Characterization And Function Of TolC In Extraintestinal Pathogenic Escherichia Coli

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are zoonosis pathogens that cause a variety of clinical syndromes, including urinary tract, central nervous, circulatory, and respiratory system infections in humans and animals. Infections of ExPEC were increased year by year, and which lead to increase of medical cost, reducing of animal production and economic losses. It is difficult to control and treat ExPEC infected due to multi-drug resistance, biofilm formation and lacking effective vaccine.TolC, belonged to the outer membrane efflux protein (OEP) family, is involved in the assembly of many multidrug efflux pump systems in E. coli and other gram-negative bacteria. To date, the structure and function of TolC was studied over 30 years. Some previous studies demonstrated that some tolC mutants without TolC expression are tolerant of colicin E1; hypersensitive to certain dyes, drugs, and detergents; have altered bacterial virulence; and pump the biomolecules derived from bacterial self-metabolism. However, the biological function of TolC in ExPEC remains unknown, especially in biofilm formation, pathogenicity and resistance to external environment, which could require to further study.Hence, the roles of TolC in ExPEC for biofilm formation, pathogenicity and survival to resistant to hypertonic conditions were researched in detail in this study. The research was following as:1. Effects of TolC in ExPEC to biofilm formationIn order to study the role of TolC protein for biofilm formation in ExPEC, we used the suicide vector to successfully construct tolC gene mutant(△tolC) and complementation strain (Cm-tolC) from ExPEC PPECC42 WT strain. The results showed that the △tolC mutant lost the ability to form mature biofilm in M9 medium, but the complementation strain containing plasmid pSH396::tolC partly restored the ability to form biofilm. Biofilms were grown on glass coverslips in M9 medium, and attached bacteria were visualized using scanning electron microscopy. The results showed that the WT strain attached the coverslips, formed the cell-cell interactions and generated large amounts of extracellular fimbriae, while △tolC mutant was unable to attach the coverslips, and did not form biofilms and extracellular fimbriae. In E.coli, the strains can produce the curli and cellulose as two important extracellular matrixs that takes part in biofilm formation and binds the Congo red of calcofluor. In our study, WT and Cm-tolC strain produced curli on M9-CR plates at 28℃, but △tolC did not. Whereas △tolC mutant increased the production of the cellulose. The biogenesis of curli and cellulose required two divergently transcription operon, the csgABC and csgDEFG operon, encoding the structural components of curli, and bcsABZC operon, involved in cellulose biosynthesis. Here, the mRNA levels of csgB or csgD and bcsA gene were tested for all strains grown on M9 medium at 28℃. The mRNA level of csgB and csgD gene was significantly decreased in △tolC mutant compared to that levels in the wild-type strain (p<0.01), but the mRNA level of bcsA gene was significantly higher in △tolC mutant for middle and later stage in biofilm formation (p<0.01). These results demonstrated that TolC was essential for the formation of mature biofilm and production of extracellular matrix in ExPEC and were coincident with the morphotypes described as above. It is reported that the E.coli biofilm formation and synthesis of curli was controlled by osmolarity, we carried on the biofilm formation assay and staining of curli in 1/2 M9 medium under 28℃ to explore the reasons of TolC affecting biofilm formation and synthesis of curli. The data showed that the △tolC mutant was restored the ability of biofilm formation and synthesis of curli and cellulose in 1/2M9 medium to that of WT strain. Additionally, the mRNA levels of csgB, csgD and bcsA gene were restored in △tolC grown in middle and later stages. In order to further confirm that the osmolarity can affect biofilm formation of △tolC mutant, biofilm formation of WT and △tolC mutant was detected, which grown on 1/2 M9 medium added a series of concentrations of NaCl and sucrose. The results demonstrated that the abilities of biofilm formation was reduced or inhibited in 1/2 M9 medium added NaCl and depended on concentration for WT and AtolC mutant; but △tolC mutant was susceptible to osmolarity in biofilm formation and significantly decreased or lost the biofilm formation compared WT strain in same concentration of NaCl or sucrose. Hence, the difference of forming biofilm for △tolC mutant between M9 and 1/2 M9 medium may be probably due to different osmolarity between M9 and 1/2 M9 medium. On the one hand, the △tolC mutant decreased biofilm formation was not related to small signal molecules