Qualitative and quantitative proteomic analyses of Schistosoma japonicum eggs and egg-derived secretory-excretory proteins.

BACKGROUND: Schistosome parasites lay up to a thousand eggs per day inside the veins of their mammalian hosts. The immature eggs deposited by females against endothelia of venules will embryonate within days. Approximately 30% of the eggs will migrate to the lumen of the intestine to continue the parasite life-cycle. Many eggs, however, are trapped in the liver and intestine causing the main pathology associated with schistosomiasis mansoni and japonica, the liver granulomatous response. Excretory-secretory egg proteins drive much of egg-induced pathogenesis of schistosomiasis mansoni, and Schistosoma japonicum induce a markedly distinct granulomatous response to that of S. mansoni. METHODS: To explore the basis of variations in this responsiveness, we investigated the proteome of eggs of S. japonicum. Using mass spectrometry qualitative and quantitative (SWATH) analyses, we describe the protein composition of S. japonicum eggs secretory proteins (ESP), and the differential expression of proteins by fully mature and immature eggs, isolated from faeces and ex vivo adults. RESULTS: Of 957 egg-related proteins identified, 95 were exclusively found in S. japonicum ESP which imply that they are accessible to host immune system effector elements. An in-silico analysis implies that ESP are able of stimulating the innate and adaptive immune system through several different pathways. While quantitative SWATH analysis revealed 124 proteins that are differentially expressed by mature and immature S. japonicum eggs, illuminating some important aspects of eggs biology and infection, we also show that mature eggs are more likely than immature eggs to stimulate host immune responses. CONCLUSIONS: Here we present a list of potential targets that can be used to develop better strategies to avoid severe morbidity during S. japonicum infection, as well as improving diagnosis, treatment and control of schistosomiasis japonica.

In vitro cultivation of Schistosoma japonicum-parasites and cells.

Schistosomiasis is a serious parasitic zoonosis caused by blood-dwelling flukes of the genus Schistosoma. Understanding functions of genes and proteins of this parasite is important for uncovering this pathogen's complex biology, which will provide valuable information to design new strategies for schistosomiasis control. Effective applications of molecular tools reported to investigate schistosome gene function, such as inhibitor studies and transgenesis, rely on the developments of in vitro cultivation system of this parasite and cells. Besides the in vitro culture studies dealing with Schistosoma mansoni, there are also numerous excellent studies about the in vitro cultivation of Schistosoma japonicum, which were performed by Chinese researchers and published in Chinese journals. Nearly every stage of the life-cycle of S. japonicum, including miracidia, mother sporocysts, cercariae, schistosomula, and egg-laying adult worms, was employed for developing in vitro cultivation methods, being accompanied by the introduction of several media and supplements that helped to improve culture conditions. It was not only possible to generate mother sporocysts from miracidia in vitro, but also to obtain adult worms from cercariae through in vitro cultivation. The main obstacles to complete the life cycle of S. japonicum in the lab are the transition from mother sporocysts to cercariae, and the production of fertilized and completely developed eggs by adult worms generated in vitro. With regard to cells from S. japonicum, besides established isolation protocols and morphological observations, media optimizations were conducted by using different chemical reagents, biological supplements and physical treatment. Among these, mutagens like N-methyl-N-nitro-N-nitrosoguanidine and the addition of extracellular matrix were found to be able to induce mitogenic activities. Although enzyme activities or the level of silver-stained nucleolar region associated protein in cultured cells indicated still suboptimal conditions, the achievements made point to the possibility of reaching the aim of establishing cell lines for S. japonicum. Both the improvements of the in vitro culture of larval and adult worms of S. japonicum as well as the access of cells of this parasite provide excellent advances for research on this important parasite in the future.

Synthesis of fluorescent derivatives of praziquantel: cell-imaging and interaction with Schistosoma japonicum cercariae.

