Study On The Antifungal Activity Of Antimicrobial Peptide ZY4 Against Candida Albicans

With the extensive application of broad-spectrum antibiotics,antitumor drugs,hormones and immunosuppressive agents,the incidences of fungal disease are constantly increased in recent years.For the reasons of limited antifungal drugs and the side effects,treatment for fungal infection is difficult than bacterial infection.Moreover,because fungal pathogens are eukaryotes,and therefore share many of their biological processes with humans,many antifungal drugs can cause toxicity when used therapeutically.Thus,new agents containing antifungal activity but low toxicity are needed for fungal disease treatment.A designed peptide named ZY4 with 15 amino acid residues contains strong antimicrobial activity against Candida albicans,especially the clinically isolated strains with durg-resistance.ZY4 showed no cytotoxicity on normal human cells,hemolytic activity on human blood red cells,and serine protease-inhibiting activity.Furthermore,ZY4 is very stable in human plasma.It might be an ideal candidate for the treatment of fungal infection.In hydrophilic environment,ZY4 adopts a random-coil conformation.While in hydrophobic or membrane-mimetic environments,it adopts a typical amphipathic a-helical conformation.The result of bacteria-killing kinetics experiment showed that ZY4 could rapidly exert its antimicrobial activity in 30 min at the concentration of 1×MIC(Minimal inhibitory concentration,2.35 ?g/ml),and the antimicrobial activity is proved to be lethal.It could kill all the C.albicans at the concentration of 5×MIC and 10×MIC in 30 min.The result of Scaning Electron Microscopy(SEM)showed that the cell shapes of ZY4 treated C.albicans are significantly changed?Cells expanded and deformed and a large number of bleb-like structures formed and protruded from the surface of bacteria cells.Majority of bacterial cells dissolved and the contents of the cells leaked out.Combined with its circular dichroism(CD)and SEM results,we deduce that after attaching to fungal membrane,ZY4 transforms to a typical amphipathic a-helical conformation and inserts into the fungal membrane,and ultimately the transmembrane pores form,leading to the death of flungal cells.ZY4 showed weak toxicity to mice,but no lethality,and the median lethal dose(LD50)cannot be detected.After 50 min of intravenous injection(I.V.)of ZY4,the mice showed symptoms such as drowsy,laid sprawled on their stomach,abdominal collapse when breathing,and the probability of happening and duration is dose-dependent,all the symptoms disappeared in 50 min.The mice showed little weight increase after I.V.injection of ZY4 in 14 days,but there was no significant difference by comparing with the control mice.The maximum tolerable dose of ZY4 in mice was 500 mg/kg.More importantly,ZY4 also showed therapeutic potential for systemic fungal infection induced by C.albicans.It decreased the number of colony-forming units in main infected organs,and the infiltrated inflammatory cells were decreased markedly by HE staining.After administration of ZY4,four important inflammatory cytokines were significantly inhibited by the peptide.Combined its simple primary structure with only 15 amino acid residues,which facilitates to produce,ship and store,little hemolytic activity,little cytotoxicity,and high stability in human plasma,ZY4 might be an excellent therapeutic agent for the treatment of fungal disease.