Characterization of the DNA-binding properties and the transactivation activity of Schistosoma mansoni nuclear receptor fushi tarazu-factor 1alpha (SmFTZ-F1alpha)

A FTZ-F1-related orphan nuclear receptor SmFTZ-F1alpha was previously identified from Schistosoma mansoni. The deduced SmFTZ-F1alpha protein contains a highly conserved DNA binding domain (DBD, C domain), a less conserved ligand binding domain (LBD, E domain) and three highly variable regions, the N-terminal A/B domain (108 aa), a large hinge region (D domain, 1027 aa) and an F domain (220 aa). Herein, we characterize the DNA binding properties and the transactivation activity of SmFTZ-F1alpha. In in vitro assays, SmFTZ-F1alpha bound as a monomer to a response element (FF1RE: TCAAGGTCA) recognized by mammalian steroidogenic factor 1 (SF-1), and to related sequences (p14: TTAAGGTCA and SmFF1a-2: CGAAGGTCA) derived from known schistosome gene promoters. Competition assays with p14 oligonucleotides containing a single mutation at each nucleotide position defined the optimum DNA sequence required for SmFTZ-F1alpha binding. The optimal consensus sequence for SmFTZ-F1alpha binding is TN(A/G)AGGTC(A/G) (N: any base). This sequence is similar but not identical to the SF-1 response element (SFRE) consensus sequence [(T/C)CAAGG(T/C)C(A/G)]. By performing yeast one-hybrid assays, the ability of SmFTZ-F1alpha to bind productively to a p14-derived 9-base pair sequence was demonstrated in vivo. The ability of the full-length SmFTZ-F1alpha to transactivate reporter gene expression was shown to be A/B domain-dependent in a yeast system. In addition, the hinge region contained an unexpected activation function (AF) domain, termed AF-3, while no transactivation activity was detected within the E/F domain. This AF-3 region (from aa 982 to aa 1110) revealed a strong autonomous transactivation activity, which was masked when it was present in the full-length SmFTZ-F1alpha. Taken together, our results suggest that SmFTZ-F1alpha possesses the characteristic DNA binding specificity of FTZ-F1 subfamily members and the capacity to transactivate a reporter gene.     

Isolation and characterization of Schistosoma mansoni constitutive androstane receptor

Full length cDNA clones encoding a Schistosoma mansoni homologue of vertebrate CAR/PXR/VDR group nuclear receptor, termed SmCAR were isolated from screening a S. mansoni adult worm cDNA library. SmCAR is a 702 amino acid protein which retains a typical domain organization of nuclear receptor superfamily members. A homology search demonstrated that SmCAR exhibits the highest homology with mouse constitutive androstane receptor (CAR). Like its orthologues from invertebrates, SmCAR contains a P box sequence of ESCKA in the DNA binding domain. The P box is important in determining the DNA binding specificity for nuclear receptors. SmCAR mRNA is expressed in every stage of S. mansoni life cycle with an elevated expression level in egg and cercaria stages. Two forms (78 and 81 kDa) of SmCAR protein were detected in schistosome worm extract by Western blot analysis. SmCAR protein was demonstrated to be widely distributed in adult worms by immunolocalization studies, being found in the subtegument in both male and female worms and in the ovaries, vitellaria and eggs in female worms. In vitro DNA binding assays demonstrated that SmCAR binds to the hsp27 ecdysone response element (EcRE) as well as schistosome p14 gene upstream region. The AGTGCA half site is essential for binding of SmCAR to the p14 gene upstream region. Therefore, AGTGCA probably serves as a high affinity binding half site for ESCKA containing nuclear receptors.     

Schistosoma mansoni tetraspanning orphan receptor (SmTOR): a new vaccine candidate against schistosomiasis

One approach to fight against schistosomiasis is to develop an efficient vaccine. Schistosoma mansoni tetraspanning orphan receptor (SmTOR) might be a vaccine candidate, as it is a tegument membrane protein expressed most highly in cercariae. In this study we characterized the recombinant first extracellular domain of SmTOR (rSmTORed1) as having the expected property to bind C2 of complement similarly to a smaller peptide of the same domain, and to produce specific and high-titre antibodies in BALB/c mice immunized using complete Freund''s adjuvant/incomplete Freund''s adjuvant (CFA/IFA). Immunization was protective against parasite infection, as demonstrated by a significant decrease in worm burden in immunized BALB/c mice versus the control groups over two independent trials [64 and 45% reduction for mean adult worm burden in immunized versus phosphate-bufferd saline (PBS) injected mice]. Interestingly, infection by itself did not lead to the generation of anti-rSmTORed1 antibodies, corresponding to the low frequency of specific anti-rSmTORed1 antibodies detected in the sera of patients infected with S. mansoni (2/20; 10%). These data suggest that, as opposed to the natural infection during which SmTOR induces antibodies only rarely, immunization with its smaller first extracellular domain might be more efficient.     

