E.coli Outer Membrane Vesicles Induce DNA Damage In Intestinal Epithelial Caco-2 Cells

Research BackgroundThe outer membrane vesicles?OMV?are membrane vesicles of phospholipid bilayer structure that are detached from the outer membrane of the bacterial cell wall.They are composed of outer membrane proteins,lipopolysaccharides,periplasmic proteins,enzymes,DNA,toxins,etc.Composition that transports these substances to other cells to mediate cell-to-cell communication.Both Gram-negative bacteria and a small number of Gram-positive bacteria secrete OMV.Studies have shown that when the intestinal flora is disordered or unbalanced,E.coli-induced gastrointestinal diseases such as diarrhea,intestinal bleeding,and intestinal adhesion may be associated with cell damage induced by adhesion of OMV and intestinal epithelial cells.In this study,E.coli OMV and intestinal epithelial Caco-2 cells were used as materials to analyze the DNA damage of intestinal epithelial Caco-2cells induced by E.coli OMV.From the perspective of OMV intercellular communication,the mechanism of causing digestive tract diseases is explained,which provides a new idea for seeking ways to control digestive diseases.Purposes1.Establish a method for separation and purification of E.coli OMV.2.To compare the differences in the ability of EMV-induced DNA damage in intestinal epithelial Caco-2 cells,and to analyze the possible mechanisms of DNA damage induction.3.Analyze the viability and possible mechanism of E.coli in pure water.Methods1.E.coli OMV was prepared by 20,000xg and 50,000xg centrifugal force respectively.The size distribution of OMV was analyzed by particle size analyzer.The ultrastructure of OMV was observed by transmission electron microscope.The protein content of OMV was determined by BCA protein assay.2.OMV and Caco-2 cells were co-cultured for 24 h at 37°C and 5%CO₂.The uptake of OMV by intestinal epithelial Caco-2 cells was observed by fluorescence confocal microscopy.The expression of?-H2AX protein in intestinal epithelial Caco-2 cells treated with OMV for 24 h was detected by Western-blot.The intestinal epithelial Caco-2 was detected by single cell gel electrophoresis.The extent of DNA double-strand break damage in cells treated with OMV for 24 h.3.Collect a logarithmic proliferating phase of E.coli to prepare a pure aqueous suspension,and store it at 37°C.The survival curve of the plate was counted in 3 months;the LIVE/DEAD bacterial activity was detected by Calcein-AM and PI;the bacterial ultrastructure was observed by transmission electron microscope;the relative expression of E.coli LexA protein was detected by Western-blot;Precipitation was used to detect the relative expression of the E.coli RecA-LexA protein complex.Results1.Ultrastructural display OMV is a lipid bimolecular membrane microcapsule structure of varying sizes.The average OMV diameters of20,000xg and 50,000xg were 217.5±7.29 nm and 186.3±6.59 nm?P<0.05?,respectively.The OMV protein content of 20,000xg and 50,000xg was?1.88±0.01?x10³?g/Ml,?2.09±0.09?x 10³?g/ml?P<0.05?.2.Confocal microscopy showed that OMV almost completely entered Caco-2 cells at 24h.The ratios of the?-H2AX protein bands with 20,000xg OMV,50,000 xg OMV,and control?no OMV?Caco-2 cells were 2.23±0.18,1.58±0.20,1±0.30,respectively?P<0.05?.The DNA content of Tail DNA?Tail DNA%,TDNA%?was?72.21±14.61?%,?23.11±4.98?%,?1.02±1.41?%?P<0.05?,and the corresponding DNA damage was high?3?.Level),Moderate?Level 2?,No Damage?Level 0?.3.The number of live bacteria in E.coli cultured in pure water for 0,1,2,and 3 months was?3.85±0.07?x 10¹⁴,?4.82±3.02?x 10⁷,?1.69±0.55?x 10⁷,?2.61±0.04?X10⁶ CFU/ml,compared with the 0 month group,the number of bacteria decreased significantly in January,February and March?P<0.05?;The ratio of the normalized data analysis of the LexA protein band gray scale values from 0 to 3 was 1±0.08,0.83±0.08,0.73±0.04,and 0.53±0.06,respectively.Compared with the 0 month group,the bacterial expression of LexA in the 1st-3 month group was significant.The difference was significantly decreased with the prolongation of preservation time?P<0.01,P<0.001?.The relative gray values of the protein of RecA binding to LexA and the protein of LexA binding RecA in the 0 month group were73274.4±2191.36,32097±2422.27,respectively,and RecA in the 1,2,and 3months group combined with LexA?95604.6±10712.48,120245.8±5078.71,129991?The relative gray value of±2251.07)and LexA combined with RecA?51587.6±2288.72,61851.8±3089.31,73558.6±6120.73?was significantly higher than that of the 0 month group?P<0.001,P<0.01,P<0.05?,and the preservation time Positive correlation.The ultrastructure showed that E.coli survived in January,February and March with intact cell walls,but the internal structure was relatively simple.Conclusions1.OMV induces DNA double-strand break damage in intestinal epithelial Caco-2 cells,and the degree of damage is related to the size of OMV.Larger OMVs induced greater DNA damage in intestinal epithelial Caco-2 cells than smaller OMVs.2.The survival of E.coli in pure water is related to the bacterial SOS response system.