Influence Of Pseudomonas Aeruginosa Quorum Sensing Signal Molecule N-(3-oxododecanoyl) Homoserine Lactone On Mast Cells

Objective:To chemically synthesis Pseudomonas aeruginosa quorum-sensingautoinducer 3OC₁₂-HSL and check it's biological activity.To investigate cell viability,apoptotic effects,intercellular calcium mobilization as well as cytokine secretion profileand degranulation effect of mast cells in response to different concentrations of3OC₁₂-HSL.Methods:The molecule 3OC₁₂-HSL is synthesized by following methylataion ofL-methionine by iodomethane,hydrolization of S-methionine methylsulphonium,lactonization of S-homoserine in acidic condition,aminolysis by acyl chloride or acylatedMeldrum's acid.Its biological activity was checked using a quorum sensing sensorbacterial strain containing the reporter gene of gfp (E.coli MT102 (pJBA132))and lacZ(E.coli MG4(pKDT17)).Different concentrations of 3OC₁₂-HSL were used to incubatewith murine mast cell P815.At different time points,cell viabilities were assayed throughWST-1 method.Cell apoptosis were determined by flow cytometry after staining withAnnexin V/PI.Moreover,intracellular calcium mobilization was investigated through laserscanning confocal microscope according to calcium indicator Fluo-3 AM.In cytokinessecretion assay,different concentrations of molecule 3OC₁₂-HSL were used to stimulatemurine mast cell line P815 and human mast cell line HMC-1.The releases of cytokines(IL-4,IL-6)were determined in P815 cells by ELSIA.Histamine induction (ELIAS)anddegranulation (transmission electron microscopy)were investigated in both P815 andHMC-1 cells.Results:The yield of 3OC₁₂-HSL was 67.31%,the structure of synthesized3OC₁₂-HSL were confirmed by ultraviolet,mass spectrum and proton nuclear magneticresonance (NMR).The purity of compound power was 99.6% as showed by high-pressureliquid chromatography.The molecule structure and purity of synthesized.The sensor strainE.coli MT102 (pJBAI32)positively showed AHLs produced by P.aeruginosa on agarplate through"T"stick after 16h,and the strain E.coli MG4(pKDT17)showed similarsensitivity.Synthesized 3OC₁₂-HSL induced same result as P.aeruginosa in E.coliMG4(pKDT17).In cell viability assay,at concentration of 100μM,3OC₁₂-HSL showed drastically inhibitory effect on P815 cells when stimulated for 2h (45%)to 24h (27%).Afterincubation with 50-100μM concentrations of 3OC₁₂-HSL for 12h,P815 cell exhibitedtypical morphological change as shrinkage.In apoptotic aspect,at 4h of treatment,about 20and 40% of apoptotic cells were observed in response to 50 and 1001μM 3OC₁₂-HSL,respectively.About twofold increase of calcium were observed in P815 cells treated with100μM 3OC₁₂-HSL,while a relatively milder increase after 50μM 3OC₁₂-HSL stimulation.About the cytokine release,at 12h or 24h incubation,no significant changes of IL-4 releasewere observed in P815 cells in response to different concentrations (6.25-100μM)of3OC₁₂-HSL.After 24h stimulation,3OC₁₂-HSL (6.25-12μM)increased IL-6 release indose-dependent fashion (P＜0.05),however,at higher concentrations (25-100μM),a steadilydrop of IL-6 production were observed.At 12h,no changes were found in IL-6 release.Inhistamine release assay,drastically increase in HMC-1,but not in P815,after treated with100μM 3OC₁₂-HSL.Typical degranulation was observed in HMC-1 after stimulation.Conclusion:The biological active 3OC₁₂-HSL was synthesized successfully.3OC₁₂-HSL shows inhibitory effect of cell viability in murine mast cell P815.Cellapoptosis contribute to viability decrease and intracellular calcium increase may beinvolved during this process.3OC₁₂-HSL induce P815 cells to release IL-6 as well asstimulate HMC-1 to produce histamine and degranulation,indicating that 3OC₁₂-HSL mayplay a role in Pseudomonas aeruginosa infection and mast cell involved in this situation.