Schistosomiasis is one of the most burdensome of the neglected tropical diseases. Praziquantel is a recommended drug for treatment against all forms of schistosomiasis. To investigate the interaction between praziquantel and Schistosoma japonicum cercariae, two praziquantel derivatives (PZQ-2 and PZQ-3) and one praziquantel fluorescent derivative (PZQ-5) have been synthesized and characterized using nuclear magnetic resonance spectroscopy (NMR) and MS spectra. The cytotoxicity of PZQ-2, PZQ-3 and PZQ-5 was measured by performing the methyl thiazolyl tetrazolium (MTT) assay. The cell viability for them shows that the three compounds exhibit low cytotoxicity to HeLa cells. Cell imaging experiments demonstrate that PZQ-5 is biocompatible and cell-permeable, which indicates that PZQ-5 is suitable for studying their interaction. Confocal fluorescence microscopy revealed that PZQ-5 is mainly located at the cercarial tegument, which leads to the death of cercariae with the increase in time.

3-oxoacyl-ACP reductase from Schistosoma japonicum: integrated in silico-in vitro strategy for discovering antischistosomal lead compounds.

BACKGROUND: Schistosomiasis is a disease caused by parasitic worms and more than 200 million people are infected worldwide. The emergence of resistance to the most commonly used drug, praziquantel (PZQ), makes the development of novel drugs an urgent task. 3-oxoacyl-ACP reductase (OAR), a key enzyme involved in the fatty acid synthesis pathway, has been identified as a potential drug target against many pathogenic organisms. However, no research on Schistosoma japonicum OAR (SjOAR) has been reported. The characterization of the SjOAR protein will provide new strategies for screening antischistosomal drugs that target SjOAR. METHODOLOGY/PRINCIPAL FINDINGS: After cloning the SjOAR gene, recombinant SjOAR protein was purified and assayed for enzymatic activity. The tertiary structure of SjOAR was obtained by homology modeling and 27 inhibitor candidates were identified from 14,400 compounds through molecular docking based on the structure. All of these compounds were confirmed to be able to bind to the SjOAR protein by BIAcore analysis. Two compounds exhibited strong antischistosomal activity and inhibitory effects on the enzymatic activity of SjOAR. In contrast, these two compounds showed relatively low toxicity towards host cells. CONCLUSIONS/SIGNIFICANCE: The work presented here shows the feasibility of isolation of new antischistosomal compounds using a combination of virtual screening and experimental validation. Based on this strategy, we successfully identified 2 compounds that target SjOAR with strong antischistosomal activity but relatively low cytotoxicity to host cells.

Apoptosis phenomenon in the schistosomulum and adult worm life cycle stages of Schistosoma japonicum.

Apoptosis is an important aspect of a number of biological processes, from embryogenesis to the stress-injury response. It plays a central role in balancing cell proliferation and tissue remodeling activity in many organisms. In the present study, apoptosis in 14 days post infection schistosomula was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays and DAPI staining. Additionally, flow cytometry using the Annexin V-FITC/propidium iodide (PI) (Annexin V/PI) assay confirmed the percentage of early apoptotic, late apoptotic, and necrotic cells in 14 and 23 days post infection worms. Conserved Domain Database (CDD) BLAST analysis and alignment analysis of known schistosome proteins demonstrated the feasibility of detecting the activity of caspase-3 and -7 using the caspase-3/7 Glo analysis assay. Analysis of caspase-3 and -7 activities in schistosome demonstrated that both caspases were active in each developmental stage of Schistosoma japonicum, but was highest in the 14 days post infection schistosomula. Additionally, the caspase peptide inhibitor (Z-VAD-FMK) inhibited the caspase-3/7 activity at all developmental stages examined. Therefore, we hypothesized that two main signaling pathways are involved in apoptosis in S. japonicum, the caspase cascade and the mitochondrial-initiated pathway. We have constructed a model of these two pathways, including how they may interact and their biological outcomes. qRT-PCR analyses of the gene expression profiles of apoptosis-related genes supported our hypothesis of the relationship between the apoptotic pathway and parasite development. The data presented here demonstrates that apoptosis is an important biological process for the survival and development of the schistosome, and identifies potential novel therapeutic targets.

Schistosoma japonicum ova maintains epithelial barrier function during experimental colitis.

