Comparative Metabonomic Investigations of Schistosoma japonicum From SCID Mice and BALB/c Mice: Clues to Developmental Abnormality of Schistosome in the Immunodeficient Host.

The growth and development of schistosome has been affected in the immunodeficient hosts. But it remains unresolved about the molecular mechanisms involved in the development and reproduction regulation of schistosomes. This study tested and compared the metabolic profiles of the male and female Schistosoma japonicum worms collected from SCID mice and BALB/c mice at 5 weeks post infection using liquid chromatography tandem mass spectrometry (LC-MS/MS) platform, in which the worms from SCID mice were the investigated organisms and the worms from BALB/c mice were used as the controls. There were 1015 ion features in ESI+ mode and 342 ion features in ESI- mode were identified after filtration by false discovery rate. Distinct metabolic profiles were found to clearly differentiate both male and female worms in SCID mice from those in BALB/c mice using multivariate modeling methods including the Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA). There were more differential metabolites in female worms than in male worms between SCID mice and BALB/c mice. And common and uniquely perturbed metabolites and pathways were identified among male and female worms from SCID mice when compared with BALB/c mice. The enriched metabolite sets of the differential metabolites in male worms between SCID mice and BALB/c mice included bile acid biosynthesis, taurine and hypotaurine metabolism, sphingolipid metabolism, retinol metabolism, purine metabolism, etc. And the enriched metabolite sets of differential metabolites in female worms included retinol metabolism, alpha linolenic acid and linoleic acid metabolism, purine metabolism, sphingolipid metabolism, glutamate metabolism, etc. Further detection and comparison in transcript abundance of genes of the perturbed retinol metabolism and its associated meiosis process in worms identified clues suggesting accumulated retinyl ester and perturbed meiotic process. These findings suggested an association between the schistosome with retarded growth and development in SCID mice and their perturbed metabolites and metabolic pathways, and provided a new insight into the growth and development regulation of S. japonicum worms from the metabolic level, which indicated great clues for discovery of drugs or vaccines against the parasites and disease with more researches.

Influence of Schistosoma japonicum programmed cell death protein 10 on the growth and development of schistosomula.

BACKGROUND: Schistosomiasis caused by Schistosoma japonicum is among the most serious endemic zoonoses in China. To study interactions between schistosomula, the pre-adult juvenile stage, and hosts, it is important to study the functions of key genes involved in schistosomula growth and development. Programmed cell death protein 10 (pcdp10) is an important apoptosis-related gene with various biological functions. This study described the molecular characterization of S. japonicum PCDP10 (SjPCDP10) and evaluated its functions in schistosomula. METHODS: Real-time quantitative polymerase chain reaction (qPCR) and western blot were used to detect Sjpcdp10 mRNA and protein levels, respectively, at different developmental stages. Immunolocalization was performed to determine SjPCDP10 expression in the parasite. RNA interference (RNAi) experiments were used to assess gene functions associated with SjPCDP10 in schistosomula growth and development. RESULTS: Real-time qPCR revealed that Sjpcdp10 was expressed during all investigated developmental stages and upregulated during schistosomula growth and development. Histochemical localization showed that SjPCDP10 was mainly distributed in the teguments of schistosomula in all investigated stages and part of the parenchymal area of 14-, 18-, and 21-day-old schistosomula. Following Sjpcdp10 knockdown by RNAi, the lengths, widths, areas, and volumes of schistosomula were significantly lower than those in the control group. Scanning electron microscopy showed that the body surfaces of schistosomula subjected to RNAi were seriously damaged, with few tegumental spines and sensory papillae. Transmission electron microscopy indicated that the teguments of Sjpcdp10-knockdown schistosomula were incomplete, the number of layers was reduced, and the thickness decreased significantly as compared with those in the control group. Furthermore, terminal deoxynucleotidyl transferase dUTP nick-end labelling results showed that the rate of apoptosis in Sjpcdp10-knockdown schistosomula was significantly higher than that in the control group. CONCLUSIONS: Sjpcdp10-knockdown influenced the growth and development of schistosomula. Therefore, our results indicated that SjPCDP10 contributes to the regulation of cell apoptosis and is essential for schistosomula growth and development.

