Comparative Proteomics Study Of Exosomes In Vibrio Harveyi And Vibrio Anguillarum

In recent years,with the continuous development of aquaculture industry,the problem of vibrio infection has been on the rise,which has caused serious threatens to the ecological environment of healthy marine aquaculture,also caused serious influence and economic loss to the marine aquaculture industry,so it is of great significance to reveal the pathogenic mechanism of bacteria for its prevention and treatment.In this study,the exosomes of Vibrio harveyi and Vibrio anguillarum,which pose a threat to the health of marine culture,were isolated with supercentrifugation and identified by transmission electron microscope and nanoflow detector.Then the exosome proteins of the two vibrio species were analyzed by proteomics technique.Finally,acute exposure experiments were carried out to detect the corresponding virulence proteins of Vibrio harveyi and Vibrio anguillarum exosomes in Ruditapes philippinarum by real-time fluorescence quantitative PCR.The results of this study are as follows:(1)Successfully obtaining exosomes of Vibrio harveyi and Vibrio anguillarum:The exosomes of the two bacteria were obtained by ultra-centrifugation method.After observation and detection by transmission electron microscope and flow particle size analyzer,it was confirmed that the separation state of exosomes was good and the shape was almost round.The average diameter of exosomes of Vibrio harveyi and Vibrio anguillarum were 78.16 ± 0.71 nm and 77.11 ± 0.14 nm,30-150 nm particles accounted for 99.57 ± 0.21% and 99.31 ± 0.13%,and the concentrations were(8.37 E + 10)±(1.7E + 9)and(3.63 E + 12)±(4E + 11)respectively.The data showed that exosomes of the two bacteria were successfully extracted,which laid a good foundation for subsequent comparative proteomics experiments.(2)Screening and comparison of differentially expressed proteins in exosomes:Although Vibrio harveyi and Vibrio anguillarum both belong to vibrio genus,the expression of exosome proteins in these two bacteria are still different.There were2016 differentially expressed proteins in the exosomes of Vibrio harveyi and Vibrio anguillarum,of which 1000 proteins were up-regulated and 1016 proteins were down-regulated.In GO enrichment analysis,2165 proteins,197 cellular components and 1479 molecular functional proteins related to biological processes were enriched in the exosomes of Vibrio harveyi and Vibrio anguillarum.Our study in GO enrichment analysis showed that exosomes of both Vibrio harveyi and Vibrio anguillarum were involved in important pathogenic processes of bacteria.KEGG functional annotation indicated exosomes of the two vibrio species were different in metabolic pathways.(3)Discovery of secretory systems and virulence factors related to toxicity:Through the analysis of differentially expressed proteins,some secretory systems(such as Vibrio anguillarum mainly contains type II and type VI secretory system proteins,and Vibrio harveyi mainly contains type VI secretory system proteins)and virulence factors(such as hemolysin and metalloproteinase in Vibrio anguillarum,lipase and phospholipase in Vibrio harveyi and chitinase in both vibrio species)were found in the exosomes of the two vibrio species.These secretory systems and virulence factors in exosomes of both vibrio species played important roles in bacterial-host antagonism.(4)Verification of virulence protein at gene level: In order to verify whether exosome proteins of Vibrio harveyi and Vibrio anguillarum were involved in vibrio toxicity,we selected some virulence proteins for verification in gene level.After the acute challenge experiment of two vibrio was carried out in Ruditapes philippinarum,eight virulence genes(type VI secretion systems effectors Tss I1,type VI secretion systems effectors Tss I2,type III secretion system protein,lipase in Vibrio harveyi and metalloprotease Pmb A,hemolysin,type VI secretion system baseplate subunit Tss F,type II secretion system protein Gsp L in Vibrio anguillarum)of the two vibrio exosomes in blood cells of Ruditapes philippinarum were detected by real-time quantitative PCR.The data showed eight genes were up-regulated to varying degrees,revealing that exosomes were involved in vibrio toxicity.In conclusion,exosome proteins released by Vibrio harveyi and Vibrio anguillarum not only contained virulence factors,but also participated in the complex biological process of bacteria.There were also differences in the expression of exosome proteins in the two bacteria.In addition,the exosomes of both bacteria were involved in vibrio toxicity.The results of this study could provide effective theoretical data for understanding the pathogenesis of vibrio from the perspective of exosomes.