Research On Mechanism Of Biofilm Formation Mediated By Core Extracellular Polysaccharide Biosynthetic System In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a common opportunistic pathogen,which can not only cause serious water pollution,but also cause various chronic or acute diseases in human.P.aeruginosa is easy to form biofilm and is considered as a model strain for biofilm research.Biofilm is a multicellular aggregate formed by bacterial cells and their secretions adhering to the surface of biological or non-biological materials,which enhance the pathogenicity,drug resistance,and host resistance of bacteria,seriously endangering food safety and public health.Extracellular polysaccharides are important component in biofilm,which is capable of boosting bacterial resistance against the external environment,antibacterial agents,and host defense.P.aeruginosa can produce three core extracellular polysaccharides closely related to biofilm formation:alginate,Psl,and Pel.Their synthesis and transport in bacterial cells depend on the corresponding alginate,Psl,and Pel biosynthetic systems,respectively.However,up to now,there are few studies on the synthesis and transport mechanism of extracellular polysaccharides in P.aeruginosa biofilm,resulting in a blank in the study of biofilm formation mechanism.Therefore,in this study,the mutant strains P.aeruginosa PAO1 of three core extracellular polysaccharide biosynthetic systems were selected,PAO1-Δpel F,PAO1-ΔpslAB and PAO1-Δalg8 were used as the research object.Firstly,quartz crystal microbalance with dissipation（QCM-D）,crystal violet staining,and microscopic imaging techniques were used to conduct multidimensional characterization of the biofilm of the studied strain.Then,transcription-metabolism combined omics technology was used to analyze the internal regulatory mechanism of the biofilm of P.aeruginosa,aiming to systematically revealing the role of three extracellular polysaccharide biosynthetic systems in the biofilm formation of P.aeruginosa,and elucidating the dynamic mechanism of biofilm formation mediated by extracellular polysaccharide biosynthetic systems from the perspectives of phenotypic changes,transcriptional regulation,and metabolic differences.The detailed research content and results are as follows:1.Research on phenotypic changes of P.aeruginosa biofilm mediated by core extracellular polysaccharide biosynthetic systemAlginate,Psl,and Pel are the core extracellular polysaccharides in the biofilm of P.aeruginosa,which are secreted out of the cell by alginate biosynthetic system,Pslbiosynthetic system and Pel biosynthetic system,respectively.Alg8,PslAB,and Pel F are key enzymes in the biosynthesis of extracellular polysaccharides,respectively.The deletion of these genes will lead to the abnormal synthesis of extracellular polysaccharides.Therefore,in this article,the PAO1-Δpel F、PAO1-ΔpslAB and PAO1-Δalg8 biofilm was qualitatively and quantitatively characterized by QCM-D,crystal violet staining,electron scanning microscopy（SEM）,and laser confocal scanning microscopy（CLSM）.The results show that compared with wild type PAO1,the amount and thickness of biofilm of PAO1-ΔpslAB strain decreased by nearly 1/3,and the roughness was higher.The biomass and thickness of biofilm of PAO1-Δpel F decreased,and there was no significant difference between the biofilm of PAO1-Δalg8 and WT strain.This indicates that Pslplays the most important role in the formation of PAO1biofilm,increasing the biomass,thickness,and elasticity of biofilm;Pel is the extracellular polysaccharide second only to Pslin the PAO1 biofilm,and participates in the formation of biofilm in a redundant manner;Alginate is not expressed or slightly expressed in PAO1,and has no significant contribution to the formation of PAO1biofilm.2.Research on transcription regulation of P.aeruginosa biofilm mediated by core extracellular polysaccharide biosynthetic systemIn order to study the internal regulatory mechanism of the core extracellular polysaccharide biosynthetic system on the formation of P.aeruginosa biofilm,transcriptome analysis of the biofilm of the studied strain was conducted.The results showed that after screening for significantly differential genes（DEGs）using a screening threshold of|log₂FC|>=1,p adjust<0.05,there are 36 DEGs inΔpel F vs control,1293 DEGs inΔpslAB vs control,and no DEGs inΔalg8 vs control.The enrichment results of KEGG pathway showed that,enrichment pathways in theΔpel F vs control are flagellar assembly and biofilm formation.The differential genes inΔpslAB vs control are mainly enriched in bacterial chemotaxis,two-component system,flagellar assembly,biofilm formation,quorum sensing,cationic antimicrobial peptide resistance,valine,leucine,and isoleucine