Fonsecaea monophora黑素对巨噬细胞自噬的影响及机制研究

Fonsecaea monophora is the etiologic agent of chromoblastomycosis in southern China. We obtained the melanin-deficient mutation of F. monophora （SUMS0505）by inoculating with 10 transfers. We initial demonstrated that TLRs, Dectin-1 are the major receptors involved in host immune response to melanin strain of F. monophora and result in an inhibition of the Th1 response, which may lead to different in pathogenicity between melanin strain and melanin-deficient mutant. Recently, the recruitment of proteins of the autophagy in response to TLR activation was found to be important for pathogen elimination by macrophage. As a result, melanin is associated with virulence, but how it interacts with immune response and antifungal mechanisms remains unknown. Herein, we would investigate whether F. monophora cell wall melanin blocks autophagy to promote pathogenicity. So we conduct experiments extend on our previous findings to get the transformation of the melanin- deficient mutant with the Alternaria melanin gene ALM to find whether it can restore pathogenicity to the mutant. For evaluating the role of macrophage autophagy in the response to F. monophora melanin, we compare autophagosome formation, PAMPs exposure, PRRs expression, NADPH oxidase and ROS production among different strains. These studies will clarify the role of melanin in modulating host macrophage autophagy and the mechanisms for the process, which would provide evidence in understanding the melanin in pathogenesis of F. monophora infection.

Fonsecaea monophora是我国南方着色芽生菌病的主要病原菌。我们前期将临床分离株SUMS0505多次传代获得白化突变株，发现色素株与白化突变株均通过巨噬细胞TLRs、Dectin-1受体免疫识别，但色素株毒力强且引起Th1/Th2细胞免疫偏倚，证实黑素为重要毒力因子，但其如何影响宿主对F.monophora的免疫杀伤机制尚不明确。TLR通路激活对自噬激活及病原体清除起重要作用，我们推测黑素可能通过干扰自噬下调免疫反应。拟在前期确定突变位点基础上，构建色素回复株；比较菌株间毒力及细胞壁主要成分表达差异，与巨噬细胞PRRs活化、诱导NADPH氧化酶活化、ROS生成及巨噬细胞自噬体形成差异中的关系。旨在阐明F. monophora黑素是否通过改变菌细胞壁成分下调免疫识别、影响NADPH氧化酶活化下调自噬的发生及其分子机制，以进一步揭示着色芽生菌病的发病机制。

F. monophora是我国南方着色芽生菌病的主要病原菌。毒力因子和硬壳细胞形成及细胞壁黑素皆有关。此研究主要探讨黑素如何影响宿主对F. monophora的免疫杀伤。旨在阐明黑素是否通过改变菌细胞壁成分下调免疫识别、影响NADPH氧化酶活化下调自噬的发生及其分子机制。主要研究内容包括：1）采用‘共生法’获得白化株的色素回复株，分析色素回复后菌株对各种环境因素的抵抗力及其毒力；2）对色素株、白化株细胞壁几丁质含量进行代谢组学分析；3）构建色素株、白化株、色素回复株大蜡螟和 Balb/c小鼠感染模型，比较不同菌株的体内毒力差异；4）使用人源和鼠源的巨噬细胞 （THP-1、RAW264.7）和色素株、白化株及标准株共培养，观察巨噬细胞自噬体形成差异；5）通过敲除黑素合成过程中的关键基因pks1验证黑素对巨噬细胞自噬的影响。.结果发现：采用’共生法’可恢复白化株色素，且色素回复株和色素株抵抗各种应激压力能力明显强于白化株。在成功构建的大蜡螟和小鼠感染模型中发现色素株及色素回复株毒力强于白化株。细胞壁几丁质含量分析发现黑素能够改变细胞壁几丁质的含量；代谢组学分析结果发现白化株还原型谷胱甘肽表达量较色素株明显上升，可能是导致菌株丢失色素的潜在原因。使用来源人源和鼠源的巨噬细胞与 F. monophora 色素株、白化株及标准株共培养结果显示，各组菌均不能明显诱导自噬形成，但发现色素株比白化株更易引起巨噬细胞凋亡；且色素株更能引起巨噬细胞脂滴的形成，可能和F. monophora的毒力有关，相关研究还在进行中。该研究对揭示着色芽生菌病的病原菌的致病机制提供了一新的思路。