| Infectious disease is the leading cause of mortality and morbidity worldwide,claiming about 15 million lives each year.Among the pathogenic bacteria,Staphylococcus aureus(S.aureus)is a major community and nosocomial pathogen,which seriously threatens human health and causes huge economic losses.Bovine mastitis caused by S.aureus is a common and frequent cause of economic losses in the dairy industry,which not only leads to a decrease in milk production but also debases the the milk quality.Exosomes are 20-200 nm vesicles secreted by many types of cells.Exosomes are found in a number of body fluids,and regarded as important signalosomes mediating cellular communication.Exosomes contain a large number of cargos(proteins,lipids and nucleic acids),which can be internalized into target cells to regulate the physiological and biochemical processes.On the other hand,the composition and quantity changes of exosomal cargos have the potential to reflect physiological and biochemical status of donor cells.In this study,the exosomes derived from milk with S.aureus infection were used as a model to reveal the immune function of exosomal non-coding RNAs(miRNAs and circRNAs)in response to bactieral infection.In order to verify the prevalence of milk mastitis caused by S.aureus,we detected S.aureus infection in 31.58% of dairy cows(24/76)and 13.95% of milk samples(42/301),respectively.Then,we collected exosomes from 3 control groups and 6 SA groups(milk samples infected by S.aureus)by ultracentrifugation.Each exosome samples were identified by transmission electron microscope,dynamic light scattering,and flow cytometry.The results showed that the average particle diameter of milk-derived exosomes was ～119.4nm,and the positive rates of exosome surface molecular markers CD63 and CD81 were 72.0% and 77.9%,respectively.In order to detect the functions of milk-derived exosomal miRNAs and circRNAs in response to S.aureus infection,we extracted total RNA from milk-derived exosomes and detected them by next-generation sequencing.A total of 290 miRNAs were obtained from milk-derived exosomes,of which 221 were known miRNAs and 69 were novel miRNAs.Among the exosomal miRNAs,bta-mi R-148 a was the highest expressed.Known miRNAs found in milk exosomes were mainly derived from dairy cow chromosome(Chr)X,19,and 21,while novel miRNAs are mainly derived from dairy cow chromosome Chr 15.Compared with the control group,37(22 known and 15 novel)miRNAs had significant expression changes after S.aureus infection,of which 28 were significantly up-regulated and significantly down-regulated,and the result had been verified in vitro by q RT-PCR.These results indicated that S.aureus infection can cause miRNA expression changes in milk-derived exosomes.The target genes of miRNAs with significantly different expression levels were predicted and analyzed by GO and KEGG.The results showed that the target genes were enriched in 121 GO pathways,and the “lysosome pathway” was the molecular KEGG pathway with the most significant enrichment.In order to explore the molecular biomarkers of milk quality in milkderived exosomes,we arranged the expression levels and fold changes of exosomal miRNAs.The results showed that bta-mi R-378 and bta-mi R-185 had the potential to be the biomarkers of mastitis caused by S.aureus.The predicted target genes of bta-mi R-378 and bta-mi R-185 were detected using the double luciferase reporter vector,and VAT1 L was the target gene of bta-mi R-378,while DYRK1 B,MLLT3,HP1BP3,NPR2 and PGM1 were the target genes of bta-mi R-185.Although previously circRNAs are proved to be present in exosomes,studies on circRNAs in milk-derived exosomes have not been reported.This study confirmed for the first time that milk-derived exosomes contained abundantly expressed circ RNA.S.aureus infection could significantly reduce the total amount of circRNAs in milk-derived exosomes,but increase the abundance of circRNAs on the other hand.The circRNAs in milk-derived exosomes covered a wide range,and they are distributed differently on bovine chromosomes.The expressions of 290 exosomal circRNAs were significantly changed by S.aureus infection,of which 169 were significantly down-regulated and 121 were significantly up-regulated.The GO and KEGG analysis results of the original genes of these circRNAs showed that many of the original genes were enriched in the pathways related to immunity.In addition,we analyzed the miRNAs that can be adsorbed by the eight circRNAs with the highest up-regulation folds,and analyzed the target genes of these miRNAs.The result showed that multiple target genes are related to immunity.In order to further explore the functions of exosomal circRNAs in respond to pathogenic infections,we selected three types of circRNAs(the eight types of circRNAs with the most significant up-regulation,the six types of circRNAs with the most significant down-regulation and expression The amount of 8 kinds of circRNAs without significant change).These circRNAs were confirmed by PCR amplification with specific primers and RNase R resistance.More importantly,we confirmed that the process of circRNAs into exosomes were through selective package mechanism by comparing the relative expression levels of circ RNA and linear homologous genes in milk-derived somatic cells and exosomes.Additionally,the S.aureus infection could change the types of circRNAs which were packaged into exosomes or remained in doner cells.In summary,in the present research we used dairy cow mastitis caused by S.aureus infection as a model to study the immune functions of exosomal miRNAs and circRNAs in respond to pathogen infection.The results show that the body can actively regulate the package mechanism of exosomal functional non-coding RNAs to resist infection by pathogenic bacteria.Even more,this active regulation may achieve intergenerational or even species through media,such as milk exosomes.This research provides a new theoretical basis for our understanding of bacterial infections. |
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