Proteomics And Small RNA-Omics Analysis Of Extracellular Vesicles Isolated From Adult Female And Male Schistosoma Japonicum

Schistosomiasis caused by parasitic schistosome in humans and animals is mainly distributed in Asia,Africa and Latin America,involving 78 countries and regions,affecting the health and safety of 200 million people,is an zoonotic parasitic disease.Unlike other trematode parasites,schistosomes have evolved separate sexes.Pairing of males and females is a prerequisite for female sexual development and subsequent egg production.However,the mechanisms of these processes remain poorly understood.Extracellular vesicles(EVs)is a collective term for tiny vesicles with membrane structure actively secreted by cells.EVs are rich in a variety of biologically active ingredients,such as proteins,nucleic acids and lipids.These vesicles can be used as intercellular communication media and play an important role in multiple biological processes such as reproductive development,antigen presentation,immune response and tumorigenesis.In the present study,based on the unique development process of Schistosoma japonicum and the important physiological functions of EVs,the following studies were conducted for the EVs isolated from adult S.japonicum:Ⅰ.The EVs of male and female S.japonicum were isolated.In this study,centrifugation and dialysis combined with isolation kit were used to isolate and purify the EVs of female adult(28d)and male adult(28d).Transmission electron microscopy showed that the shape of EVs is around or oval.The vesicle particles are ranged from 100 to 400 nm in size.The Z-Average(d.nm)of female EVs is 111.9±2.081,and the Z-Average(d.nm)of male EVs is 127.6±0.3464.Western blot analysis indicated the EVs marker proteins such as HSP70 and GAPDH are associated wih these isolated EVs.This study provides a fundamental information for further understanding the composition and physiological functions of EVs of S.japonicum adult females and males.Ⅱ.Proteomics analysis of EVs of male and female S.japonicum.In this study,proteomics technology was used to identify and analyze EVs proteins of male and female S.japonicum.The results showed that a total of 1252 proteins were identified in the EVs isolated from male and female S.japonicum,of which 558 were identified in the male EVs and 894 were identified in the female EVs.There are 200 proteins co-identified in male and female EVs.Bioinformatics analysis shows that EVs proteins putatively play roles in catalytic activity,binding and antioxidant activity and other physiological functions.The molecular function of female EVs proteins is significantly enriched in cofactor binding,suggesting that thioredoxin glutathione reductase(TGR)and serine hydroxymethyl transferase(SHMT)may be involved in this process and play an important role.Ⅲ.Small RNA-omics analysis on EVs of male and female S.japonicum.The results of small RNA sequencing showed that the length of small RNA in EVs was mainly 20-24 nt,and tRNA/rRNAs,repeat-associated small RNAs and miRNAs were the main categories.In female EVs,the proportions of tRNA/rRNAs,repeat-associated small RNAs and miRNAs are 57%,5%and 10%,respectively;in males EVs,the proportion of these three is 57%,7%and 5%.A total of 38 known miRNAs of Schistosoma japonicum were identified and in which 18 miRNAs with reads≥500 in at least two biological replicates were validated by RT-qPCR.Both sequencing and RT-qPCR confirmed that Sja-miR-750 was significantly enriched in female EVs,suggesting that female EVs may be selectively loaded with Sja-miR-750.Ⅳ.Sja-miR-750 affects the development of female S.japonicum.Sja-miR-750 is significantly expressed in female ovaries.miR-750 inhibitor is introduced into in vitro cultured S.japonicum females by electroporation.It can be observed that female ovaries led to vacuoles and the number of eggs and the activity of eggs were significantly reduced.Bioinformatics analysis shows that the potential target genes of Sja-miR-750 mainly perform binding and catalytic functions.After inhibition of Sja-miR-750 in vitro,RT-qPCR results showed that Rab10 expression was significantly increased,suggesting that it may be a target of Sja-miR-750.In summary,the study conducted a comparative analysis of proteins and small RNA in EVs of male and female S.japonicum,which laid a foundation for a further understanding of the composition and function of EVs of S.japonicum.It is also helpful for understanding the the reproduction,parasite-host interaction and drug/vaccine design.