Evaluation And Mechanism Investigation Of The Effects Of Protease Treatment On Acinetobacter Baumannii Biofilm Formation On The Abiotic Surface

The bacterial biofilm is a complex structure formed by cells attaching to the biological or abiotic surfaces,which proliferate and secret a large amount of extracellular matrix.The extracellular matrix is primainly comprised of exopolysaccharides,proteins and extracellular DNA,The bacterial biofilm is highly resisitant to the stress conditions and withstands adverse environments such as dryness,ultraviolet rays,disinfectants and antibiotic treatments.Acinetobacter baumannii is a common oppotunistic pathogen in the clinical environment.It has a strong tendency to form biofilm,which makes it spread widely across various hospital surfaces and cause a high number of infectious deseases.Changing the chemical properties of material surface can eliminate biofilm by making the cells difficult to adhere,such as by anchoring toxic chemicals to kill cells,or anchoring biofilm matrix hydrolytic enzymes to hydrolyze the biofilm.Previous studies have shown that the biofilm formation of A.baumannii is mediated by Csu pili.At the tip of the pili,CsuE is composed of three finger-like structures that dominate the cell adhesion and subsequent formation of biofilm on the hydrophobic surface.The absence of Csu pili or CsuE component leads to the complete loss of the ability to form biofilm.Therefore,in the curerent study,we sought to employ different proteases to inhibit the biofilm formation of A.baumannii.First,we evaluated the effects of different protease treatments on the biofilm formation of A.baumannii on different abiotic surfaces.The results showed that protease K significantly inhibits A.baumannii biofilm on both hydrophilic and hydrophobic surfaces.Interestingly,trypsin treatment is not able to inhibit A.baumannii biofilm,while promote its biofilm formation on hydrophilic surface.The results of initial adhesion and mature biofilm degradation experiments indicate that protease K efficiently diminishes A.baumannii biofilm by inhibiting the initial cell attachment on the solid surface.By conducting the isolation of outer membrane proteins(OMPs)and proteomic mass spectrometry identification,it was found that CsuE and CsuA/B proteins in the Csu pili are hydrolyzed by proteinase K.As a powerful proteolytic enzyme,proteinase K effectively hydrolyzes the Csu pili,and the loss of tip protein reults in the decrease of the initial cell attachment,which eventually leads to the inhibition of biofilm formation.The isolated OMPs were treated with both proteinase K and trypsin,and the two enzymes both hydrolyzed the Omp38,CsuE and CsuA/B protein to a certain extent.In the proteinase K treatment group,the degradation of related proteins was relatively thorough,while trypsin partially hydrolyzed these proteins.After knocking out omp38 or csuE gene,it was found that the biofilm formation abilities are diminished in the mutants,and transmission electron microscope observation shows that the stability of the connection between the outer membrane and peptidoglycan layer is impaired in omp38 mutant.According to our observations and previous published results,we speculated a structural and assembling model for A.baumannii Omp38 protein and Csu pili,and proposed that the deletion of Omp38 affects the connecting stability between outer membrane and peptidoglycan layer,subsequently interferes with the assembly of Csu pili,and eventually diminishes the formation of biofilm.Furthermore,the absence of Omp38 leads to the excessive production of exopolysaccharides that is the major component of A.baumannii biofilm’s extracellular matrix,which exhibits a supermucoid phenotype in colony morphology.As a hydrophilic polymer,exopolysaccharides in A.baumannii plays a significat role in the formation of biofilm on the hydrophilic surface.In addition,the BfmR/S twocomponent system,GGDEF/EAL domain-containing proteins responsible for the synthesis and degradation of bacterial second messenger c-di-GMP,and the K locus gene cluster of A.baumannii have also been reported to be involved in the production of exopolysaccharides and biofilm formation.We propsed a possible regulatory model ofA.baumannii exopolysaccharides production and biofilm formation by using Omp38 as a sensor:mediating by Omp38,the signal of membrane stress pressure is transmitted to BfmR/S system,then to GGDEF/EAL proteins,and the assembly and production of Csu pili and exopolysaccharides is regulated,which eventually controls A.baumannii biofilm formation.After excluding the possibility of trypsin affecting the protein in the culture medium or the hydrophobicity of the surface,it was assumed that our observation that trypsin promotes biofilm formation on hydrophilic surface may be due to its incomplete degradation of the cell surface protein.The ability of trypsin to hydrolyze Csu pilirelated proteins is not strong,and the pili on the cell surface is not completely degraded,which is the reason that trypsin does not significantly inhibit A.baumannii biofilm,especially the initial adhesion of cells.At the same time,the partial hydrolysis of Omp38 by trypsin leads to the change of membrane stress pressure.Through the regulatory components as BfmR/S system and GGDEF/EAL proteins et al.,the expression of K locus gene clusters is up-regulated to produce more hydrophilic exopolysaccharides,resulting in a significant increase in the formation of mature biofilm on hydrophilic surface.In the case of proteinase K treatment,while this Omp38mediated regulatory pathway also responds,the absence of Csu pili leads to the complete elimination of the initial biofilm adhesion,therefore,the prmotion of A.baumannii formation on the hydrophilic surface has not been observed.In the current study,the effects of different protease treatments on the biofilm formation process of A.baumannii on different hydrophilic and hydrophobic nonbiological surfaces were evaluated.The results show that proteinase K completely hydrolyzes the CsuA/B and CsuE proteins in the Csu pili and the outer ring of Omp38 protein,thereby effectively inhibits the formation of A.baumannii biofilm.It provides a potential method to inhibit and eliminate the spread of A.baumannii biofilm and its resulting infectious deseases.At the same time,a possible molecular model using Omp38 protein as a sensor to regulate A.baumannii exopolysaccharides production and biofilm formation was proposed,which provided a reasonable explaination for the obervation that trypsin treatment promotes A.baumannii biofilm formation on hydrophilic surface.Next,the function interaction of each key component in the proposed regulatory pathway will be investigated through systematic construction of mutant strains and precise protein activity analysis.