Digest the Sugar, Kill the Parasite: A New Experimental Concept in Treating Alveolar Echinococcosis.

INTRODUCTION: The E. multilocularis laminated layer (LL) is a heavily glycosylated parasitic structure that plays an important role in protecting the larval stage (metacestode) of this parasite from physiological and immunological host reactions. We elaborated an experimental design with the idea to modify the (glycan) surface of the LL by a targeted digestion. This should allow the host defense to more easily recognize and attack (or kill) the parasite by immune-mediated effects. METHODS: Experimentally, E. multilocularis (clone H95) metacestodes were cultured in vitro with or without addition of α1-3,4,6-galactosidase or ß1-3-galactosidase in the medium. Morphological changes were subsequently measured by microscopy at different time points. Parasites were then recovered at day 5 and reinjected into mice for assessing their viability and infectious status. For finally recovered parasites, the respective load was assessed ex vivo by wet weight measurement, and host-related PD1 and IL-10 levels were determined as the key immunoregulators by using flow cytometry. RESULTS: Our experiments demonstrated that the parasite vesicular structure can be directly destroyed by adding galactosidases into the in vitro culture system, resulting in the fact that the parasite metacestode vesicles could not anymore infect and develop in mice after this glycan digestion. Moreover, when compared to the mice inoculated with E. multilocularis metacestode without galactosidases, PD1 expression was upregulated in CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with ß1-3-galactosidase, with a lower IL-10 secretion from CD4+ Teffs; there was no difference of PD1 and IL-10 expression levels regarding CD4+ Teff from mice inoculated with E. multilocularis metacestode pretreated with α1-3,4,6-galac-tosidase. DISCUSSION: We raised our hypothesis that this "aborting" effect may be linked to an altered PD1 and IL-10 response fine-tuning between immunopathology and immune protection. These findings justify a continuation of these experiments upon therapeutical in vivo administration of the enzymes.

Expansion of Host Regulatory T Cells by Secreted Products of the Tapeworm Echinococcus multilocularis.

Background: Alveolar echinococcosis (AE), caused by the metacestode larval stage of the fox-tapeworm Echinococcus multilocularis, is a chronic zoonosis associated with significant modulation of the host immune response. A role of regulatory T-cells (Treg) in generating an immunosuppressive environment around the metacestode during chronic disease has been reported, but the molecular mechanisms of Treg induction by E. multilocularis, particularly parasite immunoregulatory factors involved, remain elusive so far. Methodology/Principal Findings: We herein demonstrate that excretory/secretory (E/S) products of the E. multilocularis metacestode promote the formation of Foxp3+ Treg from CD4+ T-cells in vitro in a TGF-ß-dependent manner, given that this effect was abrogated by treatment with antibody to mammalian TGF-ß. We also show that host T-cells secrete elevated levels of the immunosuppressive cytokine IL-10 in response to metacestode E/S products. Within the E/S fraction of the metacestode we identified an E. multilocularis activin A homolog (EmACT) that displays significant similarities to mammalian Transforming Growth Factor-ß (TGF-ß/activin subfamily members. EmACT obtained from heterologous expression failed to directly induce Treg expansion from naïve T cells but required addition of recombinant host TGF-ß to promote CD4+ Foxp3+ Treg conversion in vitro. Furthermore, like in the case of metacestode E/S products, EmACT-treated CD4+ T-cells secreted higher levels of IL-10. These observations suggest a contribution of EmACT to in vitro expansion of Foxp3+ Treg by the E. multilocularis metacestode. Using infection experiments we show that intraperitoneally injected metacestode tissue expands host Foxp3+ Treg, confirming the expansion of this cell type in vivo during parasite establishment. Conclusion/Significance: In conclusion, we herein demonstrate that E. multilocularis larvae secrete factors that induce the secretion of IL-10 by T-cells and contribute to the expansion of TGF-b-driven Foxp3+ Treg, a cell type that has been reported crucial for generating a tolerogenic environment to support parasite establishment and proliferation. Among the E/S factors of the parasite we identified a factor with structural and functional homologies to mammalian activin A which might play an important role in these activities.

Dogs as victims of their own worms: Serodiagnosis of canine alveolar echinococcosis.

