Molecular Design And Mechanism Of Action Of Leucocin A

The rapid emergence and prevalence of drug-resistant pathogens have posed a great threat to the global healthcare,food and animal husbandry industries.It is urgent to develop new antimicrobial agents which can kill bacteria quickly,but not induce drug resistance as fast as conventional antibiotics.Recently,antimicrobial peptides(AMPs)have attracted much attention due to their broad-spectrum antimicrobial activities,and have been considered as one of the most promising alternatives to conventional antibiotics.Leucocin A is a cationic AMPs with 37 amino acids produced by Leuconostoc gelidum UAL 187.Leucocin A has low hemolytic activity,strong thermal stability and anti-Listeria activity,but its antibacterial spectrum is narrow and the antibacterial activity is lower.Therefore,we optimized the structure of Leucocin A via two methods to improve its antibacterial effects.Firstly,we modified the amphiphilic parameters based on the C-terminal(18-35 amino acids)of Leucocin A.A large number of antibacterial tests and mechanism were conducted.WRL3,with the optimal activity,was selected.Furthermore,we evaluated the antibacterial efficacy of WRL3 against methicillin-resistant Staphylococcus aureus(MRSA)in vivo and in vitro.Secondly,a series of novel ?-hairpin AMPs were designed by combining the ?-turn of Leucocin A with the repeated sequence.The anti-Salmonalla activities and mechanisms of these ?-hairpin peptides were studied.The main findings are as follows:1.Design of ?-helical amphiphilic AMPs with potent antibacterial activityThe 18-35 amino acid sequence at C-terminal of Leucocin A was analyzed and identified as an ?-helical structure,mainly consisting of negatively-charged amino acids and hydrophilic amino acids.However,previous studies have shown that positively-charged and hydrophobic amino acids have a greater impact on the antimicrobial activities of AMPs.Therefore,a series of ?-helical amphiphilic AMPs derivatives were obtained by replacement of negatively-charged and non-charged hydrophilic amino acids with arginine(Arg)and leucine(Leu).Circular dichroism showed that the structure of these novel AMPs is similar to original peptide LeuA,showing the ?-helix structure.Among these novel AMPs,WRL3 showed the strongest antimicrobial activity and cell selectivity,while maintaining lower cytotoxicity and hemolytic activity.The results from fluorescence detection and microscopy observation showed that these new AMPs could permeabilize inner and outer membranes of E.coli O157,depolarizing the plasma membrane and further causing to the loss of membrane integrity and leakage of intracellular contents,and eventually leading to the cell death.The above results showed that the amphiphilic properties of AMPs could effectively improve their antibacterial activity and bactericidal efficiency.This study provides a new and effective strategy for design and development of high efficiency and broad-spectrum AMPs.2.Biofilm formation of Shiga-toxin producing Escherichia coli in beef juice and their resistence to WRL3In this study,biofilm formation of six top STEC in beef juice at 22? or 13? were evaluated.In addition,the effect of WRL3 on STEC biofilm was investigated.The results showed that,at 22?,O113,O145 and O91 formed strong biofilm after incubation in M9 for 24 hours,but the level of biofilms decreased over time(P<0.05).The biofilm-forming ability of O113,O145 and O91 significantly reduced at 24 hours in M9 mixed with 25%,50%and 100%beef juice.However,after 48 and 72 h of incubation,the level of biofilms increased significantly(P<0.05),especially in 100%beef juice.At 13? O113,O145,O91 showed weak biofilm formation in all media after 24 hours of incubation.However,biofilm-forming ability of O113 and O145 increased(P<0.05)in 25%and 50%beef juice with incubation time increased.Moreover,the results from plate counting showed that numbers of O113 biofilm cells decreased by 1.6,2.3 and 3.7 log CFU/coupon after treatment with WRL3 for 5,30 and 60 min,respectively.Therefore,WRL3 showed a potent killing effect on O113 biofilm cells.Token tegather,low temperature can effectively inhibit STEC biofilm formation in the food processing environment,and the development of WRL3-based antimicrobials will provide novel alternatives to conventional sanitizers for removing the formed biofilm.3.Antibacterial activity in vitro and in vivo of WRL3 against methicillin-resistant Staphylococcus aureus(MRSA)WRL3 not only showed strong antibacterial activity against Gram-negative bacteria,but also indicated high antibacterial activity against Gram-positive bacteria.In this study,we investigated the antibacterial activity and mechanism of WRL3 against S.aureus and its drug-resistant bacteria.We also studied the therapeutic effects of WRL3 on MRSA-induced infection in vivo.The results showed that WRL3 exhibited strong inhibitory activity against S.aureus,comparable to polylysine,ceftriaxone and methicillin,but lower than