Mechanism Of Antimicrobial Peptides P255 And P256 Against Candida Albicans

Candida albicans is the main opportunistic pathogen of human beings.When the host immune state is disturbed,C.albicans can cause local or systemic diseases.With the increase of incidence rate and drug resistance of C.albicans,new antifungal agents are urgently needed.Antimicrobial peptides(AMPs)are considered to be one of the most potential antifungal agents.In this study,antimicrobial peptides P255 and P256 were designed based on PAF26.Their antifungal activities were determined by micro-dilution method,and the synergistic effects of peptides with Amphotericin B and EDTA were determined.Mode of action of P255 and P256 against cell wall,cell membrane and intracellular targets were systematically studied.The antimicrobial activity of P255 and P256 in vivo was evaluated by using a Galleria mellonella-C.albicans infection model.The main results were as follows:(1)Both P255 and P256 have six amino acids,which are cationic peptides.Structural simulation and helical wheel diagram prediction showed that P256 had typical amphiphilic structur with cationic amino acids aggregated on the same side.The MICs of PAF26,P255 and P256 to C.albicans were 300 μg/m L,125 μg/m L and 50 μg/m L,respectively.P255(3.90625 μg/m L–31.25 μg/m L)and P256(1.5625 μg/m L–12.5 μg/m L)presented synergistic effects when combined with Amphotericin B(1.25 μg/m L).(2)The cell wall and cytoplasm of C.albicans treated with P255 and P256 showed dark purple after crystal violet staining,and the cell wall integrity of P256 treatment was significantly lower than that of P255 treatment.Both P255 and P256 increased the cell membrane permeability of C.albicans in a short time.Results of scanning electron microscopy showed that the structure of the cells treated with P255 and P256 was abnormal and irregular,and leaked cell contents and cell debris were observed around the cells.Results of transmission electron microscopy showed that the cytoplasm of the cells treated by P255 and P256 was disordered,the electron density was uneven,and multiple vacuoles appeared inside.RT-q PCR results showed that after treatment with P255 and P256,the expression levels of cell wall synthesis-related genes GSL1 and GSL2 were significantly up-regulated.While after treatment with P255,the cell membrane ergosterol synthesis-related gene ERG1 was significantly down-regulated and ERG11 was significantly up-regulated.Cell membrane ergosterol synthesis-related genes(ERG1,ERG5 and ERG11)were significant up-regulation when treated by P256.(3)The cell adhesion rates of C.albicans treated with 2 MIC of P255 and P256 were62.3% and 78.01%,respectively.2 MIC of P255 and P256 could inhibit more than 95% of C.albicans biofilm formation.The damage rates of C.albicans biofilm treated by 2 MIC P255 and P256 were 43% and 56.57%,respectively.Results of the combined effects assay showed that the combined effects of P255 and P256 with Amphotericin B or EDTA were superior to that of the AMP alone.(4)P255 and P256 could bind to C.albicans genomic DNA,and the variation of DNA electrophoretic mobility showed a concentration dependent relationship.RT-q PCR results showed that the expression of DNA replication gene PSF1 and DNA repair gene RAD16 of C.albicans treated with P255 was significantly up-regulated,while the expression of DNA replication gene PSF1,PSF2 and DNA repair gene RAD16 in C.albicans treated with P256 was significantly down-regulated.In addition,treatment with P255 and P256 could induce the increase of intracellular ROS in C.albicans.(5)Results of resistance assay showed that C.albicans did not develop resistance to P255 and P256 after 14 d of continuous treatment.The hemolytic activity of P255 and P256 against fresh rabbit erythrocytes was low at the tested concentration.(6)Compared with untreated group,different doses of P255 and P256 could significantly improve the survival rate of larvae infected with C.albicans,and the survival rate of P256 treatment was higher than that of P255 at low dose(10 mg/kg).Treatment with P255(40 mg/kg)and P256(20 mg/kg)for 72 h could completely eliminate C.albicans from the larvae.After treatment with P255 and P256,the melanin content in the haemolymph decreased.Histopathological analysis showed that the infected C.albicans cells treated with P255 and P256 were less than those in untreated group.This research will theoretically help us to understand the antifungal activity and mechanism of P255 and P256,and provide technical support for the development of P255 and P256 as new antimicrobial agents in application.