granulosus) protoscolices and their relationship with their host. METHODS: Proteins from the hydatid-cyst fluid (HCF) from E. granulosus were identified by proteomics. An inductively coupled plasma atomic emission spectrometer (ICP-AES) was used to determine the elements, an automatic biochemical analyzer was used to detect the types and levels of biochemical indices, and an automatic amino acid analyzer was used to detect the types and levels of amino acids in the E. granulosus HCF. RESULTS: I) Approximately 30 protein spots and 21 peptide mass fingerprints (PMF) were acquired in the two-dimensional gel electrophoresis (2-DE) pattern of hydatid fluid; II) We detected 10 chemical elements in the cyst fluid, including sodium, potassium, calcium, magnesium, copper, and zinc; III) We measured 19 biochemical metabolites in the cyst fluid, and the amount of most of these metabolites was lower than that in normal human serum; IV) We detected 17 free amino acids and measured some of these, including alanine, glycine, and valine. CONCLUSIONS: We identified and measured many chemical components of the cyst fluid, providing a theoretical basis for developing new drugs to prevent and treat hydatid disease by inhibiting or blocking nutrition, metabolism, and other functions of the pathogen.
Assuntos
Líquido Cístico/química , Equinococose , Echinococcus granulosus/química , Animais , Eletroforese em Gel de Poliacrilamida , Proteínas de Helminto/análise , HumanosRESUMO
Introduction The aim of this study was to explore the environment of Echinococcus granulosus (E. granulosus) protoscolices and their relationship with their host. Methods Proteins from the hydatid-cyst fluid (HCF) from E. granulosus were identified by proteomics. An inductively coupled plasma atomic emission spectrometer (ICP-AES) was used to determine the elements, an automatic biochemical analyzer was used to detect the types and levels of biochemical indices, and an automatic amino acid analyzer was used to detect the types and levels of amino acids in the E. granulosus HCF. Results I) Approximately 30 protein spots and 21 peptide mass fingerprints (PMF) were acquired in the two-dimensional gel electrophoresis (2-DE) pattern of hydatid fluid; II) We detected 10 chemical elements in the cyst fluid, including sodium, potassium, calcium, magnesium, copper, and zinc; III) We measured 19 biochemical metabolites in the cyst fluid, and the amount of most of these metabolites was lower than that in normal human serum; IV) We detected 17 free amino acids and measured some of these, including alanine, glycine, and valine. Conclusions We identified and measured many chemical components of the cyst fluid, providing a theoretical basis for developing new drugs to prevent and treat hydatid disease by inhibiting or blocking nutrition, metabolism, and other functions of the pathogen. .
Assuntos
Animais , Humanos , Líquido Cístico/química , Equinococose , Echinococcus granulosus/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Helminto/análiseRESUMO
There is substantial evidence suggesting that certain parasites can have antitumor properties. We evaluated mucin peptides derived from the helminth Echinococcus granulosus (denominated Egmuc) as potential inducers of antitumor activity. We present data showing that Egmuc peptides were capable of inducing an increase of activated NK cells in the spleen of immunized mice, a fact that was correlated with the capacity of splenocytes to mediate killing of tumor cells. We demonstrated that Egmuc peptides enhance LPS-induced maturation of dendritic cells in vitro by increasing the production of IL-12p40p70 and IL-6 and that Egmuc-treated DCs may activate NK cells, as judged by an increased expression of CD69. This evidence may contribute to the design of tumor vaccines and open new horizons in the use of parasite-derived molecules in the fight against cancer.
Assuntos
Antineoplásicos/administração & dosagem , Mucinas/administração & dosagem , Neoplasias/tratamento farmacológico , Peptídeos/administração & dosagem , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Echinococcus granulosus/química , Humanos , Subunidade p40 da Interleucina-12/imunologia , Interleucina-6/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Lectinas Tipo C/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Mucinas/química , Mucinas/isolamento & purificação , Neoplasias/imunologia , Neoplasias/patologia , Peptídeos/química , Peptídeos/isolamento & purificação , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
The glycobiology of the cestodes, a class of parasitic flatworms, is still largely unexplored. An important cestode species is Echinococcus granulosus, the tissue-dwelling larval stage of which causes hydatid disease. The E. granulosus larva is protected from the host by a massive mucin-based extracellular matrix termed laminated layer (LL). We previously reported ( Díaz et al. 2009. Biochemistry 48:11678-11691) the molecular structure of the most abundant LL O-glycans, comprising up to six monosaccharide residues. These are based on Cores 1 and 2, in cases elongated by a chain of Galpß1-3 residues, which can be capped by Galpα1-4. In addition, the Core 2 GlcNAcp residue can be decorated with the Galpα1-4Galpß1-4 disaccharide. Larger glycans also detected contained additional HexNAc residues that could not be explained by the structural repertoire described above. In this work, we elucidate, by mass spectrometry (MS) and nuclear magnetic resonance (NMR), six additional glycans from the E. granulosus LL between six and eight residues in size. Their structures are related to those already described but in cases bear GlcNAcpß1-6 or Galpα1-4Galpß1-4GlcNAcpß1-6 as ramifications on the core Galpß1-3 residue. We also obtained evidence that noncore Galpß1-3 residues can be similarly ramified. Thus, the new motif together with the previous information may explain all the glycan compositions detected in the LL by MS. In addition, we show that the anti-Echinococcus monoclonal antibody E492 (Parasite Immunol 21:141, 1999) recognizes Galpα1-4Galpß1-4GlcNAcp (the blood P(1)-antigen motif). This explains the antibody's reactivity with a range of Echinococcus tissues, as the P(1)-motif is also carried on non-LL N-glycans and glycolipids from this genus.
