Isolation And Charaterization Of Extracellulr Vesicles Of Mycoplasma Bovis And Bomac Cell Infected With Mycoplasma Bovis

Mycoplasma bovis(M.bovis)is the main pathogen of many diseases,such as mastitis,pneumonia and arthritis.Extracellular vesicles(EVs)produced by pathogenic microorganisms are a way of secreted proteins that are a kind of virulence factors to enter the external environment,which can participate in the pathogenic processes,as well as trigger or inhibit the host immune responses.EVs produced by eukaryotic cells are important agents for signal transduction in normal physiological processes and pathological processes,but infection of pathogenic microorganisms can affect the secretory capacity of extracellular vesicles to interfere those processes.These two types of EVs have important value in the study of pathogenic mechanisms.In this study,we isolated and characterized EVs which were secreted by M.bovis and bovine macrophage(Bomac)cells infected by M.bovis,intending to explore the role of them in the pathogenic process of M.bovis based on mass spectrometry identification and bioinformatics analysis.The study results are as follows:1.Isolation and identification of EVs of M.bovis:After centrifugation,filtration,ultrafiltration and sucrose density gradient ultracentrifugation(S-DGUC),EVs were isolated from culture supernatants of M.bovis,whose colony counting of M.bovis was<1000 CFU / m L.The particle size of EVs secreted by M.bovis,which appeared as a circular sphere with membrane structure under TEM,was distributed between 7.53nm-225.00 nm,and concentrated in 58.77 nm.2.Determination on secretion mechanism of EVs secreted by M.bovis: Under the addition of two inhibitors,the number of EVs secreted by M.bovis was reduced severally by 2.50% and 4.50% respectively.Meanwhile,the protein concentration was decreased by 44.80% and 71.68%.The secretion of EVs from M.bovis was driven by PADs and associated with change of cell membrane curvature.3.Identification proteins in EVs secreted by M.bovis:There were 78 proteins which was identified in EVs secreted by M.bovis through Mass spectrometry,and they were consist of 36 cytosolic proteins,30 classical secreted proteins,7 non-classical secreted proteins,and 5 proteins undefined their secretion types.In all the proteins,36 were localized on the cytoplasm,10 were on the outer membrane,9 were on the plasma membrane,and 23 with unknown localization based on prediction of subcellular location.In addition,41 virulence proteins were predicted from the 78 proteins.Compared to the secreted proteins of M.bovis previously isolated in our laboratory,only 13 were identified in EVs.To learn about main functions and major metabolic pathways of EVs,the 78 identified proteins were further performed COG function annotation and KEGG signaling pathway analysis.4.Isolation and identification of EVs secreted from Bomac cells infected by M.bovis:EVs secreted from Bomac cells infected with M.bovis was isolated,and the partical size of EVs were distributed between 21.04 nm-615.10 nm.The peak size were 78.82 nm.There was no significant reduction in secretion of EVs,while the protein concentration of EVs decreased by 55.30% after infecting with M.bovis,which indicated that infection with M.bovis didn’t affect the number of EVs secreted by Bomac cells but reduced protein concentrate.5.Mass spectrometry analysis of proteins in EVs secreted from Bomac cells infected by M.bovis: A total of 941 proteins were identified in EVs secreted from Bomac cells infected by M.bovis.There were 136 proteins identified only in EVs secreted from Bomac cells infected by M.bovis.The 136 proteins identified only in EVs secreted from Bomac cells infected by M.bovis performed GO function annotation and KEGG signal pathway analysis to learn about main function and pathway.To summarize,in this study,EVs secret by M.bovis were isolated through S-DGUC,meanwhile they were identified to determine that the secretion of EVs from M.bovis is associated with the change of membrane curvature and drived by the PADs.Besides,it was also confirmed that EVs secreted by M.bovis enrich virulent proteins,and can send secreted proteins of M.bovis into the extracellular environment.About EVs secreted from Bomc cells and Bomc cells infected by M.bovis,they were isolated and identified to prove that the composition of proteins in EVs secreted by Bomac cells will change with infection.