Effect Of Escherichia Coli-derived OMVs On Mitochondrial Function And Immune Effects In Macrophages

Objective:In view of the important significance of OMVs for bacterial pathogenesis,we isolated and enriched OMVs by using ultrafiltration concentration and ultracentrifugation methods,and analyzed the purification efficiency.Then,taking the interaction between E.coli-OMVs and macrophages as a starting point,we clarified the impact of E.coli-OMVs on the mitochondrial function and immune regulation of macrophages,providing new ideas for further research on the pathogenic mechanism/infection prevention and control of Escherichia coli.Methods:1.Extraction and identification of the E.coli-OMVs Inoculated bacteria were cultured in LB liquid medium until the OD600 was 0.8.E.coli-OMVs were extracted and purified by ultrafiltration concentration and differential centrifugation.The protein concentration of E.coli-OMVs was determined by BCA method,the morphology of E.coli-OMVs was determined by TEM,and the particle size distribution and concentration were analyzed by nano-particles tracking(NTA).2.E.coli-OMVs on mitochondrial function and immune regulation in macrophages(1)Quantitative and proteomic analysis of E.coli-OMVs The LPS content of E.coli-OMVs was detected,and the protein of E.coli-OMVs was extracted for SDS-PAGE and mass spectrometry analysis.(2)Effect of E.coli-OMVs on the survival rate of RAW264.7 cells CCK-8 assy was used to determine the effect of E.coli-OMVs(1,5,10,20,50,100μg/ml)on cell survival,and appropriate stimulation concentration of E.coli-OMVs for subsequent tests was selected.(3)OMVs internalization test Di I-OMVs were co-cultured with RAW264.7 cells.After the culture,the cells were stained with fluorescence and observed under fluorescence focal microscope.(4)Transmission electron microscopy was used to observe the effect of E.coliOMVs on the morphology of RAW264.7 cells.(5)Effect of E.coli-OMVs on mitochondrial function of RAW264.7 cells The changes of mitochondrial membrane potential were detected by JC-1 staining;The changes of intracellular ATP level and ROS level were detected.(6)Effect of E.coli-OMVs on the cellular immune function of RAW264.7The immunophenotype of RAW264.7 cells was detected by flow cytometry;The changes of cytokines were detected by RT-PCR.Results:1.Both ultrafiltration and differential centrifugation can successfully extract E.coli-OMVs,which is a spherical solid membrane structure with a diameter of about20~250 nm.However,the membrane of OMVs extracted by differential centrifugation is incomplete.The average particle size of OMVs extracted by ultrafiltration concentration method is more concentrated,the proportion of vesicles less than 250 nm is higher than that prepared by differential centrifugation method,and the protein concentration is also higher.2.SDS-PAGE analysis was performed on the isolated E.coli-OMVs,and silver staining showed that there were a large number of protein bands,which showed a significant difference compared to the whole bacterial protein bands.Through proteomic analysis,it has been proved that E.coli-OMVs have rich signal transduction,immune response,and virulence related proteins,including omp C,omp A,and other pathogenic factor components.3.E.coli-OMVs produced negligible RAW264.7 cytotoxicity at the concentrations of 1~50 μg/ml for the 24 h exposure.However,a significant decrease in relative survival rate of cells treated with E.coli-OMVs was only observed at the concentrations of 100 μg/ml.Subsequent experiments used OMV at concentrations of20μg/ml and 50μg/ml.4.There was a prompt uptake of OMVs by RAW264.7 cells.Cells show changes such as increased volume,decreased surface dendritic structure,mitochondrial swelling,and abundant intracellular organelles.5.After 24 hours of exposure to E.coli-OMVs at concentrations of 20μg/ml and 50μg/ml,the levels of MMP and ATP in RAW cells decreased,ROS increased in a dose-dependent manner,and pro-inflammatory cytokines increased.The expression of cell marker CD206 increased slightly while the level of CD86 increased significantly.Conclusion:1.Ultrafiltration concentration method has higher extraction efficiency.The integrity,purity and protein concentration of extracted OMVs are higher than that of differential centrifugation method.2.E.coli-OMVs are rich in signal transduction,immune response,and virulence-related proteins,including pathogenic factor components such as omp C,omp A.They can directly interact with macrophages and participate in immune responses,causing oxidative stress in host cells and inflammatory reactions in the body.