Studies on the establishment of a co-culture system of lung stage Schistosoma japonicum with host cells.

Due to their role in eliciting protective Th1 cell-mediated immune responses in definitive hosts lung stage schistosomula are in the focus of intensive research. In vitro culture approaches in the past exhibited significant differences in gene expression profiles between lung stage schistosomula isolated from hosts and those cultured conventionally. Therefore, new approaches to culture schistosomula are of broad interest. In the present study, co-culture systems of schistosomula of Schistosoma japonicum and different vertebrate host cells were tested. Among these, human hepatic venous endothelial cells (ED25) turned out to be very suitable and interesting feeder cells. Compared with controls cultured in vitro or co-cultured with other cells, schistosomula co-cultured with ED25 cells shared more similarities in morphology and tegumental structures with schistosomula directly obtained from infected mice as microscopically determined. According to results from a suppression subtractive hybridization approach to compare transcriptional differences of co-cultured and host group or control group parasites, four candidate transcripts encoding cathepsin L precursor, heat shock protein 70, glyceraldehyde 3-phosphate dehydrogenase, and programmed cell death protein 10 were shown to be differently expressed among the three groups by real-time PCR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis finally confirmed not only congruent protein patterns but also interesting differences among the compared schistosomula groups. The co-culture system between schistosomula of S. japonicum and ED25 cells established in the present study improved existing cultivation attempts. Although some differences to host-derived schistosomula were still observed, co-culture with ED25 cells positively influenced parasite morphology and gene expression in a more host-like manner.

Effects of protein extract from head-foot tissue of Oncomelania hupensis on the growth and gene expression of mother sporocysts of Schistosoma japonicum.

Oncomelania hupensis is the intermediate host of Schistosoma japonicum. In the present study, we investigated the effects of protein extracts from head-foot or gland tissue of O. hupensis on mother sporocysts of S. japonicum cultured in vitro. In the presence of head-foot protein extract of snails from the native province Hunan, in-vitro-transformed mother sporocysts presented not only a longer survival time and stronger motility, but also a bigger size than parasites cultured with protein extracts of glands of the same snail or head-foot tissue of a non-native snail from the Hubei province. Using suppression subtractive hybridization, two subtractive libraries were constructed on the basis of RNA of sporocysts cultured with or without native snail head-foot protein extract. A number of 31 transcripts were found to be up-regulated. Sequence analyses revealed that they represented genes involved among others in metabolic process, electron transport chain, response to chemical stimulus, and oxidation-reduction processes. Opposite to that 20 down-regulated transcripts were among others related to pseudouridine synthesis, RNA processing, and ribosome biogenesis. The differential expression of three of these transcripts, encoding cytochrome c oxidase subunit 2 (Cox2), NADH-ubiquinone oxidoreductase (ND1), and dyskeratosis congenita 1 protein (DKC1), were confirmed by real-time PCR. The promoted development and the differential gene expression of cultured sporocysts under the influence of head-foot protein extract of native O. hupensis implied not only its ability to improve in vitro culture conditions for intramolluscan stages, it may also represent a priming result with respect to the identification and characterization of factors involved in the parasite-host interplay between S. japonicum and O. hupensis.

[Effects of male worm extraction on ultrastructure of cultured vitelline cells from Schistosoma japonicum].

