Study On Anti-quorum Sensing Activity And Self-assembly Properties Of Antimicrobial Peptides

In recent years,infections caused by bacterial multi-drug resistance（MDR）have increased rapidly.For traditional antibiotics,they mostly act on protein,DNA,RNA synthesis,or inhibit the metabolic pathways of bacteria.However,due to the unreasonable use and even abuse of antibiotics,bacteria will develop resistance to these antibiotic mechanisms and thus develop resistance.Therefore,it is of great significance to find new antibacterial drugs.Antimicrobial peptides（AMPs）,as a new type of antibacterial drugs,have most of their targets on the cell membrane.They mainly contact and bind to the negatively charged cell membrane through electrostatic interaction,destroying the integrity of the cell membrane structure,and ultimately leading to cell death.But at the same time,there are also shortcomings such as low antibacterial efficacy and high cytotoxicity to host cells.Therefore,finding new antibacterial drugs has become a top priority.Quorum sensing（QS）is a communication system that coordinates the expression of bacterial biofilms and virulence factors.It can increase bacterial resistance and pathogenicity.Therefore,interfering with QS-mediated signals to disrupt the communication between bacteria and thereby weaken their virulence is a new method and strategy for the development of new antibacterial drugs.In this paper,the laboratory AMPsG（IIKK）₃I-NH₂,C₈G（IIKK）₂I-NH₂,LL-37 are the research objects to evaluate its anti-QS activity,cell selectivity,toxicity and self-assembly performance,and preliminary exploration of its antibacterial The mechanism has laid the foundation for the development of new antibacterial drugs.The main work of this paper is as follows:First,we evaluated the antibacterial activity of AMPsG（IIKK）₃I-NH₂,C₈G（IIKK）₂I-NH₂,LL-37 and the QS quenching ability of bacteria,and selected a variety of experimental strains for qualitative and quantitative analysis.First,the gram-positive bacteria S.aureus,S.pneumonia;the gram-negative bacteria C.violaceum CV026,P.aeruginosa PAO1 And E.coli are experimental strains to evaluate the antibacterial activity of AMPs.The results showed that the minimum inhibitory concentration（MIC）of AMPs for the five experimental strains was low（≤500μΜ）,indicating that these AMPs have broad-spectrum antibacterial activity.Next,Staphylococcus aureus,C.violaceum CV026,P.aeruginosa PAO1 were used as models to evaluate the ability of AMPs to quench the bacterial QS.The results showed that AMPs can regulate the biofilm and virulence of the bacterial QS system without affecting bacterial growth.Factors have obvious inhibitory effects.Secondly,in order to explore the specific antibacterial mechanism of AMPs,we expressed and purified the important QS-regulated protein LasR in P.aeruginosa PAO1through protein engineering.The LasR gene was amplified by PCR technology and cloned into the p ET-28a^（+）plasmid to construct the recombinant expression vector p ET-28a^（+）-LasR.The plasmid was transformed into E.coli BL21（DE3）engineering bacteria,and used IPTG induces the soluble expression of LasR,and the expression conditions are as follows:culture at 37°C to OD600≈0.6,add 1 m M IPTG,and induce 20 h at 18°C.The recombinant protein LasR was purified by affinity chromatography on a nickel column,and the concentration of the recombinant protein was determined by the Lowry method.The final protein concentration was 0.7 mg/m L.Finally,based on the results of the evaluation of the ability of AMPs to quench bacterial QS,the cytotoxicity,selectivity,self-assembly properties,and antibacterial mechanisms of AMPs were initially explored.First,we use hemolysis experiments,circular dichroic spectroscopy,and Zeta potential to explore the cytotoxicity and selectivity of AMPs.The results show that,compared with G（IIKK）₃I-NH₂,C₈G（IIKK）₂I-NH₂ has lower cytotoxicity and High membrane rupture selectivity,so we choose C8G（IIKK）2I-NH2 as the object of further research.The interaction between AMPs and LasR was measured by circular dichroism spectroscopy,and the results showed that C₈G（IIKK）₂I-NH₂significantly changed the secondary structure of LasR.Therefore,we speculate that C₈G（IIKK）₂I-NH₂ can regulate the secondary structure of the important protein LasR by changing QS and block its downstream signaling pathways,thereby failing to initiate the expression of related genes controlled by QS and reducing its virulence.SEM,TEM,and AFM were used to characterize the self-assembly performance of AMPs.The self-assembly performance of AMPs was tested at the QS concentration of 5μM and 2 m M higher than its concentration.The results showed that C₈G（IIKK）₂I-NH₂ is at two concentrations.There are two different self-assembly morphologies of vesicles and fibers at different concentrations.Combined with the SEM characterization of the morphology of the PAO1cell,it was found that at a concentration of 5μM,the PAO1 cell was intact without rupture;at a concentration of 2 mM,the cell membrane of the PAO1 cell was collapsed or even ruptured.This result indicates that self-assembly has a significant effect on the antibacterial mechanism of C₈G（IIKK）₂I-NH₂.