Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype.

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.

The laminated layer: Recent advances and insights into Echinococcus biology and evolution.

The laminated layer is the unique mucin-based extracellular matrix that protects Echinococcus larvae, and thus to an important extent, shapes host-parasite relationships in the larval echinococcoses. In 2011, we published twin reviews summarizing what was known about this structure. Since then, important advances have been made. Complete genomes and some RNAseq data are now available for E. multilocularis and E. granulosus, leading to the inference that the E. multilocularis LL is probably formed by a single type of mucin backbone, while a second apomucin subfamily additionally contributes to the E. granulosus LL. Previously suspected differences between E. granulosus and E. multilocularis in mucin glycan size have been confirmed and pinned down to the virtual absence of Galß1-3 chains in E. multilocularis. The LL carbohydrates from both species have been found to interact selectively with the Kupffer cell receptor expressed in rodent liver macrophages, highlighting the ancestral adaptations to rodents as intermediate hosts and to the liver as infection site. Finally, LL particles have been shown to possess carbohydrate-independent mechanisms profoundly conditioning non-liver-specific dendritic cells and macrophages. These advances are discussed in an integrated way, and in the context of the newly determined phylogeny of Echinococcus and its taenid relatives.

Understanding the laminated layer of larval Echinococcus I: structure.

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.

14-3-3 proteins in Echinococcus: their role and potential as protective antigens.

14-3-3 Proteins are a family of highly conserved proteins among all eukaryotic organisms studied so far. As basically intracellular proteins, they play a key role in basic cellular events related to cellular proliferation, including signal transduction, cell-cycle control, cell differentiation and cell survival. The 14-3-3 proteins have been described and characterized in several parasites, and mostly studied in Echinococcus granulosus and Echinococcus multilocularis. Here, we review the discoveries regarding this protein family in the genus Echinococcus, describing new data about specific aspects related with their implication in the parasite biology and immunology in the frame of the host-parasite relationship.

The Taenia saginata homologue of the major surface antigen of Echinococcus spp. is immunogenic and 97% identical to its Taenia solium homologue.

The TEG-Tsag gene of Taenia saginata is homologous to the genes expressing the two major surface antigens of Echinococcus spp. (EM10 and EG10). Surface antigens of parasites are logical candidates for vaccines, and in this paper we demonstrate that cattle vaccinated with the recombinant TEG-Tsag protein, either used singly or in conjunction with the recombinant HP6-Tsag protein, the major 18 kDa surface/secreted antigen of T. saginata oncospheres, produce excellent antibody responses to both these recombinant proteins. Thus TEG-Tsag may have utility as a vaccine and also as a diagnostic tool for bovine cysticercosis. In addition, as we now demonstrate a 97% homology between TEG-Tsag and its Taenia solium homologue, TEG-Tsol, this latter molecule may have similar potential in the control of human and porcine cysticercosis. The TEG molecule is characterized by an N-terminal FERM domain and a C-terminal ERM domain which are found in a number of cytoskeletal-associated proteins located at the interface between the plasma membrane and the cytoskeleton and in proteins that interact with lipid membranes. The FERM domain is also postulated to bind to adhesion proteins, in a PIP2-regulated fashion, providing a link between cytoskeletal signals and membrane dynamics. Thus TEG protein may play a role in tegument function and interaction with the host.

Echinococcus multilocularis: identification and molecular characterization of a Ral-like small GTP-binding protein.

In mammals, Ral (Ras-like) GTPases have been implicated in the regulation of several cellular key processes such as oncogenic transformation, endocytosis, and actin-cytoskeleton dynamics. Here we provide, for the first time, molecular data on a Ral homologue from a parasitic helminth. We have cloned and characterized the complete cDNA molecule and the chromosomal locus encoding a novel GTP binding protein, EmRal, of the human parasite Echinococcus multilocularis. The encoded protein contained all highly conserved amino acid residues of the protein family at corresponding positions and shared significant sequence homologies with human RalA (53% identity) and RalB (54%). Upon heterologous expression of EmRal in Escherichia coli, the recombinant protein was able to bind GTP, thus indicating functionality of the Echinococcus factor. Using an in vitro prenylation assay, the purified protein was shown to be geranylgernylated, but not farnesylated, in both rabbit reticulocyte and Echinococcus cell extracts. The EmRal mRNA was found to be processed via trans-splicing and, using RT-PCR and virtual Northern blot experiments, expression of the factor could be demonstrated for the larval stages metacestode and protoscolex during an infection of the intermediate host. The data presented herein provide a solid basis for further investigations on Ras-Ral signaling mechanisms in Echinococcus.

