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.

Use of individual polymorphism to validate potential functional markers: case of a candidate lectin (BgSel) differentially expressed in susceptible and resistant strains of Biomphalaria glabrata.

BgSel has been identified in Biomphalaria glabrata as a candidate adhesion molecule exhibiting both an Ig-like domain and a carbohydrate recognition domain showing similarities with the l domain of C-type lectins. As susceptibility or resistance of B. glabrata to the trematode Echinostoma caproni correlates with a differential hemocytic adhesive behavior, we investigated the expression of BgSel in snails selected for their susceptibility or resistance. Semi-quantitative RT-PCR analysis of BgSel expression revealed that (i) BgSel expression level was high in susceptible snails and almost undetectable in resistant snails, and that (ii) exposure to the parasite did not affect the expression level of BgSel in either strain. In order to validate this apparent association between low levels of BgSel expression and resistance, we used Real-Time PCR to characterize the relative expression of BgSel in individual snails segregating for susceptibility/resistance. Results established that differential expression of BgSel represents a functional strain marker, but is not a marker of resistance/susceptibility. It is suggested that this correlative approach may be a rapid and efficient alternative to complete functional analyses, and may facilitate the validation of candidate transcripts potentially identified through the numerous differential analyses of animal transcriptomes.

Schistosoma mansoni and Echinostoma caproni excretory-secretory products differentially affect gene expression in Biomphalaria glabrata embryonic cells.

Biomphalaria glabrata embryonic (Bge) cells have been shown to be a valuable in vitro cellular model for the study of snail host-parasite interactions. They both promote the growth and differentiation of various trematode species including Schistosoma mansoni, and Echinostoma caproni and share some morphological and functional features with circulating haemocytes. As an approach to investigate snail genes potentially regulated following exposure to trematode excretory-secretory (ES) products, we compared gene expression profiles of Bge cells exposed to saline solution, or saline solution containing ES products from S. mansoni or E. caproni, two trematode species parasitizing B. glabrata. Following differential display RT-PCR analysis we characterized 23 differentially displayed cDNAs and we focussed on the 5 cDNAs showing sequence similarity to known genes for expression validation. Using RT-PCR, we confirmed that ES products from S. mansoni and E. caproni differentially affect the expression levels of 4 out of the 5 transcripts. These partial transcripts corresponded to novel B. glabrata sequences, and showed significant sequence similarity to genes coding for (i) cytochrome C, (ii) methyl-binding proteins, (iii) glutamine synthetases, and (iv) protease inhibitors from the Kunitz family. The possible significance of these gene expression changes in host-parasite molecular interactions is discussed.

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.

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.

Biomphalaria glabrata embryonic cells express a protein with a domain homologous to the lectin domain of mammalian selectins.

We have cloned from Biomphalaria glabrata, the intermediate host of the helminth parasite Schistosoma mansoni, a 36-kDa apparent-molecular-mass molecule (BgSel) that shares sequence identity with selectins of the cell-adhesion-molecule superfamily. BgSel exhibited in its C-terminal part a putative C-type lectin domain similar to the selectin lectin domain. Using antibodies to the recombinant BgSel protein, we demonstrated the presence of BgSel in snail hemocytes as well as in the cell line derived from B. glabrata embryos (Bge). Anti-BgSel antibodies specifically recognized a 79-kDa component in Bge-cell-secreted products that was supposed to represent the native form of BgSel, as was confirmed after glycosidase treatment. Lectins are known to be implicated in recognition mechanisms participating in humoral and cellular immunity in molluscs. The possible role of BgSel in the interaction between sporocysts and Bge cells, particularly in the in vitro model of sporocyst development dependent on Bge cell factors, remains to be determined.

Conservation and divergence of NF-Y transcriptional activation function.

The CCAAT-binding protein NF-Y is involved in the regulation of a variety of eukaryotic genes and is formed in higher eukaryotes by three subunits NF-YA/B/C. We have characterized NF-Y of the trematode parasite Schistosoma mansoni and studied the structure and the function of the SMNF-YA subunit. In this work, we present the cloning and sequence analysis of the B subunit of the parasite factor. SMNF-YB contains the conserved HAP-3 homology domain but the remaining part of the protein was found to be highly divergent from all other species. We demonstrated by transfections of GAL4 fusion constructs, that mouse NF-YB does not contain activation domains while the C-terminal part of SMNF-YB has transcriptional activation potential. On the other hand, the N-terminal parts of SMNF-YA and mouse NF-YA were shown to mediate transactivation; the integrity of a large 160 amino acid glutamine-rich domain of NF-YA was required for this function and an adjacent serine- and threonine-rich domain was necessary for full activity in HepG2, but redundant in other cell types. Transactivation domains identified in SMNF-YB are also rich in serine and threonine residues. Our results indicate that serine/threonine-richsequences from helminth parasites potentiate trans-cription and that such structures have diverged during evolution within the same transcription factor.

Characterization of an intracellular receptor for activated protein kinase C (RACK) from the mollusc Biomphalaria glabrata, the intermediate host for Schistosoma mansoni.

