Effect Of Overexpression Of Five Outer Membrane Proteins On Drug Resistance Of ExPECΔtolC Strain And Study On The Mechanism

Extraintestinal pathogenic Escherichia coli(ExPEC)is an important zoonotic pathogen.AcrAB-TolC pump is the main active efflux system of E.coli,mediating the transport of antibiotics and other substrates,which is closely related to drug resistance.As a transmembrane protein,TolC is responsible for transmembrane transport of substrate to extracellular.Previous studies in our laboratory found that the deletion of tolC gene significantly reduced ExPEC drug resistance,while the overexpression of outer membrane protein X(OmpX)in ΔtolC strain restored bacterial drug resistance,while the mechanism remains unclear.OmpX and TolC share the same transmembrane β-barrel structure,so we expect to know if OmpX can replace TolC to form active efflux pump with AcrA and AcrB for the efflux of antimicrobial agents,and if other OMPs sharing the similar structures have the same function.In order to solve these questions,five OMPs with β-barrel structure,OmpX,OmpC,OmpN,OmpW and PhoE were selected as targets to analyze the effect of their overexpression on drug resistance of ExPEC ΔtolC strain,and to explore the mechanism of action by such experiments as efflux inhibition test and drug accumulation test.The main study contents and results are shown as follows:1.Construction of the experimental strains and study on their drug resistancePorcine ExPEC PPECC042 strain(WT)or its ΔtolC strain was used as parent strain,ΔacrA strain and ΔacrAΔtolC strain were constructed by homologous recombination method.The recombinant expression plasmid of ompC,ompN,ompW or phoE gene was respectively prepared and electro transformed into competent cells of ΔtolC,ΔacrA,ΔacrB and ΔacrAΔtolC strains to construct the experimental strains overexpressing various OMPs.The results of growth curve showed that the overexpression of various OMPs did not affect the growth rate of their parental strain.The minimal inhibitory concentration(MIC)of different antimicrobials was determined by microdilution broth method to evaluate the drug sensitivity of the experimental strains.The results showed that the deletion of tolC gene significantly increased the sensitivity of ExPEC to macrolides,quinolones and tetracyclines,while the overexpression of various OMPs in ΔtolC strain could all restore the resistance of ExPEC to three kinds of drugs to the level of WT strain.2.Study on the mechanism of restoring ExPEC resistance of ΔtolC strain by the overexpression of five outer membrane proteinsThe efflux activity of the experimental strains was detected by adding sub-inhibitory concentration of carbonyl cyanobenzene Hydrazone(CCCP),an efflux pump inhibitor,in the sensitivity test of three kinds of antimicrobial drugs.The results showed that CCCP did not affect the drug sensitivity of ΔtolC strain,but reduced the drug sensitivity of WT strain and ΔtolC strain overexpressing various OMPs to different extent,suggesting that efflux ability of ExPEC to different antimicrobials was lost by the deletion of tolC gene,and was compensatorily restored by the overexpression of various OMPs.The drug sensitivity tests of ΔacrA,ΔacrB and ΔacrAΔtolC strains and their derived strains overexpressing various OMPs.The results showed that:(1)compared with WT,the MIC values of ΔacrA,ΔacrB and ΔacrAΔtolC strains decreased significantly to the levels similar to the ΔtolC strain,indicating that the deletion of tolC gene the decrease of ExPEC drug resistance by destroying the structure of AcrAB-TolC efflux pump;(2)the overexpression of various OMPs did not affect the MIC values of ΔacrA,ΔacrB and ΔacrAΔtolC strains,indicating that in mediating the active efflux resistance mechanism to antimicrobial drugs,all of the five OMPs can not compensate the inactivation of AcrA and AcrB proteins and the compensation to the inactivation of TolC depend on the presence of AcrA and AcrB proteins.Ciprofloxacin accumulation test was used to further verify the drug efflux ability of WT,ΔtolC,ΔacrB strains and the ΔtolC strain overexpressing various OMPs.The results showed that:(1)the drug concentration in cytoplasm was the lowest for the WT strain and the highest for the ΔacrB strain,while the drug concentration in periplasm was the lowest for the ΔacrB strain and the highest for the ΔtolC strain.(2)All the overexpression strains of five OMPs could significantly decrease the drug concentration in cytoplasm and periplasm that the parental ΔtolC strain(p<0.001).The results suggest that the inactivation of AcrB protein leads to the fact that the drug accumulates in the cytoplasm and can not enter the periplasm,while the inactivation of TolC protein leads to the fact that the drug enters the periplasm and can not be excreted out of the cell.All of five OMPs can replace TolC protein to assemble the active efflux pump with AcrA and AcrB proteins to transport drugs into the periplasm and then out of the cell.Nile red dye was used as substrate for real-time efflux analysis,and the efflux activity of the experimental strains was detected.The results showed that the fluorescence intensity of all the strains did not change within 100 s when the energy was not provided at the beginning.After energy stimulation,the fluorescence intensity of WT strain and the ΔtolC strain overexpressing various OMPs decreased sharply,while that of ΔtolC strain,ΔacrB strain and the ΔacrB strain overexpressing five various OMPs decreased slightly,further confirmed that all of five OMPs can replace TolC protein to assemble the efflux pump with AcrA and AcrB proteins and recover the efflux activity.