AIM: To evaluate the impacts of Schistosoma japonicum (S. japonicum) ova on the tight junction barriers in a trinitrobenzenesulfonic acid (TNBS)-induced colitis model. METHODS: Balb/c mice were randomly divided into three groups: control group; TNBS(+)ova(-) group and TNBS(+)ova(+) group. TNBS was used intracolonic to induce colitis and mice of the TNBS(+)ova(+) group were pre-exposed to S. japonicum ova as a prophylactic intervention. Colon inflammation was quantified using following variables: mouse mortality, weight loss, colon extent and microscopic inflammation score. Serum expression of tumor necrosis factor-α and interferon-γ were assessed to evaluate the systemic inflammatory response. NOD2 and its mRNA were also tested. Bacterial translocations were tested by culturing blood and several tissues. ZO-1 and occludin were chosen as the representations of tight junction proteins. Both the proteins and mRNA were assessed. RESULTS: Ova pre-treatment contributed to the relief of colitis and decreased the mortality of the models. NOD2 expression was significantly downregulated when pretreated with the ova. The TNBS injection caused a significant downregulation of ZO-1 and occludin mRNA together with their proteins in the colon; ova pre-exposure reversed these alterations. Treatment with S. japonicum ova in the colitis model caused lower intestinal bacterial translocation frequency. CONCLUSION: S. japonicum ova can maintain epithelial barrier function through increasing tight junction proteins, thus causing less exposure of NOD2 to the luminal antigens which may activate a series of inflammatory factors and induce colitis.

[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.

Apoptosis governs the elimination of Schistosoma japonicum from the non-permissive host Microtus fortis.

The reed vole, Microtus fortis, is the only known mammalian host in which schistosomes of Schistosoma japonicum are unable to mature and cause significant pathogenesis. However, little is known about how Schistosoma japonicum maturation (and, therefore, the development of schistosomiasis) is prevented in M. fortis. In the present study, the ultrastructure of 10 days post infection schistosomula from BALB/c mice and M. fortis were first compared using scanning electron microscopy and transmission electron microscopy. Electron microscopic investigations showed growth retardation and ultrastructural differences in the tegument and sub-tegumental tissues as well as in the parenchymal cells of schistosomula from M. fortis compared with those in BALB/c mice. Then, microarray analysis revealed significant differential expression between the schistosomula from the two rodents, with 3,293 down-regulated (by ≥ 2-fold) and 71 up-regulated (≥ 2 fold) genes in schistosomula from the former. The up-regulated genes included a proliferation-related gene encoding granulin (Grn) and tropomyosin. Genes that were down-regulated in schistosomula from M. fortis included apoptosis-inhibited genes encoding a baculoviral IAP repeat-containing protein (SjIAP) and cytokine-induced apoptosis inhibitor (SjCIAP), genes encoding molecules involved in insulin metabolism, long-chain fatty acid metabolism, signal transduction, the transforming growth factor (TGF) pathway, the Wnt pathway and in development. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and PI/Annexin V-FITC assays, caspase 3/7 activity analysis, and flow cytometry revealed that the percentages of early apoptotic and late apoptotic and/or necrotic cells, as well as the level of caspase activity, in schistosomula from M. fortis were all significantly higher than in those from BALB/c mice.

In vitro and in vivo evaluation of small interference RNA-mediated gynaecophoral canal protein silencing in Schistosoma japonicum.

BACKGROUND: Schistosomiasis causes liver and intestinal damage and can be very debilitating. The pairing of a male worm with a female worm residing in the gynaecophoral canal of male plays a critical role in the development of female parasite. Because the male specific gynaecophoral canal protein of Schistosoma japonicum (SjGCP) is found in significant quantities in the adult female worm after pairing, it could play an important role in parasite pairing. METHODS: In the present study, three small interfering (si)RNA duplexes targeting the SjGCP gene were designed, synthesized and the silencing effects were evaluated in vitro as well as in mice infected with S. japonicum in vivo. RESULTS: In vitro studies using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR revealed the reduction of SjGCP at the transcript level. Similarly, western blotting and immunofluorescence studies showed its reduction at the protein level after treatment of parasites with siRNAs. At a concentration of 200 nM, two siRNAs totally abolished the parasite pairing. To evaluate such a pairing inhibitory effect in vivo, mice infected with S. japonicum were treated with siRNA and both parasite pairing and burden were evaluated. In vivo tests confirmed the in vitro silencing effect of SjGCP siRNA and revealed that the systemic delivery of siRNA significantly inhibited early parasite pairing and the associated burden. CONCLUSIONS: Our preliminary results demonstrated that the SjGCP plays an important role in pairing and subsequent development in S. japonicum, and its silencing might have potential as a therapeutic approach for controlling schistosomiasis.

