Possible eggshell protein gene from Schistosoma mansoni

We have identified and sequenced a cDNA clone of a mRNA found only in mature female schistosomes. This mRNA is not detectably synthesized by female worms from single sex infections (unisexual females), by males or by the developing miracidia in the eggs. The clone hybridises to a highly abundant polyadenylated mRNA of approximately 1500 nucleotides. The nucleotide sequence of the clone predicts a polypeptide comprising two repetitive regions. A pentapeptide repeat with the consensus sequence Gly-Tyr-Asp-Lys-Tyr, and a region rich in histidine residues. Hybrid selected mRNA translated in vitro with [3H]tyrosine as labelled amino acid yields a polypeptide of 48 kDa (p48) that corresponds to the major [3H]tyrosine labelled translation product of female worm total mRNA. p48 does not label with [35S]methionine and is absent from the translation products of male and unisexual female mRNAs. The amino acid sequence of p48 has significant homologies to silk moth chorion proteins and we suggest that it is one of the major components of the schistosome eggshell probably accounting for the high level of [3H]tyrosine incorporation into the vitellaria of Schistosoma mansoni. The tyrosine content of the polypeptide suggests that it may play a role in phenol oxidase mediated cross-linking of the schistosome eggshell and in support of this we find that mushroom phenol oxidase will cause the specific cross-linking of p48 in in vitro translation products.     

Bacteriophage T3 DNA binding protein interaction with DNA

Single-stranded DNA binding protein (DBP), coded by T3 phage, is essential for concatemer formation (H. Fujisawa, M. Yamagishi, H. Matsuo-Kato, and T. Minagawa, 1980, Virology, 105, 480–489.) T3 DBP was purified to homogeneity and found to exist as a dimer. The structure of the T3 DBP-fd DNA complex appears as a condensed and beaded ring structure by electron microscopy. As judged by sucrose gradient centrifugation, DBP binds to single-stranded, but not to double-stranded DNA; at saturation, one protein monomer is bound per every 100 nucleotides. The T3 DBP has a strong ability to catalyze renaturation of DNA even in the absence of Mg. The results suggest that the T3 DBP acts in concatemer formation by stimulating pairing of the single-stranded redundant termini of T3 DNA.     

Acetylcholinesterase from Schistosoma mansoni: immunological characterization

The enzyme acetylcholinesterase (AChE) is present in the trematode Schistosoma mansoni, which infects humans and causes a severe disease called schistosomiasis or Bilharzia. We have purified this enzyme and raised polyclonal antibodies against it. The specificity of these antibodies against the schistosome enzyme was demonstrated by their capacity to precipitate exclusively AChE activity from cercariae extract and to recognize the 8S molecular form of the parasite's AChE. On the other hand, they did not cross-react at all with AChE from human erythrocytes. By employing immunogold electron microscopy, AChE was located on the surface, in the membranal bodies of the tegument and in the muscles of schistosomula. The antibodies raised against the purified AChE of S. mansoni are of protective value, as they led to efficient complement-mediated killing of schistosomula in vitro. It was also demonstrated that antibodies specific towards S. mansoni AChE are present in the sera of mice and of human patients infected with the parasite, suggesting that this enzyme partakes in the immune response towards the parasite during infection. These cumulative data, particularly the schistosomicidal activity of the antibodies and their lack of cross-reactivity with human AChE, are of significance in the consideration of the S. mansoni AChE for vaccination purposes.     

Organization of the ribosomal RNA genes in Schistosoma mansoni

The organization of the rRNA genes of Schistosoma mansoni has been determined by Southern blot analysis of genomic DNA digested with restriction enzymes, by isolation of the entire repeat on a single fragment of about 11 kilobase pairs from a genomic DNA library constructed in bacteriophage lambda and by characterization of three cloned EcoRI fragments which span the entire repeat. The segments encoding both the large and small rRNA subunits have been identified using specific cloned yeast rDNA fragments as probes and EcoRI, HindIII and BamHI restriction enzyme maps of the rRNA genes were constructed. The ends of the RNAs have been precisely mapped on the genomic DNA by S1 protection experiments. Our data indicate that the rRNA genes are present as a tight cluster. The total length of the rDNA repeat is about 10 kilobase pairs. There appears to be no variation in the size of transcribed and non-transcribed spacer DNA. At the RNA level we have characterized and mapped a small gap in the 28S RNA molecule. The interruption causes the RNA to dissociate into two equal sized fragments when analyzed under denaturing conditions.     

