Mechanism Of Antimicrobial Peptide Merecidin Inhibiting The Biofilm Of Pseudomonas Aeruginosa

Background The previous research results of the research team showed that the antimicrobial peptide Merecidin can inhibit the biofilm formed by clinical isolates Pseudomonas aeruginosa PA03,and the prokaryotic transcriptome sequencing analysis of the biofilm formed by the antimicrobial peptide Merecidin before and after PA03.Objective To analyze the results of PA03 biofilm transcriptome sequencing before and after the Merecidin of antimicrobial peptides and to screen the differentially expressed genes in order to find the mechanism of antimicrobial peptides merecidin inhibit the biofilm PA03 Pseudomonas aeruginosa.Methods1.Screen the target genes based on the sequencing results of the prokaryotic transcriptome of the biofilm formed by PA03 before and after the antimicrobial peptide Merecidin treatment.2.On the basis of the results screened in the first part,the PA4781 genes in the PA03 strain were amplified PCR inserted into the p MP2444 plasmid,the overexpression vector was constructed,which was transformed into PA03 competent cells,and a PA4781 overexpression strain was constructed.The colony PCR verified its correctness.Three target sg RNAs were designed according to the PA4781 gene sequence.Using pn Cas PA-BEC system,with a view to the expected base mutation at the target to produce a termination codon,early termination of PA4781 transcription,so as to achieve the goal of knockout PA4781 gene,and the correctness of the knockout detection by Sanger sequencing method.3.Crystal violet staining was used to observe the growth of PA03 strain,PA4781 overexpressing strain,PA4781 knockout strain for 24 hours,and the development of biofilm of each strain under the action of antimicrobial peptide Mericidin 24,48,72 μmol/L Dihydroxybiphenyl solution chromogenic method was used to detect alginate production under interference from antimicrobial peptide Mericidin 48,72 μmol/L to the PA03 strain,PA4781 overexpressing strain and PA4781 knockout strain.Alginate is an exopolysaccharide polymer composed of mannituronic acid and guloruronic acid which produced by various bacteria.It is an important component of pseudomonas aeruginosa biofilm.HPLC was used to detect the biofilm content Merecidin each strain under the action of antimicrobial peptide.the microscopic changes of biofilm of each strain under the action of antimicrobial peptide Merecidin were observed by scanning electron microscope(SEM).All the tested bacteria were placed on the swimming、swarming moving plate to observe the change of the movement ability of each strain.Detection of optical density after extraction of chloroform hydrochloride detection of the expression of Pseudomonas aeruginosa in the tested bacteria.Results1.After analysis and screening,it was verified by real time PCR that the gene expression of PA4781 gene was 2.8,3.3,and 3.9 times that of the control group PA03 under the effect of the antimicrobial peptide Merecidin 12,24 and 48 μmol / L.PA4781 was used as our target gene for subsequent experimental research.As a phosphodiesterase,PA4781 has function to degrade c-di-GMP,which is bacterial second messenger molecule.2.PCR screening and Sanger sequencing results proved that PA4781 was overexpressed and the knockout strain was successfully constructed.3.(1)The results of crystal violet staining showed that under the treatment of 24 μmol/L antimicrobial peptide Merecidin,there was no significant difference in the formation of biofilm between the three groups(P>0.05).Under the treatment of 48 μmol/L and 72 μmol/L antimicrobial peptide Merecidin,there was a significant difference between the overexpression group and the normal group and the knockout group(P<0.05),the biofilm was significantly reduced,and the biofilm thickness of the knockout group was higher than that of the PA03 group(P<0.05).(2)With the increase of the concentration of the antimicrobial peptide Mericidin,the alginate content of each group decreased,and the overexpression strain had the highest inhibition rate of alginate production under the action of the antimicrobial peptide Merecidin,which reached to 65%.(3)The c-di-GMP content of PA4781 knockout strain was higher than that of PA03 and PA4781 overexpression strains(P<0.05).The biofilm c-di-GMP content of PA03 strains,PA4781 overexpressing strains,and PA4781 knockout strains all decreased after the antimicrobial peptide Merecidin treatment.Among them,the PA4781 overexpressing strain had the greatest decrease.When treated with the antimicrobial peptide Merecidin 72 μmol / L,the overexpressing strain The content of c-di-GMP is only 37.2% of the amount produced without adding drugs,while the knockout strain pn Cas PA-BEC-ΔPA4781 is 66.9%,and the PA03 strain is 58.8%.(4)Scanning electron microscopy observed the microcosmic changes of the biofilms of each strain,and found that the over-expression strain biomembrane morphology damage degree was the most obvious after the antimicrobial peptide Merecidin treatment,and the cell damage was the most serious.(5)Through the observation of the motility of each strain,it was found that the antimicrobial peptide Merecidin can inhibit the motility of each strain.The effect of the overexpressing strain was the most obvious.The overexpressing strain of Merecidin 72μmol / L had lost the motility.(6)Pyocyanin was significantly inhibited by the antimicrobial peptide Merecidin.Conclusion1.The analysis of prokaryotic transcriptome sequencing before and after the action of antimicrobial peptide Merecidin showed that there were many related pathways affecting the action of antimicrobial peptide biofilm A target gene screened in this experiment suggested that the action of antimicrobial peptide on Pseudomonas aeruginosa PA03 biofilm might be related to the c-di-GMP of bacterial second messenger molecule.2.The antimicrobial peptide Merecidin up-regulates the expression of PA4781 gene and enhances its role in degrading the second messenger molecule c-di-GMP of the bacteria,thereby amplifying the effect of the c-di-GMP system in inhibiting P.aeruginosa biofilm.Moreover,the antimicrobial peptide Merecidin may affect the formation of biofilms by inhibiting bacterial movement and the production of the virulence factor pyocyanin.