Schistosoma japonicum: protective immunity induced by schistosomulum-derived cells in a mouse model.

We previously reported that immunization with intact live cells from schistosomula of Schistosoma japonicum (S.j) partially protected the Kunming strain of mice from challenge infection. In the present work, 2 immune protective experiments were designed to further validate the protective effect induced by this type of vaccine and to optimize the immunization protocol, including the number of inoculations and parasite stages from which immunogenic cells were derived. Three antigens derived from 18-day-old postinfection live (LLC) and dead (DLC) larval worm cells and from dead 42-day-old postinfection adult worm cells (DAC) were used as immunogens. Our results demonstrate that live cells from 18-day-old worms are capable of inducing significant protection in mice using a murine-Sj challenge model as shown by reduction rates of worm recoveries and egg burdens. The development of adult worms was stunted. A Th1-biased immune response was reflected in the protected groups as evidenced by the ratio of IgG2a/IgG1. A 38-kDa polypeptide was recognized by sera from LLC immunized animals. We demonstrate that live parasite cells are a source of novel protective antigens that can be exploited for vaccine development.

Diagnosis of Schistosomiasis japonica infection coincident with hepatocellular carcinoma by fine-needle aspiration.

Schistosomiasis japonica is not an endemic parasite in Taiwan. Previously, there have been no primarily infected cases reported with the exception of Chinese war veterans who had immigrated from Mainland China. We reported a 79-year-old patient who was diagnosed as having non-B, non-C liver cirrhosis complicated with a 1.2 x 1.2 x 1.2 cm liver tumor in the S5 of the liver. For a definitive diagnosis, we performed ultrasonographic-guided fine needle (21 gauge) aspiration biopsy. In the smear of liver aspiration, a calcified fragment of a parasite egg with foreign body reaction adjacent to the crowding cancer cells of hepatocellular carcinoma was found. To our knowledge, parasite eggs are difficult to find within the fibrotic bands using fine needle aspiration biopsy. The etiologic relationship between Schistosomiasis japonica infection and the hepatocellular carcinoma is also discussed in this report.

In vitro and in vivo activities of synthetic trioxolanes against major human schistosome species.

Schistosomiasis is a parasitic disease that remains of considerable public health significance in tropical and subtropical environments. Since the mainstay of schistosomiasis control is chemotherapy with a single drug, praziquantel, drug resistance is a concern. Here, we present new data on the antischistosomal properties of representative synthetic 1,2,4-trioxolanes (OZs). Exposure of adult Schistosoma mansoni for 24 h to a medium containing 20 mug/ml OZ209 reduced worm motor activity, induced tegumental alterations, and killed worms within 72 h. While exposure of S. mansoni to OZ78 had no apparent effect, addition of hemin reduced worm motor activity and caused tegumental damage. Administration of single 200-mg/kg of body weight oral doses of OZ78, OZ209, and OZ288 to mice harboring a juvenile S. mansoni infection resulted in worm burden reductions of 82.0 to 95.4%. In the adult infection model in mice, single 400-mg/kg doses of these compounds resulted in a maximum total worm burden reduction of 52.2%. High worm burden reductions (71.7 to 86.5%) were observed after administration of single 200-mg/kg doses of OZ78 and OZ288 to hamsters infected with either juvenile or adult S. mansoni. A single 200-mg/kg dose of OZ78 to hamsters infected with adult Schistosoma japonicum resulted in total and female worm burden reductions of 94.2 to 100%. Our results, along with the low toxicity, metabolic stability, and good pharmacokinetic properties of the OZs, indicate the potential for the development of novel broad-spectrum antischistosomal OZ drug candidates.

Schistosoma japonicum and S. mansoni cercariae: different effects of protein in medium, of mechanical stress, and of an intact complement system on in vitro transformation to schistosomula.

