Screening And Inhibitory Mechanism Of Quorum Sensing Inhibitors Of Pseudomonas Fluorescens P07

Pseudomonas fluorescens are the specific spoilage bacteria of various refrigerated aquatic products.It can grow and reproduce at low temperature and produce extremely heat-resistant protease and lipase,which leads to the spoilage of food.P.fluorescens can also produce a large number of biofilms,which help bacteria effectively escape the killing of antibiotics and produce resistance to antibiotics.Once biofilms are formed,they are difficult to remove completely from the adhesion site,causing a potential threat to food safety.Common disinfection is useless to remove biofilms in food processing equipments because bacteria will increase their resistance to disinfectants,thus,a new method is urgently needed to control the biofilm formation and food corruption caused by P.fluorescens.Studies have shown that certain characteristics of P.fluorescens are related to food corruption.And these characteristics are regulated by their quorum sensing?QS?system.Therefore,finding quorum sensing inhibitors?QSIs?to interfere the bacterial QS and destroy its biological function will provide a possible way to solve bacterial drug resistance and prolong the shelf life of food.However,traditional way to search for QSIs is often random,low in efficiency,high in cost,and requires a large number of experiments.Computer aided virtual screening of QSIs can just solve this problem.For this reason,P.fluorescens P07 was taken as the research object in this study.Their QS system was taken as the target to intuitively and conveniently discover novel QSIs through homologous modeling,virtual screening and molecular docking methods.And the inhibition mechanism of QSIs is further explored.Finally,the selected QSIs and electrospun technology were combined to be applied for the preservation of aquatic products,providing a reference for exploring the QS system of P.fluorescens and finding new ways to promote the shelf life and safety of aquatic products.The main results are as follows:?1?The amino acid sequences of LuxI-and LuxR-type protein in the QS system of P.fluorescens P07 were obtained through whole-genome sequencing and gene annotation.The three-dimensional?3D?structure of the receptor proteins were obtained by homology modeling.The quality of the constructed protein models was evaluated by SAVES and QMEAN methods.Results showed that the constructed LuxI-and LuxR-type proteins were of good quality.?2?The LuxI-and LuxR-type protein were used as targets to screen for potential QSIs from a traditional Chinese medicine compound database?Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP?.The results showed that 10substances had QS inhibitory activities?hit rate:66.67%?.Benzyl alcohol,rhodinyl formate,ethyl linalool and houttuynine have potent QS inhibitory activities.Benzyl alcohol with the strongest activity had significant inhibitory effects on the QS-regulated phenotypes of P.fluorescens P07.The inhibition mechanism of benzyl alcohol on the QS system of P.fluorescens P07 was analyzed by molecular docking.Results showed that the inhibition mechanism of benzyl alcohol might be that benzyl alcohol closely combined to LuxI-type protein,blocking the production of AHLs and,thus,eventually interfering with the QS circuit of P.fluorescens P07.?3?In order to screen for safer and more efficient QSIs,LuxI-and LuxR-type protein were used as docking targets for virtual screening from a food-derived compound database.Results showed that 19 compounds were identified to have QS inhibitory activities?hit rate:76.00%?.Among them,?+?-catechin,?-?-epicatechin,propyl gallate,hesperidin,lycopene and solanesol have strong QS inhibitory activities.Results also showed that?+?-catechin with the strongest QS inhibitory activity had significantly inhibited the production of extracellular protease,swimming motility,biofilm formation and EPS of P.fluorescens P07.?4?QSIs of P.fluorescens P07 were screened from ChEBI,ChEMBL,Traditional Chinese Medicine database?TCM Database@Taiwan?and ZINC natural product database by molecular similarity-based and pharmacophore-based virtual screening,respectively.Results showed that phenyle,honokiol,magnolol,ethyl,vanillylacetone,guaiacol,cinchonidine,2-?Hydroxymethyl?phenyl-beta-D-glucopyranoside,citral,vanillin,melatonine,undecenoic acid and L?+?-Diethyl L-tartrate had strong QS inbitory activity.?5?P.fluorescens P07 was treated with vanillin which has strong QS inhibitory activity.Its inhibitory effects on swarming motility,biofilm formation and EPS were determined,and its inhibition mechanism was explored by transcriptomics.Results showed that vanillin had a significant inhibitory effect on the swarming motility,biofilm formation and EPS of P.fluorescens P07.Transcriptomics test showed that 92 differentially expressed genes were up-regulated and 87 differentially expressed genes were down-regulated after vanillin treatment.The reduction of biofilm of P.fluorescens P07 after vanillin treatment was the effect of vanillin on a variety of factors of bacteria,such as bacterial mobility,adhesion,chemotaxis,the content of EPS and bacterial QS system.?6?By combining the electrospun technology with the selected QSIs,PLA–propyl gallate and PCL–?-?-epicatechin coaxial electrospun fibers were prepared,and the prepared nanofibers were characterized and tested for their inhibitory effect on decaying phenotypes of P.fluorescens P07 in salmon slices during cold storage.Results showed that the electrospun fibers were of small diameter,and they interlaced with each other and had smooth surface without beads and other defects.By characterizing the properties of the electrospun fibers,it was found that the properties of the fibers,such as thermal stability and tensile strength met the requirements of refrigerated conditions.And the QSIs were well wrapped by the fibers and had sustained release behavior.The electrospun fibers carrying QSIs did not inhibit the growth of bacteria in salmon slices,and the PLA–propyl gallate fibers and PCL–?-?-epicatechin electrospun fibers had significant inhibitory effect on the production of TVB-N,TMA and water soluble extracellular polysaccharide of bacteria in the salmon slices.And they also significantly delayed the decrease of Ca²⁺-ATPase activity as well as the deterioration rate of muscle tissue of the salmon slices.The electrospun fibers carrying QSIs had a significant inhibitory effect on the decay ability of bacteria.