In Vitro Potent Activity Of ε-poly-lysine Against Candida Albicans And The Underlying Mechanisms

ObjectiveTo understand the antifungal activity ofε-poly-lysine on C.albicans,as well as the effects on the main virulence hyphae and biofilm,and further explore the antifungal mechanism ofε-poly-lysine on C.albicans.MethodsThe MIC,MFC and SMIC₅₀ values ofε-poly-lysine against C.albicans were determined by micro-dilution method according to CLISI-M27 file.The time-growth curve of planktonic strains within 48h was determined by microturbidimetry.Determination ofε-poly-lysine by crystal violet method inhibits the formation of C.albicans biofilm and mature biofilm.Laser confocal microscopy observed morphology ofε-poly-lysine intervention in C.albicans biofilm.Light microscopy was used to observe and record the formation rate of germ tubes and the length of germ tubes within 4 hours,and to photograph the formation of hyphae in C.albicans-induced mycelial medium containing different concentrations of drugs.The damage of C.albicans cell membrane before and after drug treatment was detected by PI staining.Fluorescence spectrophotometer was used to determine the ROS reactive oxygen species in C.albicans before and afterε-poly-lysine treatment.Malondialdehyde was used to determine the degree of lipid oxidation of C.albicans cell membrane before and afterε-poly-lysine treatment.The content of cytochrome C in intracellular and mitochondria of C.albicans was determined by spectrophotometer.The changes of oxidative stress pathway and main virulence genes of C.albicans were detected by Real-time quantitative PCR.The change of protein profile before and after the action of different concentrations of C.albicans was detected by Time of Flight Mass Spectrometer.ResultsThe MIC of ε-poly-lysine against C.albicans was 512μg/m L,MFC was 1024μg/m L,and the SMIC₅₀ was 512μg/m L.The inhibitory effect ofε-poly-lysine on C.albicans biofilm was positively correlated with drug concentration,and significant inhibition was observed in the early stage of biofilm formation.Biofilm-related experiments show thatε-poly-lysine has significant inhibitory effects on the formation of C.albicans biofilm and mature biofilms.Laser scanning confocal microscopy experimental results show thatε-poly-lysine can inhibit the proliferation of biofilms.High concentration ofε-poly-lysine has obvious inhibitory effect on the formation rate of germ tube and length of germ tube of C.albicans.The damage ofε-poly-lysine on the cell membrane of C.albicans is positively correlated with the concentration of drug.The action of ε-poly-lysine on C.albicans resulted in the accumulation of ROS and oxygen oxidation in the Fungs,which is the positive correlation with the concentration of the drug.ε-poly-lysine induced the mitochondria of C.albicans Pigment C was released into the cells.The q RT-PCR showed thatε-PL induced the high expression of oxidative stress gene and low virulence gene expression of C.albicans.The main peak in the mass spectrum ofε-poly-lysine acting on C.albicans showed a clear change.Conclusionε-PL has significant inhibitory effect on C.albicans phytoplankton and biofilm in vitro.In vitro experiments show thatε-poly-lysine exerts antifungus action by inhibiting mycelial formation,destroying biofilm structure,and impairing the cell membrane of fungus.Molecular mechanisms have shown thatε-poly-lysine releases cytochrome C by inducing ROS reactive oxygen species,attacking C.albicans cell membranes to aggravate lipid oxidation,and inhibiting C.albicans-associated virulence the expression of genes and proteins plays a bacteriostatic role.