MicroRNA-1 targets ribosomal protein genes to regulate the growth, development and reproduction of Schistosoma japonicum.

Eggs laid by mature female schistosomes are primarily responsible for the pathogenesis of schistosomiasis and critical for transmission. Consequently, elucidating the mechanism of sexual maturation as well as egg production may lead to new strategies for the control of schistosomiasis. MicroRNAs (miRNAs) are involved in multiple biological processes including reproduction in many organisms, yet their roles have not been well characterized in schistosomes. Here, we investigated microRNA-1 (miR-1), which was downregulated gradually in both male and female Schistosoma japonicum after they reached sexually maturity. The expression of miR-1, as shown with quantitative reverse transcription PCR (qRT-PCR), was lower in the reproductive organs of adult females compared with the somatic tissues. Overexpression of miR-1 in adult worms destroyed the morphological architecture of reproductive organs and reduced the subsequent oviposition, which may be due to the activation of apoptosis pathways. Through in silico analysis, 34 potential target genes of miR-1 were identified, including five ribosomal protein genes, called rp-s13, rp-l7ae, rp-l14, rp-l11 and rp-s24e. In vitro dual-luciferase reporter gene assays and miRNA overexpression experiments further validated that these ribosomal protein genes were directly regulated by miR-1. In contrast to the gene expression of miR-1, qRT-PCR and in situ hybridization experiments demonstrated these ribosomal protein genes were enriched in the sexual organs of adult females. Using RNA interference to silence the ribosomal protein genes in different developmental stages in a mouse model system, we demonstrated that these miR-1 target genes not only participated in the reproductive development of S. japonicum, but also were required for the growth and survival of the parasite in the early developmental stages. Taken together, our data suggested that miR-1 may affect the growth, reproduction and oviposition of S. japonicum by targeting the ribosomal protein genes, which provides insights for exploration of new anti-schistosome strategies.

Inhibition of signal peptidase complex expression affects the development and survival of Schistosoma japonicum.

Background: Schistosomiasis, the second most neglected tropical disease defined by the WHO, is a significant zoonotic parasitic disease infecting approximately 250 million people globally. This debilitating disease has seriously threatened public health, while only one drug, praziquantel, is used to control it. Because of this, it highlights the significance of identifying more satisfactory target genes for drug development. Protein translocation into the endoplasmic reticulum (ER) is vital to the subsequent localization of secretory and transmembrane proteins. The signal peptidase complex (SPC) is an essential component of the translocation machinery and functions to cleave the signal peptide sequence (SP) of secretory and membrane proteins entering the ER. Inhibiting the expression of SPC can lead to the abolishment or weaker cleavage of the signal peptide, and the accumulation of uncleaved protein in the ER would affect the survival of organisms. Despite the evident importance of SPC, in vivo studies exploring its function have yet to be reported in S. japonicum. Methods: The S. japonicum SPC consists of four proteins: SPC12, SPC18, SPC22 and SPC25. RNA interference was used to investigate the impact of SPC components on schistosome growth and development in vivo. qPCR and in situ hybridization were applied to localize the SPC25 expression. Mayer's carmalum and Fast Blue B staining were used to observe morphological changes in the reproductive organs of dsRNA-treated worms. The effect of inhibitor treatment on the worm's viability and pairing was also examined in vitro. Results: Our results showed that RNAi-SPC delayed the worm's normal development and was even lethal for schistosomula in vivo. Among them, the expression of SPC25 was significantly higher in the developmental stages of the reproductive organs in schistosomes. Moreover, SPC25 possessed high expression in the worm tegument, testes of male worms and the ovaries and vitellarium of female worms. The SPC25 knockdown led to the degeneration of reproductive organs, such as the ovaries and vitellarium of female worms. The SPC25 exhaustion also reduced egg production while reducing the pathological damage of the eggs to the host. Additionally, the SPC-related inhibitor AEBSF or suppressing the expression of SPC25 also impacted cultured worms' pairing and viability in vitro. Conclusions: These data demonstrate that SPC is necessary to maintain the development and reproduction of S. japonicum. This research provides a promising anti-schistosomiasis drug target and discovers a new perspective on preventing worm fecundity and maturation.

