The Mechanism Of Antibacterial Peptide AMP-17 From Musca Domestica Against Candida Albicans

Objective:On the basis of confirming the antifungal activity of AMP-17,the antifungal mechanism of AMP-17 was further explored from both intracellular and extracellular levels.Methods: 1.Effects on fungal morphology and structure: The effects of AMP-17 on the morphology and structure of Candida albicans were observed by microscopy,scanning electron microscopy and transmission electron microscopy.2.Effects on fungal cell wall: Cell wall specific staining method was used to detect the change of cell wall integrity rate of Candida albicans after AMP-17 treatment.3.Effects on fungal cell membrane: The fluorescent dye DPH was used as a probe to detect the change of cell membrane structure of Candida albicans after AMP-17 treatment;The fluorescent dye PI was used as a probe to detect the effect of AMP-17 on the cell membrane permeability of Candida albicans.Determination of changes in glycerol content in Candida albicans cells after AMP-17 intervention,and response to changes in intracellular osmotic pressure.4.Effects on the expression of genes related to fungal cell wall and cell membrane synthesis: qRT-PCR was used to detect the expression changes of Candida albicans FKS2,ERG1,ERG5,ERG6,ERG11 and MET6 after AMP-17 treatment.5.Effect on the cell cycle of Candida albicans: Using PI as a probe,flow cytometry was used to detect the change of cell cycle of Candida albicans after AMP-17 intervention.6.Effect on reactive oxygen species in Candida albicans cells: DCFH-DA was used as a probe to detect the changes of reactive oxygen species in Candida albicans cells by multi-function microplate reader.The correlation between the accumulation of ROS in Candida albicans and cell necrosis was analyzed by DCFH-DA(probe for detecting intracellular ROS content)and PI(indicative dye for cell necrosis).7.Effect on mitochondrial membrane potential in Candida albicans cells: The JC-1 kitmeasures intracellular mitochondrial membrane potential levels after AMP-17 treatment.8.Effect on apoptosis of Candida albicans: Detection of apoptosis of Candida albicans cells after AMP-17 intervention by AnnexinV/FITC apoptosis assay kit.Results: 1.Microscopic observation showed that the budded cells of Candida albicans and pseudohyphae in the AMP-17 group were significantly reduced,and the internal substances of diseased cells were disordered,and the cells were aggregated and dissolved.The results of scanning electron microscopy showed that the morphology of Candida albicans in the AMP-17 group showed irregular changes,and the cells showed aggregation and dissolution.Some lesions had gaps and tailing.Transmission electron microscopy showed that the antibacterial peptide AMP-17 acted on Candida albicans for 16 h,and the shape of the cells was abnormal.There are vacuoles at the junction of some cell membranes and cytoplasm,and there are vacuoles in the cytoplasm and at the edge of the nucleus.2.After the action of the antibacterial peptide AMP-17,most of the Candida albicans cells were stained dark purple,the cell wall was not obvious,and the cell integrity rate was 26.7%,which was much lower than that of the control group,with significant difference(P<0.01).3.The relative fluorescence intensity of dye DPH decreased from 100% to 39%,which was a dose-dependent decrease.Laser confocal microscopy showed positive correlation between the number of Candida albicans cells stained with PI and the concentration of antimicrobial peptide AMP-17.The antibacterial peptide AMP-17 was able to induce the intracellular glycerol content of Candida albicans increased,but it decreased when the concentration of antimicrobial peptide reached 80 mg/l.4.After AMP-17 intervention,the cell wall synthesis-related gene FKS2 of C.albicans was up-regulated by 3.46 times,and the cell membrane ergosterol synthesis related genes ERG1,ERG5,ERG6 and MET6 were down-regulated by 5.88 times,17.54 times,13.33 times and 7.14 times,respectively.5.With the increase of AMP-17 concentration,the number of cells arrested in S phase gradually increased from30.94% to 64.63% in the control group,suggesting that Candida albicans cells were arrested in S phase.6.AMP-17 can induce the accumulation of active oxygen ROS in Candida albicans without antioxidant VC and GSH,and it is positively correlatedwith the dose.In the presence of two antioxidants VC and GSH,the ROS induction of AMP-17 is inhibition.7.Antimicrobial peptide AMP-17 can reduce the mitochondrial membrane potential of Candida albicans in a dose-dependent manner and induce mitochondrial membrane potential depolarization.8.With the increase of the concentration of antimicrobial peptides AMP-17,the number of green fluorescent cells increased gradually and there were also a small amount of red fluorescent cells,that is to say,after AMP-17 intervention,a large number of apoptotic C.albicans cells and a small amount of necrotic Candida albicans cells appeared.Conclusion: 1.AMP-17 can exert antifungal effect by destroying fungal cell wall structure,interfering with cell membrane structure,changing fungal cell membrane permeability,increasing intracellular osmotic pressure and inhibiting the expression of cell membrane synthesis related genes.2.AMP-17 can cause damage to Candida albicans cells by inducing intracellular ROS accumulation by Candida albicans,depolarization of mitochondrial membrane potential,and alteration of the cell cycle.