Construction Of An In Vivo Delivery System For Antimicrobial Peptides And Its Mechanism For Promoting Autophagic Clearance Of Intracellular Staphylococcus Aureus

Mastitis is one of the most common diseases in dairy cows,which can lead to a decrease in milk production and quality,causing serious economic losses.Staphylococcus aureus is one of the main pathogenic bacteria causing mastitis in dairy cows.It can invade mammary epithelial cells and replicate and survive intracellularly.However,traditional antibiotics are difficult to treat intracellular Staphylococcus aureus infection,and new treatments are urgently needed.However,antimicrobial peptides have shortcomings such as poor stability,short half-life,and easy degradation by proteases.They have low oral bioavailability and difficulty in in vivo delivery,which limits their clinical application.In addition,the in vivo bactericidal mechanism of antimicrobial peptides is not fully understood.The purpose of this study:(1)To construct an in vivo delivery system of antimicrobial peptides based on probiotic EcN 1917;(2)To explore the clearing effect and mechanism of antimicrobial peptides on intracellular Staphylococcus aureus.(1)Construction of an antimicrobial peptide in vivo delivery system based on probiotic EcN 1917 and observation of its prevention and treatment effectsThe bicistronic gene sequence containing the antimicrobial peptide Cecropin A-27 was fused to the Yeb F gene,cloned into the p ET-HITES vector through homologous recombination technology,and transfected into EcN 1917.SDS-PAGE was used to detect the expression of the target protein,and the growth curve of the recombinant bacteria was determined by the turbidimetric method.In vitro antibacterial activity of recombinant antimicrobial peptide Cecropin A-27 and EcN1917 against Staphylococcus aureus(S.aureus)and Klebsiella pneumoniae(K.pneumoniae)was characterized by agarose diffusion method.And determine the minimum inhibitory concentration of recombinant antimicrobial peptides against S.aureus and K.pneumoniae.KM female mice aged 4 to 6 weeks were selected and fed with EcN1917 recombinant bacteria(2×107 CFU/mouse/day)every day.After 5 days,they were infected with S.aureus subcutaneously and K.pneumoniae intraperitoneally.The back skin tissues infected by S.aureus were collected 3 days later,and the lungs and kidneys infected by K.pneumoniae were collected 7.5 hours later.The bacterial load of each tissue was detected,and paraffin sections were made for H&E staining and pathological scoring.The results showed that the antimicrobial peptide Cecropin A-27 and EcN1917 recombinant bacteria had inhibitory effects on both S.aureus and K.pneumoniae;the in vivo application of recombinant bacteria EcN1917 could significantly reduce the loads of S.aureus and K.pneumoniae in tissues,and alleviate the pathological changes and pathological scores of lung and kidney.(2)Mechanism of antimicrobial peptides promoting autophagic clearance of intracellular Staphylococcus aureusMac-T cells were pre-incubated with antimicrobial peptides for 12 hours and transfected with p CMV-N-m Cherry-C27 plasmid for 48 hours and then infected with Staphylococcus aureus.The results showed that both the pre-incubation of antimicrobial peptides and the transfection of antimicrobial peptide plasmids showed significant Significantly inhibited the survival of intracellular Staphylococcus aureus.In order to explore the relationship between the elimination effect of antimicrobial peptides on intracellular Staphylococcus aureus and autophagy,a Mac-T intracellular infection model after antimicrobial peptide plasmid transfection was constructed.Western blot was used to detect LC3-II levels,and si RNA was used to knock down autophagy-related proteins ATG5 and TAX1BP1 to block the autophagy pathway.The results showed that antimicrobial peptides dose-dependently enhanced the autophagic flow of Staphylococcus aureus-infected cells,and blocked autophagy to inhibit the clearance of intracellular Staphylococcus aureus by antimicrobial peptides,suggesting that Antimicrobial peptides may mediate the clearance of intracellular S.aureus by promoting autophagy.In order to explore the molecular mechanism of antimicrobial peptides promoting the autophagic clearance of intracellular Staphylococcus aureus,a Mac-T intracellular infection model after antimicrobial peptide plasmid transfection was constructed.Observe the co-localization of antimicrobial peptides with m Cherry fluorescence and Staphylococcus aureus,and use Western blot to detect antimicrobial peptide protein levels and ubiquitination of intracellular Staphylococcus aureus.Detection of ubiquitination of antimicrobial peptide proteins using co-immunoprecipitation.The results showed that antimicrobial peptides can target intracellular Staphylococcus aureus and promote ubiquitination of Staphylococcus aureus.In order to explore the relationship between bacterial autophagy mediated by antimicrobial peptides and autophagy receptors,si RNA was used to knock down the expression of autophagy receptors p62,NDP52,NBR1 and OPTN,and the internalization and activation of Staphylococcus aureus after antimicrobial peptide plasmid transfection were detected.Intracellular survival,it was found that knocking down autophagy receptors p62,NDP52,NBR1 and OPTN did not affect bacterial autophagy mediated by antimicrobial peptides.In summary,this study successfully constructed probiotic EcN 1917 that can secret e and express antimicrobial peptides.This recombinant bacteria has a preventive and th erapeutic effect on experimental Staphylococcus aureus and Klebsiella pneumoniae infect ions;Antimicrobial peptides can be taken up by host cells and target intracellular Staphy lococcus aureus,promoting its ubiquitination modification,thereby promoting the elimin ation of Staphylococcus aureus by the autophagy mechanism.