Antibacterial Activity And Mechanism Of Antimicrobial Peptide Jelleine-?

The increasing resistance of pathogens to antibiotics and chemotherapeutic drugspromotes the research and development of new antimicrobial substances,and antimicrobial peptides(AMPs)are considered to be one of the most potential antimicrobial agents.Jelleine-? is a member of the natural Jelleines antimicrobial peptide family,which exists in bee royal jelly.At present,the research of Jelleine-? only involves the activity research,and its specific mechanism of action on bacteria is not further explored.In view of this,the antibacterial activity of Jelleine-? against several tested bacteria were determined.Subsequently,the action mechanism of the peptide against bacteria was studied systematically from cell membrane and intracellular targets.The antibacterial activity of the peptide on bacterial infected Galleria mellonella and the preservation of chilled pork were also evaluated.Finally,the safety of the peptide were analyzed.The main results were as follows:(1)The sequence of Jelleine-? is PFKLSLHL-NH₂.Its molecular weight is 953.19 Da,net charge is+2.1,the proportion of hydrophilic residues is 25%,and isoelectric point is10.4.It belongs to cationic antibacterial peptide.The results of antibacterial activity showed that the MIC of Jelleine-? against Staphylococcus aureus,Escherichia coli,Listeria monocytogenes,and Salmonella enterica were 100,50,12.5,and 50?g/m L,respectively.The MBC was 200,100,50,and 100?g/m L,respectively.The peptide with lower concentration(1/4 MIC?1/16 MIC)had synergistic effect with EDTA(1/4 MIC).(2)Compared with normal cells,the membrane permeability of four tested bacteria were increased after treatment with Jelleine-?;Results of scanning electron microscopy showed that L.monocytogenes treated by Jelleine-? exhibited different degrees of deformation,fold and hollow phenomenon,and even cell membrane rupture and pore formation.Results of transmission electron microscope showed that L.monocytogenes treated by Jelleine-? exhibited obvious membrane lysis,cell content leakage,uneven electron density,and cavity.When treated with Jelleine-?(1 MIC),the initial adhesion inhibition rates of L.monocytogenes and E.coli were 43.04%and 51.03%,respectively.The inhibition rates to biofilm formation were 94.73%and 96.05%,respectively.The destruction rates to formed biofilm were 62.53%and 63.99%,respectively.(3)Jelleine-? could bind to bacterial DNA and inhibit the migration of bacterial DNA,and the migration changes of DNA showed a concentration dependence relationship.RT-q PCR results showed that Jelleine-? could affect the expression of DNA replication genes and repair genes.Jelleine-? also induced bacteria to release intracellular ROS.(4)The results of hemolytic activity and cytotoxicity showed that Jelleine-? had low hemolytic activity on rabbit erythrocytes,and had not cytotoxic to mouse fibroblast L-929cells,and could promote cell growth and proliferation.The results of microbial resistance showed that L.monocytogenes treated by different concentrations of Jelleine-? for 14 days had no drug resistance.(5)The survival rate of larvae infected with bacteria treated by Jelleine-? was significantly higher than the untreated group,and the survival rate of larvae in high-dose group was increased by 30%compared with the untreated group.The therapeutic effect of Jelleine-?(40 mg/kg)was similar to Ampicillin(2 mg/kg).After 72 h of Jelleine-? treatment,the infected bacteria disappeared.The results of histopathology showed that Jelleine-? could significantly attenuate the infection of L.monocytogenes on G.mellonella.(6)The results of sensory index,bacterial count,p H value,and TVB-N showed that Jelleine-? could effectively inhibit the growth of bacteria in chilled pork and prolong the shelf life of chilled pork for 3 days.It has obvious preservative effect on chilled pork.This study will theoretically help us to clarify the antibacterial activity of Jelleine-? and its specific action mechanism on bacteria,and provide a theoretical basis for the development of Jelleine-? as a new antimicrobial agent.