Study On The Activity And Mechanism Of An Antibacterial Peptide From Spodoptera Litura

Antimicrobial peptides(AMPs)are a class of small molecule peptides commonly found in organisms.It is an important effector molecule to produce by the body's immune system and an important part of the insect's innate immune system.With people's understanding of the side effects and drug resistance of traditional antibiotics,antimicrobial peptides have received more and more attention.Some insect antibacterial peptides have a special inhibitory or killing effect on animal and plant pathogenic microorganisms(such as bacteria,fungus and viruses).Compared with traditional antibiotics,antibacterial peptides have the advantages of low toxicity and resistance to drug resistance.We identified a sequence encoding a proline-rich antimicrobial peptide from Spodoptera litura transcript data,named it Spodoptera litura Lebocin1(Sl Leb1),and carried out research on it.Firstly,the tissue distribution and induced expression of the gene in Spodoptera litura were analyzed.Secondly,the amino acid sequence of Sl Leb1 was analyzed and aligned;then several potential polypeptide fragments with antibacterial activity were synthesized and their antibacterial activity.Finally,several peptide fragments with potential antibacterial activity were synthesized,and the activity and effect on antibacterial peptides were analyzed by bacterial growth inhibition experiment,agglutination experiment,scanning electron microscopy,transmission electron microscopy,laser scanning confocal microscopy and flow cytometry analysis the mechanism has been preliminarily studied.The main findings are as follows:(1)Its nucleotide sequence consists of a 477-bp ORF that encodes a polypeptide of 158 amino acids.The proprotein contains a signal sequence of 21 residues.The mature proprotein region consists of residues 22 to 158,which contains 3 RXXR motifs(RARR,RTAR,RNAR)and high content of proline residues in some regions.Therefore,this precursor protein may be cleaved into 4 short peptides by Furin protease at the position of RXXR motif.We named these short peptides according to the numbers of the first and last amino acid residues(22-45,46-88,89-123 and 124-158),and synthesized them by solid-phase synthesis.(2)By constructing a phylogenetic tree,it was found that Sl Leb1 has a paralog in S.litura and two orthologs in Spodoptera exigua.The full-length Sl Leb1 was aligned with 5 characterized lebocins to compare their sequence features,Sl Leb1 and Ms Leb B/C have 3 RXXR motifs,while Bm Leb1/3/4 have 4 RXXR motifs.The first and second RXXR motifs in all sequences aligned perfectly,while the third RXXR motifs of Ms Leb B/C didn't align with those of Sl Leb1/Bm Leb1/3/4.The alignment of mature peptides shows that 124-158 is similar to the mature peptides of B.mori,honeybees,Trichoplusia,and Chrysodeixis.4 proline and 1 phenylalanine residues are conserved.(3)Sl Leb1 is mainly expressed in the hemocytes and midgut of the larva,and the expression level in the midgut is significantly higher than that of hemocytes.Through q PCR experiments,it was found that significantly up-regulation was induced in hemocytes 3 hours after S.aureus injection,but significantly down-regulation was induced in the midgut 3 and 6 hours after E.coli and S.aureus injection,and 3 hours after E.coli and S.aureus injection post fat body induced significantly down-regulation.(4)The antibacterial results show that 50 ?M 124-158 has strong antibacterial activity against E.coli and B.subtilis,each short peptide shows partial activity against S.aureus,but no small peptide shows activity of S.marcescens.Scanning electron microscopy results showed that the E.coli cells treated with the peptide 124-158 became extremely elongated,the cell membranes of B.subtilis and B.bassiana were severely disrupted,the S.aureus cells became significantly larger with distinct septa in the middle of cells.Laser scanning confocal microscopy results showed that the fluorescent peptide Sl Leb1(124-158)could be localized on E.coli,B.subtilis and S.marcescens and could bind to the surface of B.bassiana spores.The results of transmission electron microscopy showed that 124-158 can cause rupture of E.coli cell membrane and leakage of contents.(5)In order to further study the function of 124-158 active small peptides,we further synthesized 4 shorter peptides based on 124-158,which were at the N-terminus(127-158,135-158)or C-terminus(124-155,124-149)lack of some amino acid residues,and then carried out antibacterial,agglutination and flow cytometry analysis.Their antibacterial experiment results showed that the 124-149 peptide has the highest antibacterial activity.The results of the agglutination experiment showed that only 124-158 and 127-158 peptides could agglutinate bacteria and B.bassiana spores.The results of flow cytometry analysis showed that after treatment with small peptides,the distribution of S.aureus cells changed significantly,that is,cells with larger diameters or more complicated surface features increased,and cells with higher DNA content also increased accordingly.This study helps to increase our understanding of proline-rich antimicrobial peptides,and lays a foundation for the further study of the antibacterial mechanism of the Lebocin family antimicrobial peptides in Lepidoptera.