Japonicum Schistosomula Research Articles

Glyceraldehyde-3-phosphate dehydrogenase affects the growth of Schistosoma japonicum schistosomula

This study was conducted to evaluate the effect of glyceraldehyde-3-phosphate dehydrogenase from Schistosoma japonicum (SjGAPDH) on the growth of schistosomula. Quantitative reverse transcription PCR and immunohistochemical analysis were performed to analyze the mRNA levels and immune localization of SjGAPDH. RNA interference experiments were conducted to further examine the role of SjGAPDH in the schistosomula growth of S. japonicum. The results demonstrated that SjGAPDH mRNA was expressed during all stages of S. japonicum development, with its expression gradually increasing over time. SjGAPDH was mainly distributed on the surface and in some parenchymal cells of S. japonicum. Double-stranded RNA-mediated GAPDH knockdown reduced SjGAPDH expression by approximately 59%. Light microscopic observations revealed that the size, length, width, volume, and area of schistosomula in the SjGAPDH interference group were significantly lower than those in the enhanced green fluorescent protein control group. These findings indicate that SjGAPDH may affect the growth of S. japonicum schistosomula and could be a useful target for treating schistosomiasis.

Involvement of the fatty acid-binding protein in the growth of Schistosoma japonicum schistosomula.

This study aimed to explore the effect and mechanism underlying the role of the Schistosoma japonicum antigen of fatty acid-binding protein (SjFABP) on the growth of the schistosomula. SjFABP levels were evaluated by quantitative real-time polymerase chain reaction of samples of mice infected with S. japonicum; SjFABP was expressed and its levels gradually increased during all stages of S. japonicum schistosomula, including on 3, 10, 14, and 21days of the growth process. Immunohistochemistry results demonstrated that SjFABP was distributed in the parenchyma, especially in the digestive tract of the S. japonicum schistosomula. RNA interference resulted in more than 60% knockdown of SjFABP leading to a reduction in length, volume, width, and area of the schistosomula as compared to control samples, as determined by light microscopy. Terminal deoxynucleotidyl transferase dUTP nick-end labeling detection further suggested that SjFABP knockdown resulted in increased apoptosis of schistosomes. Taken together, these results suggest that SjFABP may be related to the growth and survival of S. japonicum schistosomula, thereby representing a potential target for the treatment of schistosomiasis.

EFFECT OF ADENYLATE KINASE 1 ON THE GROWTH AND DEVELOPMENT OF SCHISTOSOMA JAPONICUM SCHISTOSOMULUM.

We investigated the effect of Schistosoma japonicum adenylate kinase 1 (Sjak1) on the growth and development of schistosomula. Quantitative real-time PCR showed that Sjak1 mRNA was expressed in 3-, 10-, 14-, 18-, and 21-day-old schistosomula, and its levels increased gradually with the development of S. japonicum. Using immunohistochemical techniques, ak1 protein was found to be mainly distributed in the tegument and some parenchymal tissues of the schistosomula. Double-stranded RNA-mediated knockdowns of ak1 decreased ak1 mRNA transcripts by more than 90%, and western blot results showed that expression of ak1 protein was decreased by 66%. Scanning electron microscopy following the RNA-mediated ak1 knockdown showed that the sensory papillae did not develop. Transmission electron microscopy showed a lower mean thickness of the tegument in the Sjak1 interference group than in the negative control group. Terminal deoxynucleotidyl transferase dUTP nick-end labeling suggested higher apoptosis in the interference group than the negative control group. These results showed that ak1 may be involved in the growth and development of S. japonicum schistosomula and especially in the development of the integument. Consequently, ak1 may be a potential target in developing prevention methods for schistosomiasis in the future.

