Fonsecaea monophora色素毒力性研究

目的探讨黑色素是否为Fonsecaea monophora的一个重要毒力因子。方法从Fonsecaea monophora的分生孢子突变株(CBS122845)传代接种产生白色突变株(CBS 125149)。透射电子显微镜(TEM)下观察到黑色素是位于分生孢子细胞壁表面上的电子致密颗粒。通过碱-酸法提取来自两个不同菌株的细胞壁色素颗粒。建立不同菌株或色素颗粒与活化巨噬细胞(RAW264.7)共培养体系,通过实时荧光相对定量PCR检测i-NOS基因的表达,格里斯法检测一氧化氮(NO)的表达结果,ELISA检测IL-12、TNF-α、IL-10的表达结果。结果色素型分生孢子和其色素颗粒能够降低巨噬细胞诱导型一氧化氮合酶(I-NOS)基因的表达和抑制一氧化氮的合成(P<0.05)。提高Th2细胞因子表达,同时抑制Th1细胞因子表达(P<0.05)。结论黑色素可能是Fonsecaea monophora逃避巨噬细胞对其氧化应激的重要机制。同时黑色素下调Th1免疫应答,可能利于真菌的持续感染。

Study on the pathogenesis of melanin in Fonsecaea monophora

Objective To know whether melanin contributes to disease pathogenesis of Fonsecaea monophora.Methods In this study,one albino mutant(CBS125149)was generated from a parent meristematic mutant(CBS122845)of Fonsecaea monophora.Transmission electron microscopy(TEM)profiles showed that melanin in the parent strains appeared as electron-dense granules which located on the cell wall surface.The cell wall fractions from the two different strains were extracted by an alkali-acid method.The different strains or its cell wall fractions were interacted with the activated RAW264.7.Gene expression of i-NOS gene was detected by real time PCR.Expression of nitric oxide(NO)was detected by Griess method.Otherwise,expression results of IL-12,TNF-α,IL-10 was detected by ELISA.Results The pigmented strain and its cell wall fraction could reduce the expression of inducible nitric oxide synthase(i-NOS)gene and inhibit the synthesis of nitric oxide(NO)in vitro(P<0.05).An exacerbated Th2 response and inhibited Th1 response occurred in the interaction between activated RAW264.7 with the pigmented strain or its cell wall fraction.Conclusion Our results suggest that melanin plays an importantrole in fungal escaping from the oxidative burst of macrophages.Melanin causes a negatively Th1 immune response and favors the persistence of the fungal infection.