Human Blood Fluke Schistosoma Japonicum Research Articles

A novel coagulation inhibitor from Schistosoma japonicum.

Little is known about the molecular mechanisms whereby the human blood fluke Schistosoma japonicum is able to survive in the host venous blood system. Protease inhibitors are likely released by the parasite enabling it to avoid attack by host proteolytic enzymes and coagulation factors. Interrogation of the S. japonicum genomic sequence identified a gene, SjKI-1, homologous to that encoding a single domain Kunitz protein (Sjp_0020270) which we expressed in recombinant form in Escherichia coli and purified. SjKI-1 is highly transcribed in adult worms and eggs but its expression was very low in cercariae and schistosomula. In situ immunolocalization with anti-SjKI-1 rabbit antibodies showed the protein was present in eggs trapped in the infected mouse intestinal wall. In functional assays, SjKI-1 inhibited trypsin in the picomolar range and chymotrypsin, neutrophil elastase, FXa and plasma kallikrein in the nanomolar range. Furthermore, SjKI-1, at a concentration of 7·5 µ m, prolonged 2-fold activated partial thromboplastin time of human blood coagulation. We also demonstrate that SjKI-1 has the ability to bind Ca(++). We present, therefore, characterization of the first Kunitz protein from S. japonicum which we show has an anti-coagulant properties. In addition, its inhibition of neutrophil elastase indicates SjKI-1 have an anti-inflammatory role. Having anti-thrombotic properties, SjKI-1 may point the way towards novel treatment for hemostatic disorders.

An integrated immunoproteomics and bioinformatics approach for the analysis of Schistosoma japonicum tegument proteins

Schistosomiasis remains one of the major neglected tropical diseases (NTDs) causing morbidity of humans residing in the tropical countries. Much effort has been devoted to the development of vaccines, since it is recognized that vaccines can be served as an important supplementary component alongside chemotherapy for the future control and elimination of schistosomiasis. To accelerate digging new potential target antigens, it is essential to extensively and intensively search immunogenic proteins in a high-throughput manner using proteomics-microarray techniques. In the present study, an integrated immunoproteomics and bioinformatics approach was used to profile the tegument of the human blood fluke Schistosoma japonicum. Results showed that the full-length tegument proteins were high-throughput cloned and expressed and screened with sera from S. japonicum-infected patients and normal subjects using protein arrays. Here, thirty highly immunoreactive tegument proteins and 10 antigens with an AUC value greater than 0.90 were identified at first time. In particularly, STIP1, the highest immunoreactive tegument protein has been shown good antigenicity and immunogenicity, and thus makes it to be a potential target for designing anti-parasite drug or vaccine. The schistosome tegument plays a crucial role in host-parasite interactions and there are several tegument proteins that proved to be potential vaccine candidates. However, vaccines are not yet available, thus it is important to identify new target antigens from schistosome tegument proteome. Herein, we demonstrate that the S. japonicum tegument proteins were analyzed by an integrated immunoproteomics and bioinformatics approach. We found that thirty highly immunoreactive tegument proteins and 10 antigens with an AUC value greater than 0.90 were identified for the first time. In particularly, we found 17 of tegument immunoproteomes having putative interaction networks with other proteins of S. japonicum. The results will provide clues of potential target molecules for vaccine development and biomarkers for diagnostics of schistosomiasis.

Approaches to genotyping individual miracidia of Schistosoma japonicum

Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where human infections have reemerged, and to characterize infrapopulations in individual hosts. The life stage that interests us as a target for collecting genotypic data is the miracidium, a very small larval stage that consequently yields very little DNA for analysis. Here, we report the successful development of a multiplex format permitting genotyping of 17 microsatellite loci in four sequential multiplex reactions using a single miracidium held on a Whatman Classic FTA indicating card. This approach was successful after short storage periods, but after long storage (>4years), considerable difficulty was encountered in multiplex genotyping, necessitating the use of whole genome amplification (WGA) methods. WGA applied to cards stored for long periods of time resulted in sufficient DNA for accurate and repeatable genotyping. Trials and tests of these methods, as well as application to some field-collected samples, are reported, along with the discussion of the potential insights to be gained from such techniques. These include recognition of sibships among miracidia from a single host, and inference of the minimum number of worm pairs that might be present in a host.