Chromatin from two classes of platyhelminthes display both protist H1 and higher eukaryote core histones.

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.

The crystal structure of Echinococcus granulosus fatty-acid-binding protein 1.

We describe the 1.6 A crystal structure of the fatty-acid-binding protein EgFABP1 from the parasitic platyhelminth Echinococcus granulosus. E. granulosus causes hydatid disease, which is a major zoonosis. EgFABP1 has been implicated in the acquisition, storage, and transport of lipids, and may be important to the organism since it is incapable of synthesising most of its lipids de novo. Moreover, EgFABP1 is a promising candidate for a vaccine against hydatid disease. The crystal structure reveals that EgFABP1 has the expected 10-stranded beta-barrel fold typical of the family of intracellular lipid-binding proteins, and that it is structurally most similar to P2 myelin protein. We describe the comparison of the crystal structure of EgFABP1 with these proteins and with an older homology model for EgFABP1. The electron density reveals the presence of a bound ligand inside the cavity, which we have interpreted as palmitic acid. The carboxylate group of the fatty acid interacts with the protein's P2 motif, consisting of a conserved triad R em leader R-x-Y. The hydrophobic tail of the ligand assumes a fairly flat, U-shaped conformation and has relatively few interactions with the protein.We discuss some of the structural implications of the crystal structure of EgFABP1 for related platyhelminthic FABPs.

Characterization of carbohydrates of adult Echinococcus granulosus by lectin-binding analysis.

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.

Alternative mRNAs arising from trans-splicing code for mitochondrial and cytosolic variants of Echinococcus granulosus thioredoxin Glutathione reductase.

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.

A histochemical study on the hydatid cyst and electron microscopy of the hydatid sand of Echinococcus granulosus.

The histochemistry of the hydatid cyst wall of E. granulosus from goat and sheep were studied. The cyst wall contains a carbohydrate-protein substrate complex, collagen and possibly calcium. Calcium is also reported in protoscolices of hydatid sand. Tegumental projections on free brood capsules and protoscolices were viewed by scanning electron microscope (SEM) and the tegument of protoscolices was revealed by transmission electron microscopy (TEM).

myo-Inositol hexakisphosphate is a major component of an extracellular structure in the parasitic cestode Echinococcus granulosus.

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.

Complete mitochondrial genomes confirm the distinctiveness of the horse-dog and sheep-dog strains of Echinococcus granulosus.

Unlike other members of the genus, Echinococcus granulosus is known to exhibit considerable levels of variation in biology, physiology and molecular genetics. Indeed, some of the taxa regarded as 'genotypes' within E. granulosus might be sufficiently distinct as to merit specific status. Here, complete mitochondrial genomes are presented of 2 genotypes of E. granulosus (G1-sheep-dog strain: G4-horse-dog strain) and of another taeniid cestode, Taenia crassiceps. These genomes are characterized and compared with those of Echinococcus multilocularis and Hymenolepis diminuta. Genomes of all the species are very similar in structure, length and base-composition. Pairwise comparisons of concatenated protein-coding genes indicate that the G1 and G4 genotypes of E. granulosus are almost as distant from each other as each is from a distinct species, E. multilocularis. Sequences for the variable genes atp6 and nad3 were obtained from additional genotypes of E. granulosus, from E. vogeli and E. oligarthrus. Again, pairwise comparisons showed the distinctiveness of the G1 and G4 genotypes. Phylogenetic analyses of concatenated atp6, nad1 (partial) and cox1 (partial) genes from E. multilocularis, E. vogeli, E. oligarthrus, 5 genotypes of E. granulosus, and using T. crassiceps as an outgroup, yielded the same results. We conclude that the sheep-dog and horse-dog strains of E. granulosus should be regarded as distinct at the specific level.

