Targeting Control And Mechanism Of Pseudomonas Biofilm By Extracellular Polysaccharide Hydrolase PelAN

Food-borne pathogens have caused great harm to human health and public safety,and the formation of biofilm has intensified their risk of disease and antibiotic resistance.Enzymes are highly specific and can target specific substances in the biofilm,so as to clear the biofilm of foodborne pathogens,which has important scientific research value and wide application prospect.Pseudomonas spp.,a common food-borne pathogenic bacteria and spoilage bacteria,is regarded as a model strain in biofilm research.It widely exists in all kinds of food and environment,causing serious impact on food safety and human health.Pel is a cationic extracellular polysaccharide composed of N-acetyl-D-glucosamine and N-acetyl-D-galactosamine,which is an essential component for the formation of Pseudomonas biofilm,and is closely related to the thickness,adhesion and cell aggregation of the biofilm.Therefore,targeted inhibition of Pel of Pseudomonas can completely destroy the "core skeleton" of its biofilm,fundamentally disintegrate the formation of biofilm,and thus effectively reduce the risk of drug resistance of the bacterium.Therefore,in this study,bioinformatics analysis was firstly conducted on Pel polysaccharide biosynthesis system(PelABCDEFG),and it was found that the n-terminal of chaperone protein PelA of Pel system contains a glycoside hydrolase domain,which has the potential of targeted hydrolysis of Pel extracellular polysaccharides.The recombinant protein was further expressed and purified from the glycoside hydrolase domain of PelA(named PelAN in this study),and the enzymatic properties,safety and removal effect of the extracellular polysaccharide hydrolase PelAN on biofilm on the surface of different types of materials were investigated.Combined with gene editing technology,the mechanism of action of extracellular polysaccharide hydrolase PelAN targeted to control foodborne Pseudomonas biofilm was studied.The specific research contents and results are as follows:1.Mining and analysis of PelAN bioinformatics data of extracellular polysaccharide hydrolaseIn this study,three common Pseudomonas aeruginosa,Pseudomonas fluorescens and Pseudomonas putida were selected as research objects.The physical and chemical properties,Signal peptides,transmembrane regions,and third-order structures and functions of pelA-G,a protein related to the Pel transport system of Pseudomonas sp.,were analyzed by using bioinformatics techniques combined with Ex PASy online tool,Signal P4.0Server,TMHM-2.0 and Phyre2 software.The results showed that the physical and chemical properties of the Pel system of the three Pseudomonas were basically the same except that the theoretical isoelectric points of Pel E and Pel G proteins were different.Pseudomonas fluorescens PelA protein did not contain transmembrane region,and Pseudomonas aeruginosa Pel E protein had one more transmembrane region than the other two strains,and there was no significant difference in the transmembrane region of other proteins.Homology modeling results showed that the pelA-G proteins of the three strains had the same folding pattern and highly overlapped three-dimensional structures,suggesting that they played the same role in the transport of Pel extracellular polysaccharides.This study revealed the structural and functional commonality of Pel system of three Pseudomonas from the perspective of bioinformatics,providing important data basis for further study on the transport mechanism of Pel system.In addition,the chaperone PelA of the Pel system was found to have two specific functional domains,including one glycoside hydrolase domain at the N-terminal and one carbohydrate lipase domain at the C-terminal.These results suggest that PelA can not only assist pseudomonas to transport exopolysaccharides,but also have the potential of targeted hydrolysis of Pel exopolysaccharides.2.Expression and purification of extracellular polysaccharide hydrolase PelANIn this study,Pseudomonas aeruginosa PelA protein sequence(amino acid 48-303)and Pseudomonas fluorescens PelA glycoside hydrolase domain protein sequence(amino acid 37-288)were further selected,and their Codon was optimized by E.coli Codon Usage Analyzer 2.1.The optimized sequence was cloned into p ET-28 b expression vector containing T7 promoter,and recombinant extracellular polysaccharide hydrolases PelAN-PA and PelAN-PF containing 6 N-terminal histidine residues were constructed.E.coli BL21(DE3)cells were induced to express the two enzymes with 0.2m M IPTG at 20℃.Extracellular polysaccharide hydrolases PelAN-PA and PelAN-PF were purified with different ni-nta resins and eluted in a buffer containing 300 m M imidazole.High purity PelAN-PA and PelAN-PF proteins were obtained by AKTA purification system.The obtained high purity proteins were confirmed to be extracellular polysaccharide hydrolases PelAN-PA and PelAN-PF by mass spectrometry.The results showed that PelAN-PA and PelAN-PF proteins had the advantages of good quality,high purity and no obvious aggregation.In this chapter,the expression and purification side rates of extracellular polysaccharide hydrolases PelAN-PA and PelAN-PF were successfully constructed,and high purity PelAN-PA and PelAN-PF proteins were obtained,which provided high-quality research materials for the follow-up study on the effect and mechanism of extracellular polysaccharide hydrolase PelAN on the removal of biofilm.3.Study on enzymatic properties,effects and preliminary mechanism of extracellular polysaccharide hydrolase PelANIn order to promote the application of extracellular polysaccharide hydrolase PelAN in food industry,the enzymatic characteristics,safety and effects of PelAN were further explored in this study,and the mechanism of action was preliminarily revealed.The results showed that the optimal p H and temperature of extracellular polysaccharide hydrolases PelAN-PA and PelAN-PF were 8.5 and 40℃ respectively.It was found that both extracellular polysaccharide hydrolases had no toxicity to macrophage RAW254.7and human colon epithelial cell line HT-29.It was further proved that reducing Na Cl concentration in the enzyme system or adding exogenous metal chelating agent EDTA could improve the effect of the enzyme.Crystal violet staining combined with laser confocal scanning microscopy(CLSM)and scanning electron microscopy(SEM)were used to quantitatively analyze the effect of the enzyme on different materials(non-biological materials: glass,stainless steel and polypropylene;Biological materials:Celery,apricot bao mushroom scales and surface Pseudomonas biofilm inhibition,the results showed that after PelAN extracellular polysaccharide hydrolase treatment,Pseudomonas aeruginosa,Fluorescent pseudomonas and Pseudomonas putida biofilm biomass(BV)and mean thickness(MT)were significantly decreased,and the roughness(BR)increased significantly,It can effectively inhibit the formation of pseudomonas biofilm.Finally,CAZymes Analysis Toolkit was used to predict the enzyme activity site of extracellular polysaccharide hydrolase PelAN-PA.The results showed that glutamate(E)located at PelA 218 th was the key active site of the enzyme.It plays an important role in the activity of extracellular polysaccharide hydrolase.This study provides a new targeted elimination strategy for the precise control of pseudomonas biofilm,which helps to reduce the risk of biofilm antibiotic resistance and effectively guarantee food quality and safety.