Schistosoma mansoni shares a protective carbohydrate epitope with keyhole limpet hemocyanin.

The glycanic epitope of the 38,000 Mr Schistosoma mansoni schistosomula major immunogen defined by the IPLSm1 protective mAb was identified in the hemocyanin of the marine mollusc Megathura crenulata, better known as KLH. This antigenic community was exploited to investigate further the biological properties of this epitope. KLH was shown to strongly inhibit the binding of IPLSm1 mAb to its 38,000 Mr target antigen. Immunization of naive LOU rats with KLH elicited the production of anti-S. mansoni antibodies capable of immunoprecipitating the 38,000 Mr schistosomulum antigen. Antibodies to KLH mediated a marked eosinophil-dependent cytotoxicity and passively transferred immunity towards S. mansoni infection. Finally, rats immunized with KLH were significantly protected against a challenge with S. mansoni cercariae. The deglycosylation of KLH completely abolishes its immunological and functional KLH properties, indicating the participation of an oligosaccharidic epitope of the native KLH that is also recognized by the sera of S. mansoni-infected patients. These observations provide new opportunities of access to the well-defined structure of a glycanic epitope potentially available for the immunoprophylaxis and seroepidemiology of schistosomiasis, and a new approach to the isotypic response towards a well-chemically defined epitope.

Schistosoma mansoni: signal transduction processes during the development of the reproductive organs.

Among the topics of considerable interest concerning our understanding of the unusual biology of schistosomes is the sexual maturation of the female. The identification of genes coding for signal transduction proteins controlling essential steps of the pairing-dependent differentiation of the reproductive organs, vitellarium and ovary will help to substantiate our knowledge about this unique parasite. Furthermore, such signalling proteins could be potential targets to interfere with the development of this parasite to combat schistosomiasis since its pathology is caused by the eggs. This review summarises first post-genomic steps to elucidate the function of gonad-specific signalling molecules which were identified by homology-based cloning strategies, by in silico identification or by yeast two-hybrid interaction analyses, using a combination of novel techniques. These include the in vitro culture of adult schistosomes, their treatment with chemical inhibitors to block enzyme activity, the use of RNAi to silence gene function post-transcriptionally, and confocal laser scanning microscopy to study the morphological consequences of these experimental approaches. Finally, we propose a first model of protein networks that are active in the ovary regulating mitogenic activity and differentiation. Some of these molecules are also active in the testes of males, probably fulfilling similar roles as in the ovary.

Insulin receptors and glucose uptake in the human parasite Schistosoma mansoni.

Very little is known about insulin signalling in schistosomes despite its potential importance in host-parasite molecular dialogue and parasite growth and development. The recent characterization of two insulin receptors (SmIR-1 and SmIR-2) in Schistosoma mansoni has led us to reconsider the question of the potential importance of insulin in host-schistosome interactions. In this work, we demonstrated that insulin could regulate glucose uptake in schistosomes and we investigated the implication of SmIR-1 and SmIR-2 in this process. The possibility that specific inhibitors of SmIR-1 and SmIR-2 tyrosine kinase activities could be developed to target schistosomes is discussed.

Diagnostic value of a synthetic peptide derived from Echinococcus granulosus recombinant protein.

A specific monoclonal antibody (MAb; EG 02 154/12) directed against a protein epitope of Echinococcus granulosus antigen 5 was used to screen a cDNA library constructed from E. granulosus protoscoleces RNA. One clone designated Eg14 was selected and shown to code for an amino acid sequence partially homologous to that of the clone Eg6 previously identified with the same MAb. Hydrophobic cluster analysis showed that both recombinant antigens may adopt a similar alpha-helical organization and share a common conformational epitope. A synthetic peptide (89-122) mimicking the conformational site of Eg6 and Eg14 was constructed and demonstrated to be able to inhibit binding of the MAb and human hydatid sera to the Eg6 fusion protein (FP6) or to native hydatid antigens. To assess the diagnostic value of the peptide 89-122, we tested sera from patients infected with different parasites for their antibody reactivity with this peptide in ELISA. A high binding sensitivity and specificity of IgG-A-M antibodies were obtained with E. granulosus-infected patient sera. Moreover, the peptide 89-122 was found to be specifically recognized by IgE antibodies from patients with hydatid disease. These results indicate the particular interest of this synthetic peptide as a standardized antigen in diagnosis and treatment surveillance of hydatidosis.