Binding properties of Echinococcus granulosus fatty acid binding protein.

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.

Echinococcus granulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein.

We report the isolation and characterization of an Echinococcus granulosus gene that codes for a protein with actin filament fragmenting and nucleating activities (EgAFFP). The genomic region corresponding to the EgAFFP gene presents a coding sequence of 1110 bp that is interrupted by eight introns. The EgAFFP deduced amino acid sequence is about 40% homologous to those of several members of the gelsolin family, such as Physarum polycephalum fragmin, Dictyostelium discoideum severin, and Lumbricus terrestris actin modulator. As do other proteins of the same family, EgAFFP presents three repeated domains, each one characterized by internal conserved amino acid motifs. Assays with fluorescence-labeled actin showed that the full-length recombinant EgAFFP effectively binds actin monomers in both a calcium-dependent and calcium-independent manner and also presents actin nucleating and severing activities.

Characterization of the laminated layer of in vitro cultivated Echinococcus vogeli metacestodes.

The metacestode (larval) stages of the cestode parasites Echinococcus vogeli and E. multilocularis were isolated from the peritoneal cavity of experimentally infected C57BL/6 mice and were cultured in vitro for a period of up to 4 mo under conditions normally applied for the in vitro cultivation of E. multilocularis metacestodes. In contrast to E. multilocularis, E. vogeli did not exhibit extensive exogenous budding and proliferation but increased in size with a final diameter of up to 10 mm. Most metacestodes contained protoscoleces, singly or in groups, either associated with brood capsules or growing directly out of the germinal layer. Each individual metacestode was covered by an acellular translucent laminated layer that was considerably thicker than the laminated layer of E. multilocularis metacestodes. The ultrastructural characteristics, protein content, and carbohydrate composition of the laminated layer of in vitro cultivated E. vogeli and E. multilocularis were assessed using transmission electron microscopy, lectin fluorescence labeling, and lectin blotting assays. The laminated layer of E. vogeli is, as previously described for E. multilocularis metacestodes, largely composed of N-acetyl-beta-D-galactosaminyl residues and alpha- and beta-D-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine-beta-1,3-galactose. However, in contrast to E. multilocularis, N-linked glycopeptides and alpha-D-mannosyl and/or glucosyl residues were also associated with the laminated layer of E. vogeli. The laminated layer from both species was isolated from in vitro cultivated metacestodes, and the purified fractions were comparatively analyzed. The protein:carbohydrate ratio (1:1) was similar in both parasites; however, the protein banding pattern obtained by silver staining following sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested intrinsic differences in protein composition. A polyclonal antiserum raised against the E. multilocularis laminated layer and a monoclonal antibody, G11, directed against the major E. multilocularis laminated layer antigen Em2 did not cross-react with E. vogeli, indicating distinct compositional and antigenic differences between these 2 parasites.

Immunofluorescent localization of intermediate filaments (IFs) in helminths using anti-mammalian IFs monoclonal antibody.

Intermediate filaments (IFs) make up the cytoskeleton of most eukaryotic cells. In vertebrates, a number of IF proteins have been identified, showing distributions unique to tissue or cell type. Information on helminth IFs is limited to some nematode species. To observe immunofluorescent localization of IFs in helminth tissues, we selected a murine hybridoma clone producing IgM antibody to multiple types of mammalian IF proteins and examined cross-reactivity to helminth proteins. The selected monoclonal antibody (HUSM-9) cross-reacted well with IFs from nematode species such as Toxocara canis, Dirofilaria immitis, Anisakis simplex, and Trichinella britovi; strong immunofluorescence on cryostat sections was detected in the hypodermis, cords, body muscle, smooth muscle of the uterus, and other epithelial structures. In platyhelminths, i.e., adult Schistosoma mansoni, larval Taenia taeniaeformis, adult Taenia crassiceps, and Echinococcus multilocularis protoscolex, the reactivity was weaker than in nematodes, and localized in the body wall muscle and subtegumental tissue. Western blotting of 8 M urea extracts of parasites with the antibody detected a pair of clear bands in nematodes but not in S. mansoni or the cestodes. These results might be explained by sparse distribution of IFs in platyhelminths, or low affinity of the used antibody to platyhelminth IF proteins, or both.