Prediction of Major Histocompatibility Complex Binding Peptides and Epitopes from Fatty-Acid-Binding Protein of the Human Blood Fluke Schistosoma Japonicum

Schistosoma japonicum are blood flukes of humans that cause chronic, highly debilitating diseases involving extensive liver damage. In the present study, fatty-acid-binding protein of the human blood fluke Schistosoma japonicum is being used to find out highly suitable MHC binding peptides and epitopes. MHC molecules are cell surface proteins, which take active part in host immune reactions and involvement of MHC class in response to almost all antigens and it give effects on specific sites. Predicted MHC binding regions acts like red flags for antigen specific and generate immune response against the parent antigen. So a small fragment of antigen can induce immune response against whole antigen. This theme is implemented in designing subunit and synthetic peptide vaccines. Fragments identified through this approach tend to be high efficiency binders, in which larger percentage of their atoms are directly involved in binding as compared to larger molecules. Binding ability prediction of peptides to major histocompatibility complex (MHC) class I & II molecules is important in vaccine development from fatty-acid-binding protein of the human blood fluke Schistosoma japonicum.

Identification and characterization of six novel tetraspanins from Schistosoma japonicum

BackgroundTetraspanins (TSPs), also known as members of the trans-membrane 4 super-family (TM4SF), comprise an assemblage of surface antigens reported in eukaryotic organisms. In the work presented here, six novel TSP proteins from the human blood fluke Schistosoma japonicum (S. japonicum) were produced and analyzed through a combination of bioinformatics and experimental approaches.ResultsSix novel TSP proteins of Schistosoma japonicum (designated as Sj-TSP-#1~6) contained four trans-membrane regions and one large extracellular loop (LEL) with a conserved CCG motif. Size of the proteins varied from 227 to 291 amino acid residues. All the six proteins were produced in E.coli and immune sera to each protein were prepared. Analysis of transcription profiles of the proteins by RT-PCR showed that Sj-TSP-#4 was transcribed only in the egg stage while transcription of the Sj-TSP-#2 was detected in female worms but not in males. The similar results were obtained by Western blot. Immunolocalization of the TSP proteins by immunofluorescence assay showed that the Sj-TSP-#2, Sj-TSP-#5 and Sj-TSP-#6 were located in the tegument of worms.ConclusionsThis study provided six novel TSP members of S. japonicum including their sequences and recombinant proteins. Availability of the novel proteins and information on their expression profile and location provided a basis for further investigation of the TSP proteins for their biological functions and as vaccine candidates.

Effects of low temperature on the schistosome-transmitting snail Oncomelania hupensis and the implications of global climate change

The impact of climate change on schistosomiasis transmission has attracted considerable attention in recent years. As the inter-mediate hosts for schistosomes, snails play an obligatory role in schistosomiasis transmission. In order to determine the impact of low temperature on snail survival, we studied crystallization temperature (Tc) and lower lethal temperature (LLT) of the snail Oncomelania hupensis, the intermediate host of human blood fluke Schistosoma japonicum, under different physiological conditions. The mean Tc of ‘dry’ snails was -12.79 ± 1.17°C, which is significantly lower than that of ‘wet’ snails (-5.36 ± 2.11°C). Survival of ‘dry’ snails was high (92% after 24 h) when the temperature was higher than -7°C, then decreased rapidly between -7°C and -12°C (92% to 0 % after 24h). LT50 between 0.25 h and 24 h exposure was between -10.8 °C and -9.4°C. Our data suggest that O. hupensis out of water could be avoiding freezing. As winter temperatures continue to rise due to global warming, O. hupensis may increase its range, thereby spreading schistosomiasis to the northern part of China.

