Mechanism Of TolC-mediated Tolerance Of ExPEC Biofilm Formation To Hyperosmotic Environment

Biofilm is a bacterial anti-stress survival strategy by adhering to the surface of living or inanimate substrates.It can also cause the multi-drug resistance,immune evasion,chronic and persistent infection.Hypertonic treatment is one of the main measures to inhibit the growth and reproduction of foodborne pathogens in meat processing,and can also inhibit the formation of bacterial biofilms.Therefore,it is of great significance to strengthen the study of hypertonic environment in bacteriostatic effect and the control of biofilms.Extraintestinal pathogenic Escherichia coli(ExPEC)is an important foodborne zoonotic pathogen that can cause human and animal diseases by contaminated food and water and poses serious threats to animal health and public health.The outer membrane protein TolC is a major component of many different families of efflux pumps in Gram-negative bacteria.Our previous studies confirmed that the deletion of tolC decreased the tolerance of ExPEC to hypertonic environment and activated CpxAR two-component system,further inhibiting Curli biosynthesis and biofilm formation.However,the mechanism of action remains unclear.However,the outer membrane protein OmpW associated with hyperosmotic responses was identified to be significantly up-regulated by the deletion of tolC in hypertonic environment.Therefore,this study aims to explore the molecular mechanism of TolC-mediated tolerance of ExPEC biofilm formation to hypertonic environment from both TolC-dependent efflux pumps and OmpW.The main results are shown as follows:1.Effect of the efflux pump inhibitor on ExPEC biofilm formation.The growth and biofilm formation of ExPEC PPECC42 strain were determined in the 1/2M9 medium with 0.8% of sucrose or 0.06mol/L of NaCl or without,respectively,supplemented with different sub-inhibitory concentrations of carbonyl cyanide m-chlorophenylhydrazone(CCCP).The results showed that sub-inhibitory concentrations of CCCP could inhibit ExPEC biofilm formation,as well as the growth ability.2.Role of TolC-dependent efflux pumps in biofilm formation of ExPEC under hypertonic environment.In order to analyze the role of TolC-dependent efflux pumps in ExPEC biofilm formation under hypertonic environments,we constructed ten deletion mutants of different genes(macB,acrB,emrY,mdtC,mdtB,acrD,mdtE,emrB,mdtF and acrF)encoding the TolC-dependent efflux pump chaperone proteins derived from ExPEC PPECC42 WT strain.The results showed: 1)the deletion of the efflux pump genes didn'taffect the growth ability of the strain in three media;2)specific TolC-dependent efflux pumps were related to the resistance of ExEPC to erythromycin,tetracycline,ciprofloxacin,chloramphenicol,and rifampicin;3)the tolerance to hyperosmotic environment and the decrease of biofilm formation ability under hypertonic environment of ?tolC strain were linked to the functional impairment of some TolC-dependent efflux pumps.3.Role of OmpW in ExPEC biofilm formation under hypertonic environment.We constructed the ?ompW,?tolC?ompW and WT::ompW strains derived from the parental ExPEC PPECC42 WT or ?tolC strain,and then explored the effects of ompW deletion and overexpression on bacterial growth,Curli biosynthesis and biofilm formation under NaCl-or sucrose-induced hypertonic environment.The results showed that the deletion of ompW did not affect the growth and biofilm formation of the strain.However,similar to the deletion of tolC,overexpression of ompW significantly decreased the ability to form biofilms by ExPEC under the NaCl-induced but not the sucrose-induced hyperosmotic environment,and led to the down-regulated transcriptional expression of csgB and up-regulated transcriptional expression of cpxP.Considering the fact of up-regulated transcriptional expression of ompW in ?tolC mutant determined by RNA-seq and RT-qPCR,it is proposed that under the NaCl-induced hypertonic environment,the deletion of tolC decreased the tolerance of ExPEC biofilm formation by up-regulating the expression of ompW,and the hyperosmotic signal within periplasmic space activated the CpxAR two-component system futher inhibiting Curli biosynthesis and biofilm formation.Overexpression of ompW under the sucrose-induced hyperosmotic environment did not affect ExPEC biofilm formation,suggesting that the decreased tolerance to sucrose-induced hypertonic environment by the deletion of TolC was due to other complex mechanisms.In conclusion,some TolC-dependent efflux pumps may be involved in the tolerance of ExPEC biofilm formation to hypertonic environments.OmpW overexpression can compromise the tolerance of ExEPC biofilm formation to high NaCl environment through the regulation of CpxAR two-component system and Curli biosynthesis.