Transcriptome sequencing and analysis of the zoonotic parasite Spirometra erinacei spargana (plerocercoids).

BACKGROUND: Although spargana, which are the plerocercoids of Spirometra erinacei, are of biological and clinical importance, expressed sequence tags (ESTs) from this parasite have not been explored. To understand molecular and biological features of this parasite, sparganum ESTs were examined by large-scale EST sequencing and multiple bioinformatics tools. METHODS: Total RNA was isolated from spargana and then ESTs were generated, assembled and sequenced. Many biological aspects of spargana were investigated using multi-step bioinformatics tools. RESULTS: A total of 5,634 ESTs were collected from spargana. After clustering and assembly, the functions of 1,794 Sparganum Assembled ESTs (SpAEs) including 934 contigs and 860 singletons were analyzed. A total of 1,351 (75%) SpAEs were annotated using a hybrid of BLASTX and InterProScan. Of these genes, 1,041 (58%) SpAEs had high similarity to tapeworms. In the context of the biology of sparganum, our analyses reveal: (i) a highly expressed fibronectin 1, a ubiquitous and abundant glycoprotein; (ii) up-regulation of enzymes related with glycolysis pathway; (iii) most frequent domains of protein kinase and RNA recognition motif domain; (iv) a set of helminth-parasitic and spargana-specific genes that may offer a number of antigen candidates. CONCLUSIONS: Our transcriptomic analysis of S. erinacei spargana demonstrates biological aspects of a parasite that invades and travels through subcutaneous tissue in intermediate hosts. Future studies should include comparative analyses using combinations of transcriptome and proteome data collected from the entire life cycle of S. erinacei.

Molecular characterization of a Spirometra mansoni antigenic polypeptide gene encoding a 28.7 kDa protein.

The Spirometra mansoni antigenic polypeptide (SmAP) gene was expressed in Escherichia coli, and its characteristics and value as an antigen for the serodiagnosis of sparganosis were investigated. The recombinant SmAP protein (rSmAP) has the molecular weight of 28.7 kDa. On Western blotting analysis, the rSmAP strongly reacted with the sera of mice infected with spargana, but not with normal sera; the anti-rSmAP serum obviously recognized the 28.7-kDa band in the crude antigens and excretory-secretory (ES) antigens of spargana. The immunofluorescence test (IFT) results showed that the positive staining was observed at different stages of spargana from the infected frogs and mice, but not adult worm of S. mansoni. An immunolocalization analysis identified SmAP in the teguments and parenchymal tissues of spargana. ELISA with rSmAP antigen or sparganum ES antigens were evaluated for the serodiagnosis of sparganosis. The results showed that the sensitivity of rSmAP-ELISA and ES-ELISA was 83.3% (25/30) and 100% (30/30), respectively, for the detection of anti-sparganum IgG antibodies in sera of the experimentally infected mice (P > 0.05), the specificities of both ELISA were 100% (67/67). It is suggested that the rSmAP might be a potential candidate antigen for serodiagnosis of sparganosis.

Bioinformatic analysis for structure and function of TCTP from Spirometra mansoni.

OBJECTIVE: To predict structure and function of translationally controlled tumor protein (TCTP) from Spirometra mansoni by bioinformatics technology, and to provide a theoretical basis for further study. METHODS: Open reading frame (ORF) of EST sequence from Spirometra mansoni was obtained by ORF finder and was translated into amino acid residue by DNAclub. The structure domain was analyzed by Blast. By the method of online analysis tools: Protparam, InterProScan, protscale, SignalP-3.0, PSORT II, BepiPred, TMHMM, VectorNTI Suite 9 packages and Phyre2, the structure and function of the protein were predicted and analyzed. RESULTS: The results showed that the EST sequence was Sm TCTP with 173 amino acid residues, theoretical molecular weight was 19 872.0 Da. The protein has the closest evolutionary status with Clonorchis sinensis, Schistosoma mansoni, and Schistosoma japonicum. Then it had no signal peptide site and transmembrane domain. Secondary structure of TCTP contained two α -helices and eight ß -strands. CONCLUSIONS: Sm TCTP was a variety of biological functions of protein that may be used as a vaccine candidate molecule and drug target.

Identification, structure and function of a novel tetraspaninhomologue from Spirometra erinaceieuropaei.