Inhibition of lymphocyte proliferation by factor(s) produced by Schistosoma mansoni

Normal spleen cells of CBA mice or Fischer rats were cultured with mitogens or allogeneic cells, together with various substances of Schistosoma mansoni origin, and thymidine uptake was measured. The proliferation (DNA synthesis) of normal lymphocytes was inhibited by the incubation product of the parasite as well as by cell-free supernatant of schistosome culture. Inhibition was obtained only when active materials were added at the beginning of the culture. Both T and B cell proliferation were inhibited. The inhibitory activity found in cell-free supernatant suggested the release by the parasite of some factor(s) interfering with lymphocyte proliferation. Moreover, serum from rats infected by S. mansoni inhibited lymphocyte proliferation also. The inhibitor(s) appeared heat resistant, dialyzable and of low molecular weight (500–1000). Incubation of normal spleen cells with S. mansoni inhibitor(s) did not enhance the release of nonspecific suppressor cell factor. The inhibition product(s) released by the parasite could explain part of the immunosuppression status found in schistosomiasis.     

Cytoarchitecture and the patterning of fushi tarazu expression in the Drosophila blastoderm

In the Drosophila embryo at the blastoderm stage, the segmentation gene fushi tarazu (ftz) is expressed in a seven-banded pattern. The generation of this pattern, like many other segmentation gene expression patterns, coincides with the formation of cell membranes around the blastoderm nuclei. To test the role of cellularization in resolving the banded ftz pattern, we used cytoskeletal inhibitors (colcemid and cytochalasin B) to block cellularization. We found that banded ftz RNA and protein patterns can form without cellular structure. We also tested the importance of rapid degradation of the ftz RNA, using cycloheximide to block degradation. RNA degradation is essential to maintain the banded ftz pattern in a syncytium, but is not required to maintain the pattern in a cellularized embryo. A latticework of cytoskeletal microtubules that forms during cellularization appears to be a key component in localizing the ftz mRNA. We conclude that RNA degradation and cellular structure normally work together to localize ftz RNA to its sites of synthesis.     

Effect of a novel benzimidazole derivative in experimental Schistosoma mansoni infection

Currently, praziquantel is the only drug of choice for treatment of schistosomiasis. Reports of praziquantel resistance raise concerns about future control of the disease. Therefore, the search for new schistosomicidal drugs is eminent. In this study, the effect of a novel benzimidazole-derived compound (compound BTP-Iso) was assessed in mice harboring adult Schistosoma mansoni (Egyptian strain). Mice were treated 42 days p.i. with compound BTP-Iso using two treatment regimens (200 or 300 mg/kg). In both regimens, there were significant reductions in the number of recovered S. mansoni worms especially females and in immature ova, in addition to a significant reduction in the number and size of hepatic granulomata. A dose of 300 mg/kg resulted in a significant decrease in intestinal and hepatic tissue egg loads. Effect on schistosomes was confirmed by scanning electron microscopy, where adult worms recovered from mice treated with 200 mg/kg of compound BTP-Iso revealed tegumental alternations, characterised by swelling of tegumental ridges, bleb formation, and mild erosion in male worms; however in females, there were extensive erosion and destruction of the tegumental surface. These promising results may encourage future use of compound BTP-Iso in the treatment of schistosomiasis. However, more research is needed to detect the effect of compound BTP-Iso on early developmental stages of S. mansoni and on other species of human schistosomes.     

Identification by monoclonal antibody of a major (28 kDa) surface membrane antigen of Schistosoma mansoni

Monoclonal antibody M.1 was generated from mice immunized with membrane enriched extracts of mechanically transformed schistosomula. M.1 bound to the surface membranes of cercariae and young (0-24 h post-transformation) schistosomula but did not bind to older schistosomula or cultured worms. M.1 immunoprecipitated an antigen of approximate molecular weight 27-28 kDa from schistosomula. Experiments using metabolic labeling showed that the antigen was actively synthesized by developing schistosomula. Further M.1 immunoprecipitated a similar 27-28 kDa antigen from membrane-enriched extracts of miracidia, lung and adult worms as well as from schistosomula.     

Expression, modification, and localization of the fushi tarazu protein in Drosophila embryos

The fushi tarazu (ftz) protein of Drosophila is required during embryogenesis for the process of body segmentation. To study the biochemical properties of the ftz protein, ftz cDNA was expressed in Escherichia coli and the protein was purified to homogeneity. Polyclonal antibodies raised against the purified protein were used to localize and quantitate the protein during embryogenesis. Three temporally and spatially distinct phases of expression were observed, which include a previously undetected period later in embryogenesis. During this last phase, the protein is localized predominantly in the developing hindgut. Analysis of embryonic ftz protein on Western blots permitted us to approximate the number of protein molecules per nucleus. During the blastoderm phase of development, when ftz protein is most abundant, we estimate that there are approximately 20,000 molecules of protein per ftz-expressing nucleus. The embryonic ftz protein migrates more slowly on SDS-polyacrylamide gels than protein made either in E. coli or in a reticulocyte lysate system in vitro, indicating that it is modified in the embryo. To facilitate characterization of ftz protein made in embryos, an ftz overexpression system functional in Drosophila was developed. When fused to an hsp70 heat shock promoter and introduced into the germ line by P-element-mediated transformation, ftz could be overexpressed at all stages of development by heat shock. This protein is localized in the nucleus comigrates on SDS-polyacrylamide gels with endogenous ftz protein. Two-dimensional gel electrophoresis followed by Western blotting resolves the overexpressed protein into a series of isoforms that differ in charge and electrophoretic mobility. Post-translational modification may influence the biochemical properties and functions of the ftz protein during embryogenesis.