associated biofilm by TRANS-WELL assay. On the other hand, structural integrity of the outer membrane was detected by transmission electron microscopy (TEM) and ultramicrotomy, and it was damaged in △tolC strain compared with WT strain grown on M9 medium at 28℃. Altogether, our observations suggest that TolC is essential not only to drug resistance and virulence, but also to biofilm formation and expression of curli. Furthermore, TolC mutant losing biofilm formation and curli was due to alteration in response to stress, which was caused by outer membrane structure damaged.2. Effects of ToIC in ExPEC to pathogenicityIn this section study, the virulence of △tolC, △csgD and WT strains was tested in vivo or in vitro. Firstly, it is found that WT strain did only produced curli in M9 medium, but △tolC and △csgD strain did not produce curli in M9-CR plates assay and all strains did not producing curli in LB-CR plates. Secondly, the adhesion and invasion of ExPEC to pulmonary epithelial cells (A549) and mouse macrophage (RAW264.7) was determined. The results showed a significant reduction of 50-80% in the adhesion and invasion of AtolC mutant compared with that of the WT strain and its complementation strains grown on M9 medium (P<0.01), ant that the abilities of adhesion and invasion of △tolC mutant were similar with that of the △csgD mutant. However, the abilities of adhesion and invasion were no difference between WT strain and △tolC mutant strain grown on LB medium. Lastly, survivals of all strains grown in M9 medium in RAW264.9 cells were tested, and the data demonstrated that the survival ability of △tolC and △csgD was similar and significantly decreased compared with WT strain. Curli plays important roles in adhesion, invasion and colonization for bacteria to host cells, so these results suggested that adhesion, invasion and survival of △tolC was reduced due to △tolC mutant lost curli production. The virulence of the WT stain and △tolC mutant and complementation strains was examined with mouse intraperitoneally infected, and the results showed △tolC mutant attenuated virulence compared with ExPEC WT and its complementation strain in mice. Additionally, the damages of visceral organs infected △tolC mutant were compromise compared with WT strain, and bacteria loaded in organs were also decreased in △tolC mutant than WT strain. Taken together, ToIC taken part in pathogenicity for ExPEC in vivo and in vitro, and the reduction in virulence of △tolC mutant may due to its less curli production. Moreover, membrane proteomics analysis showed that differentially expressed proteins included metabolic enzymes, regulatory protein and virulence associated proteins between WT and △ tolC mutant by 2D electrophoresis and mass spectrum. Based on the above results, it is speculated that reduction of pathogenicity for △tolC mutant in mice was due to decreasing colonization and proliferation in host cells, which reasons may be that △tolC mutant lost curli production and changed expression of metabolic enzymes, regulatory protein and virulence associated proteins.3. Effects of ToIC in ExPEC to survival in hypertonic conditionsExPEC WT, △tolC deletion mutant and its complement strains were cultured in M9 media containing varying concentrations of NaCl or sucrose. Noticeably, the growth of the △tolC mutant strain in different high-osmolarity media was severely inhibited during the whole experimental period, with significantly lower bacterial count than the WT and complement strains after 24 h under the hypertonic conditions (P< 0.05 or P< 0.01). Additionally, the populations of △tolC mutant strain significantly reduced under hypertonic conditions induced by NaCl and sucrose in a time dependent manner (P< 0.05 or P< 0.01). The results showed that the growth and survival of △tolC in ExPEC was decreased in high-osmolarity conditions, and it was found that TolC was essential for ExPEC to resist to hypertonic conditions. Therefore, this phenomenon is regarded highly valuable and interesting for food preservation due to ExPEC TolC mutant reducing the survival in high-osmolarity conditions. To further elucidate the role of TolC, the survival of WT ExPEC treated efflux pump inhibitor CCCP under the hypertonic conditions was evaluated. The growth and survival of WT strain in varying high-osmolarity solution containing 12.5μg/ml CCCP significantly reduced than that without CCCP treatment. In addition, CCCP inhibited ExPEC biofilm formation at concentrations 4,8 and 16mg/L, it was shown that CCCP can effectively abolish biofilm formation of ExPEC. Due to EPIs enhancing bacteriostasis and bactericidal effects in hypertonic conditions, and reducing or inhibiting the biofilm formation, therefore, EPIs derived from plants or plant extracts as additives for food industry may have a meaningful, effective and potential to persevere food.