Oncomelania hupensis (Gastropoda: Rissooidea), Intermediate Host Of Schistosoma japonicum In China: Genetics and Molecular Phylogeny Based On Amplified Fragment Length Polymorphisms

Schistosomiasis japonica is one of the most serious parasitic diseases in China. Subspecies of the pomatiopsid snail species Oncomelania hupensis transmit the human blood fluke Schistosoma japonicum, the parasite causing the disease. In at least one study involving the subspecies Oncomelania hupensis robertsoni, there was no clear phylogenetic concordance between the mitochondrial and nuclear data, and it was strongly recommended that future studies incorporate more data from nuclear loci in order to better understand phylogeography and host-parasite coevolution (Wilke et al., 2006). In this paper, we explore genetic diversity based AFLP data involving 25 populations throughout mainland China. AFLP is a DNA fingerprinting technique that detects the polymorphism of the whole genome without prior knowledge of the nucleotide sequence, with the high degree of reproducibility and small amount of template DNA needed.Our AFLP data show that: (1) the genetic diversity within an O. hupensis population increases gradually with snail sample size, and when the sample size is more than 30 individuals, the genetic variation within one O. hupensis population trends to stabilization. (2) There is high intra-population genetic variation on the mainland of China, and these intra-population genetic variations from different areas differ significantly. (3) The considerable genetic differentiation occurs throughout China. (4) The genetic variation among populations of O. h. hupensis is higher than that of O. h. robertsoni. (5) The patterns of genetic differentiation are basically consistent with geographical distribution of snail populations. Our results are similar to the results of allozymes and COI gene sequences, excluding a snail population from Guangxi Province (Gx-1), indicating that there are three distinct subspecies in mainland China, namely O. h. hupensis, O. h. robertsoni, and O. h. tangi. However, our results do not support that the snail population from Guangxi Province belongs to O. h. hupensis.

The cellular distribution and stage-specific expression of two dynein light chains from the human blood fluke Schistosoma japonicum

Schistosomes are pathogenic helminth parasites of human portal veins. Their body wall is a highly active syncytial tegument involved in an array of host interactions. The cytoskeletal organization and dynamics of this syncytium are poorly understood, but predominant motor components are the LC8 class of cytoplasmic dynein light chains (DLC). Four LC8 members occur in schistosomes, two of which are expressed in the tegument. Here, we describe the cytoplasmic distribution, stage-specific expression and cellular location of two diverse LC8 molecules of Schistosoma japonicum. SjDLC1 was detected in surface-membrane specific extracts of adult worms and was shown by quantitative immuno-electron microscopy to predominate along heptalaminate membranes of the worm surface. SjDLC3 also occurs in the tegument, but was shown to be present in basal layers of the tegument and did not preferentially co-localize with particular membrane components. SjDLC3 was also detected in the gastrodermis. SjDLC1 is expressed only in mammalian–parasitic stages, whereas SjDLC3 is expressed throughout the life-cycle. The data suggest that SjDLC1 is preferentially located to the host-interactive distal parasite membrane, and plays a role in surface membrane dynamics, while SjDLC3 is a ubiquitous motor component of schistosome epithelia of all stages.

Sj-FABPc fatty-acid-binding protein of the human blood fluke Schistosoma japonicum: structural and functional characterization and unusual solvent exposure of a portal-proximal tryptophan residue.

