Effect Of OmpX Overexpression On Biofilm Formation By The △tolC Mutant Of Extraintestinal Pathogenic Escherichia Coli(ExPEC)

Extraintestinal pathogenic Escherichia coli(Ex PEC), as important zoonotic and foodborne pathogens, have attracted great attention. ExPEC can spread by the contamination of food, water, and feed, and thus result in extraintestinal infections in humans and animals. Biofilm formation increases bacterial resistance to multiple drugs and adverse environments in vitro and in vivo, so it must be strengthened to explore biofilm formation mechanism and its influence factors in order to develop efficient prevention and control measures.Outer membrane protein TolC, a major component in multidrug efflux pumps of Gram-negative bacteria, mediates the efflux of exogenous elements, metabolites and toxins inside bacteria, as well as plays an important role in maintaining the integrity of envelope and biofilm formation. Our previous studies showed that TolC involved in ExPEC responsibility to environmental hyper-osmotic condition, and bacterial biofilm formation by regulating the production of Curli fimbriae. Furthermore, outer membrane protein X(OmpX) was identified to decrease by the inactivation of TolC. OmpX has been reported as a virulence factor involved in bacterial pathogenesis by mediating the adhesion to and invasion of host cells. OmpX can also participate in bacterial adaptability to environmental osmotic stress. It remains unclear whether OmpX mediates the role of TolC in ExPEC responsibility to hypertonic environment and biofilm formation. It had been confirmed that ompX deletion didn’t affect Ex PEC biofilm formation. Therefore, this study was designed to investigate the effect of OmpX overexpression on biofilm formation by the △tolC mutant. The predominant results are shown as follows:1. Expression of OmpX in ExPEC strain grown in hyper-osmotic media. The expression of OmpX in Ex PEC strain grown in NaCl- and sucrose-induced hypertonic media significantly increased detected by qRT-PCR method, indicating that OmpX may be implicated in ExPEC response to environmental hyper-osmotic stress.2. Construction of the mutants highly expressing OmpX and study on their biological characteristics. The expression plasmid pHSG396::ompX with the full-length ompX of ExPEC was transferred into the Ex PEC WT and △tolC strains to acquire the WT::ompX and △tolC::ompX strains highly expressing OmpX, respectively. Bacterial growth curve showed that overexpression of OmpX didn’t affect the growth kinetics of ExPEC strains grown in different media. Inactivation of TolC obviously relieved the resistance of ExPEC to such antimicrobials as ciprofloxacin, cefotaxime, tetracycline, amikacin and sodium dodecyl sulfonate(SDS), while the △tolC::ompX strain restored the multidrug resistance to the levels of WT strain. Moreover,the △tolC:: ompX strain can partially relieve the compromised ability to adhere to, and invasion of, human pulmonary epithelial A549 cells, as well as to survive in mouse macrophage RAW264.7 cells, resulted from TolC inactivation in ExPEC.3. Effect of OmpX overexpression on bacterial biofilm formation and Curli production. Crystal violet assay indicated that all the strains tested formed strong biofilms in 1/2M9 medium. However, the △tolC mutant exhibited a weak ability to form biofilms, and the △tolC::ompX strain regained the strong ability to form biofilms similar to the level of WT strain. Further study indicated that biofilm formation of the △tolC mutant was more easier to be inhibited by the NaCl- and sucrose-induced hypertonic conditions compared to of the WT and △tolC::ompX strains.Curli fimbriae are the key component in ExPEC biofilm formation. So Curli production of the experimental strains was investigated using Congo red assays. The results showed that all the strains presented the rdar morphology(Curli production) on 1/2 M9 agar plates. On M9 agar plates,although the WT and △tolC::ompX strains presented the rdar morphology,the △tolC mutant presented a saw morphology(no Curli). Moreover, further study showed that the compensation of OmpX expression to Curli production in the △tol C mutant was due to the transcriptional regulation of Curli biosynthesis-related genes including csgD and csgB detected by real-time qRT-PCR.4. Screening of the targets by TolC and OmpX in regulating ExPEC biofilm formation using transcriptomic technique. In order to explore the molecular mechanism that TolC and OmpX participate in ExPEC resistance to hyper-osmotic environment and biofilm formation, the genes differentially transcriptional expression among the WT, △tolC and △tolC::ompX strains grown in 1/2M9 medium containing high concentrations of NaCl or sucrose were analyzed using RNA-seq technique. Series of genes related to bacterial biofilm formation and resistance to extracellular osmotic stress were identified, including many transcriptional regulatory factors,multiple stress reactive protein YifR(BhsA), and cpx two-component signaling system accessory protein CpxP.In conclusion, the OmpX may participate in Ex PEC biofilm formation by responding environmental stress. OmpX overexpression can make up the defect in Curli fimbriae and biofilm formation caused by the deletion of TolC. According to the results of transcriptomic analysis, the effect of overexpressing OmpX on △tolC mutant may be mainly mediated by regulating two-component system and stress response factors.