ε-多聚赖氨酸和谷胱甘肽影响抗真菌药物对烟曲霉作用效果

Role and Effect of ε-Polylysine and Glutathione Affecting Antifungal Medicine against Aspergillus fumigatus in vitro

为了解多肽ε-多聚赖氨酸和谷胱甘肽对常见抗真菌药物体外抗人类病原真菌烟曲霉效果的影响,利用平板点菌实验、最低抑菌浓度实验中的微量液基法和E-test法,以及ROS检测等方法研究ε-多聚赖氨酸和谷胱甘肽在抗真菌药物体外抗烟曲霉过程中的作用以及可能的作用机制。通过基因定点敲除谷胱甘肽合成酶基因gcsA和gshA观察细胞内谷胱甘肽对于烟曲霉生长的重要性。结果显示,ε-多聚赖氨酸与抗真菌药物尤其是卡泊芬净和伊曲康唑呈协同作用;而谷胱甘肽则能够明显地拮抗伊曲康唑和卡泊芬净的抗烟曲霉作用。ε-多聚赖氨酸与伊曲康唑联用时菌体内的ROS含量比单独使用伊曲康唑时显著增多,而谷胱甘肽与伊曲康唑联用时菌体内的ROS含量比伊曲康唑单独使用时减少。基因敲除实验结果显示γ-L-谷氨酰-L-半胱氨酸合成酶基因gcsA为烟曲霉生长必需基因,而谷胱甘肽合成酶基因gshA对生长没有明显影响。结果表明,ε-多聚赖氨酸与谷胱甘肽影响了伊曲康唑和卡泊芬净对烟曲霉的作用。ε-多聚赖氨酸通过促进伊曲康唑刺激细胞产生ROS诱导烟曲霉死亡从而实现协同作用。谷胱甘肽为细胞内必需还原型多肽,通过消除胞内伊曲康唑诱导产生的ROS从而产生拮抗作用。

In order to understand the role and effects of ε-polylysine and glutathione in common antifungal medicines against human pathogenic fungus Aspergillus fumigatus in vitro and its possible mechanism of action during the fighting against A.fumigatus,plate test,micro liquid based method,and E-test method in antifungal susceptibility test,and reactive oxygen species(ROS)determination were adopted in minimum inhibition concentration experiments.Gene targeting knockout of gcsA and gshA in glutathion synthase was adopted to observe the importance to glutathion in the cell growth of A.fumigatus.The results showed that ε-polylysine and antifungal medicine are antagonistic,and ε-polylysine assumed synergistic action with antifungal medicines especially itraconazole and caspofungin,and glutathion could obviously antagonize against A.fumigatus.The ROS content in mycelia increased significantly when ε-polylysine was used united with itraconazole than when itraconazole was used by itself,and the ROS content in mycelia was attenuated when glutathione was used united with itraconazole.Gene knockout experiments results showed thatγ-L-glutamyl-L-cysteine synthetase gene gcsA is an essential gene to the growth of A.fumigatus,and glutathione synthase gshA gene had no significant effect on the growth of A.fumigatus.The results showed that ε-polylysine affected the effects of itraconazole and caspofungin against A.fumigatus. ε-polylysine stimulated itraconazole to irritate cells producing ROS and induce the death of A.fumigatus,accordingly realized the synergistic action.Glutathione is an essential intercellular reduction polypeptide,and produce ROS induced by eliminating intercellular itraconazole and produced antagonistic effect.