The Research Of Bacterial Resistance Based On Lactase AidH And Efflux Pump System AcrAB-TolC

Quorum Sensing（QS）and multidrug efflux pump AcrAB-TolC are important factors in the formation of bacterial resistance.Understanding and inhibiting the two effects is of great significance for understanding bacterial resistance.Quorum sensing is a way of information communication between bacterial communities,and it is an important group behaviour mode by the release of QS signal molecules by bacteria.Among them,the formation of bacterial biofilm mediated by QS signal molecules acyl-homoserine lactone（AHL）in gram-negative bacteria is an important factor in the formation of bacterial resistance,and also an important problem in clinical practice.INP-AidH is constructed by recombination ofα/βhydrolase AidH from Ochrobactrum sp.,which can hydrolyze AHL lactone ring,with ice nucleation protein（INP）.It can be immobilized on silica gel materials by cell membrane chromatography（CMC）and still maintain the activity of hydrolyze QS signal molecules C6-HSL and inhibit biofilm formation.It provides a new strategy for obtaining new materials to solve bacterial infection.AcrAB-TolC,a bacterial multidrug efflux pump,exists on the cell membrane and is driven by proton exchange in the form of a trimer to achieve the transport and efflux of antibiotics and other substances,leading to the formation of bacterial resistance.The organization and function of AcrAB-TolC play an important role in the transport of QS signal molecules and the formation of bacterial biofilm.However,the dynamic assembly process of AcrAB-TolC and the transport process of QS signal molecules remain unclear.In view of the above problems,this study will use a series of characterization methods to study bacterial resistance based on lactase AidH and efflux pump system AcrAB-TolC.The specific research contents are as follows:Using the constructed plasmid p ET28a[INP-AidH]transformed into E.coli BL21（DE3）for induce the recombinant protein INP-AidH,and the correct expression and high purity（>95%）protein was obtained.The cell membrane fragments containing the recombinant protein INP-AidH were immobilized on silica gel materials by CMC.SEM and EDS characterization results showed that the immobilized silica gel particles were successful.By discussing the functional activity of degradation of QS signal molecules after immobilized,it was found that silica gel particles with 10μg immobilized cell membrane could degrade about 80%of 2mmol/L C6-HSL within 30 minutes.Through the study of the function of inhibiting the formation of biofilm by silica gel particles after immobilization,it was found that containing 20μg cell membrane protein of silica particles can prevent about 60%of biofilm formation,as well as the immobilized silicone catheter can inhibit the formation of biofilm formation on the surface of catheter and the surrounding culture;The plasmid p ET28a[Acr A],p ET28a[Acr B]and p ET28a[Tol C]were constructed based on the genomic sequence of efflux pump system AcrAB-TolC in E.coli K-12 by molecular biological operation.The induction conditions of Acr A were determined as follows:when the strain was cultured to OD₆₀₀=0.4-0.8,IPTG was added to the final concentration of 1.0 mmol/L and induced at 16℃for 12-16hours.The induction conditions of Acr B and Tol C were as follows:when the strains were cultured to OD₆₀₀=1.0,IPTG was added to 0.2 mmol/L and induced at 37℃for12-16 hours.Through purification and ultrafiltration,the protein with high concentration and high purity（>98%）was obtained.EB efflux experiment and ITC experiment result analysis confirmed its transport activity and interaction with QS signal molecules,and observed the dynamic assembly process of efflux pump subunit through TIRFM,which is of great significance for the study of bacterial resistance and antibacterial strategy generated by multidrug efflux pump.