Involvement of the Glyceraldehyde-3-Phosphate Dehydrogenase on the Schistosomula Growth of <i>Schistosoma japonicum</i>

We evaluated the effect of the Schistosoma japonicum antigen of glyceraldehyde-3-phosphate dehydrogenase (SjGAPDH) on the growth of schistosomula. Quantitative real-time PCR and immunohistochemical analysis were performed to evaluate the level and immunolocalization of SjGAPDH mRNA and protein, respectively. RNA inference was conducted to further study the role of SjGAPDH on the schistosomula growth of S. japonicum. The results demonstrated that SjGAPDH mRNA was expressed during all stages of S. japonicum development, during which it gradually increased over time. SjGAPDH protein was mainly distributed on the surface and in some parenchyma of S. japonicum. Double-stranded RNA-mediated GAPDH gene knockdown reduced SjGAPDH mRNA transcription by more than 90%, and western blot analysis showed that SjGAPDH protein expression was decreased by more than 60%. Light microscopy observation revealed that the size of schistosomula including the length, width, volume, and area of schistosomula in the SjGAPDH interference group was significantly lower than those in the enhanced green fluorescent protein control group. Thus, SjGAPDH may be involved in the growth of S. japonicum schistosomula and a useful target for treating schistosomiasis.

Role of adenylate kinase 1 in the integument development of Schistosoma japonicum schistosomula

Schistosomula antigens play an important role in the growth and development of Schistosoma japonicum. We investigated the role of S. japonicum adenylate kinase 1 (SjAK1) in the growth and development of schistosomula. Quantitative real-time PCR showed that SjAK1 mRNA was expressed in all schistosomula stages, but increased gradually with the development of S. japonicum schistosomula. Using immunohistochemical techniques, the AK1 protein was found to be mainly distributed in the tegument and in some parenchymal tissues of the schistosomula. Double-stranded RNA-mediated knockdown of AK1 reduced AK1 mRNA transcripts by more than 90%; western blot analysis demonstrated that AK1 protein expression decreased by 66%. Scanning electron microscopy following RNA-mediated AK1 knockdown demonstrated that the sensory papillae degenerated significantly. Transmission electron microscopy demonstrated that the mean thickness of the tegument in the SjAK1 interference group was lower than that in the negative control group. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) suggested that, compared with the negative control group, apoptosis increased in the interference group. These results show that AK1 may be involved in the growth and development of S. japonicum schistosomula, and thus may be a target when developing treatments for schistosomiasis.

Genome-wide miRNAs expression profiles of Schistosoma japonicum schistosomula in response to artesunate.

miRNAs play a significant role in pharmacogenomics and are likely to be important in the molecular mechanism of atesunate (ART) effects on Schistosoma japonicum. We sequenced the RNAs using an Illumina (Solexa) DNA sequencer and compared the relative expression levels of the miRNAs in 10-day-old schistosomula from ART and the parallel control group. We characterized 95 known miRNAs from S. japonicum schistosomula individuals, including 38 novel miRNA families. Among the detectable 134 miRNAs differentially expressed (>2.0-fold change, p < 0.01) after ART treatment in schistosomula, a total of seven known or novel 3p- or 5p- derived S. japonicum miRNAs were characterized. We propose that sja-miR-125b may regulate the expression of ART metabolizing enzymes, glutathione synthetase or heme-binding protein 2 to help S. japonicum resists or adapts to drug stress and also ART may significantly inhibit sexual maturation of female worms mediated by mir-71b/2 miRNA cluster. This was the first comprehensive miRNAs expression profile analysis of S. japonicum in response to ART, and provides an overview of the complex network of the mechanism of action of ART on S. japonicum.

Molecular characterization and identification of Th1 epitopes of a Schistosoma japonicum protein similar to prosaposin

The tegument of schistosomula contains T cell antigens that might simulate the protective mechanisms of the radiation-attenuated vaccine in a mouse model of schistosomiasis. Immune mechanisms mediated by the CD4+ Th1 response are important in the RAV model. To rapidly identify Th1 epitopes in molecules from the Schistosoma japonicum schistosomula tegument, this study analyzed S. japonicum proteomics data. Preliminary experiments identified a protein similar to prosaposin (SjPSAP) from the tegument of schistosomula. We confirmed that SjPSAP was present in the tegument of the parasite using an indirect immunofluorescence assay. We then identified Th cell epitopes in SjPSAP using in silico prediction combined with experimental validation. From the SjPSAP sequence, we used several algorithms to predict 11 promiscuous Th cell epitopes that might bind to both murine and human MHC class II molecules. To validate the in silico predictions, proliferation and cytokine production profiles of spleen lymphocytes from BALB/c mice immunized with the 11 predicted peptides were measured in vitro using a modified methyl thiazolyl tetrazolium assay and flow cytometry. The results showed that 4 of the 11 predicted peptides induced a recall CD4+ Th1 response in vitro. We measured direct binding of the four peptides predicted to induce a response to antigen-presenting cells from BALB/c mice using a fluorometric method and found that the peptides bound to both I-Ad and I-Ed mouse molecules. These results demonstrated that potentially protective Th1-type epitopes in SjPSAP molecules could be identified rapidly by combining in silico prediction with experimental validation. This strategy could be a fast method for identifying Th1 epitopes in a schistosoma antigen with features such as large size or poor expression of recombinant antigens.