BACKGROUND: Besides acting as definitive hosts for Echinococcus multilocularis, dogs can become infected by the larval form of this parasite and thereby develop life-threatening alveolar echinococcosis (AE). Although AE is a zoonotic disease, most therapeutic and diagnostic approaches have been developed for human patients. In dogs, AE is typically diagnosed in the advanced stage of the disease when the parasitic mass has already caused abdominal distension. At that stage, complete resection of the parasitic mass is often impossible, leaving a guarded prognosis for the affected dogs. For humans, sensitive and specific diagnostic protocols relying on serology have been validated and are now widely used. In contrast, sensitive and specific laboratory diagnostic tools that would enable early diagnosis of canine AE are still lacking. The aim of the current study was to establish a serological protocol specifically adapted to dogs. METHODS: We tested several native and recombinant antigens (EmVF, Em2, recEm95, recEm18) in in-house ELISA, an in-house Western blot (WB), as well as a commercially available WB developed for serodiagnosing human AE (Anti-Echinococcus EUROLINE-WB®), using a panel of known status dog sera. RESULTS: RecEm95-antigen was revealed to be the most promising antigen for use in ELISA, demonstrating 100% (95% CI: 72-100%) sensitivity and 100% (95% CI: 93-100%) specificity in our study. The in-house WB using EmVF antigen performed as well as the recEm95-ELISA. The commercial WB also correctly identified all infected dogs, coupled with a specificity of 98% (95% CI: 91-100%). CONCLUSION: The recEm95-ELISA alone or in combination with either the in-house WB or the Anti-Echinococcus EUROLINE-WB® (IgG) with a minor modification should be considered as the best current approach for the serological diagnosis of dogs infected with the larval stage of E. multilocularis. However, larger studies with a focus on potentially cross-reacting sera should be undertaken to verify these findings.

Comparative proteomics of hydatid fluids from two Echinococcus multilocularis isolates.

The hydatid fluid (HF) that fills Echinococcus multilocularis metacestode vesicles is a complex mixture of proteins from both parasite and host origin. Here, a LC-MS/MS approach was used to compare the HF composition of E. multilocularis H95 and G8065 isolates (EmH95 and EmG8065, respectively), which present differences in terms of growth and fertility. Overall, 446 unique proteins were identified, 392 of which (88%) were from parasite origin and 54 from culture medium. At least 256 of parasite proteins were sample exclusive, and 82 of the 136 shared proteins presented differential abundance between E. multilocularis isolates. The parasite's protein repertoires in EmH95 and EmG8065 HF samples presented qualitative and quantitative differences involving antigens, signaling proteins, proteolytic enzymes, protease inhibitors and chaperones, highlighting intraspecific singularities that could be correlated to biological features of each isolate. The repertoire of medium proteins found in the HF was also differential between isolates, and the relevance of the HF exogenous protein content for the parasite's biology is discussed. The repertoires of identified proteins also provided potential molecular markers for important biological features, such as parasite growth rate and fertility, as well potential protein targets for the development of novel diagnostic and treatment strategies for alveolar echinococcosis. BIOLOGICAL SIGNIFICANCE: E. multilocularis metacestode infection of mammal hosts involve complex interactions mediated by excretory/secretory (ES) products. The hydatid fluid (HF) that fills the E. multilocularis metacestode vesicles contains complex repertoires of parasite ES products and host proteins that mediate important molecular interactions determinant for parasite survival and development, and, consequently, to the infection outcome. HF has been also extensively reported as the main source of proteins for the immunodiagnosis of echinococcosis. The performed proteomic analysis provided a comprehensive profiling of the HF protein composition of two E. multilocularis isolates. This allowed us to identify proteins of both parasite and exogenous (medium) origin, many of which present significant differential abundances between parasite isolates and may correlate to their differential biological features, including fertility and growth rate.

Synthesis and Antigenicity against Human Sera of a Biotin-Labeled Oligosaccharide Portion of a Glycosphingolipid from the Parasite Echinococcus multilocularis.

Synthesis of a biotinylated analog of the carbohydrate portion of a glycosphingolipid from the parasite Echinococcus multilocularis has been achieved. We synthesized ß-D-Galp-(1→6)-ß-D-Galp-(1→6)-[α-L-Fucp-(1→3)]-ß-D-Galp-(1→R: biotin probe) (1) and compared the antigenicity by an enzyme linked immunosorbent assay (ELISA) with biotinylated trisaccharide α-D-Galp-(1→4)-ß-D-Galp-(1→3)-α-D-Galp-(1→R: biotin probe) (F), which has been shown to have significant antigenicity. Both of the oligosaccharides reacted with sera of alveolar echinococcosis (AE) patients, but showed different reactivity. Among the 60 sera of AE patients, more sera reacted with the linear sequence Galα1→4Galß1→3GalNAcα1→R of oligosaccharide (F) than for branched compound 1. Some sera showed high specificity to one of the compound, indicating that the antibodies in the sera of AE patients differ in their specificity to recognize carbohydrate sequences of glycosphingolipids. Our results demonstrate that both of the biotinylated oligosaccharides 1 and F have good serodiagnostic potential and are complementary to detect infections caused by the parasite Echinococcus multilocularis.