OBJECTIVE: To study the effects of male worm extraction on the proliferation and metabolic activity of cultured vitelline cells from Schistosoma japonicum. METHODS: The 28-day S. japonicum worms were harvested by perfusion. The male and female of them were isolated after asepsis separately. The vitelline glands of female worms were isolated, and the vitelline cells were harvested by the cold digestion, then they were inoculated with the moist system method on the walls of culture flasks. The cultured vitelline cells were randomly divided into test and control groups. The cells in the control group were cultured in routine media and those in the test group were cultured in routine media containing male worm extraction of the concentration of 100 microg/ml. When cultured for 7 days, the cells in both groups were prepared for observation under a transmission electron microscope. RESULTS: In the test group, the numbers of mature vitelline cells were more than those in the control group; the cytoplasm and nucleus of mature vitelline cells were homogeneous stain. The nucleolus and rough-surfaced endoplasm reticula were clear, the intervals of vitelline globules were clear and their numbers could be counted. The number of mitochondria was small and the electron density was low; abundant rough-surfaced endoplasm reticula were found in the immature vitelline cells. There were more immature vitelline cells in the control group. The cytoplasm of the cultured vitelline cells took changes of balloon, especially in mature vitelline cells, vitelline globules fused each other, no mitochondria were found; in immature vitelline cells, the space between vitelline globules and the membrane surrounding them broadened gradually and vitelline globules were released and uncovered; rough-surfaced endoplasmic reticula enlarged, space vacuolated and the ribosomes dropped; and the number of lipid increased. CONCLUSION: The S. japonicum male worm extraction can stimulate the development and survival of the cultured vitelline cells.

Ultrastructure of cultured cells from Schistosoma japonicum.

Ultrastructures and their dynamic changes of the cultured cells from Schistosoma japonicum were observed in the present experiments. Several types-including polygonal, round granular, deltaic fan-shaped and flagellated cells-were found in the cultures. The polygonal cells took a major ratio in the cultures from adult S. japonicum, while the majority from schistosomula was round granular cells. The ultrastuctures on the cell surface were different between the cells from adults and schistosomula. Some papilla-like tubercula, microvilli and pinocytotic vesicles were observed on the surface of adult cells, but none were found on schistosomula cells. However, more or less mitochondria, endoplasmic reticula, ribosomes and glycogen were observed in the cytoplasm of the cultured cells from both adults and schistosomula. Golgi complexes were rarely found. The nucleus was round, with round nucleolus inside and clear pores on the unit membrane. There was much lumpish heterochromatin located near to the nuclear membrane. Cells from different worm tissues had their own organelles. The germ cells, vitelline cells, flame cells, multinucleate subtegumental cells and nerve cells could be observed in the cultures from adults. The vitelline cells were the greatest in number and nerve cells were the least in number among them. Similarly, there were germ cells, sustentacular cells, flame cells, nerve cells, mast cells, muscle cells, multinucleate subtegumental cells, interstitial cells and penetration gland cells in the cultures from the schistomomula. In addition, a few division cells were also found. It indicated that the schistosomula cells had greater potential ability to proliferate than the adult cells in in vitro culture. Along with the prolongation of the culture time, degeneration of schistosomal cell occurred more and more. Generally, the electron density of cultures gradually got lower, the cristae of mitochondria blurred and disappeared and the mitochondria themselves swelled and finally vacuoled completely. Vitelline cells were most sensitive to the changes of the in vitro condition in all cultures. Their degeneration showed the following characteristics: (1) vitelline globules fused each other, the space between vitelline globules and the membrane surrounding them broadened gradually and vitelline globules were released and uncovered; (2) rough-surfaced endoplasmic reticula enlarged, vacuolated and the ribosomes dropped; and (3) the number and volume of lipid increased. The ultrastructural changes of most of the cultures from schistosomula had the following trends: (1) heterochromatin increased and euchromatin decreased gradually; and (2) endoplasmic reticula changed into short tubes and vacuoles and disappeared finally. The degenerative process of the cultures from S. japonicum consisted of necrosis according to the ultrastructural changes of the mitochondria, vitelline globules, chromatin and endoplasmic reticula within the cells. The changes of the above structures could be used to estimate whether the culture conditions were appropriate.