Sj-FABPc of the blood fluke of humans, Schistosoma japonicum, is a member of the FABP/P2/CRBP/CRABP family of beta-barrel cytosolic fatty-acid-binding and retinoid-binding proteins. Sj-FABPc has at least eight different variants encoded by a single-copy polymorphic gene. In fluorescence-based assays, recombinant Sj-FABPc was found to bind 11-(dansylamino)undecanoic acid (DAUDA), inducing a shift in peak fluorescence emission from 543 to 493 nm. A similar spectral change was observed in dansyl-amino-octanoic acid (in which the dansyl fluorophore is attached at the alpha-carbon rather than the omega-carbon of DAUDA), indicating that the ligand enters entirely into the binding site. Sj-FABPc also bound the naturally fluorescent cis-parinaric acid, as well as oleic acid and arachidonic acid, by competition, but not all-trans-retinol. Dissociation constants were, for cis-parinaric acid, K(d)=2.5+/-0.1 microM (mean+/-S.E.M.) and an apparent stoichiometry consistent with one binding site per molecule of Sj-FABPc and, for oleic acid, K(i) approximately 80 nM. A deletion mutant from which alpha-II was absent failed to bind ligand. Sj-FABPc modelled well to known structures of the protein family; an unusually solvent-exposed Trp side chain was evident adjacent to the presumptive portal through which ligand is thought to enter and leave. Intrinsic fluorescence analyses of Sj-FABPc and of the deletion mutant (from which Trp-27 is absent) confirmed the unusual disposition of this side chain. Virtually all members of the FABP/P2/CRBP/CRABP protein family have prominent hydrophobic side chains in this position, with the exception of liver FABP and ileal FABP, which instead have charged side chains. Liver FABP is known to be distinct from other members of the protein family in that it does not seem to contact membranes to collect and deposit its ligand. It is therefore postulated that the unusually positioned apolar side chains in Sj-FABPc and others in the family are important in interactions with membranes or other cellular components.

Sj-FABPc fatty-acid-binding protein of the human blood fluke Schistosoma japonicum: structural and functional characterization and unusual solvent exposure of a portal-proximal tryptophan residue

Sj-FABPc of the blood fluke of humans, Schistosoma japonicum, is a member of the FABP/P2/CRBP/CRABP family of β-barrel cytosolic fatty-acid-binding and retinoid-binding proteins. Sj-FABPc has at least eight different variants encoded by a single-copy polymorphic gene. In fluorescence-based assays, recombinant Sj-FABPc was found to bind 11-(dansylamino)undecanoic acid (DAUDA), inducing a shift in peak fluorescence emission from 543 to 493 nm. A similar spectral change was observed in dansyl-amino-octanoic acid (in which the dansyl fluorophore is attached at the α-carbon rather than the Ω-carbon of DAUDA), indicating that the ligand enters entirely into the binding site. Sj-FABPc also bound the naturally fluorescent cis-parinaric acid, as well as oleic acid and arachidonic acid, by competition, but not all-trans-retinol. Dissociation constants were, for cis-parinaric acid, Kd = 2.5±0.1 μM (mean±S.E.M.) and an apparent stoichiometry consistent with one binding site per molecule of Sj-FABPc and, for oleic acid, Ki≈ 80 nM. A deletion mutant from which α-II was absent failed to bind ligand. Sj-FABPc modelled well to known structures of the protein family; an unusually solvent-exposed Trp side chain was evident adjacent to the presumptive portal through which ligand is thought to enter and leave. Intrinsic fluorescence analyses of Sj-FABPc and of the deletion mutant (from which Trp-27 is absent) confirmed the unusual disposition of this side chain. Virtually all members of the FABP/P2/CRBP/CRABP protein family have prominent hydrophobic side chains in this position, with the exception of liver FABP and ileal FABP, which instead have charged side chains. Liver FABP is known to be distinct from other members of the protein family in that it does not seem to contact membranes to collect and deposit its ligand. It is therefore postulated that the unusually positioned apolar side chains in Sj-FABPc and others in the family are important in interactions with membranes or other cellular components.