OBJECTIVE: To identify a full length c DNA sequence of a novel tetraspanin (TSP) homologue from Spirometra erinaceieuropaei and to predict the structure and function of its encoding protein using bioinformatics methods. METHODS: Using the NCBI, EMBI, Expasy and other online sites, the open reading frame (ORF), conserved domain, physical and chemical parameters, signal peptide, transmembrane domain, epitope, topological structures of the protein sequences were predicted. And Vector NTI software was used for multiple sequence alignment and phylogenetic tree construction. RESULTS: The target sequence was 1132 bp length with a 681 bpbiggest ORF encoding 226 amino acids protein with typical TSP conserved domain. It was confirmed as full length c DNA of TSP16 from Spirometra erinaceieuropaei and named as SeTSP16 (GenBank accession number: JF728872). The predicted molecular weight and isoelectric point of the deduced protein were 24 750.5 Da and 7.88 Da, respectively. Compared with TSP16s from Schistosoma japonicum and Schistosoma mansoni, it showed similarity of 59% and 59%, respectively. SeTSP16 contained four transmembrane domains (TM1-4), intracellular N and C-termini, one short small extracellular loop and one large extracellular loop. Four major epitopes that were significant different from the corresponding epitope regions of TSP16 from Schistosoma mansoni and Schistosoma japonicum were predicted. CONCLUSIONS: The full length c DNA sequences of SeTSP16 are identified. It encodes a transmembrane protein which might be an ideal diagnosis antigen and target molecule for antiparasitic drugs.

Differential protein expression in Spirometra erinacei according to its development in its final host.

This study was undertaken to identify genes involved in the growth and development of Spirometra erinacei larvae, an intestinal tapeworm of cats and dogs, within the final host. The differential protein expression at three different stages of S. erinacei, the plerocercoid larvae, 8-day-old juveniles, and adults, was compared using two-dimensional electrophoresis. Specifically or highly expressed proteins in juvenile worms were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MS)/MS. The proteome map of larvae showed fewer protein spots than juveniles or adults, whereas juveniles or adults revealed a similar protein expression profile. Eight juvenile-specific and five upregulated proteins of juveniles were identified and matched to proteins of known biological functions. These were grouped into several categories of functionally related proteins: DNA/RNA metabolism, cell trafficking, cytoskeleton, protein processing and degradation, energy metabolism, and oxidative stress. Our results give an overview of the growth and development mechanisms of cestodes within the final host and extend our understanding of parasite biology in the host-parasite relationship.

Partial purification and characterization of a cysteine protease inhibitor from the plerocercoid of Spirometra erinacei.

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.

Partial Purification and Characterization of a Cysteine Protease Inhibitor from the Plerocercoid of Spirometra erinacei

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.

A new method for concentration of proteins in the calcareous corpuscles separated from the spargana of Spirometra erinacei.

Calcareous corpuscles are a characteristic structure found in larval and adult stage cestodes. These corpuscles are known to contain several protein components and to possess protein-binding activity. However, the proteins bound to calcareous corpuscles in situ have not been studied. The present study was undertaken to identify the proteins on calcareous corpuscles. Calcareous corpuscles were purified from the plerocercoids (= spargana) of Spirometra erinacei, and serially dissolved using 0.1 M sulfamic acid solution. Collected supernatants were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The results showed that only the fraction remaining after the 19th dissolved fraction contained proteins. A total of 20 protein molecules were detected in gel, with major bands at 56, 53, 46, 40, 35, 29, 28, 24.5, 21, 19, 16, 13, 10 and 8 kDa. In particular, the proteins corresponding to the 21 and 16 kDa bands were most abundant. Our results demonstrated for the first time the protein contents of the calcareous corpuscles of spargana. Further studies on the functions of these proteins are required.

A new method for concentration of proteins in the calcareous corpuscles separated from the spargana of Spirometra erinacei

Calcareous corpuscles are a characteristic structure found in larval and adult stage cestodes. These corpuscles are known to contain several protein components and to possess protein-binding activity. However, the proteins bound to calcareous corpuscles in situ have not been studied. The present study was undertaken to identify the proteins on calcareous corpuscles. Calcareous corpuscles were purified from the plerocercoids (= spargana) of Spirometra erinacei, and serially dissolved using 0.1 M sulfamic acid solution. Collected supernatants were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The results showed that only the fraction remaining after the 19th dissolved fraction contained proteins. A total of 20 protein molecules were detected in gel, with major bands at 56, 53, 46, 40, 35, 29, 28, 24.5, 21, 19, 16, 13, 10 and 8 kDa. In particular, the proteins corresponding to the 21 and 16 kDa bands were most abundant. Our results demonstrated for the first time the protein contents of the calcareous corpuscles of spargana. Further studies on the functions of these proteins are required.

Enzymatic N-glycan analysis of 31 kDa molecule in plerocercoid of Spirometra mansoni (sparganum) and its antigenicity after chemical oxidation.

