The Effect And Regulatory Mechanism Of Sweat Exosome And Outer Membrane Vesicle In Brucella Intracellular Survival

Brucella is a genus of zoonotic pathogens,that causes brucellosis,results in substantial infertility,abortion,stillbirth and mummy in livestock and wild animal,and lead to huge economic losses to global livestock industry.Brucella is a facultative intracellular bacteria,that doesn't have any classical virulence factor,or capsule,but it is high virulent to eukaryotic cell.Brucella can be endocytosed by macrophage and located in lysosome,generated Brucella-containing vesicle(BCV).BCV inhibits autophagy pathway,regulates the release of immune factors,and replicates in macrophage.The mechanism of intracellular survival plays an important role in brucella infection.Our study demonstrated that extracellular vesicles promoted the intracellular replication of brucella in macrophages,which provides fundamental basis for developing new vaccines,discovering drug targets and diagnostic markers,provides reference for the prevention and control of other intracellular bacteria.Patients infected brucellosis would be sick with sweat and perspire during sleep,that make it difficult to diagnosis and miss their chance of correct treatment.In order to discover biomarkers of sweat,sweat exosome was isolated and purified via differential ultracentrifugation,observed its teacup structure under transmission electron microscopy(TEM),measured its size by dynamic light scattering(DLS),and confirmed by immunoblot.DLS shows show that vesicles exhibit a z-average size between 50 and 200 nm.PCR of 16 s r DNA confirmed the remove of bacteria in sweat exosome.Fat Gas chromatography confirmed the remove of fatty acids in sweat exosome.Then shotgun liquid chromatography(LC)-mass spectrometry(MS)/MS analysis was conducted to investigate the proteomic profiling of sweat exosome.A total of 1,062 proteins were identified in sweat exosome,including 997 different proteins compared with sweat proteomics,which contained 13 antimicrobial peptides and immunological factors.Except for 10 reported antimicrobial peptides and immunological factors,histone H2 B 2-C?histone H2B?histone H2 A and histone H1.3 were first discovered in sweat.In the analysis of subcellular localization,most proteins in sweat exosome locates in extracellular,cytoplasm and plasma membrane;in the analysis of biological progress,most proteins in sweat exosome play roles in cellular process and metabolic process;in the analysis of molecular function,most proteins in sweat exosome have catalytic activity,binging activity,structural molecule activity and transporter activity.Compared with sweat,977 new proteins were discovered in sweat exosome;compared with saliva exosome,urine exosome and plasma exosome,there are 5 common proteins and 896 unique proteins in sweat exosome,including reported biomarkers of schizophrenia,ectodermal dysplasia and Alzheimer's disease.RNA from sweat exosome was identified by RNA-seq.There are 284 mi RNAs in sweat exosome.mi RNA-203 a and mi RNA-1246 were confirmed by reverse transcriptionPCR(RT-PCR).Our study confirmed the exist of sweat exosome and identified the proteomics and mi RNA profiling of sweat exosome,which discovered a large number of antimicrobial peptides and biomarkers and helped the diagnosis of patients with brucellosis at early stage.Meanwhile,outer membrane vesicles(OMV)from brucella in minimal medium at neutral condition and acid condition were isolated by ultracentrifugation with sucrose cushion,observed by TEM,measured by nanoparticle tracking analysis(NTA)and DLS.Western blot confirmed periplasmic protein and outer membrane protein in brucella OMV.Label free quantification identified 310 proteins in brucella OMV.Gene Ontology analysis showed that most proteins in brucella OMV located in cell part and membrane;most proteins in brucella OMV play roles in cellular process and metabolic process;most proteins have catalytic activity and binging activity.RNA-seq of brucella OMV identified three abundant small RNAs.Brucella OMV at neutral condition or acid condition can promote brucella intracellular replication in macrophage.After incubated macrophage with increased dose of brucella OMV,ELISA date showed that the release of IFN-? and TNF-? were upregulated,q RT-PCR data displayed that the levels of AIM2,STING-?,STING-?,IFN?,p65,IL-10 and IL-6 m RNA were increased.All proteome and coculture experiments revealed that brucella OMV can efficiently promote brucella intracellular survival.In conclusion,our study investigated the protein and mi RNA profiling of sweat exosome,identified biomarkers of sweat exosome from host perspective;meanwhile,our study analyzed proteome and small RNAs of brucella OMV,and evaluated the effect of brucella OMV on the infection,intracellular replication,interaction and regulating immune system of brucella in macrophage from perspective of OMV,which provide theoretical reference for mechanism of pathogenic infection,intracellular replication and interaction between brucella and host cell.