Characterisation of a family of Schistosoma japonicum proteins related to dynein light chains

Dynein light chains (DLC) are components of dynein, an enzyme complex involved in various aspects of microtubule-based motility. We report here the molecular cloning and sequencing of cDNAs encoding a family of DLC-like polypeptides (SjcDLC1–5) from the human bloodfluke Schistosoma japonicum with open reading frames of 87–104 amino acids and deduced molecular masses ranging from 10.5 to 12.3 kDa. Two-dimensional Western blot analysis confirmed the presence of several S. japonicum DLC isoforms with differing p I values and molecular sizes. We also describe the molecular characterisation, genomic organisation and expression of clone SjcDLC1, and the immunological characterisation and localisation of its encoded protein. Northern blot analysis of adult worm RNA indicated SjcDLC1 is encoded by a single message of approximately 650 bp and Southern analysis suggested one SjcDLC1 gene exists in the S. japonicum genome. Immunolocalisation studies demonstrated that the SjcDLC1 protein is present in the tegument of the adult and cercarial stages of S. japonicum. SjcDLC1 and the other SjcDLC may function in the transport of specialised organelles, comprising membranous and discoid bodies, through the tegument to the schistosome-unique heptalaminate tegumental membrane at the external surface of the adult worm. As a consequence, they may provide novel targets for anti-schistosome vaccine and/or drug development.

Characterization of cDNAs encoding a new family of tetraspanins from schistosomes--the Sj25 family.

The tetraspanins, also known as members of the transmembrane 4 superfamily (TM4SF), comprise an assemblage of surface antigens reported from mammalian and other vertebrate cells, from schistosomes, from fruit flies and from Caenorhabditis elegans. Tetraspanins are characterized by the presence of four hydrophobic domains, which are presumed to be membrane-spanning, and specific conserved motifs. A novel cDNA encoding a new tetraspanin, termed TE736 (tetraspanin 736), was isolated and characterized from the human blood fluke Schistosoma japonicum. Nucleotide and deduced amino acid sequences of the cDNA revealed that TE736 was similar to the previously characterized Sm23/Sj23/Sh23 species homologues and to Sj25/TM4 from schistosomes, and to other tetraspanins. Comparison of hydropathicity profiles of TE736, Sj25/TM4 and two novel tetraspanin-like sequences from S. mansoni showed that they contain four potential transmembrane domains like other tetraspanins. Sequence alignments showed there are four highly conserved, cysteine residues on the second predicted extracellular loop of TE736. A phylogenetic comparison of the relationship of approximately 30 tetraspanins from mammals and other groups revealed that TE736, Sj25/TM4 and the two sequences from S. mansoni formed an independent family of tetraspanins. We have termed these tetraspanins from schistosomes the Sj25 family. TE736 appeared to be encoded by a single gene and to be expressed in at least two life cycle stages of S. japonicum.

Gene cloning, overproduction and purification of a functionally active cytoplasmic fatty acid-binding protein (Sj-FABPC) from the human blood fluke Schistosoma japonicum.

We report the gene cloning, molecular characterisation and purification of a 14.7-kDa functionally active recombinant (re) cytoplasmic fatty acid-binding protein (Sj-FABPC) from the Chinese strain of the human bloodfluke Schistosoma japonicum (Sj). As schistosomes are unable to synthesise long chain fatty acids and sterols de novo and must, therefore, take up these lipids from the host, Sj-FABPC is an attractive vaccine and/or drug target. Clone 39 (C39), which contains the entire Sj-FABPC gene, was isolated from a Sj lambda ZAPII cDNA expression library immunoscreened with hyperimmune rabbit serum (HRS) raised against soluble adult Sj proteins. The complete ORF (open reading frame) of Sj-FABPC encodes a protein of 132 amino acids (aa) of 14.7 kDa. The aa sequence of Sj-FABPC exhibits 91% identity to a FABP of S. mansoni (Sm14) and 45% identity to a FABP of Fasciola hepatica (Fh15), putative vaccine candidates for schistosomiasis. Sj-FABPC was subcloned into the QIAexpress vector, pQE-10, and subsequently expressed in Escherichia coli. The re-Sj-FABPC, purified under non-denaturing conditions, was recognized by sera from patients with acute and chronic schistosomiasis japonica. The purified re-Sj-FABPC was also shown to bind to palmitic acid with high affinity. The functional expression of Sj-FABPC will facilitate studies on re-Sj-FABPC to assess its potential as a drug and/or vaccine candidate.