Cellular immune response of lymph nodes from dogs following the intradermal injection of a recombinant antigen corresponding to a 66 kDa protein of Echinococcus granulosus.

A recombinant polypeptide (referred to as EgA31), which represents a 66kDa protein, was prepared from an Echinococcus granulosus cDNA library. In order to assess its potential to induce cellular immune responses, dog popliteal and prescapular lymph nodes were sensitized with this recombinant polypeptide. Subpopulations of lymphocytes were then analyzed by flow cytometry and immunohistochemistry on lymph node sections. Five days after the sensitization, the paracortical areas of the lymph nodes appeared hypertrophic, the number of CD3+, CD4+, CD8+ and CD5+ cells increased, the number of B-cells began to augment and some secondary follicles occurred, and a number of CD4+ cells appeared in germinal centers. Many large secondary follicles and a significantly augmented number of CD5+ cells in cords of medullae were observed 10 days after the sensitization. These active cellular responses strengthen the interest for further studies on the development of a vaccine with this recombinant polypeptide.

High molecular mass glycans are major structural elements associated with the laminated layer of in vitro cultivated Echinococcus multilocularis metacestodes.

The laminated layer of the larval stage (metacestode) of the cestode parasite Echinococcus multilocularis is composed largely of carbohydrates, which form a tight microfibrillar meshwork around the entire metacestode. Since this laminated layer is the only parasite structure which is in constant contact with host immune and non-immune cells, and appears largely resistant to physiological and immunological reactions of the host, it most likely carries out important functions with regard to host-parasite interactions. In infected hosts, the metacestode is usually concentrically covered by host connective tissue cells and large amounts of collagen, causing a dense scar-like fibrosis, and it is likely that host-derived components are incorporated into the laminated layer at the host-parasite interface. Therefore, in order to obtain information on the molecular composition of this structure, we used parasite larvae which were generated through in vitro cultivation and thus were largely devoid of interfering host components. Lectin fluorescence on section-labelling of metacestodes embedded in LR-White suggested that the laminated layer is largely composed of N-acetyl-beta-D-galactosaminyl, and alpha- and beta-D-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine-beta-1.3-galactose, while N-linked glycopeptides and alpha-D-mannosyl residues and/or glucosyl residues were found mainly within the germinal layer, and within the cellular mass and the surface of developing protoscoleces. Lectin-gold EM confirmed these findings. The laminated layer was isolated from in vitro cultivated metacestodes by urea extraction, and the ultrastructure of the purified laminated layer was assessed comparatively with respect to the laminated layer of intact parasites. The glycan composition was determined using SDS-PAGE and lectin blotting. This work has laid the basis for a more detailed dissection of the molecular composition of the laminated layer.

Synthetic studies on glycosphingolipids from the parasite Echinococcus multilocularis.

Novel neutral glycosphingolipids isolated from the metacestodes of Echinococcus multilocularis by Persat, may be expected to be involved in host-parasite interactions. We have synthesized these glycosphingolipid analogues containing 2-branched fatty alkyl residues in place of ceramide. The glycosylation of galactosyl donors 4 and 5 with each of the acceptors 2 and 11 in the presence of N-iodosuccinimide (NIS)/TfOH, and the glycosylation of fucosyl donor 13 with acceptors 12 and 20 in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) gave the desired oligosaccharide derivatives at good yield. The fully per-O-acylated 2-(trimethylsilyl)ethyl glycosides 6, 15, 21, and 26 were converted to glycosylimidates 7, 16, 22, and 27, which were condensed with 2-(tetradecyl)hexadecanol and subsequently deacylated give four target glycosphingolipid analogues.