The Roles Of Outer Membrane Channel Protein TolC In Biofilm Formation Of Gallibacterium Anatis

Gallibacterium anatis(G.anatis)is a representative species of the genuse Gallibacterium in Pasteurella family,which resides normally in the upper respiratory tract and the lower genital tract in poultry.G.anatis infection can cause oviduct cysts,salpingitis,sepsis,oophoritis,and peritonitis,which lead to decrease egg production and increase mortality in laying hens.At present,the multi-drug resistance isolates are found around the world,which can reduce the effect of drug control and be easy to mix infection with other pathogens,resulting in huge losses to the poultry industry.TolC family protein(TolC)is an outer membrane channel protein,which can mediate the extracellular transport of various substances and play important roles in the bacterial biological functions.The formation of bacterial biofilms is not only important for pathogen colonization and infection,but also mediates bacterial drug resistance.Therefore,it is important in preventing and control G.anatis infection to analyze the role of TolC protein in mediating biofilm formation of G.anatis.In this study,markerless deletion mutants of tolC(ΔtolC)was constructed by natural transformation using a clinical isolate G.anatis strain,Yu-PDS-RZ-l-SLG(WT).The biological function of TolC protein in G.anatis was studied primarily by comparing the biological characteristics of WT and ΔtolC,and the function and mechanism of TolC protein mediating biofilm formation of G.anatis were further discussed.The main works are presented below:1.The construction and biological characteristics of seamless deletion mutants of tolC in G.anatisThe G.anatis seamless deletion mutant ΔtolC was obtained by homologous recombination technology,and identified by PCR and Western blotting analysis.The biological characteristics of WT and ΔtolC were compared and analyzed to speculate the biological functions of TolC in G.anatis.Compared with WT strain,the results of ΔtolC showed that,bacteria colonies and zone of hemolysis were smaller,the logarithmic growth phase was delayed 2 h,and it was with a slower growth rate,a weakened biofilm formation ability,and significantly reduced survival rates under high temperature,oxidative stress and osmotic pressure circumstance,increasing sensitivity to some antibacterial drugs in planktonic state and biofilm state,and a weak adhesion ability to Hela cells.The construction and analysis of biological characteristics of seamless deletion mutant deletionΔtolC laid a foundation for studying the biological functions,pathogenesis and drug resistance mechanism of TolC protein in G.anatis.2.Effect of Outer membrane channel protein TolC on G.anatis biofilm formationIn this study,the biofilm formation cycle,morphology and the amount of biofilm formation of G.anatis WT and ΔtolC biofilms were determined by crystal violet staining,silver staining,Congo red staining and scanning electron microscopy on qualitative or quantitative aspects,and the auto-agglutination activity and hydrophobicity were analyzed and the amount of biofilm formation in different nutrients medium were determined.Enzyme treatment,confocal laser scanning microscope(CLSM)and RT-qPCR were used to analyze the extracellular polymeric substances(EPS)composition of G.anatis WT andΔtolC biofilms and the expression levels of genes related to biofilm formation.Compared with WT strain,the results showed that the change trend of biofilm formation curves ofΔtolC was consistent with that of WT,but the amount of biofilm formation were decreased significantly,and less biofilm microcolonies were observed by silver staining.Congo red staining results showed that ΔtolC could not secrete mucopolysaccharide to form black complexes.Scanning electron microscope showed that the ΔtolC cells were shrunken and encapsulated with polymer.There was no significant difference between ΔtolC and WT in auto-agglutination activity,but surface hydrophobicity of ΔtolC was decreased significantly.In BHI medium with different concentrations of NaCl and saccharides,the amount of biofilm formation of WT was significantly higher than that of ΔtolC.And it was found that in the BHI medium supplemented with 3%NaCl and 2%and 3%glucose,the deletion of TolC protein significantly affected the biofilm formation ability of G.anatis under hypertonicity.However,BHI medium supplemented with different concentrations sucrose,the amount of biofilm formation of ΔtolC was not significantly affected was significantly.The results of enzyme treatment experiments showed that the proportion of DNA and protein in biofilm of ΔtolC was higher than that of WT at different time points,and the proportion reached a maximum at 24 h.In biofilm staining experiment,the fluorescence intensity of dead bacteria of ΔtolC strain was higher than that of WT strain at 12 h,but its polysaccharide,protein and DNA were lower the latter.Interestingly,the polysaccharide ofΔtolC strain remained significantly lower than that of WT strain at 24h.The results of RT-qPCR indicated that after tolC gene deleted,the expression levels of quorum-sensing factor luxS,fimbria-related genes flfA,flfC,flfD and flfG were up-regulated at various degrees in the early(8 h)and middle(12 h)stages of biofilm formation,and then gradually decreased.The expression levels of two-component system response regulator cpxR at 8 h and global regulator arcA and elongation factor EF-Tu at 12 h were significantly up-regulated,while the expression levels at other time points were down-regulated.In summary,the changes of ΔtolC biofilm formation were studied,it was preliminarily revealed that TolC protein positively regulates the formation of G.anatis biofilm through a complex mechanism affecting multiple functions such as polysaccharide synthesis,quorum sensing system and pressure stress and other function,which laid a foundation for exploring the mechanism of TolC protein in biofilm formation in G.anatis and provided a theoretical basis for studying TolC as a potential anti-biofilm target protein.