De novo discovery of neuropeptides in the genomes of parasitic flatworms using a novel comparative approach.

Neuropeptide mediated signalling is an ancient mechanism found in almost all animals and has been proposed as a promising target for the development of novel drugs against helminths. However, identification of neuropeptides from genomic data is challenging, and knowledge of the neuropeptide complement of parasitic flatworms is still fragmentary. In this work, we have developed an evolution-based strategy for the de novo discovery of neuropeptide precursors, based on the detection of localised sequence conservation between possible prohormone convertase cleavage sites. The method detected known neuropeptide precursors with good precision and specificity in the models Drosophila melanogaster and Caenorhabditis elegans. Furthermore, it identified novel putative neuropeptide precursors in nematodes, including the first description of allatotropin homologues in this phylum. Our search for neuropeptide precursors in the genomes of parasitic flatworms resulted in the description of 34 conserved neuropeptide precursor families, including 13 new ones, and of hundreds of new homologues of known neuropeptide precursor families. Most neuropeptide precursor families show a wide phylogenetic distribution among parasitic flatworms and show little similarity to neuropeptide precursors of other bilaterian animals. However, we could also find orthologs of some conserved bilaterian neuropeptides including pyrokinin, crustacean cardioactive peptide, myomodulin, neuropeptide-Y, neuropeptide KY and SIF-amide. Finally, we determined the expression patterns of seven putative neuropeptide precursor genes in the protoscolex of Echinococcus multilocularis. All genes were expressed in the nervous system with different patterns, indicating a hidden complexity of peptidergic signalling in cestodes.

Negligible elongation of mucin glycans with Gal ß1-3 units distinguishes the laminated layer of Echinococcus multilocularis from that of Echinococcus granulosus.

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.

Identification and characterisation of Emp53, the homologue of human tumor suppressor p53, from Echinococcus multilocularis: its role in apoptosis and the oxidative stress response.

Larvae of the fox tapeworm, Echinococcus multilocularis, cause alveolar echinococcosis, which is considered to be the most lethal helminthic infection in humans. Since it develops in host organs, the parasite must have evolved a stress defense system to cope with various genotoxic and cellular stresses that may cause DNA damage and genomic instability. Tumor suppressor p53, well known as the "guardian of the genome", plays a vital role in response to many types of stress and damage. In the present study, we describe the characterisation of Emp53 from E. multilocularis and demonstrate that it is a structural and functional homologue of mammalian tumor suppressor p53. We show that Emp53 binds specifically to oligonucleotides containing conventional p53 binding sites, indicating that it exhibits a function as a DNA binding transcription factor. Inhibition of Emp53 function can suppress UV irradiation-induced apoptosis in the E. multilocularis metacestode, indicating an important role of Emp53 in the induction of apoptosis following DNA damage. We also reveal that Emp53 plays important roles in resistance to oxidative stress and regulation of oxidative stress-induced apoptosis. Our results suggest that, similar to its human counterpart, Emp53 plays a central role in the network of DNA damage responses and apoptosis in E. multilocularis. These results may help in exploring stress defense mechanisms of parasitic helminths and may provide useful information for the development of new interventions and therapeutic drugs for the control of alveolar echinococcosis.

Molecular cloning and characterization of major vault protein of Echinococcus multilocularis.

The cDNA clone coding a major vault protein (MVP)-like protein was derived from Echinococcus multilocularis cysts. MVP is a main component of vault particles, which are the largest cytoplasmic ribonucleoprotein particles in eukaryotic cells. We sequenced and characterized E. multilocularis MVP (EmMVP). The nucleotide sequence of the emmvp cDNA clone was 2607 bp in the full length open reading frame and its deduced amino acid sequence had several signature motifs which were specific to MVP families. Immunoblot analysis with mouse anti-EmMVP antiserum revealed that crude antigens of E. multilocularis included EmMVP protein. Furthermore, our results showed that the expression of EmMVP protein in an Sf9 insect cell line using a baculovirus vector directed the formation of particles that shared similar biochemical characteristics with other vault proteins and the distinct vault-like morphology when negatively stained and examined by electron microscopy.