The cercariae of Schistosoma japonicum were subjected in vitro to treatments known for Schistosoma mansoni to generate schistosomula-like organisms. As a technical prerequisite to pipette or to otherwise handle the sticky cercariae of S. japonicum, the addition of protein to water or medium was found to abolish the stickiness of cercariae of this species. Shearing forces exerted in vitro by syringe (22 G) passage are known since long to fully transform S. mansoni cercariae, but this treatment was found to be much less efficient with S. japonicum. Thus, even with very narrow needles (27 G), complete transformation of cercariae was not obtained with S. japonicum. Complement, provided by fresh human serum, is also well known to induce rapid transformation of S. mansoni cercariae with subsequent killing of the schistosomula. This treatment of S. japonicum cercariae induced degeneration of the tails and strongly promoted the transformation to schistosomula-like organisms, but at a much slower pace. These effects were absent from sera either heat-inactivated or depleted of factor B or of complement component C8, but were restored after adding the purified respective complement components. The schistosomula-like organisms of S. japonicum were not susceptible to lysis after 1 day of in vitro culture in the presence of 50% fresh human serum, although both cercariae and schistosomula of S. mansoni were killed under these conditions. In conclusion, the dynamics of in vitro transformation of S. japonicum cercariae differ significantly from those of S. mansoni, and complement has a major transformation-promoting activity.

The effect of a mutagen (N-methyl-N-nitro-N-nitrosoguanidine) on cultured cells from adult Schistosoma japonicum.

The transforming effect of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) on cultured cells from Schistosoma japonicum (S. japonicum) was studied using mono-factor and orthogonal tests. Under the influence of MNNG, cultured cells grew well, and cell survival time was more than 246 days in low-serum medium. When treated with 3 mug/ml MNNG for 48 h, the number of dividing cells increased significantly as determined by bright-field and scanning electron microscopy (SEM). Under these conditions, abundant microvilli, ruffles, microridges, papillae and blebs were observed on the surface of the induced cells. Treatment with MNNG may overcome existing limitations to get continually proliferating schistosome cells and open the possibility to immortalize isolated cells.

[Biological identification on sub-cultivation cells of Schistosoma japonicum adult worms in vitro].

Cultivation of cells from 30-day old Schistosoma japonicum (S.j) adult worms showed that the growth features of the cells were semi-floating and accumulative. The survival rate of the primary cells, passage cells prior to the 5th generation and recovered cells was all up to 90%. Phases of cell division were observed during cultivation. Chromosome karyotype of the 5th generation cells possessed diploid feature of the blood-flukes (2n=8 in number). Ultrastructure of the 5th generation cells showed that four types of cells in normal morphology and three types of cells in abnormal morphology were both viewed. It is suggested that some of the cells from S.j adult worms were subcultured successfuly in the 1640-40 defined medium.

[Identification of the source and types of cells of Schistosoma japonicum by histochemical staining].

OBJECTIVE: To study the possibility on in vitro identifying the source or kinds of cells from Schistosoma japonicum (S.j). METHODS: The cells from digested tissues of 18 days old schistosomula were smeared on slides. The adult worms of S.j were used for making paraffin sections. The cell smears and tissue sections were stained with 6 different methods of histochemical staining including Periodic Acid-Schiff (PAS), Argyrophil reaction (by Grimeliu's), Picric acid-acid Fuchsin (by Van Gieson, VG), Thionin, Toluidine blue (TB) and Hematoxylin-Eosin (HE) staining parallelly. The results were judged through inspecting the specific color of the cells on smears referring to location of corresponding staining of paraffin sections of adult worm tissues under light microscopy. RESULTS: Vitelline gland cells, mother germ cells, nerve cells, digestive epithelial cells, muscular cells and mast cells were shown clearly. The stainings of VG, PAS and Thionin demonstrated cell types coinciding to histological location. The TB staining did not find red-purple mast cell in tissue sections but one in cell smears. The Grimeliu's argyrophil reaction displayed that intestine wall of tissure sections and stained cells of cell smears were in black clearly. CONCLUSION: HE staining together with histochemical staining can reliably and rapidly distinguish the cell types of Schistosoma japonicum.

[Effect of MNNG on ornithine decarboxylase activity in cells from adult Schistosoma japonicum].

OBJECTIVE: To observe the change of ornithine decarboxylase(ODC) activity in cells from adult Schistosoma japonicum after the cells were treated with N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG). METHODS: The cells were treated with MNNG at a concentration of 3 micrograms/ml for 48 hours after the cells being incubated for one week. The cells were then cultured with RPMI-1640 containing 10% calf serum. ODC activity was detected with spectrophotography. RESULTS: ODC activity rose significantly in two to three weeks after the cells were treated with MNNG. CONCLUSION: There was ODC activity in cells from adult S. japonicum and MNNG has an effect to reinforce ODC activity in the cells.