Transgene expression in Schistosoma mansoni: introduction of RNA into schistosomula by electroporation

Despite their significance in human and veterinary medicine, and the ability to maintain the parasites in the mouse, relatively little functional detail is available regarding the biology of schistosomes. This deficit is due largely to the lack of well-developed molecular tools for manipulating gene expression in these parasites. Here, we describe an electroporation protocol that provides a routine approach for efficiently introducing nucleic acids into schistosomes. Using luciferase-encoding RNA for electroporation, and luciferase activity as a read-out, we established 400 microg/ml of RNA, and a 20 ms pulse at 125 V using a square wave electroporation generator to be optimal for electroporating schistosomes. Under these conditions schistosomula from 1 hr to 18 hr old could be successfully electroporated, the majority of parasites within a population expressed the introduced RNA, and acute mortality was negligible. Electroporation, as described here, makes possible experimental studies using transiently expressed constitutively active and/or dominant negative mutant proteins, etc. In addition, the finding that electroporation can be used to introduce RNA into schistosomula raises the possibility of using this approach to introduce either DNA constructs or dsRNA sequences, both of which might be expected to have longer-term, ideally inheritable, effects.     

Surfactant protein D binding to terminal alpha1-3-linked fucose residues and to Schistosoma mansoni

Pulmonary surfactant protein (SP)-D is an important component of the innate immune system of the lung, which is thought to function by binding to specific carbohydrates on the surface of viruses and unicellular pathogens. SP-D has been shown to have a relatively high affinity for the monosaccharides mannose, glucose, and fucose. However, there is limited information on SP-D binding to complex carbohydrate structures, and binding of SP-D to fucose in the context of an oligosaccharide has not yet been investigated. In this study, we used surface plasmon resonance spectroscopy to examine the potential of SP-D to bind to various synthetic fucosylated oligosaccharides, and identified Fucalpha1-3GalNAc and Fucalpha1-3GlcNAc elements as strong ligands. These types of fucosylated glycoconjugates are presented at the surface of Schistosoma mansoni, a parasitic worm that, during development, transiently resides in the lung. In line with the findings by surface plasmon resonance, we found that SP-D can bind to larval stages of S. mansoni, demonstrating for the first time that SP-D interacts with multicellular lung pathogens.     

Predicted structure of a major Schistosoma mansoni eggshell protein

The complete sequence for a major Schistosome mansoni eggshell protein gene has been determined from a genomic DNA fragment. The use of an open reading frame encoding a glycine-rich polypeptide was confirmed by in vitro translation of schistosome mRNA in the presence of [3H]glycine and comparison with the amino acid composition of purified, schistosome eggshells. Apart from the extraordinary abundance of glycine and tyrosine which are evenly distributed throughout the polypeptide chain, the most striking features of the deduced amino acid sequence are the presence of five well-conserved tandem repeats of 16-18 residues in the N-terminal region and the asymmetrical distribution of charged residues. Acidic residues (Asp) are confined to the N-terminal region, while basic residues (Lys, His), with the exception of a single histidine, are found in the C-terminal region. A model structure composed of short anti-parallel beta-strands is proposed, in which glycines and residues with small side chains lie within the strands and tyrosines and cysteines are arranged at the bends, where they would be available for cross-linking. Four such strands form one of the tandem repeats which are predicted in turn to form a stack of five closely packed beta-sheets, each of three strands and linked by the more variable fourth strand. The C-terminal region may form a similar but less compact structure. The ordered structure demonstrated by birefringence studies of the schistosome eggshell [Kusel, J. (1970) Parasitology 60, 79-88] could be formed by packing of the polypeptides such that the N-terminal domain contributes counter ions or cross-links to the C-terminal domain of adjacent molecules.     

The interaction of human serum with the surface membrane of schistosomula of Schistosoma mansoni

After contact with human serum, a series of proteins become exposed on the surface membranes of schistosomula of Schistosoma mansoni as revealed by radioiodination of the intact parasites. Among the proteins, a doublet (Mr 45 000) is particularly prominent. These doublet proteins, which are believed to be parasite-derived, become apparent after a very short time of incubation with human serum (10 min or less) and are expressed on the surface membranes after contact with a high molecular weight component of human serum (Mr greater than 80 000). Pretreatment of the parasites with 1.25 mM colchicine or fixation with 2% glutaraldehyde does not prevent the serum-induced expression of the doublet proteins. Extraction of the parasites with chloroform:methanol 2:1 (v/v), however, blocks the human serum effect. Affinity chromatography using immobilized low density lipoproteins (LDL) from human serum shows a tight binding between the 125I-labelled 45 kDa doublet to the LDL. The possible role of the 45 kDa doublet as a receptor for LDL is discussed.     