A highly specific antigenic protein of 31 kDa from plerocercoid of Spirometra mansoni (sparganum) was obtained by gelatin affinity and Mono Q anion-exchange column chromatography. The purified 31 kDa protein was subjected to N-glycan enzymatic digestion for structural analysis. The relative electrophoretic mobility was analyzed by SDS-PAGE, before and after digestion. On SDS-PAGE after enzymatic digestion, the 31 kDa protein showed a molecular shift of approximately 2 kDa, which indicated the possession of complex N-linked oligosaccharides (N-glycosidase F sensitive) but not of high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H, non-sensitive). Chemically periodated 31 kDa protein showed statistically non-significant changes with human sparganosis sera by enzyme linked immunosorbent assay (ELISA). Therefore, the dominant epitopes of the 31 kDa molecule in human sparganosis were found to be mainly polypeptide, while N-glycans of the antigenic molecule in sparganum was minimal in anti-carbohydrate antibody production.

Comparison of carbohydrate moieties of sparganum proteins of the snake, mouse and those of adult worm.

The carbohydrate moieties of larval sparganum proteins in two different species, the snakes, Elaphe rufodorsata, the Balb/c mouse and those of the adult worm, Spirometra erinacei, were compared using five different lectins including GNA, SNA, MAA, PNA and DSA. The GNA positive 53 kDa molecule, which is excretory-secretory protease in the sparganum from the snake showed a stage specific and developmental regulation. We also suggested that sparganum glycosylation may be involved in immune evasion and differentiation into an adult worm.

Comparison of carbohydrate moieties of sparganum proteins of the snake, mouse and those of adult worm

The carbohydrate moieties of larval sparganum proteins in two different species, the snakes, Elaphe rufodorsata, the Balb/c mouse and those of the adult worm, Spirometra erinacei, were compared using five different lectins including GNA, SNA, MAA, PNA and DSA. The GNA positive 53 kDa molecule, which is excretory-secretory protease in the sparganum from the snake showed a stage specific and developmental regulation. We also suggested that sparganum glycosylation may be involved in immune evasion and differentiation into an adult worm.

Cestode parasites: application of in vivo and in vitro models for studies on the host-parasite relationship.

Cestode worms, commonly also known as 'flat' worms or tapeworms, are an important class of endoparasitic organisms. In order to complete their life cycle, they infect intermediate and definitive hosts in succession, through oral ingestion of eggs or larvae, respectively. Serious disease in humans or other mammalian hosts is mostly caused by the larval stages. Echinococcus spp. and Taenia spp. have been extensively investigated in the laboratory due to the fact that they represent important veterinary medical challenges and also cause grave diseases in humans. In contrast, Hymenolepis spp. and Mesocestoides spp. infections are relatively rare in humans, but these parasites have been extensively studied because their life cycle stages can be easily cultured in vitro, and can also be conveniently maintained in laboratory animal hosts. Thus they are more easily experimentally accessible, and represent important models for investigating the various aspects of cestode biology. This review will focus on in vitro and in vivo models which have been developed for studies on the host-parasite relationship during infection with Echinococcus, Taenia, Hymenolepis, Mesocestoides and Spirometra, and will cover the use of these models to investigate the morphology and ultrastructure of respective genera, the immunological relationship with the host and the development of vaccination approaches, as well as applications of these models for studies on parasite metabolism, physiology and gene expression. In addition, the use of these models in the development of chemotherapeutic measures against cestode infections is reviewed.

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress the TNF-alpha gene expression by reducing phosphorylation of ERK1/2 and p38 MAPK in macrophages.

We previously reported that excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expression and production of tumour necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide. The present study investigated the suppressive mechanisms of tumour necrosis factor-alpha mRNA by excretory/secretory products in lipopolysaccharide-stimulated murine macrophages. Electrophoretic mobility shift assay and supershift assay revealed that neither nuclear translocation of nuclear factor-kappa B nor conformation of the p50/p65 nuclear factor-kappa B subunits was affected by the treatment of excretory/secretory products in lipopolysaccharide-stimulated macrophages. Inhibition of extracellular signal-regulated protein kinase 1/2 with PD98059 or p38 mitogen-activated protein kinase with SB203580 partially reduced tumour necrosis factor-alpha mRNA expression, and a combination of the two inhibitors additionally suppressed the level of tumour necrosis factor-alpha mRNA, revealing that both pathways are crucial for full induction of the gene. Northern blot analysis showed that excretory/secretory products additionally suppressed tumour necrosis factor-alpha mRNA expression in cells treated with PD98059 or SB208530 and, in turn, we found that excretory/secretory products reduced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated macrophages by Western blot analysis. This is the first report demonstrating that excretory/secretory products from parasites suppress tumour necrosis factor-alpha mRNA expression by reducing phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase without any effect on nuclear factor-kappa B activity in macrophages stimulated with lipopolysaccharide. We hypothesise that excretory/secretory products may enable this parasite to survive within the host.

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expressions and production of tumor necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide or lipoteichoic acid.