Synthesis of the carbohydrate moiety from the parasite Echinococcus multilocularis and their antigenicity against human sera.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions with a Galß1-3GalNAc core of the Em2 glycoprotein antigen obtained from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Galß1-3(GlcNAcß1-6)GalNAcα1-R (G), tetrasaccharide Galß1-3(Galß1-4GlcNAcß1-6)GalNAcα1-R (J) and pentasaccharide Galß1-3(Galα1-4Galß1-4GlcNAcß1-6)GalNAcα1-R (K) (R=biotinylated probe) were synthesized by block synthesis by the use of 5-(methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl-(1→3)-2-azido-4-O-benzyl-2-deoxy-α-d-galactopyranoside as a common glycosyl acceptor. Moreover, linear trisaccharide Galα1-4Galß1-3GalNAcα1-R (H) and branched tetrasaccharide Galα1-4Galß1-3(GlcNAcß1-6)GalNAcα1-R (I) were synthesized by stepwise condensation. We examined the antigenicity of these five oligosaccharides by an enzyme linked immunosorbent assay (ELISA). Our results demonstrate that biotinylated oligosaccharides H, I and K show good serodiagnostic potential to detect infections caused by the parasite E. multilocularis. Among them the linear sequence Galα1-4Galß1-3GalNAcα1-R in oligosaccharide (H) appears to show the highest sensitivity (95%). Moreover, our study clarified the dominant carbohydrate epitope of Em2 antigen.

Production and immunoanalytical application of 32 monoclonal antibodies against metacestode somatic antigens of Echinococcus multilocularis.

Alveolar echinococcosis is a rare but potentially fatal disease. Immunodiagnosis based on antibodies or antigens plays an important role in its diagnosis. In this study, metacestode somatic antigens of Echinococcus multilocularis were used to immunize BALB/c mice, and hybridomas were formed by cell fusion. Making use of the inherent effect of monoclonal antibody techniques to isolate different epitopes, we obtained a repertoire of 32 monoclonal antibodies against the metacestode somatic antigens. These monoclonal antibodies were used to investigate the specificity and localization of the metacestode antigens by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Nine antibodies specifically reacted with E. multilocularis, while 14 and ten cross-reacted with Echinococcus granulosus and Taenia saginata, respectively. Twenty-five antibodies stained the laminated layer. Eight reacted with the tegument of the protoscolex. Fourteen antibodies recognized the germinal layer. Most of the monoclonal antibodies can react with the antigen Em2. One antibody can react with antigen Em2 and Em10. One antibody that cross-reacted with T. saginata stained the germinal layer and protoscolex, especially its hooklets and suckers, but could not react with Em2 and Em10 antigens. It detected protein bands at 26 and 52 kDa. Two E. multilocularis-specific monoclonal antibodies stained both the germinal and laminated layers and could be used not only to purify specific antigens but also for immunohistochemical studies of E. multilocularis. In summary, these 32 monoclonal antibodies could have potential applications as useful tools in further studies of E. multilocularis antigen profiles.

Molecular cloning and characterization of a T24-like protein in Echinococcus multilocularis.

One tetraspanin, designated as E24, was cloned from a full-length enriched vector-capping cDNA library of Echinococcus multilocularis metacestode. The amino acid sequence and phylogenetic analysis suggested that E24 is a T24-like protein. The crucial, functional large extracellular loop (LEL) domain of E24 was expressed and characterized using a polyclonal antiserum by Western blot and immunohistochemistry. The results showed that anti-recombinant-E24 (anti-recE24) antibody can specifically recognize approximately 25 kDa recombinant protein and 25 kDa cyst-extracted antigen; the germinal layer of both the protoscolex-free and protoscolex-formed cysts were intensely labeled by immunofluorescent antibody. This study revealed that E24 is an antigenic, germinal layer-located protein of E. multilocularis metacestode, implying for its potential in diagnostic and vaccine development.

Echinococcus multilocularis: Proteomic analysis of the protoscoleces by two-dimensional electrophoresis and mass spectrometry.