vancomycin,gentamicin and ciprofloxacin.Interestingly,WRL3 showed the strongest bactericidal activity among these antibacterials.Moreover,WRL3 and ceftriaxone showed synergistic inhibition against S.aureus,significantly reducing the antibiotic concentration.Atomic force microscopy(AFM),transmission electron microscopy(TEM),flow cytometry,confocal microscopy and liposome leak assay showed that WRL3 probably penetrated the cell wall of S.aureus,forming holes in the membrane,and destroying the integrity of the cell membrane and eventually killing the bacteria.In vivo studies showed that WRL3 participated in the antibacterial process of wound tissue,controlled the wound infection of MRSA,and improved the tissue repair ability and wound healing.4.Designing ?-sheet peptide with enhanced antibacterial and anti-inflammatory activitiesUpon structure analysis of Leucocin A,it was found that the N-terminal sequence(1-18)of Leucocin A showed a special three-strand ?-folding structure,in which the ?-turn(9-14)had an important influence on the antibacterial activity of Leucocin A.The 1-18 amino acid sequence was truncated and its antimicrobial activity was determined.The sequence showed high thermal and ion stability,but its antibacterial activity was low and the antibacterial spectrum was narrow.Therefore,we designed a series of ?-hairpin antimicrobial peptides by combining the ?-turn(CTKSGC)with the repeat units(XK)n consisting of cationic amino acids(Lys)and hydrophobic amino acids(Val,Leu,Trp).The sequence model is as follows:(XK)nCTKSGC(KX)n,where X represents hydrophobic amino acids(Val,Leu and Trp;n=1,2 or 3).The antimicrobial activities of these novel?-hairpin peptides were tested.Compared with the parental peptide KY18,the antimicrobial activities of these ?-hairpin peptides,especially WK2,were significantly improved.The presence of salt ions and serum has little effect on the antibacterial activity of WK2,so it shows a higher physiological stability.These ?-hairpin peptides also showed low hemolytic activity and cytotoxicity.In the co-culture model,the WK2 selectively killed Salmonalla and showed no toxic effect on macrophages,so they showed high cell selectivity.The results of outer membrane permeability,plasma membrane depolarization,AFM,TEM,FITC-labeling and endotoxin binding assays showed that the novel ?-folding AMPs firstly bound to LPS by electrostatic interaction,and then enhanced the inner and outer membrane permeability by hydrophobic interaction,resulting in membrane destruction and eventually leading to bacterial cell lysis.In addition,it showed that these ?-sheet peptides can inhibit LPS-induced cytokine production,thus showing strong anti-inflammatory activity.These results suggest that these ?-sheet AMPs have great potential as new antimicrobial agents or food preservatives.5.Antibacterial activity and mechanism of WK2 against multidrug resistant S.Typhimurium biofilmAccording to the above studies,WK2 showed a strong anti-Salmonalla activity.Therefore,we further studied the anti-biofilm activity and mechanism of action of WK2 against Salmonalla.The results showed that WK2 could not only inhibit the formation of Salmonalla biofilm under the sub-inhibitory concentrations,but also killed the formed biofilm cells under higher conditions.WK2 significantly reduced the viability of biofilm cells and cell biomass.Atomic force microscopy(AFM),fluorescence quantitative PCR(RT-PCR),transmission electron microscopy(TEM)and fluorescence detection showed that WK2 might interfere with AI-2-mediated quorum sensing(QS)system by binding Salmonalla DNA,inhibit fimbriae synthesis,and eventually lead to biofilm defects.6.The effect of WK2 on intestinal tract infections caused by S.Typhimurium in mouse modelThe rapid emergence of antibiotic-resistant bacteria poses a huge challenge to current antibiotic therapy.Therefore,there is an urgent need to develop antimicrobial agents based on new mechanisms of action against drug-resistant Salmonalla.WK2 exhibits a strong anti-Salmonalla activity in vitro,but therapeutic effect on Salmonalla infection in vivo was not investigated.The results showed that WK2 could significantly reduce populations of Salmonalla in feces,liver and spleen of infected mice,inhibit the weight loss of infected mice,and significantly reduce the mortality of infected mice.TR-PCR results showed that WK2 could inhibit the expression of cytokines in intestinal mucosa and blood induced by Salmonalla.Furthermore,WK2 maintained the expression level of tight junction proteins in the intestine of infected mice,which was further confirmed by immunohistochemistry.The results of hematoxylin-eosin(HE)staining showed that the villi of small intestine of mice were demaged and shortened after infection with S.Typhimurium,but the small intestine villi treated with WK2 had a smooth surface and integrated structural system.These results suggest that WK2 can be used as a new and effective antimicrobial agent in the treatment of Salmonalla infection.