Characterization of an insulin receptor-related receptor in Biomphalaria glabrata embryonic cells.

Tyrosine kinase receptors play a key role in the communication of cells with their environment. Growth hormone receptors, such as insulin receptors, are involved in the regulation of cell growth, differentiation and metabolism in multicellular organisms. Insulin-related peptides and members of the insulin receptor subfamily have been described in a wide variety of invertebrates, including freshwater molluscs. In this paper, we describe the metabolic effect of insulin on a mollusc cell line (Bge) derived from embryos of the snail Biomphalaria glabrata. Using a PCR strategy, we have cloned from Bge cells a cDNA encoding a protein (BgIR) homologous to, and exhibiting all of the typical features of insulin receptors. Northern blot analysis confirmed the expression of BgIR in B. glabrata snails and suggested its wide distribution in the snail body. Bge cells have been shown to provide the environmental conditions necessary for the in vitro development of the sporocysts of Schistosoma mansoni, a trematode parasite that uses B. glabrata as an intermediate host. The possible implication of BgIR in the activating and proliferating processes observed in Bge cells during their coculture with S. mansoni larvae is discussed.

Cloning and characterisation of the gene encoding the 28-kDa glutathione S-transferase of Schistosoma mansoni.

The 28-kDa glutathione S-transferase (GST) of Schistosoma mansoni is considered a possible vaccine candidate for use against this medically important parasite. The gene encoding this molecule has been isolated from a lambda EMBL4 library by using the corresponding cDNA sequence as a probe. The gene contains four exons and is approximately 5.5 kb in length. Analysis of the 5' flanking region revealed the presence of a consensus AP-1 recognition site, 5'-TGACTCA, between nucleotides -231 and -225. Southern blot analysis suggested the presence of a single gene encoding the 28-kDa GST in the S. mansoni genome.

Schistosoma mansoni: antigenic community between schistosomula surface and adult worm incubation products as a support for concomitant immunity.

The use of protective monoclonal antibodies has enabled us to demonstrate antigenic community between a 38-kDa schistosomula surface molecule and a 115-kDa component derived from adult worms. Injection of adult worms in rats also led to the production of antibodies specific for the 38-kDa antigen, suggesting that the 115-kDa adult worm molecule could act as an inducer of the protective immune response raised against young invading parasites.

Use of a monoclonal antibody specific for a protein epitope of Echinococcus granulosus antigen 5 in a competitive antibody radioimmunoassay for diagnosis of hydatid disease.

A monoclonal antibody (mAb) designated as EG 02 154/12, specific for the major antigen (antigen 5) of Echinococcus granulosus was produced, and used to study the binding sites recognized by anti-antigen 5 antibodies from patients with hydatid disease. The nature of the target epitope was partially characterized. The antibody reactivity was analyzed towards sheep hydatid fluid antigens (SHF Ag) using ELISA, immunoelectrophoresis (IEP), Western blotting (WB), and immunoprecipitation (IP). In IEP, EG 02 154/12 mAb gave a single precipitin of Ag 5. The mAb and human hydatid patient sera recognized a major antigen of 64 kDa, in SHF Ag analyzed in non-reducing conditions. Both types of antibodies revealed two components of 37 and 22 kDa in reducing conditions. Deglycosylation and delipidation of SHF Ag did not affect the mAb binding. These results, together with the observation of mAb binding to in vitro translation products from protoscoleces messenger RNA, suggest the protein nature of the epitope recognized on the antigen 5. Using competitive antibody radioimmunoassay (CRIA), a competition between this mAb and hydatid patient sera, for the same epitope or closely related sites on antigen 5, was observed. No such competition was detected with the sera from other helminthiasis. The sensitivity and specificity of CRIA was compared to that of ELISA and CRIA found to be an improved diagnostic test for hydatid disease.