A new ferritin SjFer0 affecting the growth and development of Schistosoma japonicum.

BACKGROUND: Schistosomiasis, an acute and chronic parasitic disease, causes substantial morbidity and mortality in tropical and subtropical regions of the world. Iron is an essential constituent of numerous macromolecules involving in important cellular reactions in virtually all organisms. Trematodes of the genus Schistosoma live in iron-rich blood, feed on red blood cells and store abundant iron in vitelline cells. Ferritins are multi-meric proteins that store iron inside cells. Three ferritin isoforms in Schistosoma japonicum are known, namely SjFer0, SjFer1 and SjFer2; however, their impact on the growth and development of the parasites  is still unknown. In this study we report on and characterize the ferritins in S. japonicum. METHODS: A phylogenetic tree of the SjFer0, SjFer1 and SjFer2 genes was constructed to show the evolutionary relationship among species of genus Schistosoma. RNA interference in vivo was used to investigate the impact of SjFer0 on schistosome growth and development.  Immunofluorescence assay was applied  to localize the expression of the ferritins. RNA-sequencing was performed  to characterize the iron transport profile after RNA interference. RESULTS: SjFer0 was found to have low similarity with SjFer1 and SjFer2 and contain an additional signal peptide sequence. Phylogenetic analysis revealed that SjFer0 can only cluster with some ferritins of other trematodes and tapeworms, suggesting that this ferritin branch might be unique to these parasites. RNA interference in vivo showed that SjFer0 significantly affected the growth and development of schistosomula but did not affect egg production of adult female worms. SjFer1 and SjFer2 had no significant impact on growth and development. The immunofluorescence study showed that SjFer0 was widely expressed in the somatic cells and vitelline glands but not in the testicle or ovary. RNA-sequencing indicated  that, in female, the ion transport process and calcium ion binding function were downregulated after SjFer0 RNA interference. Among the differentially downregulated genes, Sj-cpi-2, annexin and insulin-like growth factor-binding protein may be accounted for the suppression of schistosome growth and development. CONCLUSIONS: The results indicate that SjFer0 affects the growth and development of schistosomula but does not affect egg production of adult female worms. SjFer0 can rescue the growth of the fet3fet4 double mutant Saccharomyces cerevisiae (strain DEY1453), suggesting being able to promote iron absorption. The RNA interference of SjFer0 inferred that  the suppression of worm growth and development may via  down-regulating Sj-cpi-2, annexin, and IGFBP.

A chromosome-level genome of the human blood fluke Schistosoma japonicum identifies the genomic basis of host-switching.

The evolution and adaptation of S. japonicum, a zoonotic parasite that causes human schistosomiasis, remain unclear because of the lack of whole-genome data. We construct a chromosome-level S. japonicum genome and analyze it together with 72 samples representing six populations of the entire endemic region. We observe a Taiwan zoophilic lineage splitting from zoonotic populations ∼45,000 years ago, consistent with the divergent history of their intermediate hosts. Interestingly, we detect a severe population bottleneck in S. japonicum, largely coinciding with human history in Asia during the last glacial maximum. We identify several genomic regions underlying natural selection, including GATAD2A and Lmln, both showing remarkable differentiation among different areas. RNAi knockdown suggests association of GATAD2A with parasite development and infection in definitive hosts, while Lmln relates to the specificity of the intermediate hosts. Our study provides insights into the evolution of S. japonicum and serves as a resource for further studies.

Comparative transcriptome profiles of Schistosoma japonicum larval stages: Implications for parasite biology and host invasion.