Lectin binding to glycoproteins in the surface membrane of Schistosoma mansoni

A number of lectins were assessed for their ability to bind to glycoproteins in the surface membrane of Schistosoma mansoni. The membrane polypeptides were separated by SDS-PAGE and the glycoproteins visualised by incubating the gel with radio-iodinated lectin followed by autoradiography. Most of the individual lectins bound to a variety of glycoproteins but peanut agglutinin and Dolichos biflorus agglutinin bound preferentially to a single glycoprotein of apparent molecular weight 170 000. This glycoprotein was subsequently shown to be exposed at the surface of the parasite and localised at the tubercles.     

Simultaneous priming with DNA encoding Sm-p80 and boosting with Sm-p80 protein confers protection against challenge infection with Schistosoma mansoni in mice

Prophylactic efficacy of Sm-p80 was tested in the mouse model using DNA priming and boosting with protein approach. However, the novelty of the approach utilized in this study is that both the DNA priming and protein boosting was performed on a single day and no further vaccine inoculations were given to mice; the animals were challenged 1 month after the initial vaccine administration. Using this approach, significant reduction in worm burden (33 to 57 %) and marked decrease in egg retention in tissues (34 to 66 %) was observed. Robust antibody titers and upregulation of cytokines (IL-1α/β, IL-12α, and IFN-γ) appears to correlate with the protection. This approach of administering vaccine on a single day could be greatly helpful in the field setting because it will eliminate the compliance issues that may arise with multiple boosters that may be required for optimal efficacy for some vaccines.     

Scanning electron microscopy of the human low-density lipoprotein interaction with the tegument of Schistosoma mansoni

The interaction between host molecules and Schistosoma mansoni has been regarded as a key feature for parasite survival. In this work, scanning electron microscopy was used to study the interaction of human low-density lipoprotein (LDL) with the tegument of the adult worm of S. mansoni. Worms were incubated in RPMI 1640 containing 10% of LPDS and 40 μg LDL/mL during 30, 60, and 120 min. Control worms were processed in the same way, without LDL. After the incubations, the samples were fixed and processed to scanning electron microscopy. The results demonstrated interaction of the LDL particles with the male parasite tegument. Male and female worms incubated without LDL from 0 (control) to 120 min did not show alterations in the tegument. It was observed a larger number of LDL particles on the dorsal region of male adult worm than others regions (anterior, posterior and gynecophoral canal). The female tegument did not show adherence of LDL. Aggregates on the tegument of the male worm were in greater number and size in the incubation times of 30 and 60 min than 120 min. The comparison between 30 and 120 min of incubation showed that the particles’ size diminished from 2,650–860 nm to 634–363 nm, respectively. Such reduction can be due to the capture and the use of the lipids by the worm. Therefore, the internalization of lipids from LDL by the male worms seems to be a mechanism independent of endocytosis. Differences between males and females suggest lipid transference from male to female through gynecophoral canal.     

A cloned DNA probe identifies the sex of Schistosoma mansoni cercariae

This report describes a method for the identification of the sex of Schistosoma mansoni cercariae using a cloned DNA probe which is female-specific. The probe is a 339 bp repeat; the sequence is presented. Cercariae from nine mono-miracidially infected snails were used in a double blind study. Mice were infected and the sex of adult worms observed. DNA was isolated from cercariae, digested with EcoRI, subjected to agarose gel electrophoresis, transferred to a matrix and hybridized with the cloned female-specific DNA probe. In all nine cases the sex of the cercariae as determined by DNA analysis agreed with the sex of the adult parasites.     

Cloning and expression of a gene encoding Sm16, an anti-inflammatory protein from Schistosoma mansoni

The gene encoding Sm16, an anti-inflammatory, immunomodulatory protein present abundantly in secretions of the infective stages of Schistosoma mansoni was cloned and partially characterized. A data base analysis showed sequence homology to an earlier reported schistosomular stathmin-like gene sequences reported in dbEST and Genbank. The putative gene coding for Sm16 is of 500 bp with an open reading frame of 117 aa that included an N-terminal signal peptide sequence of 18 aa. There are three potential sites for phosphorylation (two serine and one tyrosine residue) but no glycosylation sites in the sequence. The coding region of Sm16 was amplified from cercarial cDNA, cloned and expressed in bacterial and insect expression systems. The purified recombinant protein showed strong immunoreactivity with a polyclonal rabbit anti-Sm16 antibody raised against the native anti-inflammatory protein Sm16. Contrary to earlier report, this gene appears to be not stage-specific. Metabolic labeling studies suggested that Sm16 is phosphorylated and is synthesized by both cercariae and schistosomula of S. mansoni. Sequence homology with human stathmin, a cell cycle regulatory phospho protein, was 30%. However, when probed with specific antibodies, no cross reactivity was observed between Sm16 and human stathmin.