| Candida albicans,as one of the most common pathogenic fungi,can cause various diseases ranging from superficial to life-threatening,especially in immunocompromised patients.Because of the limitation of existing antifungal drugs and the resistance of Candida species,new antifungal drugs should be urgently developed.Antimicrobial peptides are a potential new type of antimicrobial drugs.In this study,the antifungal activity of antimicrobial peptide P107 against C.albicans was measured,and the synergistic effect of P107 with Amphotericin B(Am B)and EDTA was measured.The mechanisms of cell wall,membrane and intracellular target of P107 against C.albicans were studied.The antimicrobial activity of P107 against C.albicans in Galleria mellonella was evaluated by performing in vivo experiments.The main results were as follows:(1)The minimum inhibitory concentrations(MIC)and minimal fungicidal concentration(MFC)of P107 against C.albicans was 12.5 μg/m L and 20 μg/m L,respectively.P107(0.391 μg/m L–3.125 μg/m L)presented synergistic effects when combined with Am B(0.313 μg/m L or 0.625 μg/m L).P107(0.391 μg/m L–3.125 μg/m L)presented synergistic effects when combined with EDTA(7.813 μg/m L or 15.63 μg/m L).The killing kinetics curve showed that P107 had a significant inhibitory effect on the growth of C.albicans.P107 has a low hemolytic activity against rabbit red blood cells.(2),The cell wall of C.albicans treated with P107 was damaged,resulting in cells being stained with deep purple after staining with crystal violet.The results of sorbitol experiment revealed that P107 can affect the synthesis of C.albicans cell wall.Both PI staining and membrane permeability analysis revealed that P107 increased the cell membrane permeability of C.albicans cells in a short time.The calcium leakage experiment shows that P107 can damage the cell membrane and cause intracellular calcium leakage.The results of scanning electron microscopy showed that the C.albicans cells treated with P107 was shrinkage and the cell membrane was ruptured.Transmission electron microscopy results revealed that the structure of the cell wall of C.albicans cells treated with P107 was scattered and the integrity was lost.Results of reverse transcription-quantitative PCR(RT-q PCR)revealed that P107 significantly downregulated the expression levels of the cell wall synthesis gene GSL1 and the cell membrane ergosterol synthesis related gene ERG5.(3)DNA binding analysis revealed that P107 can bound to the genomic DNA of C.albicans,and the electrophoretic mobility of DNA exhibits a concentration-dependent manner with peptide.DAPI staining results showed that P107 can interact with the nuclei of C.albicans.Results of RT-q PCR revealed that P107 affected the expressions of DNA replication and repair related genes of C.albicans.In addition,P107 treatment can induced cellular ROS generation in C.albicans.The protective effect of adding antioxidants on C.albicans indicates that the accumulation of ROS exacerbates the damage of P107 to C.albicans.(4)P107 has good antibiofilm activity against C.albicans.At 2 MIC,the inhibition rate of P107 against C.albicans cells adhesion was 44.5%.The inhibition rate of 2 MIC of P107 against C.albicans biofilm formation was 99.24%.The desruption rate of 2 MICof P107 against C.albicans biofilm was 96.01%.(5)The antifungal activity of P107 in vivo was evaluated using a C.albicans infection model.Compared with the untreated group,different doses of P107 significantly improved the survival rate of larvae infected with C.albicans.After treatment with P107 at 20 mg/kg and 40 mg/kg for 3 d,the infected cells of C.albicans in the larvae were completely eliminated.This study will theoretically help to understand the antifungal activity and mechanism of P107,and provide technical support for the development of P107 as a new antifungal agent in application. |
PDF Full Text Request