| Rapid increase in number of multidrug-resistant bacteria and fungi has become an utmost peril to society,and at the same time,there has been a drought among the pharmaceutical companies for developing new anti-infective drugs.In spite of this growing apprehension and obvious need of novel anti-infectives,only few antibiotics have entered the market during last few decades,such as linezolid,daptomycin,tigecycline,and most recently telavancin.Therefore,there is an enormous necessity to develop new classes of antibiotics possessing novel modes of action as well as different cellular targets compared to existing antibiotics to decrease the likelihood of development of resistance.Fengycin is a member of a family of cyclic lipopeptides,and it is produced by Bacillus sp.As a fungicide,Fengycin are capable of inhibiting a variety of pathogenic fungi.Compared with traditional antimicrobial peptides,Fengycin features low hemolytic activity,besides,the combination of D-amino.acid and cyclic structure dramatically reduces the vulnerability of the compound to degradation by peptidases.Fengycin,as a biological control agent,does not have the same adverse properties as do chemical pesticides,and is not easy to develop drug resistance.Fengycin and its related compounds show good prospects to the development of antifungal drugs.In this study,C16-Fengycin A was isolated and purified from Bacillus amyloliquefaciens fmb60,and the antibacterial mechanism against Fusarium graminearum and Candida albicans were studied in vitro.At the same time,based on the linear C16-Fengycin A,a series of new antimicrobial lipopeptides were designed through amino acid substitution.The purpose was to explore the effect of ionic charge and hydrophobicity on the activity of lipopeptide.Meanwhile,we obtained a novel lipopeptide with broad-spectrum activity,high efficiency and low toxicity.This provides theoretical basis for the development and application of Fengycin as a new type of natural food preservatives or antibacterial agents for drug-resistant bacteria.1.Purification and structural characterization of Fengycin homologuesFengycin,an environmentally friendly lipopeptide of microbial origin,are a series of lipopeptides with variations in both the length and branching of the ?-hydroxy fatty acid.This study was aimed at purifying and characterizing Fengycin homologues produced by B.amyloliquefaciens fmb60.Fengycin homologues were obtained through HCl precipitation,methanol extraction,ion exchange chromatography,gel filtration chromatography,and semi-preparative high-performance liquid chromatography(HPLC).Electro-spray ionization mass spectrometry(ESI-MS)and electro-spray ionization tandem mass spectrometry(ESI-MS/MS)were employed to elucidate the chemical structures of these compounds.Compound 1 was C16-Fengycin A,and compound 2 was C16-Fengycin B.The sixth amino acid in type A is alanine(Ala),and the sixth amino acid in type B is valine(Val).The two homologues showed antifungal activity towards.Saccharomyces cerevisiae,C.albicans,Fusarium oxysporum,Fusarium gramineae,and Rhizopus stolonifer.2.Antifungal mechanism of C16-Fengycin A against F.graminearumF.graminearum causes Fusarium head blight in wheat,barley,oats and stem rot of corn,and produces mycotoxins.In this work,the antifungal activity and mechanism of C16-Fengycin A against F.graminearum were investigated.The minimum inhibitory concentration(MIC)of C16-Fengycin A against F.graminearum was 64 ?g/mL,and the growth inhibitory assay also demonstrated that C16-Fengycin A exhibited a potent antifungal activity against F.graminearum.Membrane permeability,flow cytometric analysis and transmission electron microscope demonstrated that C16-Fengycin A disrupted the membrane integrity of F.graminearum.Meanwhile,C16-Fengycin A could lower the content of ergosterol,affecting the membrane structure and stability.Moreover,a gel retardation assay,spectroscopic approaches and simulative docking were used to assess the interaction of C16-Fengycin A and genomic DNA.Overall,the results revealed C16-Fengycin A exerted its fungicidal activity by destroying cellular membranes and binding to DNA.3.Antifungal mechanism of C16-Fengycin A against C.albicansC.albicans is the most common clinical pathogenic fungus,which is highly susceptible to immunodeficiency patients.Development of novel antifungal agents has become a growing trend in the treatment of Candida infections.In the study,we explored the possible antifungal mode of C16-Fengycin A.It was predicted that C16-Fengycin A had the ability to disrupt the cell wall due to its alterations of cell ultrastructure,and reduction of cell wall hydrophobicity.This was further confirmed by the changes in the exposure of the cell wall components and down-regulation of the genes related in the cell wall synthesis.Meanwhile,with the treatment of C16-Fengycin A,the levels of reactive oxygen species(ROS)increased,resulting in mitochondrial dysfunction in the cells.We hypothesized that the antifungal mechanism of C16-Fengycin A might be via the destruction of the cell wall and the accumulation of ROS,which could activate the High-Osmolarity Glycerol Mitogen-Activated Protein Kinase(HOG-MAPK)pathway.4.Design and antimicrobial effect of novel cationic lipopeptides based on linear C16-Fengycin AAntimicrobial peptides(AMPs)are promising candidates for the development of future antibiotics.In an attempt to increase the efficacy of therapeutic AMPs,it is necessary to optimize the design of peptides.In the study,we described the design and synthesis of a new series of non-natural short cationic lipopeptides as antimicrobial agents.By systematic study of design template,we found controlling charge and hydrophobicity through sequence changes was an important strategy for developing biocompatible and potent AMPs.All of the synthesized lipopeptides were tested against Escherichia coli,Salmonella typhimurium,Staphylococcus aureus,Listeria monocytogenes,including enterohemorrhagic E.coli O157:H7 and methicillin-resistant S.aureus(MRSA).The most potent lipopeptide 3 possesses broad-spectrum antimicrobial activity and has MICs in the range of 2-32 ?g/mL against E.coli,S.typhimurium,S.aureus,and L.monocytogenes.All lipopeptides showed no or low cytotoxicity toward RAW 264.7 cells.In addition,membrane permeabilization,time-kill studies,combined with biophysical studies with lipid vesicles indicated a fast membrane permeation and further leakage of intracellular content. |
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