| Antimicrobial peptides are an important component of the natural defense against invading pathogens. They are characterized by their small size and have been demonstrated to kill fungi, bacteria, virus and even transformed or cancerous cells. They are potent candidates for new antibiotics.We adopted the M27-A2project recommended by the National Committee For Clinical Laboratory Standard(NCCLS).Thirty-one peptide were tested and selected with several clinical candida albicans with different susceptibilities to fluconazole by micro dilution test method. The susceptibility results reveal that,among the31peptides only RL4and MAP28have obvious anti-candia albicans activity,Their eighty percent of minimal inhibitory concentrations(MIC80) are12.2μM and9.4μM respectively.But by checkerboard microdilution assay,3peptide,which are just RL7、LR5、LR7,have a little synergetic effects with fluconazole against a clinical fluconazole-resistant candida albicans.The fractionary inhibitory concentration index (FICI)<0.5.The preliminary experimental results showed that RL4and MAP28have a strong antifungal activity, especially for fluconazole (FCZ)-resistant Candida albicans. The next work is to make an overall investigation of the antifungal effect of RL4and MAP28and a further preliminary research of its mechanism, to provide a new idea for the study of anti-drug-resistant fungi.First of all, our study showed that RL4and MAP28could provide an action against almost all fungi tested,the rang of them were6.1μM to48.8μM and2.32μM to18.8μM. More importantly, to some FCZ-resistant C. albicans, the action of RL4and MAP28were shown higher than that of FCZ (MIC80>208.9μM). Agar diffusion tests further confirmed the antifungal activity of them.The minimal fungicidal concentration and time-kill curve found that the certian concentrations of RL4and MAP28showed some fungicidal activity. The determination of the intracellular PI (Propidium Iodide) was confirmed that RL4and MAP28could play the role of anti-fungal in a short time.At the same time, the growth of hypha experiment proved that RL4and MAP28could inhibit the hypha formation. Next, through the study on C. albicans cell membrane permeability changes and oxidative damage effect analysis,we investigated the mechanism of action of RL4and MAP28against C. albicans. Research results showed that,RL4had no significant effect on the efflux of intracellular potassium ions.but could cause the leakage of the intracellular biological macromolecules to quickly change the permeability of the cell membrane and promote Plenter the cells.Electron microscope experiments showed that the cell membrane shrinkaged and parts of the cell wall and membrane formed gap.MAP28could promote the rapid efflux of intracellular potassium ions, at the same time could quickly cause leakage of intracellular biological macromolecules, causing changes of the cell membrane permeability.And cell membrane was destroyed more heavily through electron microscope,the intracellular FITC labeled MAP28could time-dependent enhance.Moreover MAP28was able to dose-dependent increase the level of endogenous reactive oxygen species, reduce mitochondrial membrane potential.And low concentrations of MAP28could induce fungal cell apoptosis, high concentrations of it could directly kill cells.In summary, RL4and MAP28have significant antifungal activity.antimicrobial peptides have the prospect to be new antifungal agents, but the animal experiments in vivo should to be studied further. |
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