Isolation and characterization of surface antigens from Schistosoma mansoni schistosomula.

Surface antigens of Schistosoma mansoni schistosomula were isolated using antibodies produced in rat and human schistosomiasis. Three immunoreactive surface proteins of 40 000, 37 000 and 32 000 daltons were identified by SDS-PAGE analysis of immune complexes formed by incubation of a detergent extract of surface labelled schistosomula with infected rat sera. Surface antigens of similar molecular weight were also isolated when using sera of patients with schistosomiasis. Binding of schistosomula surface antigens to specific antibodies was substantially inhibited by components released by adult worms. The results suggest that these schistosomula surface antigens could be involved in the immune response against challenge infection but their protective role in immunity still remains to be determined.

Schistosoma mansoni and its intermediate host Biomphalaria glabrata express a common 39 kilodalton acidic protein.

Two Schistosoma mansoni proteins of 43 and 39 kDa (Sm43 and Sm39) were shown to react with rabbit antibodies produced against Biomphalaria glabrata proteins. Two-dimensional gel electrophoresis of miracidial proteins indicated that Sm43 and Sm39 were acidic proteins (pI 4.8 and 4.9 respectively) and were in vitro translated from miracidial messenger RNA in the same molecular forms. Sm43 and Sm39 were expressed by all parasite stages of S. mansoni. Using anti-Sm43 and anti-Sm39 mouse sera, we demonstrated that both parasite proteins were antigenically related and cross-reacted with a unique 39 kDa (pI 4.9) protein from B. glabrata (Bg39). Cross-reactive components were found in fresh water and land snails but not in vertebrate tissues, suggesting that the 39 kDa protein was specific for invertebrates.

Stimulation of Schistosoma mansoni miracidia by a 80 kDa glycoprotein from Biomphalaria glabrata.

Soluble extracts of Biomphalaria glabrata stimulate in vitro incorporation of methionine in Schistosoma mansoni miracidia. Evidence is presented that a unique 80 kDa glycoprotein representing less than 0.01% of total snail proteins and uniformly distributed in the snail body, is responsible for the observed stimulation. This protein specifically acts on the miracidia and this observation suggests that this glycoprotein influences snail penetration and development of miracidia. However, the presence of molecules stimulating S. mansoni miracidia was also demonstrated in S. mansoni nonpermissive molluscs.

Characteristic proteins of micronemes and dense granules from Sarcocystis tenella zoites (Protozoa, Coccidia).

A subcellular fractionation procedure has been developed, which allowed the recovery of purified fractions of micronemes, dense granules and pellicles from Sarcocystis tenella zoites. As expected, sodium dodecyl sulphate polyacrylamide gels electrophoresis of the pellicles showed a fairly heterogenous protein content. In contrast, only one major omponent (42 000) daltons) was found in the dense granules, whereas two major bands (20 000 and 22 000 daltons) were observed for the micromemes. These characteristic proteins were also major components of the whole zoite and might have important functions in the physiology of the organism.

Molecular cloning of an Echinococcus granulosus protein expressing an immunogenic epitope of antigen 5.

cDNA was synthesized from RNA extracted from Echinococcus granulosus protoscoleces and cloned in the lambda gt11 expression vector. A pool of 5 E. granulosus patient sera was used to screen the library and allowed the selection of 13 clones. Ten of these were shown to be identical, among which clone 6 (Eg6) was chosen for further analysis. The nucleotide sequence (456-bp) presented an entire open reading frame coding for 152 amino acids. The fusion protein (FP6) was recognized by a mouse monoclonal antibody (EG 02 154/12) specific for E. granulosus antigen 5. Moreover, the presence of antibodies to FP6 seemed to be correlated to the ability of sera from hydatidosis patients to immunoprecipitate antigen 5. These results indicate that the cloned protein could be used as a standardized antigen for the diagnosis of hydatidosis.