Schistosoma japonicum is prevalent in Asia with a wide mammalian host range, which leads to highly harmful zoonotic parasitic diseases. Most previous transcriptomic studies have been performed on this parasite, but mainly focus on stages inside the mammalian host. Moreover, few larval transcriptomic data are available in public databases. Here we mapped the detailed transcriptome profiles of four S. japonicum larval stages including eggs, miracidia, sporocysts and cercariae, providing a comprehensive development picture outside of the mammalian host. By analyzing the stage-specific/enriched genes, we identified functional genes associated with the biological characteristic at each stage: e.g. we observed enrichment of genes necessary for DNA replication only in sporocysts, while those involved in proteolysis were upregulated in sporocysts and/or cercariae. This data indicated that miracidia might use leishmanolysin and neprilysin to penetrate the snail, while elastase (SjCE2b) and leishmanolysin might contribute to the cercariae invasion. The expression profile of stem cell markers revealed potential germinal cell conversion during larval development. Additionally, our analysis indicated that tandem duplications had driven the expansion of the papain family in S. japonicum. Notably, all the duplicated cathepsin B-like proteases were highly expressed in cercariae. Utilizing our 3rd version of S. japonicum genome, we further characterized the alternative splicing profiles throughout these four stages. Taken together, the present study provides compressive gene expression profiles of S. japonicum larval stages and identifies a set of genes that might be involved in intermediate and definitive host invasion.

Allergen-Specific Treg Cells Upregulated by Lung-Stage S. japonicum Infection Alleviates Allergic Airway Inflammation.

Schistosoma japonicum infection showed protective effects against allergic airway inflammation (AAI). However, controversial findings exist especially regarding the timing of the helminth infection and the underlying mechanisms. Most previous studies focused on understanding the preventive effect of S. japonicum infection on asthma (infection before allergen sensitization), whereas the protective effects of S. japonicum infection (allergen sensitization before infection) on asthma were rarely investigated. In this study, we investigated the protective effects of S. japonicum infection on AAI using a mouse model of OVA-induced asthma. To explore how the timing of S. japonicum infection influences its protective effect, the mice were percutaneously infected with cercaria of S. japonicum at either 1 day (infection at lung-stage during AAI) or 14 days before ovalbumin (OVA) challenge (infection at post-lung-stage during AAI). We found that lung-stage S. japonicum infection significantly ameliorated OVA-induced AAI, whereas post-lung-stage infection did not. Mechanistically, lung-stage S. japonicum infection significantly upregulated the frequency of regulatory T cells (Treg cells), especially OVA-specific Treg cells, in lung tissue, which negatively correlated with the level of OVA-specific immunoglobulin E (IgE). Depletion of Treg cells in vivo partially counteracted the protective effect of lung-stage S. japonicum infection on asthma. Furthermore, transcriptomic analysis of lung tissue showed that lung-stage S. japonicum infection during AAI shaped the microenvironment to favor Treg induction. In conclusion, our data showed that lung-stage S. japonicum infection could relieve OVA-induced asthma in a mouse model. The protective effect was mediated by the upregulated OVA-specific Treg cells, which suppressed IgE production. Our results may facilitate the discovery of a novel therapy for AAI.

Schistosoma japonicum cathepsin B2 (SjCB2) facilitates parasite invasion through the skin.

