Studies On The Mechanisms Of Parasite-host Interaction Mediated Exosomes Secreted By S. Japonicum

Dioecious flatworms of the genus Schistosoma cause schistosomiasis, which is a major public health problem in developing countries. In China, schistosomiasis japonica is a serious public health problem. Knowledges of the parasite-host interaction are still limited. In recent studies, evidences proved that secreted extracellular vesicles play an important role in pathogen-host interactions. Given this research, we hypothesized that schistosome exosomes may be involved in the regulation of schistosome-host interaction. In this study, the main composition of exosomes secreted by S. japonicum was identified. Subsequently, intercellular transfer of parasite information via S.japonicum exosomes were measured by cellular biology methods combined with microtechnology. Then cytokine assay and mRNA profile assay were used to study the influence of S. japonicum exosomes in immune response. Finally, role of miRNA Bantam in schistosome development and sexual maturation were determined by miRNA inhibition electroporation and confocal laser scanning microscopy analyses. Our results are expected to provide new insights for further understanding the mechanisms of schistosome-host interaction that may facilitate in the development of novel interventions for alleviating the symptom of S. japonicum infection as well as for preventing and treating schistosomiasis.1. Identification of S. japonicum exosome-like vesicles (EVs)We developed a protocol for exosome isolation using dialysis, ultrafiltration, and ultracentrifugation combined with a commercial kit. Isolated EVs were subjected to morphological analyses using TEM. The EVs showed round-shaped vesicles of approximately 50-100 nm in diameter. The protein components of S. japonicum EVs were determined by shotgun LC-MS/MS analyses. A total of 403 proteins were identified in S. japonicum EVs, and bioinformatics analyses indicated that these proteins were mainly involved in binding, catalytic activity, and translation regulatory activity. Next, we identified the small RNA population associated with S. japonicum EVs using Solexa deep sequencing. We found that 15 known S. japonicum miRNAs (cut-off reads>100) were present in S.japonicum EVs libraries. Interestingly, we found that there were 1,284,966 reads (3.4%,2,540 unique small RNAs) specifically mapped to the rabbit genome, including one miRNA, a ocu-miR-191-5p, with a relatively high number of reads (4,548 reads). These findings suggested that S. japonicum EVs could carry both host and schistosoma factors that could be involved in the pathogenesis of schistosomiasis and/or the regulation of schistosome development.2. S. japonicum EVs regulate the function of host liverWe labeled S. japonicum EVs with the lipid dye PKH67 and incubated the labeled exosomes with mouse liver cells (NCTC clone 1469 cells) in vitro. S. japonicum EVs were internalized by the NCTC clone 1469 cells. Further analyses demonstrated that S. japonicum EV associated miRNAs (Bantam and miR-10) were significantly detected and their potential host targets were downregulated in the RNA isolated form cells treated with labeled S. japonicum EVs, indicating that the miRNA cargo of S. japonicum EVs can transfer to recipient cells. Overall, these results indicated that uptake of S. japonicum secreted EVs could potentially regulate mouse genes via S. japonicum originating miRNAs.3. S. japonicum EVs regulate the function of host immunity responseS. japonicum exosomal protein and RNA were labeled respectively, and incubated with macrophage Raw264.7 cells in vitro. ELISA and qRT-PCR analyses were used to measure the immunity associated cytokines after S. japonicum EVs incubation. Then the mRNA profiles were analyzed by deep sequencing. In vivo, asthmatic bronchitis models were build, serum content of cytokines were measured after S. japonicum EVs injection. Further analyses demonstrated that S. japonicum EV could be internalized by Raw264.7 cells and stimulate the release of IL-13, TNF and IL-5 via the Th2 response.4. S japonicum exosomes associated Bantam directly targets CLMP to regulate TNF signaling pathway.Here, we performed bioinformatics analyses to predict the target mRNAs for a specific S. japonicum associated miRNA, Bantam miRNA.3 potential targets (CLMP, FAM212B, and PRTG of Bantam were also down-regulated in macrophages after S. japonicum EVs incubation were selected. Then, we determined the expressions of these 3 genes in the cells treated with S. japonicum EVs and in animal tissues (in the livers and spleens of mice infected with S. japonicum cercariae at 28 and 35 d post-infection). We observed significantly (P<0.05) down-regulated expression of these genes in vitro and in animal level. Further Luciferase assays revealed that transfection of bantam miRNA mimics resulted in a reduction of luciferase activity compared to transfection with scrambled miRNA mimics, indicating that this schistosoma specific miRNAs. At last, we transfected Bantam mimics to Raw264.7, and found that Bantam transfection in in vitro cultured cells could inhibits cell proliferation, promotes cell apoptosis, and down-regulates its host targets in vitro. As a TNF signaling pathway has been associated with CLMP, we examined whether miR-Bantam promotes TNF signaling pathway. We then examined whether silencing of miR-Bantam could impede the ability of cells to undergo cell death. Overall, these results indicated that S. japonicum infection regulate host immunity via schistosoma-specific miRNA Bantam mediated TNF signaling pathway pathway5. S. japonicum exosomal miRNA regulate parasite growthTo determine the role of miRNAs in schistosome development and sexual maturation, the miRNA inhibitor (Bantam inhibitor) were transfected into female schistosomes by electroporation. Worms were collected at 2d,4d, and 6d of post transfection, and the expression of Bantam and its targets were determined by qRT-PCR. In addition, the confocal laser scanning microscopy was employed to observe the morphological changes of female schistosomes. We found that inhibition of Bantam miRNA in female schistosomes resulted in the desruption in ovary, and increase in Bantam associated targets in transcription level, suggesting that miRNA Bantam and its targets might poteitial new drug targets for schitosomiasis control.