A Study Of The Expression Of PvdQ Gene In Intestinal Epithelial Cells And The Impact To The Pathogenicity Of Pseudomonas Aeruginosa

Objective:To investigate the effect and mechanism of quorum sensing autoinducer 3OC12-HSL of Pseudomonas aeruginosa on the apoptosis of Gaco-2 cells. To construct the eukaryotic expressing plasmid of pvdQ and detect the expression of pvdQ plasmid in Caco-2 cells, and observe the degradation of 3OC12-HSL molecule by Caco-2 cells expressing pvdQ gene and the effect of 3OC12-HSL on the apoptosis of Caco-2 cells with pvdQ gene.Methods:Caco-2 cells were incubated with 3OC12-HSL for 4 hours and then examined by MTT method for cytoactivities. Flow cytometry was used to analyze apoptosis rate of Caco-2 cells. Expression of apoptosis associated proteins p-p38/MAPK and NFκB were detected by western blotting investigation. The pvdQ plasmid expressed in eukaryocyte with Flag tag was constructed and identified, then transinfected into Caco-2 cells. Expression of pvdQ gene in Caco-2 cells was detected by western blotting investigation. 3OC12-HSL was utilized to interfere with Caco-2 cells expressing pvdQ gene. Then, we checked the activity of 3OC12-HSL in supernatant of Caco-2 cells by quorum sensing sensor bacterial strain E.coli MG4. We further detected the concentration of 3OC12-HSL in supernatant of Caco-2 cells expressing pvdQ gene by method of mass chromatographic analysis. At last, the apoptosis of Caco-2 cells with pvdQ gene was analyzed by flow cytometry.Results:Cytoactivity of Caco-2 cells was reduced when incubated with 3OC12-HSL for 4 hours. Meanwhile,3OC12-HSL induced the apoptosis of Caco-2 cells, and the higher dose of 3OC12-HSL, the more severe apoptosis rate of Caco-2 cells (P<0.05). The result of western blotting showed that 3OC12-HSL raised expression of apoptosis associated proteins P-p38/MAPK and NFκB. The recombinant eukaryotic expression plasmid of pvdQ gene was constructed. The result of western blotting showed that pvdQ gene could express in Caco-2 cells. The sensor strain E.coli MG4 showed 3OC12-HSL molecule was inactivated in the supernatant of Caco-2 cells expressing pvdQ gene; mass chromatographic analysis demonstrated that pvdQ gene expressed in Caco-2 cells could degrade the level of 3OC12-HSL; the result of flow cytometry showed that pvdQ gene was benefit for Caco-2 cells to resist the apoptosis effect of 3OC12-HSL molecule.Conclusion:Quorum sensing autoinducer 3OC12-HSL can affect cytoactivity of Caco-2 cells and induce its apoptosis by enhancing the expression of p-p38/MAPK and NFκB protein. pvdQ gene fully exerted its function when expressed in Caco-2 cells. It could degrade the 3OC12-HSL molecule invaded in Caco-2 cells, and protect Caco-2 cells from the apoptosis induced by 3OC12-HSL. This research could provide evidence for studying new antipathogenic drugs targeted to the pathogenicity of Pseudomonas aeruginosa.