A receptor for activated protein kinase C (RACK) was characterized from the mollusc Biomphalaria glabrata, the intermediate host for the human parasite Schistosoma mansoni. This protein was shown to possess structural and functional characteristics of other RACK proteins from various cells and organisms. Its ability to bind mammalian PKCs also confirmed the conservation of PKC and RACK interactive domains throughout evolution. Results of immunolocalization indicated the presence of Bg RACK in the cytoplasm of mollusc hemocytes and B. glabrata embryonic (Bge) cells with a more intense staining around the nucleus. These results are in agreement with the association of RACK proteins with cytoskeletal elements.

Snail control strategies for reduction of schistosomiasis transmission.

As intermediate hosts, molluscs play a major role in the transmission of schistosomes; they are the sites of an intense multiplication of parasites. Thus, snail control strategies are considered a priority for the reduction of schistosomiasis transmission. Here, Vinca Lardans and Colette Dissous review the efficacy of environmental management and the use of molluscicides and biological agents to control snail populations. They then describe the development of diagnostic tests, based on the detection of parasite antigens or specific parasite DNA sequences in snail tissues, to detect the early infection of snails. Finally, they discuss progress in studying the molecular basis of susceptibility and resistance phenotypes, and the possible application of the genetic manipulation of molluscs.

Deletion analysis of the Schistosoma mansoni 28-kDa glutathione S-transferase gene promoter in mammalian cells--importance of a proximal activator-protein-1 site.

The 1241-bp promoter region of the Schistosoma mansoni 28-kDa glutathione S-transferase gene (Sm28GST) was sequentially deleted and analyzed using the luciferase reporter gene system in different cell lines. The activator protein-1 (AP-1) site located at -231 seems to be responsible for the major part of the promoter activity. The 1241-bp Sm28GST promoter was not, in transient transfection experiments, activated by reagents generating reactive oxygen species, such as hydrogen peroxide (H2O2), 3-methylcholanthrene, and ter-methylhydroquinone, but was significantly stimulated by phorbol 12-myristate 13-acetate, a potent protein kinase C activator. The involvement of the -231 AP-1 site in phorbol 12-myristate 13-acetate stimulation was demonstrated. Moreover, evidence for in vitro and in vivo binding of the -231 AP-1 site to Jun/Fos dimers was obtained using mobility gel shift assays and co-transfection of embryonic F9 cells with Jun/Fos expression plasmids, respectively. The presence in S. mansoni nuclear extracts of components with affinity for the AP-1 site suggests conservation of this regulatory pathway in the parasite.

Nucleotide and deduced amino acid sequences of Biomphalaria glabrata actin cDNA.

The complete nucleotide sequence of Biomphalaria glabrata actin has been cloned by PCR amplification and screening of a cDNA library of Biomphalaria glabrata. The comparison of the deduced amino acid sequence with other actins suggests that a cytoskeletal form of the protein has been cloned.

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.

Expression of NF-Y nuclear factor in Schistosoma mansoni.

The A subunit of NF-Y nuclear factor from Schistosoma mansoni was expressed in E. coli fused to a histidine tag and purified by affinity chromatography using a Ni(2+)-Agarose matrix. Antibodies against the recombinant protein were prepared and used for Western blot and immunolocalization. The presence of SMNF-YA in all stages of the parasite life-cycle was determined by RT-PCR and Western blot analysis. The immunolocalization of SMNF-YA showed the presence of this factor in a parenchymal cell population of cercariae and adult worms and in embryos within eggs. The expression of SMNF-YA was demonstrated to decrease in maturating spermatozoites whereas an accumulation of this factor was observed in the nucleus from oocytes during their maturation processes.

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.

Schistosoma mansoni: the presence and potential use of opiate-like substances.

The present study demonstrates that morphine- and codeine-like molecules are present in Schistosoma mansoni following HPLC separation and identification with an appropriate commercially available antibody. Furthermore, the endogenous material, corresponding to morphine, mimics authentic morphine in its ability to induce immunocyte rounding and immobility, an action that is naloxone sensitive. The codeine-like material is not found at high concentrations compared to the morphine-like material, indicating, as in mammals and Mytilus edulis, the potential rapid conversion of codeine to morphine. Coincubation with human leukocytes increases the endogenous level of this material in adult worms, indicating the presence of a positive feedback loop. Last, EDTA, a chelator of divalent cations, has a strong stimulating effect in the synthesis of morphine-like material by the worm as noted by higher levels of this material in its presence. Taken together, the results suggest that this parasite may utilize this immune downregulating molecule in its effort to escape host immunosurveillance as well as in inhibiting an immune response directed against itself.

Schistosoma mansoni: interaction of nuclear extracts with the CCAAT-binding site revealed by the gel shift assay.

Analysis of the 5'-flanking region of the gene encoding the 28-kDa glutathione S-transferase of Schistosoma mansoni (Sm28GST) indicated the presence of motifs identical to AP-1 and CCAAT-family transcription factor recognition sequences. Gel retardation experiments showed that nuclear extracts from adult S. mansoni bound to an oligodeoxynucleotide containing at CCAAT box. A DNA fragment corresponding to the region of Sm28GST containing the CCAAT motif was demonstrated to interact with schistosome nuclear proteins. This binding was dependent on the presence of the CCAAT pentanucleotide motif. Nuclear factor Y (NF-Y) is a member of the CCAAT transcription factor family that has absolute requirement for the CCAAT sequence and that is highly conserved throughout evolution. The results of a PCR-based strategy aimed at cloning the NF-YA protein of S. mansoni are presented.