Structural Design And Antimicrobial Activity Of Lipopeptides Based On Bac8c

The increase resistance in bacterial and fungal to traditional antibiotics has been a major concern in recent years.Antimicrobial peptides（AMPs）are produced by all living species and represent key components of the innate immune system,which provide a fast-acting weapon against invasive pathogens including bacteria,fungi and yeasts.Many AMPs rapidly permeate and destroy the cell membrane,causing damage difficult to fix,thus do not induce resistance compared to conventional antibiotics.Researches focused on designing new synthetic peptides based on the characteristics of the native AMPs to achieve better performance,fewer side-effects,and a minimal peptide length have garnered significant interest in recent years.In this study,we design lipopeptides composed of a palmitic acid/lauric acid/lipoic acid tail attached to the ultrashort peptides.Antimicrobial properties,hemolytic activity,and in vitro toxicity of lipopeptides are evaluated and the mode of antimicrobial action is further investigated.The peptidic moiety Lys-Leu-Leu-Lys（KLLK）/Lys-Gly-Gly-Lys（KGGK）based on lysine was N-terminal acylated with palmitic acid/lauric acid（Palmitoyl-KLLK,Palmitoyl-KGGK,Lauryl-KLLK,Lauryl-KGGK）,aswellasArg-Trp-Arg（RWR）/Trp-Arg-Arg（WRR）based on Bac8c derived from Bactenecin（Palmitoyl-RWR,Palmitoyl-WRR,Lauryl-RWR,Lauryl-WRR）.The lipopeptides were synthesized by an Fmoc solid-phase method and characterized by electrospray mass spectroscopy and HPLC.Gold nanoparticles（Au NPs）were synthesized by tetrakis（hydroxymethyl）phosphonium chloride solution mediated reduction of HAuCl₄.The lipopeptides were then self-assembled with1-dodecanethiol-anchored Au NPs via hydrophobic interaction between the lipid tails and1-dodecanethiol molecules.Transmission electron microscope（TEM）images indicated that there was no significant difference in morphology between lipopeptides and lipopeptides/Au NPs（with small diameters ranging from 3⁵ nm）.The lipopeptides and their assemblies were tested against a variety of Gram-positive bacteria（S.aureus,S.epidermidis）,Gram-negative bacteria（E.coli,P.aeruginosa）,and yeast（C.albicans）,including meticillin-resistant S.aureus（MRSA）.Minimal inhibitory concentrations（MICs）and minimal bactericidal concentrations（MBCs）of lipopeptides and their assemblies were determined and planktonic killing kinetics was estimated by surface spread plate method.The mode of antimicrobial action was further investigated by TEM and laser scanning confocal microscope.The results showed that all lipopeptides were active toward all species tested,especially two palmitic acid modified peptides（Palmitoyl-RWR,Palmitoyl-WRR）exhibited excellent antimicrobial activity with MICs ranging from 2 to 8μg/mL.Lipopeptides and their assemblies exhibited similar antimicrobial activity,except for palmitic acid/lauric acid modified KGGK.For palmitic acid modified peptides,the reduction in cell viability against E.coli、S.aureus、S.epidermidis、MRSA was rapid and progressive（almost within 1h）.After 30min incubation with Palmitoyl-WRR,approximately 100%S.aureus cells were no longer viable.However,lipopeptide assemblies displayed slower bactericidal kinetics due to reduced interaction of lipid tails with cells,which may suggest either different affinities of binding to bacterial targets or different mechanism of action.Hemolysis analyses and in vitro cytotoxicity assays revealed that self-assembling with Au NPs would improve biological toxicity of lipopeptides with long lipid tail.Furthermore,lipoic acid was conjugated to Arg-Trp-Arg（RWR）/Trp-Arg-Arg（WRR）,followed by crosslinking via UV irradiation.Antimicrobial properties,hemolytic activity,and in vitro toxicity of Lipoic-RWR,Lipoic-WRR,Lipoic-RWR NPs,Lipoic-WRR NPs were evaluated.The results indicated that Lipoic-RWR NPs and Lipoic-WRR NPs were more active toward all species with faster bactericidal kinetics,compared to those of uncrosslinked Lipoic-RWR and Lipoic-WRR.Lipoic acid modified tripeptides exhibited no obvious hemolysis and toxicity at their respective MICs before and after crosslinking.The active antimicrobial activity characteristics,fewer biological toxity,and a minimal peptide length for low-cost production would make lipopeptides and their assemblies/crosslinked nanoparticles good candidates for various antimicrobial applications.