Genetic variation in geographically isolated populations and subspecies of Oncomelania hupensis determined by a PCR-based RFLP method

Previously, the species complex Oncomelania hupensis, individuals of which act as intermediate hosts for the human bloodfluke Schistosoma japonicum, has been characterised by morphological and isoenzyme criteria. We have examined genetic variation between and within three subspecies of Oncomelania hupensis, i.e. O. h. hupensis (China), O. h. quadrasi (Philippines) and O. h. nosophora (Japan), by direct means using a PCR-based RFLP method. The subspecies of O. hupensis were readily distinguished by their characteristic restriction patterns, supporting isoenzyme data which suggests these may warrant species-specific status. No genetic variation was observed between O. h. quadrasi from different islands within the Philippines. In contrast, geographical isolates of O. h. hupensis differed markedly by this method indicating several genetically distinct populations of O. h. hupensis occur in China.

Purification of a recombinant Schistosoma japonicum antigen homologous to the 22-kDa membrane-associated antigen of S. mansoni, a putative vaccine candidate against schistosomiasis

We describe the cDNA cloning, overproduction and purification of a 22.6-kDa antigen from the human blood fluke Schistosoma japonicum. A 777-bp cDNA (C32) was isolated from a S. japonicum λZAPII cDNA expression library immuno-screened with hyperimmune rabbit serum ( HRS) raised against soluble adult S. japonicum proteins. The open reading frame of C32 encodes a protein of 191 amino acids (aa) which exhibits 71% identity to a 22.6-kDa membrane-associated antigen of S. mansoni, a putative vaccine candidate for schistosomiasis. We have identified a sequence motif known as an EF-hand calcium-binding domain in both the S. japonicum and S. mansoni aa sequences, suggesting that the 22.6-kDa antigens are able to bind Ca 2+. Further, we have, for the first time, obtained the 22.6-kDa antigen in purified, non-denatured, recombinant form, and in sufficient quantity to assess the protective value of the molecule in vaccination/challenge experiments. This was achieved by synthesizing the schistosome antigen with a short polyhistidine tag fused to the N-terminus which was then used for subsequent affinity purification. The recombinant protein was purified under non-denaturing conditions using nickel-chelate affinity chromatography.

Cloning, molecular characterization, and functional activity of Schistosoma japonicum glyceraldehyde-3-phosphate dehydrogenase, a putative vaccine candidate against schistosomiasis japonica

We report the cloning, molecular characterization, and purification of functionally active recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the human bloodfluke Schistosoma japonicum. The GAPDH homolog from the related species Schistosoma mansoni has shown correlation of antibody titer to resistance to reinfection. A 1,164-bp cDNA (C1) was isolated from an S. japonicum lambda ZapII cDNA expression library immunoscreened with hyperimmune rabbit serum raised against soluble adult S. japonicum proteins. The open reading frame of C1 encodes a protein of 338 amino acids exhibiting 90% identity to the amino acid sequence of S. mansoni GAPDH. The inferred molecular mass of the protein is 36,589 daltons, and in vitro translation of the cDNA with [35S]methionine produced a radiolabelled band of the predicted size. Antibodies to C1 selected from hyperimmune rabbit serum by affinity purification recognized an S. japonicum protein doublet of 37 kDa but did not cross-react with a corresponding protein in S. mansoni extracts. The S. japonicum GADPH appears to be translated from a single mRNA encoded by a single-copy gene. After subcloning in the QIAexpress vector pQE-10 and subsequent expression, the recombinant protein was purified under nondenaturing conditions and shown to exhibit functional GAPDH enzymatic activity.