Messenger RNA extracted from Schistosoma mansoni larval forms codes for parasite antigens when translated in vitro.

Intact messenger RNA was extracted from three stages of development of the parasite Schistosoma mansoni, cercariae, 3 h (mechanically prepared) schistosomula and adults. Some of the in vitro translation products are precipitable with immune rat and human serum showing that these sera recognize protein determinants. Whereas the patterns of the total translation products show major proteins expressed at all three developmental stages, immunoprecipitation delineates patterns unique to each stage. Serum from chronically infected patients is likely to preferentially recognize proteins expressed by the adult parasite yet precipitates more products of translation of cercarial RNA than of adult RNA, suggesting that many of the same epitopes are already coded by cercarial messenger RNA. It seems, however, that antigens appear on lower molecular weight species in the adult. The experiments described here open the way to cloning the genes of antigens from S. mansoni.

Biochemical studies on the 30-40 kDa Schistosoma mansoni surface antigens.

Biochemical studies of the previously identified 30-40 kDa surface antigens of Schistosoma mansoni schistosomula confirmed that four molecules could be discriminated in this antigenic group. The antigens presented slightly different molecular mass in sodium dodecyl sulfate polyacrylamide gel electrophoresis (40, 38, 37 and 32 kDa) but were all found in isoelectric focusing at the same pH (6.2-6 and 7.5). The four antigens bound to concanavalin A and only the 32 kDa molecule had affinity for the Lens culinaris agglutinin. These results indicated almost similar biochemical characteristics of the 30-40 kDa antigens and partial hydrolysis of the 38 and 32 kDa antigens suggested that they were affected by a similar cleavage process. The possibility of a structural homology between these two components is discussed.

Identification of NF-AT-like transcription factor in Schistosoma mansoni: its possible involvement in the antiparasitic action of cyclosporin A.

Cyclosporin A (CsA) has been found to exert potent anti-parasite activity against a wide range of protozoan and helminth parasites. In schistosomes, evidence has been accumulated to propose that the drug damages parasites by mechanisms independent of its immunosuppressive properties. Moreover, the absence of correlation between anti-schistosomal properties and inhibition of peptidyl-prolyl cis-trans isomerase activity of cyclophilins (CsA receptors) for various drug analogs, argued against a direct implication of cyclophilins in the lethal effect of CsA. We describe, in S. mansoni, the existence of NF-AT-like transcription factors, a protein family already characterized by its sensitivity to CsA. The observation that CsA treatment of S. mansoni larvae inhibited the expression of the Sm28GST protein and the characterization of a functional NF-AT-like site in the gene encoding this protein, provide new insights in the understanding of the antischistosomal effect of CsA. Our results also support the hypothesis that the regulatory function of NF-AT-like proteins might be responsible for parasite development and survival in the host and open new perspectives in studies of helminth biology.

Structural homology of tropomyosins from the human trematode Schistosoma mansoni and its intermediate host Biomphalaria glabrata.

Molecular mimicry has been considered as a possible way for parasites to escape host immune responses. This work concerns the characterization of protein determinants shared by Schistosoma mansoni and its intermediate host Biomphalaria glabrata. Parasite (Sm39) and mollusc (Bg 39) cross-reactive proteins were identified and shown to induce in rabbit and mouse, antibodies specific for invertebrate determinants. Ultrastructural studies demonstrated that antibodies to Sm39 specifically bound to muscular structures of parasite and mollusc. Molecular cloning and sequencing indicated that Bg39 corresponded to a muscular isoform of tropomyosin. The mollusc sequence showed a 51-65% homology with seven different muscular tropomyosins from vertebrate and invertebrate species. The highest score of homology was observed with S. mansoni tropomyosin, suggesting that cross-reactive determinants could be specific for the trematode and its intermediate host. In miracidia, Sm39 epitopes were also shown to be contained in the vesicles present in epidermal ridges and cellular bodies. Such vesicles are involved in the formation of a protective tegument around sporocysts, suggesting a possible role of cross-reactive tropomyosins in miracidia and/or sporocyst-snail interactions.

