Study On Vesicles Of Pseudomonas Aeruginosa

PART1ESTABLISHMENTAND OPTIMIZATION THE METHODOF EXTRACTING PAO1’S OUTER MEMBRANEVESICLEObjective: Establish and optimize the way to extraction andpurification the outer membrane vesicles(OMV) of Pseudomonasaeruginosa(PA) in vitro so as to pave the way for study biological activityof OMV and the mechanism of infection induced by OMV.Method: The OMV of PAO1（ATCC9027） was extracted byultrafiltration concentration and Optiripe density gradientcentrifugation,respectively.The OMVs were analyzed for it’s purity bynegative-stain electron microscopy;for yield by BCA.Results: OMV exctrated by ultrafiltration concentration have moreimpurities such as flagella and debries than OMV exctrated by Optiripedensity gradient centrifugation; The yeild of exctrated by ultrafiltrationconcentration is21.3μg/1010bacteria and the yeild of OMV exctrated byOptiripe density gradient centrifugation is48.7μg/1010bacteria.Conclusion: The OMV of PA exctrated by Optiprep density gradientcentrifugation have higher purity and yeild than OMV exctrated by ultrafiltration concentration. PART2COMPOSITIONAND BIOLOGICALACTIVITY DETECTIONTHE OUTER MEMBRANE VESICLE OF PAO1Objective:Preliminary tests protein contained in OMV,research thebiological activity OMV through vivo and in vitro infection model.Method:Proteins contained in OMV were determined bySDS-PAGE;Human lung epithelial cells BASE-2B were immunized withpurified outer membrane vesicle and determined for IL-6and IL-8byELISA;Balbc mice were infected with purified OMV and observed thepathologyical change of lung tissue；detection the protective of bacterial byOMV through Polymyxin B resistance test.Results:PAO1’OMV are20to200nm in diameter and contain avarity of protein whitch molecular mass are10to100KDa; purified OMVsstimulate BASE-2B cell significant release of the immunomodulatorycytokine interleukin-6、interleukin-8(IL-6、IL-8),and lead to pathologicalchanges of lung tissue in mice.Apart from this,When add OMV in medium contained polymyxin B,the amount of viable PAO1was Significantlyincreased.Conclusion:PAO1can secret OMV which contain multiple proteinsand have multiple biologically active. PART3THE INFLUENCE OF LASB-GENE ON THE OUTERMEMBRANE VESICLE OF PAO1Objective: To investigate the influence of lasB mutations on the outermembrane vesicle (OMV) of Pseudomonasa eruginosa.Method: PAO1and PAO1lasB-were Identified by PCR, from whichOMVs were purified with Optiprep density dradient ultra_centrifugationand determined by negative-stain electron microscopy; for concentrationand contents determined by SDS-PAGE and BCA, respectively. Congo redtest the activity of elastase in OMVs.Normal human peripheral bloodmononuclear cells (PBMC) were immunized with the purified OVMs andbacteria respectively,Phagocytosis was measured as intracellularpercentages of each.Results: Lack of lasB didn’t result in morphological changes in outer membrane vesicle,Both of them are20to200nm in diameter,but decreasethe yield,and the activity of elastase as well as the ability of protectingbacteria from phagocytosis by PBMC(p<0.01)Conclusion: lasB gene play an important role in the biologicalactivities of OMV secred by pseudomonas aeruginosa.