Murine CD8+T cell cytotoxicity against schistosomula induced by inoculation of schistosomal 22.6/26GST coupled Sepharose 4B beads

Schistosomasis is a world-wide parasitic disease. Although chemotherapy is the main treatment method for schistosomasis currently, it cannot prevent schistosome reinfection. Up to now no effective vaccine is available to prevent schistosomiasis. Dendritic cells (DCs) are one of the key players in the cellular immune response and play an important role in antigen presentation as antigen-presenting cells. Here we reported a novel large particulate antigen, in which Sepharose 4B beads were coated with Sj22.6/26GST. Our results showed that this particulate antigen could be cross-presented by DCs to CD8(+)T cells. Furthermore, CD8(+)T cells stimulated by particulate antigen directly exerted cytotoxicity against Schistosoma japonicum schistosomula. We also demonstrated that S. japonicum schistosomula acquired the MHC class I molecules from host blood serum and presented the molecules at the larval surface. While it may help them escape from the host immune surveillance, these MHC I-antigen complexes presented on the surface render schistosomula the potential targets of the CD8(+)T cell cytotoxicity induced by particulate antigen-based vaccine. Finally we evaluated the protective immunity of this particulate vaccine in a mouse infection challenge model. Our data clearly showed that the particulate vaccine induced a partial reduction in both worm burdens and egg loads. Taken together, these results suggest that this large particulate vaccine could be a potential vaccine for the prevention of schistosome infection.

Identification of phage display peptides with affinity for the tegument of Schistosoma japonicum schistosomula

Peptides, bound to the tegument of live Schistosoma japonicum schistosomula, were differentially screened by phage display in vitro using three rounds of reverse absorption and bio-panning. Three M13 phage peptides were isolated and identified by determination of their recovery rate, immunohistochemical localization, immunoblot analysis, and their anti-schistosomal effects in vivo and in vitro. Of the three, M13 phage peptide ZL4 (MppZL4, YSGLQDSSLRLR, 1.4 kDa, p I 8.8) bound to the tegument of mechanically transformed schistosomula and to other developmental stages of S. japonicum from the mammalian host. By contrast, MppZL4 did not bind to the surface of cercariae. To further examine its binding properties, MppZL4 was conjugated to Rhodamine B (RhB-YSGLQDSSLRLR, RhB-ZL4) and a peptide control (RhB-AIPYFSGILQWR, RhB-12P) was similarly synthesized. The binding capacities of RhB-ZL4 to the surface membrane of S. japonicum schistosomula in vitro and of S. japonicum adult worms in vivo were examined and revealed specificity for binding. When examined for anti-parasite activity, both MppZL4 and RhB-ZL4 exhibited a potent schistosomicidal effect in vitro. Further MppZL4 also affected the growth and development of schistosomula in vivo. These findings extend previous studies showing that phage display techniques can recover polypeptides that bind specifically to living schistosomes and, moreover, that these bound peptides have the potential to inhibit key physiological processes in these parasites. Our findings suggest further that ectogenic polypeptides, which can bind to the tegument of S. japonicum, might be adapted as vectors to deliver experimental probes and/or pharmacologically relevant compounds to the schistosome tegument, including drugs and immunological mediators.

Migrating Schistosoma japonicum schistosomula induce an innate immune response and wound healing in the murine lung

The migrating schistosomulum is an important stage of the schistosome lifecycle and represents a key target for elimination of infection by natural and vaccine-induced host immune responses. To gain a better understanding of how schistosomes initiate a primary host immune response we have characterised the host lung response to migrating Schistosoma japonicum schistosomula using a combination of histopathology, microarray analysis and real-time PCR. Our findings indicate that the early pulmonary response to these migrating larvae is characteristic of innate inflammation and wound healing. This response is associated with significant up-regulation of several genes with immunoregulatory function including Ch25h, Hmox1 and Retnla which may act to control the nature or magnitude of the inflammatory response to the migrating schistosomula, promoting both parasite and host survival. These findings contribute to our understanding of host–parasite interactions associated with schistosome and, especially, S. japonicum infection, and may aid the future design of novel vaccines that target the lung stage schistosomulum.