We previously reported that the ES products from the plerocercoids of Spirometra erinaceieuropaei reduce nitric oxide synthase and chemokine gene expression in macrophages. In this study, we show that ES products suppressed tumor necrosis factor-alpha mRNA expression and tumor necrosis factor-alpha production in murine peritoneal macrophages stimulated with lipopolysaccharide or lipoteichoic acid in vitro. When macrophages from ES product-injected mice were stimulated with lipopolysaccharide in vitro, these cells produced smaller amounts of tumor necrosis factor-alpha compared with those taken from control mice. The suppressive effects of ES products were not restored by the treatment of indomethacin or anti-IL-10 antibody, and the ES products did not induce mRNA expression of secretory leukocyte protease inhibitor. Macrophages from C3H/HeJ mice, which have a single point mutation in the Toll-like receptor 4 gene, expressed tumor necrosis factor-alpha and IL-1alpha mRNA in the presence of lipopolysaccharide, but these expressions were less than those of macrophages from C3H/HeN. ES products significantly suppressed tumor necrosis factor-alpha gene expression and tumor necrosis factor-alpha production in macrophages from C3H/HeN and C3H/HeJ mice stimulated with lipopolysaccharide. However, ES products had no effect on IL-1 mRNA expression. Our data suggest that the plerocercoids secrete the tumor necrosis factor-alpha inhibitory products to evade the host's immune system, and that tumor necrosis factor-alpha mRNA expression might be inhibited downstream from Toll-like receptor 4 in the lipopolysaccharide signaling pathway.

Amino acid sequence of a trypsin inhibitor from a Spirometra (Spirometra erinaceieuropaei).

A trypsin inhibitor that is highly homologous with bovine pancreatic trypsin inhibitor (BPTI) was co-purified along with RNase from Spirometra (Spirometra erinaceieuropaei). The amino acid sequence of this inhibitor (SETI) and the nucleotide sequence of the cDNA encoding this protein were determined by protein chemistry and gene technology. SETI contains 68 amino acid residues and has a molecular mass of 7,798 Da. SETI has 31 amino acid residues that are identical with BPTI's sequence, including 6 half-cystine and 5 aromatic amino acid residues. The active site Lys residue in BPTI is replaced by an Arg residue in SETI. SETI is an effective inhibitor of trypsin and moderately inhibits a-chymotrypsin, but less inhibits elastase or subtilisin. SETI was expressed by E. coli containing a PelB vector carrying the SETI encoding cDNA; an expression yield of 0.68 mg/l was obtained. The phylogenetic relationship of SETI and the other BPTI-like trypsin inhibitors was analyzed using most likelihood inference methods.

The fatty acids of each lipid fraction and their use in providing energy source of the plerocercoid of Spirometra erinacei.

The fatty acid concentration of each lipid fraction of plerocercoids of Spirometra erinacei and the host snake serum was investigated. The major fatty acids of phospholipid of the plerocercoids were C18:1, C18:0 and C16:0, and those of the host snake serum were C16:0, C18:1 and C18:0, in order of amount in both cases. The changes of the fatty acid composition of phospholipid of the plerocercoids when they were incubated in physiological saline at 18 degrees C and at 37 degrees C for 24 h were investigated in both cases. Polyunsaturated fatty acids increased at 18 degrees C, and saturated fatty acids increased at 37 degrees C. Michaelis constants (Km) of beta-hydroxyacyl-CoA dehydrogenase (HAD), NADH: ubiquinone oxidoreductase (complex I) (NADH: ferricyanide reaction) and complex I (NADH: ubiquinone reaction) for NADH were 20.6, 50 and 13.3 microM, respectively. The ATP production in mitochondria of the plerocercoids was accelerated by adding ADP and inhibited by adding such electron transport system inhibitors as rotenone, antimycin A and sodium cyanide. These results suggested that the fatty acids in the plerocercoids played an important role in regulating the fluidity of membrane by changing the composition in membrane lipid corresponding with the change of temperature circumstance. The NADH reduced by HAD might be accepted by the complex I in the electron transport system, and thus the parasites were capable of ATP production in a classical pathway of the oxidative phosphorylation system.

The metabolism of arachidonic acid to prostaglandin E2 in plerocercoids of Spirometra erinacei.

With a simplified method of extracting and purifying prostaglandins, trace prostaglandins (nanogram order) were detected by gas chromatography-mass spectrometry. Arachidonic acid was metabolized to prostaglandin E2 (PGE2) by plerocercoids of Spirometra erinacei, the PGE2 was detected in the medium after incubation with arachidonic acid, and the role of albumin in the absorption of free arachidonic acid by plerocercoids and in the release of its metabolite was investigated. Plerocercoids absorbed arachidonic acid-binding albumin and released PGE2 efficiently. PGE2 is known to suppress the functions of mononuclear cells of the host. The selective release of PGE2 may be related to the escape mechanism of plerocercoids of S. erinacei from the host immune system to become established larva migrans, i.e., sparganosis.