degradation,and ABC transporters.There were no significantly enrichment pathway inΔalg8 vs control.The results showed that Pslbiosynthetic system greatly affects the formation of P.aeruginosa biofilm.When the system fails to function,P.aeruginosa reduces bacterial tendentiousness by downregulating the flagella synthesis efficiency,and changes the QS regulation mode,forming a QS network dominated by PQS system.At the same time,it downregulates the expression of virulence factors,weakening the colonization ability of bacteria,ultimately greatly weakening the biofilm formation ability and pathogenicity.Pel biosynthetic system also has a certain impact on the biofilm formation of P.aeruginosa.The functional defect of Pel biosynthetic system leads to an increase in bacterial flagellar synthesis,which promotes the formation of biofilm to a certain extent,but cannot fully return to normal levels.The alginate biosynthetic system has no significant contribution to the regulation of biofilm formation in P.aeruginosa.3.Research on metabolic differences of P.aeruginosa biofilm mediated by the core extracellular polysaccharide biosynthetic systemThis study further utilized metabolomics to investigate the metabolic differences of biofilm-state strains after the loss of alginate,Psl,and Pel biosynthetic systems.The results showed that the metabolism of PAO1-ΔpslAB has the greatest difference from that of PAO1.Under the screening criteria of VIP>1,P<0.05,and FC（Fold change）>1.5,there were 75 significant differential metabolites betweenΔpslAB group and control group,and most of the metabolites were in a stacked state;Δpel F vs control andΔalg8 vs control had only one significantly differential metabolite,down-regulated c-di-GMP.KEGG enrichment pathway displayed that,inΔpel F vs control,the main enrichment pathways involved in differential metabolites were purine metabolism and aminoacyl-t RNA biosynthesis;InΔpslAB vs control,the main pathways involved in differential metabolites were lysine degradation,tryptophan metabolism,lysine biosynthesis,arginine and proline metabolism,purine metabolism,and glutathione metabolism;There were no significantly enriched metabolic pathways inΔalg8 vs control.It is demonstrated that Pslbiosynthetic system had the greatest impact on the metabolism of P.aeruginosa biofilm,with significant accumulation of amino acid and purine metabolites during functional loss of the system;When the Pel biosynthetic system is deficient,the biofilm of P.aeruginosa affects purine metabolism in vivo by reducing the content of c-di-GMP;The alginate biosynthetic system does not significantly affect the physiological metabolism of P.aeruginosa biofilm.4.Combined transcription-metabolism analysis of P.aeruginosa biofilm mediated by core extracellular polysaccharide biosynthetic systemThe results of a combined transcription-metabolism analysis betweenΔpslAB group and control group showed that the lack of function of the Pslbiosynthetic system resulted in the inability to synthesize Pslexopolysaccharides.Bacteria regulated polysaccharide metabolism to increase the level of UDP-Glc NAc,and upregulated the expression of pel A for the synthesis of Pel;Bacteria mobilized organic substances such as amino acids for the synthesis of acetyl-Co A to supply ATP to produce c-di-GMP,and multiple regulation within bacteria increased the level of c-di-GMP;C-di-GMP not only activated the expression of pel operon,but also led to low expression of genes related to bacterial flagellar synthesis and regulation;The decrease in rsa L level has changed the iron uptake pattern of bacteria,using pyocyanine and entobactin as the main iron carriers;The low expression of pyoverdine and pyochelin biosynthetic genes led to an increase in muc BCD expression,which may increase the biosynthesis of alginate.In summary,this study shows that the Pslbiosynthetic system plays a key role in the formation of P.aeruginosa biofilm.The lack of the system improves the synthesis of Pel polysaccharides,increases c-di-GMP levels,and decreases bacterial chemotaxis.The pqs system is the dominant quorum sensing system,the iron ion uptake pattern is changed,virulence is weakened,and cell adhesion and biofilm formation abilities decrease.Ultimately,it results in a decrease in biofilm biomass,thickness,and elasticity.The Pel biosynthetic system has a certain effect on the formation of P.aeruginosa biofilm.When the Pel biosynthetic system fails to function,the level of c-di-GMP in bacteria decreases,contributing to an increase in the efficiency of bacterial flagellar synthesis.Finally,the mass and thickness of biofilm slightly decrease compared to normal strains.The alginate biosynthetic system had no significant effect on the formation of P.aeruginosa biofilm.