Echinococcus multilocularis is an important parasite that causes human alveolar echinococcosis. Identification and characterization of the proteins encoded by E. multilocularis metacestode might help to understand the complexity of the parasites and their interactions with the host, and to identify new candidates for immunodiagnosis and vaccine development. Here we present a proteomic analysis of E. multilocularis protoscolex (PSC) proteins. The proteins were resolved by 2-DE (pH range 3.5-10), followed by MALDI-TOF MS analysis. Fourteen known Echinococcus proteins were identified, including cytoskeletal proteins, heat shock proteins, metabolic enzymes, 14-3-3 protein, antigen P-29 and calreticulin. To construct a systematic reference map of the immunogenic proteins from E. multilocularis PSC, immunoblot analysis of PSC 2-DE maps was performed. Over 50 proteins spots were detected on immunoblots as antigens and 15 of them were defined. The results showed that cytoskeletal proteins and heat shock proteins were immunodominant antigens in alveolar echinococcosis.

Synthetic studies on the carbohydrate moiety of the antigen from the parasite Echinococcus multilocularis.

Stereocontrolled syntheses of branched tri-, tetra-, and pentasaccharides displaying a Gal beta 1-->3GalNAc core in the glycan portion of the glycoprotein antigen from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc alpha 1-OR (A), tetrasaccharide Gal beta 1-->3(Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (D), and pentasaccharides Gal beta 1-->3(Gal beta 1-->4Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (E) and Gal beta1-->3(Gal alpha 1-->4Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (F) (R = 2-(trimethylsilyl)ethyl) were synthesized by block synthesis. The disaccharide 2-(trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->3)-2-azido-4-O-benzyl-2-deoxy-alpha-d-galactopyranoside served as a common glycosyl acceptor in the synthesis of the branched oligosaccharides. Moreover, linear trisaccharide Gal beta 1-->4Gal beta 1-->3GalNAc alpha 1-OR (B) and branched tetrasaccharide Gal beta 1-->4Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc alpha 1-OR (C) were synthesized by stepwise condensation.

The vaccination potential of EMY162 antigen against Echinococcus multilocularis infection.

Alveolar echinococcosis is caused by infection with the larval stage of Echinococcus multilocularis. We recently identified a cDNA clone, designated as emy162, that encodes a putative secreted protein. EMY162 shares structural features with the EM95 antigen, which is a host-protective antigen. The amino acid sequence of EMY162 shows 31.4% identity to EM95 whereas these antigens are distinguishable with respect to their predicted secondary structure and antigenicity on Western blot analysis. RT-PCR analysis revealed that the gene expression of emy162 was significantly higher than that of em95 at each life-cycle stage. Recombinant EMY162 antigen induced a significant level of host-protection (74.3%) in experimental infection with E. multilocularis eggs in mice. Notably, recombinant EMY162 antigen showed significant reactivity to the sera from alveolar echinococcosis patients. These results may help in the development of a practical vaccine to reduce the level of alveolar echinococcosis in humans.

Synthetic studies on glycosphingolipids from Protostomia phyla: total syntheses of glycosphingolipids from the parasite, Echinococcus multilocularis.

Efficient and systematic syntheses of four neutral glycosphingolipids that have been isolated from the metacestodes of Echinococcus multilocularis have been achieved. A key step is the direct glycosylation of galactosyl donors using thioglycosides with benzoyl ceramide in the presence of N-iodosuccinimide (NIS)/TfOH, which gave the desired oligosaccharide derivatives. The fully protected glycosides 13, 20, 22 and 25 were deprotected to give four target glycosphingolipids (1-4).

Echinococcus multilocularis metacestode extract triggers human basophils to release interleukin-4.

Infections with parasitic helminths are associated with a T helper 2 (Th2) immune response and IgE production. The underlying mechanism, however, is only partially understood. Recently we have isolated a protein from extracts of Schistosoma mansoni eggs that triggers human basophils from non-sensitized donors to release interleukin-4 (IL-4), the key cytokine of a Th2 response. We called this protein IPSE (for IL-4-inducing principle from Schistosoma mansoni eggs). Supposing that IPSE-like IL-4-inducing activities might be a general principle shared among different helminth species, we investigated extracts from the cestode E. multilocularis for its effect on human basophils. Our results showed that extracts from metacestodes of E. multilocularis cause basophil degranulation, as well as the secretion of histamine, IL-4 and IL-13, in a dose-dependent manner. IgE stripping and resensitization of basophils indicated that the mechanism of IL-4 induction requires the presence of IgE on the cells. Since analogous properties have been demonstrated earlier for IPSE, we think that S. mansoni and E. multilocularis may induce a Th2 response in their hosts via a related mechanism, namely, by the induction of IL-4 release from basophils.