Assuntos
Echinococcus granulosus/química , Polissacarídeos/química , Animais , Configuração de Carboidratos , Globosídeos/imunologia , Monossacarídeos/química , Polissacarídeos/imunologiaRESUMO
Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroformâ¶methanol mixtures. This fraction constituted approximately 40-50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16â¶0, 18â¶0 and 18â¶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid metabolism and the EgAgB gene family.
Assuntos
Echinococcus granulosus/química , Lipídeos/análise , Lipoproteínas/química , Animais , Fracionamento Químico , Cromatografia Gasosa , Lipídeos/isolamento & purificação , Lipoproteínas/isolamento & purificação , Peso MolecularRESUMO
The larvae of the cestodes belonging to the genus Echinococcus are outwardly protected by the laminated layer (LL), a crucial but poorly understood carbohydrate-rich acellular structure. Carbohydrate structural data strongly suggest that the main components of the LL are mucins. The most massive LL in the genus is featured by E. granulosus, agent of cystic hydatid disease. No appropriate methods existed to date for the solubilisation of the E. granulosus LL and the electrophoretic visualisation of its proposed structural mucins. We report that reduction of disulphides greatly aids LL disassembly, resulting in almost full solubilisation in combination with moderate sonication. The structural mucins can then be visualised by agarose electrophoresis and blotting with galactose-binding lectins, which also react strongly with the LL in tissue sections. A substantial portion of the material migrates as if positively charged; since the LL glycans are neutral, this may correspond to mucins with cationic peptide backbones.
Assuntos
Echinococcus granulosus/química , Echinococcus granulosus/crescimento & desenvolvimento , Mucinas/análise , Mucinas/química , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Dissulfetos/metabolismo , Equinococose/parasitologia , Equinococose/veterinária , Echinococcus granulosus/metabolismo , Eletroforese em Gel de Ágar , Larva/química , Lectinas/metabolismo , Mucinas/metabolismo , Parasitologia/métodos , Solubilidade , SonicaçãoRESUMO
Cystic hydatid disease (CHD) is caused by infection with the Echinococcus granulosus metacestode and affects both humans and livestock. In this work, we performed a proteomic analysis of the E. granulosus metacestode during infection of its intermediate bovine host. Parasite proteins were identified in different metacestode components (94 from protoscolex, 25 from germinal layer and 20 from hydatid cyst fluid), along with host proteins (58) that permeate into the hydatid cyst, providing new insights into host-parasite interplay. E. granulosus and platyhelminth EST data allowed successful identification of proteins potentially involved in downregulation of host defenses, highlighting possible evasion mechanisms adopted by the parasite to establish infection. Several intracellular proteins were found in hydatid cyst fluid, revealing a set of newly identified proteins that were previously thought to be inaccessible for inducing or modulating the host immune response. Host proteins identified in association with the hydatid cyst suggest that the parasite may bind/adsorb host molecules with nutritional and/or immune evasion purposes, masking surface antigens or inhibiting important effector molecules of host immunity, such as complement components and calgranulin. Overall, our results provide valuable information on parasite survival strategies in the adverse host environment and on the molecular mechanisms underpinning CHD immunopathology.
