The Study On The Biological Activity Of The Antimicrobial Peptide CRAMP And Its Mechanism In The Eradication Of Pseudomonas Aeruginosa Biofilm

Pseudomonas aeruginosa(P.a)is a common zoonotic opportunistic pathogen,Increased separation rate of P.a in veterinary clinics,as a result of improper feeding management,poor e nvironment and vaccine injection.It is easy to form biofilm,which are inherently resistant to a variety of antimicrobial agents,P.a has been widely used as a model strain for studying biofil m.Antimicrobial peptides(AMPs)are oligopeptides with five to over a hundred amino acids,and it is not easy to develop drug resistance,as a candidate to replace antibiotics.Much of the p revious research about CRAMP as a member of cathelicidin-derived antimicrobial peptides ca n effectively eradicate the mature biofilm of PAO1.This subject has carried out study on the b iological activity of CRAMP,which mainly includes preliminary safety and stability evaluat ion,and research on anti-biofilm activity,and using transcriptome RNA-Seq technology to ex plore the mechanism of CRAMP to eradicate the mature biofilm of PAO1,It lays a theoretical foundation of its mechanism of action and the development of new anti-biofilm drugs.1?The preliminary evaluation of the safety and stability of antimicrobial peptides CRAMPIn order to investigate the stability of CRAMP,the effects of temperature,enzymes,metal ions and acid-base gradients on its antibacterial effect were investigated.The results showed that the minimum inhibitory concentration(MIC)of CRAMP on PAO1 was 15.625?g/mL;temperature(25-100?),two salt ions(Na⁺,K⁺)and pH value of 5-10 had no effect on its antibacterial activity.However,the pH 3-4,divalent salt ions Ca²⁺and protease all affected the antibacterial activity of CRAMP to varying degrees.In order to investigate the safety of CRAMP,CRAMP(62.5-500?g/mL)was mixed with4%Rabbit erythrocyte in equal volume to determine the hemolysis rate;according to the LDH Cytotoxicity Assay Kit method,CRAMP was used to determine the effect of CRAMP on mouse peritoneal macrophages RAW264.7 cytotoxicity.The results showed that the hemolysis rate of CRAMP to rabbits was below 2%at the tested concentration;when the test concentration range was 62.5-125?g/mL,CRAMP had no cytotoxicity to RAW264.7,and when the concentration was greater than 250?g/mL,CRAMP had no cytotoxicity to RAW264.7.It is cytotoxic.2?The effect of antimicrobial peptide CRAMP on mature biofilm of PAO1According to the results of preliminary experiments,72-hour mature biofilms of PAO1were constructed in different culture systems.In a 96-well cell culture plate,the amount of biofilm was detected by crystal violet staining,and human antimicrobial peptide LL-37 was set as the control group to screen out the best CRAMP intervention conditions.Then const ruct mature biofilms in 6-well cell culture plates and T25 cell culture flasks.After the best intervention concentration of CRAMP is used for 1 hour,the amount of biofilms is detected by crystal violet staining method,plate colony counting method and Thiazole Blue(MTT)method,The number of viable bacteria and the viability of bacteria,the laser scanning con focal microscope(CLSM)was used to observe the morphological changes of the PAO1 biofi lm by CRAMP intervention.The results of crystal violet staining method showed that in 96-well plates,the reduction rate of biofilm in CRAMP group was 56.09%(P<0.01),and the reduction rate of biofilm in LL-37 group was 46.76%(P<0.05).In 6-well plates,the reduction rate of biofilm was 56.09%(P<0.01).,The reduction rate of biofilm in CRAMP group was 67.07%(P<0.001),the reduction rate of biofilm in LL-37 group was 52.23%(P<0.05),the reduction rate of biofilm in T25 cell culture flask was 70.45%(P<0.001).The colony count results showed that CRAMP decreased by 0.87 Log10 CFU/well in 6-well plate,LL-37 decreased by 0.30 Log₁₀CFU/well,and in T25 cell culture flask,CRAMP decreased by 0.79 Log₁₀CFU/well.The results of the MTT test showed that both CRAMP and LL-37 reduced the activity of PAO1 biofilm bacteria,but the difference was not significant.CLSM results showed that compared with the control group,the CRAMP group significantly redu ced the total bacterial fluorescence intensity(PI+SYTO,P<0.05),the reduction rate was58.40%,and the total bacterial fluorescence intensity reduction rate of the LL-37 group was21.82%(P>0.05).Compared with the control group,the ratio of the fluorescence intensity of dead bacteria to the total fluorescence intensity(PI/PI+SYTO)of the CRAMP group inc reased significantly(P<0.001,the increase rate was 64.10%),and the LL-37 group increa sed by 36.36%(P<0.05).The above results indicate that CRAMP has significantly elimina ted the total amount of PAO1 biofilm and has a significant inhibitory effect on living bacteria in the biofilm,and the effect is better than that of LL-37.3?Transcriptome RNA-Seq technology explores the mechanism of CRAMP clearing the mat ure biofilm of PAO1Using the Illumina second-generation high-throughput sequencing platform,PE150 seq uencing strategy was used to analyze the difference in gene expression between CRAMP int ervention PAO1 biofilm and the control group at the transcription level,and 12 differentially expressed genes were selected for q RT-PCR verification.The results showed that after CRA MP intervention in the PAO1 biofilm,there were a total of 2914 differentially expressed genes(1489 up-regulated,1425 down-regulated).GO functional analysis mainly focused on functio nal groups such as metabolic processes,organic metabolic processes,and primary metabolic processes.KEGG functional analysis mainly Concentrate on metabolic pathways,carbon m etabolism,biosynthesis of secondary metabolites,oxidative phosphorylation and other path w ays,as well as biofilm regulation systems;using q RT-PCR to verify 12 differentially expresse d genes related to biofilms,9 genes and RNA-Seq results have the same trend,that is,75% consistency,indicating that the high-throughput sequencing results are credible.4?Effect of antimicrobial peptide CRAMP PAO1 biofilm polysaccharide contentAccording to the results of transcriptomics studies,it was found that genes related to al g al polysaccharide synthesis were significantly down-regulated.Combined with the aforemen t ioned phenotypic analysis,the effect of CRAMP on clearing the mature biofilm of PAO1 is cl osely related to the main component of extracellular polysaccharide(EPS),alginate polysacc h aride.In order to further verify its mechanism of action,the 1.3-naphthodiol method was us ed to detect the content of algae polysaccharides in the biofilm.The results showed that af ter CRAMP,the content of PAO1 biofilm algae polysaccharides was extremely significantly red uced,and the reduction rate was as high as 70.06%.In summary,the antimicrobial peptide CRAMP has high stability(except for enzyme sta bility),no hemolysis and low cytotoxicity;it has a significant clearance effect on P.a mature bi ofilm,and is closely related to the reduction of the synthesis of algal polysaccharides.