Cercariae invasion of the human skin is the first step in schistosome infection. Proteases play key roles in this process. However, little is known about the related hydrolytic enzymes in Schistosoma japonicum. Here, we investigated the biochemical features, tissue distribution and biological roles of a cathepsin B cysteine protease, SjCB2, in the invasion process of S. japonicum cercariae. Enzyme activity analysis revealed that recombinant SjCB2 is a typical cysteine protease with optimum temperature and pH for activity at 37°C and 4.0, respectively, and can be totally inhibited by the cysteine protease inhibitor E-64. Immunoblotting showed that both the zymogen (50 kDa) and mature enzyme (30.5 kDa) forms of SjCB2 are expressed in the cercariae. It was observed that SjCB2 localized predominantly in the acetabular glands and their ducts of cercariae, suggesting that the protease could be released during the invasion process. The protease degraded collagen, elastin, keratin, fibronectin, immunoglobulin (A, G and M) and complement C3, protein components of the dermis and immune system. In addition, proteomic analysis demonstrated that SjCB2 can degrade the human epidermis. Furthermore, it was showed that anti-rSjCB2 IgG significantly reduced (22.94%) the ability of the cercariae to invade the skin. The cysteine protease, SjCB2, located in the acetabular glands and their ducts of S. japonicum cercariae. We propose that SjCB2 facilitates skin invasion by degrading the major proteins of the epidermis and dermis. However, this cysteine protease may play additional roles in host-parasite interaction by degrading immunoglobins and complement protein.

Oncomelania hupensis retains its ability to transmit Schistosoma japonicum 13 years after migration from permissive to non-permissive areas.

BACKGROUND: The East Route Project (ERP) of the South-to-North Water Diversion Project (SNWDP) stretches across schistosomiasis endemic and non-endemic areas in China, which may lead to the dispersal of Oncomelania hupensis, the intermediate host of Schistosoma japonicum, from permissive areas along the Yangtze River Basin to non-permissive areas in northern China. A previous survey demonstrated that O. hupensis could survive and breed for 13 years (12 generations) after being transferred to a non-permissive area, and could be infected by S. japonicum. However, it is not clear if the migrated snails will change their ability to transmit S. japonicum. METHODS: We infected mice with the cercariae released from the infected transferred snails bred in Jining city of Shandong Province (non-permissive areas) for 13 years. The mice in the control group were infected with cercariae derived from the snails collected in their original habitat (Jiangdu county of Jiangsu Province, permissive areas). Then, we explored the pathogenicity to mice including worm burden, liver egg count and pathology. Additionally, the gene expression profiles of the adult male and female worms recovered from the infected mice were analyzed by RNA sequencing. RESULTS: The worm burden, liver egg count and pathology of the mice infected with cercariae released from transferred snails bred in non-permissive areas for 13 years showed no significant differences, when compared with the control cercariae. Slight changes occurred at the transcription level between adult male and female worms recovered from mice infected with cercariae derived from snails bred in permissive and non-permissive areas. Only fourteen genes were significantly differentially expressed in the comparison of adult female worms, and no significantly differentially expressed gene was found in the comparison of adult male worms. CONCLUSIONS: Our findings strongly suggest that transferred snails did not change their schistosomiasis transmission ability and the worms derived from them retained the original pathogenicity, even after migrating from permissive to non-permissive areas for 13 years. Therefore, a long-term surveillance system of snails along the SNWDP is urgently needed to prevent the diffusion of O. hupensis and reduce the risk of transmission of schistosomiasis.

An improved genome assembly of the fluke Schistosoma japonicum.

BACKGROUND: Schistosoma japonicum is a parasitic flatworm that causes human schistosomiasis, which is a significant cause of morbidity in China and the Philippines. A single draft genome was available for S. japonicum, yet this assembly is very fragmented and only covers 90% of the genome, which make it difficult to be applied as a reference in functional genome analysis and genes discovery. FINDINGS: In this study, we present a high-quality assembly of the fluke S. japonicum genome by combining 20 G (~53X) long single molecule real time sequencing reads with 80 G (~ 213X) Illumina paired-end reads. This improved genome assembly is approximately 370.5 Mb, with contig and scaffold N50 length of 871.9 kb and 1.09 Mb, representing 142.4-fold and 6.2-fold improvement over the released WGS-based assembly, respectively. Additionally, our assembly captured 85.2% complete and 4.6% partial eukaryotic Benchmarking Universal Single-Copy Orthologs. Repetitive elements account for 46.80% of the genome, and 10,089 of the protein-coding genes were predicted from the improved genome, of which 96.5% have been functionally annotated. Lastly, using the improved assembly, we identified 20 significantly expanded gene families in S. japonicum, and those genes were primarily enriched in functions of proteolysis and protein glycosylation. CONCLUSIONS: Using the combination of PacBio and Illumina Sequencing technologies, we provided an improved high-quality genome of S. japonicum. This improved genome assembly, as well as the annotation, will be useful for the comparative genomics of the flukes and more importantly facilitate the molecular studies of this important parasite in the future.

