基于点突变库研究丝切蛋白在烟曲霉抗逆性和致病力中的作用

Role of Cofilin in stress resistance and virulence of Aspergillus fumigatus based on Cofilin point mutation library

目的通过构建点突变库探讨丝切蛋白(Cofilin)的氨基酸位点在烟曲霉抗逆性和致病力中的作用。方法构建并筛选烟曲霉Cofilin点突变株;通过应激试验分析Cofilin点突变对烟曲霉抗氧化、抗高渗透压和抗细胞壁干扰等方面的影响;利用FITC偶联的伴刀豆蛋白荧光标记分析Cofilin点突变对烟曲霉细胞壁甘露糖合成及分布的影响;通过大蜡螟幼虫感染试验分析Cofilin点突变对烟曲霉致病力的影响。结果构建的烟曲霉Cofilin点突变库包括单突变K26A、E56A和双突变D11A-E12A、K41A-K42A和R59A-R61A;D11A-E12A、K26A、E56A和R59A-R61A导致烟曲霉菌丝生长能力减弱;D19A-R21A和K36A导致烟曲霉对过氧化氢(H_(2)O_(2))的耐受能力增强,而K36E使菌株对H_(2)O_(2)的耐受能力显著减弱;此外,K36A和E56A引起烟曲霉菌丝顶端的甘露糖聚集;D19A-R21A、K36A和K26A使烟曲霉对大蜡螟幼虫的致病力显著增强,而E56A和R59A-R61A导致菌株对大蜡螟幼虫的致病力显著减弱。结论Cofilin及其氨基酸位点在烟曲霉抗逆性和致病力等方面具有重要作用。

Objective To investigate the role of Cofilin amino acid residues in stress resistance and virulence of Aspergillus fumigatus by constructing Cofilin point mutation library.Methods The Cofilin point mutants of A.fumigatus were constructed and screened.The effects of Cofilin point mutation on some physicochemical characteristics of A.fumigatus,such as antioxidant capacity,high osmotic pressure resistance and cell wall interference resistance,were analyzed by stress test,while those on the synthesis and distribution of mannose in cell wall of A.fumigatus by FITCcoupled conca-navalin protein fluorescence markers,and those on the virulence of A.fumigatus by infection test of Galleria mellonella larvae.Results The constructed Cofilin point mutation library of A.fumigatus consisted of single mutations K26A and E56A as well as double mutations D11A-E12A,K41A-K42A and R59A-R61A.D11A-E12A,K26A,E56A and R59A-R61A decreased the growth ability of A.fumigatus filaments.D19A-R21A and K36A enhanced while K36 E significantly weakened the tolerance of A.fumigatus to hydrogen peroxide(H_(2)O_(2)).In addition,K36A and E56A caused the accu-mulation of mannose in the tip of A.fumigatus filaments.D19A-R21A,K36A and K26A significantly increased while E56A and R59A-R61A significantly decreased the virulence of A.fumigatus to Galleria mellonella larvae.Conclusion Cofilin and its amino acid sites play important roles in stress resistance and virulence of A.fumigatus.