| The excessive and prolonged use of antibiotics in large-scale animal breeding and human clinical treatment has resulted in a series of serious problems such as antibiotic contamination,drug residue,and the emergence of antibiotic-resistant bacteria.Some bacteria have become super-resistant to multiple antibiotics,seriously threatening global public health.China is one of the countries most affected by antibiotic resistance.Therefore,it is urgent to develop safer and more effective antibacterial drugs.Due to their advantages such as wide sources,high antibacterial activity,low cytotoxicity,low resistance,simple structure,and easy modification,antibacterial peptides have become a popular topic in the development of the antibacterial drugs.Insect antibacterial peptides were the earliest discovered antibacterial peptides and have long been widely studied.In this study,a novel antibacterial peptide,named LL-1,was discovered from Dichocrocis punctiferalis(Guenée).Firstly,the biochemical characteristics of LL-1 were analyzed and its biological characteristics were evaluated.Then,Escherichia coli(E.coli)was selected as a model bacterium to study the antibacterial mechanism and target of LL-1.Finally,an E.coli-infected mouse model was established to evaluate the in vivo antibacterial effect of LL-1 and analyze its impact on the intestinal flora of mice infected with E.coli.Results:1.A new antimicrobial peptide LL-1 was identified through sequence alignment analysis,and its full-length gene was amplified using RACE PCR.The ORF gene of LL-1 encodes 63 amino acid residues with a length of 192 bp.Sequence alignment analysis showed that mature LL-1 was a cecropin antibacterial peptide containing 39 amino acid residues.The antimicrobial activity of LL-1 was tested using a serial dilution method,and the results showed that LL-1 had strong antibacterial activity against gram-negative bacteria,with a minimum inhibitory concentration(MIC)of 3.125 μg/mL against E.coli(ATCC 25922).The hemolytic activity of LL-1 on mouse erythrocytes was also tested,and the results showed that the hemolysis rate of LL-1 was still less than 10%at a concentration of 250 μg/mL.Furthermore,a CCK-8 cell toxicity assay kit was used to evaluate the effect of LL-1 on cell proliferation,revealing that PK-15 cell proliferation exceeded 95%when treated with LL-1 at concentrations less than 250μg/mL.Finally,plate drilling experiments demonstrated that LL-1 possessed stability under high temperatures and salinity.2.Transmission and scanning electron microscopy observations showed that LL-1 had a good antibacterial effect on E.coli,which could cause deformation,swelling,decreased intracellular electron density,cytoplasmic dissolution,cell membrane damage,separation of cell walls,roughening of the bacterial surface,blurred contour deformation,bacterial shrinkage,and bacterial adhesion.LL-1 could significantly cause leakage of E.coli ALP(P<0.01),β-galactosidase(P<0.01),and intracellular nucleic acids and proteins(P<0.01).The results of PI staining experiments showed that the fluorescence intensity of E.coli treated with LL-1 was significantly higher than that of the untreated control group(P<0.01)in a dose-dependent manner.Additionally,LL-1 interacted with E.coli DNA as demonstrated by nucleic acid gel electrophoresis.Intracellular ATP and ROS levels were assessed,revealing that 3.125 μg/mL(MIC)of LL-1 significantly reduced the intracellular ATP content of E.coli(P<0.05)and significantly increased intracellular ROS accumulation(P<0.01),and the effect was dose-dependent.These findings suggest that LL-1 increases the permeability of the cell membrane and cell wall of E.coli,causes content leakage,disrupts DNA replication and transcription,impacts energy metabolism,and ultimately leads to bacterial apoptosis.3.LL-1-resistant E.coli was generated by continuous exposure to sub-inhibitory concentrations of LL-1,and the subsequent MIC was measured at 190 μg/mL.Genomic resequencing and SNP analysis were performed on the resistant mutants to identify potential antimicrobial targets.This analysis revealed significant mutations in both the mgrB and lpxT genes,which were confirmed through PCR amplification sequencing.Transcriptome sequencing of drug-resistant mutants and sensitive strains demonstrated significant up-regulation of the mgrB gene,and the differentially expressed genes were involved in key metabolic pathways related to the overall composition of the cell membrane and the formation of membrane molecules in E.coli.4.The disease model of E.coli-infected mice was successfully established by intraperitoneal injection,and the optimal infection dose was found to be 108 CFU/mL.The in vivo antibacterial effect of LL-1 was evaluated,revealing that an 80 mg/kg concentration of the peptide significantly reduced various clinical symptoms of E.coli-infected mice.Furthermore,LL-1 maintained their body weight,reduced mortality rates,improved their liver index,lowered kidney and spleen indices,and reduced inflammatory responses.It also significantly decreased bacterial loads in internal organs such as the heart,liver,spleen,lungs,and kidneys and mitigated histopathological changes in mouse tissues.Compared to 200 mg/kg enrofloxacin,LL-1 demonstrated a superior therapeutic effect.5.Intestinal microflora sequencing revealed that the microflora of the E.coli-infected group(POS group)deviated from that of the control group treated with PBS,whereas the microflora of the LL-1-treated group(PEPJ group)was similar to that of the control group.These results confirmed that LL-1 could inhibit the destruction of the cecal dominant bacterial community caused by E.coli and maintain normal cecal microbiota in mice.The impact of LL-1 on the cecal microbiota mainly involved increasing the number of dominant bacteria,such as lactic acid bacteria,and reducing the alpha and beta diversity of the cecal microbiota.LL-1 had no significant effect on the functional properties of the cecal microbiota in infected mice.The COG function of the cecal microbiota of the four groups of mice was similar,possibly due to the relatively short experimental duration.In conclusion,this study affirms that the novel antibacterial peptide LL-1 had high stability,low toxicity,and a potent antibacterial effect against gram-negative bacteria.LL-1 achieves its antibacterial action by significantly impacting the permeability of the E.coli cell membrane,possibly by targeting mgrB and lpxT.The peptide was effective in treating E.coli-infected mice at a dose of 80 mg/kg by increasing the number of dominant bacteria,particularly lactobacillus,in the caecum of mice.Additionally,LL-1 could prevent the destruction of the cecal bacterial community inflicted by E.coli,while also maintaining the normal cecal microbiota. |
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