Screening for biomarkers reflecting the progression of Babesia microti infection.

BACKGROUND: Babesiosis is caused by the invasion of erythrocytes by parasites of the Babesia spp. Babesia microti is one of the primary causative agents of human babesiosis. To better understand the status of the disease, discovering key biomarkers of the different infection stages is crucial. RESULTS: This study investigated B. microti infection in the mouse model from 0 to 270 days post-infection (dpi), using blood smears, PCR assays and ELISA. PCR assays showed a higher sensitivity when compared to microscopic examination. Specific IgG antibodies could be detected from 7 days to 270 dpi. Two-dimensional electrophoresis was combined with western blotting and mass spectrometric analysis to screen for specific reactive antigens during both the peak parasitaemia period (7 dpi) and IgG antibody response peak period (30 dpi) by the infected mice plasma. The 87 positive reactive proteins were identified and then expressed with the wheat germ cell-free system. Protein microarrays of all 87 targeted proteins were produced and hybridized with the serial plasma of infected mice model. Based on the antigen reaction profile during the infection procedure, 6 antigens were selected and expressed in Escherichia coli. Due to an early response to IgM, lower immunoreactivity levels of IgG after two months and higher immunoreactivity level IgG during nine months, four recombinant proteins were selected for further characterization, namely rBm2D97(CCF75281.1), rBm2D33(CCF74637.1), rBm2D41(CCF75408.1) and rBm7(CCF73510.1). The diagnostic efficacy of the four recombinant protein candidates was evaluated in a clinical setting using babesiosis patient plasma. The rBm2D33 showed the highest sensitivity with a positive rate of 62.5%. Additional characterization of the two candidate proteins using a mouse vaccination assay, demonstrated that rBm2D41 could reduce peak parasitaemia by 37.4%, indicating its efficacy in preventing severe babesiosis. CONCLUSIONS: The detection technologies of microscopic examination, PCR assays and antibody tests showed different sensitivities and accuracy during the different stages of B. microti infection. Antibody detection has a unique significance for B. microti infection in the asymptomatic stages. Using immunoreactivity profiles, biomarkers for disease progression were identified and represent useful information for future the diagnosis and vaccine development for this serious disease of public health significance.

Detection of circulating antigen in serum of mice infected with Schistosoma japonicum by immunomagnetic bead ELISA based on IgY.

We developed a novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) for detection of circulating antigen (CA) in sera of mice infected with Schistosoma japonicum. The assay involved the use of chicken polyclonal antibodies IgY against soluble egg antigens (SEA) of S. japonicum as a capture antibody and anti-SEA mouse monoclonal antibody NP28-5B labeled horseradish peroxidase (HRP) as a detecting antibody. Two groups of BALB/c mice infected with S. japonicum cercariae were used: lightly infected mice (infected with 10 S. japonicum cercariae) and heavily infected mice (infected with 30 S. japonicum cercariae). The CA was detectable as early as 4 and 5 weeks after infection in the sera of heavily and lightly infected mice, respectively. The CA levels rose rapidly and reached a peak in 8 weeks after infection and then remained a plateau for at least another 6 weeks in both groups. Moreover, the effect of praziquantel on the CA levels was also investigated. The heavily infected mice were treated with praziquantel and the CA levels in sera increased dramatically in the first week post-treatment and then decreased to the control level by 6 weeks after treatment. The novel assay appears to be sensitive for detection of schistosomal antigenemia and valuable to judge the efficacy of chemotherapy in murine schistosomiasis.