Study On The Antibacterial Activity And Mechanism Of Hermetia Illucens Cecropin

Insects are the most diverse and widely distributed species on earth.They can survive in complex environment because of their strong innate immune system.When pathogens invade insects,they can induce host insects to produce active antimicrobial peptides(AMPs).AMP is a kind of small molecular polypeptide with broad-spectrum antibacterial activity,and its are not easy to produce drug resistance.It is a potential substitute for antibiotics.In this study,we identified an antimicrobial peptide,Cecropin,from the transcriptome of the black soldier fly(Hermetia illucens)larvae.Its sequence was analyzed and the peptide was synthesized by solid phase synthesis.Its antibacterial activity against several bacteria was detected,and its mechanism of action against bacteria was studied.The main results are as follows:(1)H.illucens Cecropin mature peptide is composed of 45 amino acids with C-terminal amidation.The molecular weight of Cecropin is 4908.0 Da with 5 net charges and 12.1 p I.The proportion of hydrophilic residues is 38%.Secondary structure analysis showed that Cecropin had a typical α-helix structure.It was α-helix at N-terminal and C-terminal(50%),random coil structure at middle and C-terminal(36.96%),and the rest was extended chain.(2)The results of antibacterial activity showed that H.illucens Cecropin had good antibacterial activity against Aeromonas veronii,and the minimum inhibitory concentration(MIC)was 25 μg/m L.MIC of ampicillin against A.veronii was 100 μg/m L.Cecropin showd weak antibacterial activity against Escherichia coli,Staphylococcus aureus and Listeria monocytogenes(MIC>500 μg/m L).The hemolytic activity of H.illucens Cecropin against rabbit erythrocyte was only 5.25% at the highest concentration(500 μg/m L).(3)Cecropin(6.25 μg/m L)combined with EDTA(31.25 μg/m L～62.5 μg/m L),and EDTA(125 μg/m L)combined with Cecropin(1.5625 μg/m L～12.5 μg/m L)showed synergistic effect on A.veronii.Cecropin(1.5625 μg/m L～12.5 μg/m L)combined with Nisin(25 μg/m L)also presented synergistic effect on L.monocytogenes.(4)1.5 MIC Cecropin treatment resulted in the increase of A.veronii membrane permeability,which reached 48.94% at 8 h,which was significantly higher than that of CK.Scanning electron microscope showed that 1 MIC Cecropin treatment resulted in the destruction of the cell membrane of A.veronii,leading to the leakage of contents,irregular cells,sticky cell surface leading to cell adhesion.Transmission electron microscope showed that the cells were irregular.There were obvious cytoplasmic vacuoles,the density of electron cloud decreased and the cell membrane ruptured.H.illucens Cecropin could bind to bacterial genomic DNA and the variation of DNA electrophoretic mobility showed a concentration dependent relationship.RT-q PCR results showed that after treatment with Cecropin,the expression levels of DNA replication genes(dna A,dna B)and repair genes(Rec N)of A.veronii were significantly up-regulated.(5)Cecropin could inhibit 56.40% of the cell adhesion of A.veronii at 1/8 MIC,and ampicillin could not inhibit cell adhesion even at 2 MIC.Cecropin significantly inhibited the biofilm formation of A.veronii,and only 0.49% of the biofilm remained at 2 MIC.Even at 1/8 MIC,the inhibition rate reached 35.37%.At 1/4 MIC～2 MIC,Cecropin could significantly destroy the formed A.veronii biofilm,and the destruction rate reached46.57% at 2 MIC.This study will help us understanding the antibacterial mechanism of Cecropin,and provide a theoretical basis for the development of Cecropin as a new antimicrobia agent.