Assuntos
Equinococose/microbiologia , Echinococcus granulosus/química , Proteínas de Helminto/análise , Proteoma/análise , Animais , Bovinos , Interações Hospedeiro-ParasitaRESUMO
The cestodes constitute important but understudied human and veterinary parasites. Their surfaces are rich in carbohydrates, on which very little structural information is available. The tissue-dwelling larva (hydatid cyst) of the cestode Echinococcus granulosus is outwardly protected by a massive layer of carbohydrate-rich extracellular matrix, termed the laminated layer. The monosaccharide composition of this layer suggests that its major carbohydrate components are exclusively mucin-type O-glycans. We have purified these glycans after their release from the crude laminated layer and obtained by MS and NMR the complete structure of 10 of the most abundant components. The structures, between two and six residues in length, encompass a limited number of biosynthetic motifs. The mucin cores 1 and 2 are either nondecorated or elongated by a chain of Galpbeta1-3 residues. This chain can be capped by a single Galpalpha1-4 residue, such capping becoming more dominant with increasing chain size. In addition, the core 2 N-acetylglucosamine residue is in cases substituted with the disaccharide Galpalpha1-4Galpbeta1-4, giving rise to the blood P(1)-antigen motif. Larger, also related, glycans exist, reaching at least 18 residues in size. The glycans described are related but larger than those previously described from an Echinococcus multilocularis mucin [Hulsmeier, A. J., et al. (2002) J. Biol. Chem. 277, 5742-5748]. Our results reveal that the E. granulosus cyst exposes to the host only a few different major carbohydrate motifs. These motifs are composed essentially of galactose units and include the elongation by (Galpbeta1-3)(n) and the capping by Galpalpha1-4, novel in animal mucin-type O-glycans.
Assuntos
Equinococose/metabolismo , Equinococose/parasitologia , Echinococcus granulosus/química , Galactose/química , Mucinas/química , Polissacarídeos/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Bovinos , Cromatografia em Gel , Matriz Extracelular/química , Interações Hospedeiro-Parasita , Espectroscopia de Ressonância Magnética , Metilglicosídeos/química , Dados de Sequência Molecular , Oligossacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Álcoois Açúcares/químicaRESUMO
The abundant metabolite myo-inositol hexakisphosphate (InsP6) can form vesicular deposits with cations, a widespread phenomenon in plants also found in the cestode parasite, Echinococcus granulosus. In this organism, the deposits are exocytosed, accumulating in a host-exposed sheath of extracellular matrix termed the laminated layer. The formation and mobilization of InsP6 deposits, which involve precipitation and solubilization reactions, respectively, cannot yet be rationalized in quantitative chemical terms, as the solids involved have not been formally described. We report such a description for the InsP6 deposits from E. granulosus, purified as the solid residue left by mild alkaline digestion of the principal mucin component of the laminated layer. The deposits are largely composed of the compound Ca5H2L.16H2O (L representing fully deprotonated InsP6), and additionally contain Mg2+ (6-9% molar ratio with respect to Ca2+), but not K+. Calculations employing recently available chemical constants show that the precipitation of Ca5H2L.16H2O is predicted by thermodynamics in secretory vesicle-like conditions. The deposits appear to be similar to microcrystalline solids when analysed under the electron microscope; we estimate that each crystal comprises around 200 InsP6 molecules. We calculate that the deposits increase, by three orders of magnitude, the surface area available for adsorption of host proteins, a salient ability of the laminated layer. The major inositol phosphate in the deposits, other than InsP6, is myo-inositol (1,2,4,5,6) pentakisphosphate, or its enantiomer, inositol (2,3,4,5,6) pentakisphosphate. The compound appears to be a subproduct of the intracellular pathways leading to the synthesis and vesicular accumulation of InsP6, rather than arising from extracellular hydrolysis of InsP6.
Assuntos
Echinococcus granulosus/química , Ácido Fítico/análise , Animais , Cálcio/análise , Bovinos , Cromatografia Líquida de Alta Pressão , Echinococcus granulosus/crescimento & desenvolvimento , Exocitose , Matriz Extracelular/química , Matriz Extracelular/ultraestrutura , Hidrólise , Larva/química , Magnésio/análise , Camundongos , Ressonância Magnética Nuclear Biomolecular , Ácido Fítico/biossíntese , Ácido Fítico/isolamento & purificação , Potássio/análise , Sódio/análise , Estereoisomerismo , TermodinâmicaRESUMO
The Echinococcus granulosus actin filament-fragmenting protein (EgAFFP) is a three domain member of the gelsolin family of proteins, which is antigenic to human hosts. These proteins, formed by three or six conserved domains, are involved in the dynamic rearrangements of the cytoskeleton, being responsible for severing and capping actin filaments and promoting nucleation of actin monomers. Various structures of six domain gelsolin-related proteins have been investigated, but little information on the structure of three domain members is available. In this work, the solution structure of the three domain EgAFFP has been investigated through small-angle x-ray scattering (SAXS) studies. EgAFFP exhibits an elongated molecular shape. The radius of gyration and the maximum dimension obtained by SAXS were, respectively, 2.52 +/- 0.01 nm and 8.00 +/- 1.00 nm, both in the absence and presence of Ca2+. Two different molecular homology models were built for EgAFFP, but only one was validated through SAXS studies. The predicted structure for EgAFFP consists of three repeats of a central beta-sheet sandwiched between one short and one long alpha-helix. Possible implications of the structure of EgAFFP upon actin binding are discussed.