The Effect Of Chlorogenic Acid On Biofilm Of Pseudomonas Aeruginosa In The C-di-GMP Signaling Pathway

Part1 The effect of Chlorogenic acid on motility of PAO1Δwsp F、PAO1/P_lac-yhjH and PAO1Objective:To study the effect of Chlorogenic acid on swimming mobility、swarming mobility and twitching motility of PAO1Δwsp F、PAO1/P_lac-yhjH and PAO1.To investigate the regulatory effect of chlorogenic acid on PA in the c-di-GMP signaling pathway.Methods:The MIC of test drugs are measured by the double dilution method.The experiments divids into PAO1wild-type group,PAO1Δwsp F group and PAO1/P_lac-yhjH group.The medium has different conecntration of Meropenem.The overnight single strain in the tablet are vaccinated into different medium.Measuring the zone of mobilities to assess the motility of bacteria.Results:Chlorogenic acid can inhibit the motility of PAO1Δwsp F、PAO1/P_lac-yhjH and PAO1.Conclusions:Chlorogenic acid can inhibit the motility of pseudomonas aeruginosa in the c-di-GMP signaling pathway.The motility trace of PAO1Δwsp F、PAO1/P_lac-yhjH and PAO1 are different.And chlorogenic acid can inhibit their own motility trace,suggesting that chlorogenic acid can inhibit the activity of their flagella.Part2 The destruction of Pseudomonas aeruginosa biofilm by chlorogenic acidObjective:To study the destructive effects of chlorogen acid on prematurely and maturely biofilm of PAO1Δwsp F、PAO1/P_lac-yhjH and PAO1.And to study the destructive effects of chlorogen acid on biofilm at different stages of PA.Methods:The experiments are divided into control group,PAO1 wild-type group,PAO1Δwsp F group and PAO1/P_lac-yhjH group.The destructive effects of chlorogen acid on premature and mature biofilm was measured through Crystal violet staining.Results:Chlorogen acid can damage premature and mature biofilm of PAO1Δwsp F、PAO1/P_lac-yhjH and PAO1.Conclusions:Chlorogen acid can effect biofilm formation of Pseudomonas aeruginosa in c-di-GMP signal paythway.Chlorogen acid are moer effective on prematurely biofilm.And It didn’t show the concentration dependent in the mature biofilm.Part3 Effects of chlorogenic acid on the invasion of PAO1Δwsp F、PAO1/P_lac-yhjH and PAO1Objective: To study the effect of chlorogenic acid on invasion of alveolar epithelial cell lines with PAO1Δwsp F 、 PAO1/P_lac-yhjH and PAO1.Methods:The experiments are divided into control group,PAO1 wild-type group,PAO1Δwsp F group and PAO1/P_lac-yhjH group.The adhesion of Pseudomonas aeruginosa to alveolar epithelial cell line are observed under microscope by crystalviolet staining and fluorescence staining.Results: Chlorogenic acid inhibits the adhesion of Pseudomonas aeruginosa to alveolar epithelial cell lines.Conclusions: Chlorogenic acid can inhibit the adhesion of pseudomonas aeruginosa to alveolar epithelial cell lines in the c-di-GMP signal paythway and affect adhesion mode of PAO1Δwsp F、PAO1/P_lac-yhjH and PAO1.Part4 Molecular regulation of chlorogenic acid on the biofilm formation of PAO1Δwsp F、PAO1/P_lac-yhjH and PAO1Objective : To study the effect of chlorogenic acid on the biofilm formation of PAO1Δwsp F 、 PAO1/P_lac-yhjH and PAO1.Methods: The experiments are divided into control group,PAO1 wild-type group,PAO1Δwsp F group and PAO1/P_lac-yhjH group.The gene and c-di-GMP protein expression of PAO1Δwsp F、PAO1/P_lac-yhjH and PAO1 in prematurely and maturely biofilm were determined by real-time fluorescent quantitative PCR and microplate system.Results: In prematurely biofilm,the expressions of FLEQ、WSPR and PILZ of PAO1 and PAO1Δwsp F is decreased.The expressions of WSPR and PILZ of PAO1/P_lac-yhjH is increased.In maturely biofilm,the expressions of FLEQ and PILZ of PAO1Δwsp F is decreased.The expressions of FLEQ of PAO1 is increased.The expressions of PILZ of PAO1/P_lac-yhjH is increased.Chlorogenic acid can decrease the production of c-di-GMP protein of PAO1、PAO1△wsp F.And the production of c-di-GMP protein of PAO1/PlacyhjH is increased.Conclusions: Chlorogenic acid has different effects on the expression of genes related to biofilm formation and the generation of c-di-GMP protein in PAO1Δwsp F、PAO1/P_lac-yhjH and PAO1.And the inhibition are also differences between the premature biofilm group and the mature biofilm group.