Peroxiredoxin-1 from Schistosoma japonicum functions as a scavenger against hydrogen peroxide but not nitric oxide

Three peroxiredoxins (Prxs) are expressed during most of the developmental stages in the schistosome. Prx-1 is localized on the surface of the schistosomula and adults of Schistosoma japonicum, while Prx-2 is localized in the sub-tegumental tissues, parenchyma, vitelline glands, and gut epithelium, but not on the surface of the worms. We applied RNA interference techniques to suppress the specific genes of S. japonicum Prxs. Schistosomula of S. japonicum were cultured together with long-dsRNA encoding Prx-1 and Prx-2 of S. japonicum (the soaking method). The transcription level of each Prx gene was reduced by an RNA interference (RNAi)-mediated effect specifically. Although neither Prx was the essential protein for survival of S. japonicum schistosomula, Prx-1 dsRNA-treated larvae were susceptible to hydrogen peroxide. Moreover, these larvae were also susceptible to t-butyl hydroperoxide and cumene-hydroperoxide. However, the knockdown of neither Prx-1 nor Prx-2 influenced the resistance against nitric oxide generated from DETA/NO. Prx-1 may work as a scavenger against reactive oxygen species (ROS) generated outside of the schistosomes to prevent the oxidation of the bodies and/or the attack by immune cells producing the ROS. These findings suggest that Prx-1 may become a novel target of drugs and vaccines for schistosomiasis.

Identification and characterization of a polysaccharide-containing antigen fromSchistosoma mansonieggs which cross-reacts with the surface of schistosomula

Antisera were produced by immunizing rabbits with either a trichloroacetic acid-soluble fraction, or a high molecular weight (Mr) fraction of Schistosoma mansoni SEA (a saline-soluble fraction of homogenized egg). Both of these sera reacted monospecifically in immunoelectrophoresis against unfractionated SEA, recognizing a cathodally migrating antigen. This antigen had been identified previously as being responded to by S. mansoni-infected mouse sera, and has been designated K3 (Kappa 3). The rabbit antisera were used to partially characterize antigen K3 as having Mr in the range greater than 750-70 K, and being resistant to boiling, resistant to the action of proteases, but sensitive to periodate. It partially binds to Concanavalin A. In addition to SEA, the antigen was present in homogenized cercariae and schistosomula, but not adult worms, and it was also present in detergent extracts of intact cercariae and schistosomula. Using an antibody-dependent cell adherence assay, anti-K3 serum was found to react with the surface of live cercariae and with the surface of schistosomula recovered from the skin of mice infected up to 48 h previously. Anti-K3 serum also reacted with the surface of S. bovis, S. haematobium and to a lesser extent S. japonicum schistosomula.

Lack of resistance to Schistosoma japonicum in mice immunized with irradiated S. mansoni cercariae

Mice immunized with irradiated Schistosoma mansoni cercariae were resistant to challenge with S. mansoni cercariae (mean resistance 53%) but not to challenge with S. japonicum cercariae (mean resistance −5%). Furthermore, the antibodies induced by vaccination with irradiated S. mansoni cercariae were more reactive with S. mansoni than with S. japonicum schistosomula. These results support the concept that the resistance induced by vaccination with irradiated cercariae is immunologically specific.

Rejection of Mouse-Derived Schistosoma japonicum and Serum Lethality of Hosts Immunized with Mouse Globulin or Mouse Erythrocytes

Fourteen-day-old, mouse-derived Schistosoma japonicum schistosomula survived, matured, and deposited viable eggs following surgical transfer to the hepatic portal system of rabbits. The survival of such schistosomula is essentially eliminated in rabbits immunized with either mouse erythrocytes or mouse gamma globulin. Sera from rabbits immunized with either mouse erthrocytes or mouse gamma globulin in the presence of normal leukocytes is lethal for mouse-derived schistosomula 14 days of age or older. These sera have no significant effect on newly transformed schistosomula or those derived from rabbits. It is suggested that these results give additional evidence that immunological enhancement is one of the mechanisms involved in parasite survival.