Role Of Cpx Two-Component System In The Effect Of TolC On ExPEC Biofilm Formation

Extraintestinal pathogenic Escherichia coli(ExPEC),a special type of zoonotic and foodborne bacteria,belongs to the normal intestinal flora.But when it colonizes other parts of the body,it can cause diseases such as Urethritis,Pneumonia,Neonatal meningitis,and so on.The prevention and control of ExPEC has been difficult due to the lack of effective vaccine,increased bacterial resistance and the formation of bacterial biofilms.Biofilm is a special survival form of bacteria,distinguished from planktonic bacteria.and adapt to different living environments.When bacteria form a biofilm,it can resist immune clearance,reduce the effect of antibiotics and disinfectants,and cause the organism to infect repeatedly.Biofilms on the surface of food processing equipment may contaminate foods and cause serious harm to food safety.Therefore,it is very important to study the formation mechanism and control method of biofilm.Outer membrane protein TolC is the most important component of the multi-drug resistant efflux pumps of gram-negative bacteria,which participates in the formation of many different families of efflux pump,and causes multiple drug resistance.Recent studies have shown that TolC can participate in the formation of bacterial biofilm.Previous studies in our laboratory indicated that the loss of TolC resulted in the compromise of biofilm formation of ExPEC in M9 medium.This is due to the decreased ability of the bacterial Curli fimbriae synthesis,but the specific mechanism is unclear.Through transcriptional analysis,it is found that the deletion of TolC causes the up-regulated expression of a large number of genes,including the periplasmic interstitial protein CpxP of the Cpx two component system.Corresponding studies showed that the activation of Cpx twocomponent system can inhibit the synthesis of Curli.Therefore,we speculate that the Cpx two-component system plays a role in the biofilm formation of ΔtolC strains.So,this study was to mutate the proteins CpxA and CpxR of the Cpx two-component system in the ΔtolC strain,and study the effect of Cpx system on biofilm formation in ΔtolC strain.The main research contents and results are as follows.1.Construction of gene deletion strainsPrimers were designed to amplify cpx A upstream and downstream homology arms using PPECC42 genome as a template,and connected by Overlap PCR,then insert homologous arm into suicide vector p RE112,and transformed the recombinant plasmid into E.coli χ7213 competent cells to construct χ7213-p RE-△cpx A.The cpx A mutant was selected by biparental mating between donor strainχ7213-p RE-△cpx A and recipient strain WT.Construction method of △cpx R gene deletion strain is the same as △cpx A.Similar to single gene deletion strain construction method,through homologous recombination of Escherichia coli χ7213 and ΔtolC strains transfected with recombinant suicide plasmid p RE112,and selecte for the double gene deletion strain △tolC△cpx A or △tolC△cpx R.2.Construction of the complement strain and CpxA phosphorylation Site-point mutant strain.The full-length cpx A gene expression of cpx A.The recombinant the complement strain.inserted into the plasmid p HSG396 to acquire the vector was transferred into △tolC△cpx A to select the complement strain Cm-A-△tolC△cpx A.The same method is used to construct the Cm-R-△tolC△cpx R.The point mutation primers was designed after predicting and translating analysis.Then construct the mutant plasmid p HSG-cpx A with primers,and transferred into △tolC△cpx A to build the mutant strain of CpxA phosphorylation sites M-A-△tolC△cpx A.3.Study on some biological characteristics of test strainsFirst,the growth of test strains was determined in M9 and 1/2M9 medium.The results showed that the absence of tolC,cpx A and cpx R genes did not affect the growth rate of ExPEC,strains can reach the stable period after 8 hours.Second,MIC of the tested strains was determined using 7 antimicrobial agents.The results showed that the sensitivity of △tolC,△tolC△cpx A and △tolC△cpx R to chloramphenicol and ciprofloxacin increased significantly.While,No significant changes in the sensitivity of △cpx A and △cpx R to the 7 antimicrobial agents.4.Analysis of the ability of biofilm formation in experimental strainsDetermination of biofilm formation ability of test strain by using crystal violet staining method.The results showed that in M9 medium,the formation ability of △tolC strain was significantly decreased compared with WT,△tolC△cpx A、△tolC△cpx R and M-A-△tolC△cpx A is quite the same as the wild strains,Cm-A-△tolC△cpx A and Cm-R-△tolC△ cpx R squite the same as the △ tolC strain.The same results was found in 1/2M9+0.06mol/L Na Cl and 1/2M9+0.8% sucrose as M9 medium.Nonetheless,all strains formed strong biofilms in 1/2M9 medium.5.Observation of bacterial morphology in biofilm by scanning electron microscopyObservation of bacterial morphology in biofilm by scanning electron microscopy,△tolC△cpx A,△tolC△cpx R and M-A-ΔtolCΔcpx A resented a similar form to WT in 1/2M9+0.06mol/L Na Cl and 1/2M9+0.8% sucrose medium,which adhered to the surface of glass,and there was a lot of adhesion between the biofilm bacteria.But,△tolC were less adherent on the slide,and the adhesion between bacteria was less.6.Analysis of the synthesis ability of Curli in experimental strainsCongo red plate test shows that on M9 medium,the △tolC mutant,Cm-A-△tolC△cpx A and Cm-R-△tolC△cpx R presented a pdar(pink,dry,and rough)morphology;however,the△tolC△cpx A、△tolC△cpx R and M-A-△tolC△cpx A strain presented the rdar(red,dry and rough)morphology similar to the WT strain.On the 1/2M9-CR plate,the colony morphology of the tested strains was similar to that of WT.Our results indicates that the Cpx two-component system does play a role in the ability of biofilm formation in ΔtolC strain under hyperosmotic conditions,by inhibiting the synthesis of Curli fimbriae.