Functional analysis of the Schistosoma mansoni 28 kDa glutathione S-transferase gene promoter: involvement of SMNF-Y transcription factor in multimeric complexes.

The ability of the 5' flanking region of the gene encoding the 28 kDa glutathione S-transferase of Schistosoma mansoni gene to promote transcription, was studied in different mammalian cell lines. Results of transient transfection assays showed a strong activity of the -277 to +1 nt region of the Sm28GST gene, comparable to that of well-studied promoters. Deletion analysis indicated that an AP-1 site and two closely located CCAAT (Y1 and Y2) boxes were the principal motifs responsible for the promoter activity. Binding of the NF-Y complex to Y1 and Y2, as well as to a third CCAAT box (Y3) close to the promoter TATA box, was compared in gel shift and super-shift experiments. All of the three Y boxes bound protein complexes from S. mansoni nuclear extracts that were shown to contain the A subunit of the schistosome NF-Y complex (SMNF-YA). Competition assays revealed a differential affinity of the Y1, Y2 and Y3 sequences for NF-Y. The Y1, Y2 and Y3 regions were also shown to activate transcription when included in an heterologous promoter and data obtained strongly suggested the involvement of SMNF-Y in multimeric complexes during this process.

Cloning of Schistosoma mansoni transcription factor NF-YA subunit: phylogenic conservation of the HAP-2 homology domain.

The CCAAT-binding factor NF-Y (CBF/CP1) is a heteromeric transcription factor involved in the regulation of a variety of eukaryotic genes. We identified NF-Y as the CCAAT activity binding to the promoter region of the gene coding for the 28-kDa glutathione S-transferase of the human parasite Schistosoma mansoni (Sm28GST). We isolated the NF-YA cDNA from S. mansoni (SmNF-YA): the complete 268 amino acid sequence harbors a region in its C-terminal part that shows homology with the subunit interaction and DNA-binding domains of the mammalian NF-YA; the N-terminal region has an amino acid composition reminiscent of the mammalian and echinoderm counterparts, rich in glutamine and hydrophobic residues, but shows no sequence similarity at the primary level. In vitro synthesized SMNF-YA is able to associate with mammalian NF-YB/C subunits in the absence of DNA and to bind to the Sm28GST CCAAT box. Surprisingly, a monoclonal antibody directed against the non-conserved Q-rich activation domain of mammalian NF-YA supershifts and immunoprecipitates SMNF-YA, strongly suggesting structure conservation in the activation domain between divergent species.

Characterization of immunoreactive TNF alpha molecules in the gastropod Biomphalaria glabrata.

In recent years, several studies have demonstrated the existence of cytokine-like molecules in invertebrates, therefore suggesting that cytokines may have been conserved throughout evolution. In this study, we investigated the presence of immunoreactive TNF alpha (ir TNF alpha) in the gastropod mollusc Biomphalaria glabrata, the specific intermediate host for the trematode Schistosoma mansoni. Immunocytochemical study indicated the presence of ir TNF alpha in mollusc hemocytes corresponding to a 53-kDa molecule detected by western blot analysis. Using ELISA tests, we demonstrated the presence of substantial amounts of ir TNF alpha in hemolymph, that were significantly decreased during the S. mansoni infection. The possible role of ir TNF alpha in the regulation of mollusc immune functions and in the host-parasite relationship is discussed.