组蛋白乙酰转移酶Elp3调控烟曲霉毒力的分子机制研究

An in-depth understanding of the virulence determinants of Aspergillus fumigatus is the crucial for the treatment of A. fumigatus infection. There is emerging evidence that histone acetylation plays an important role in the virulence of animal and plant pathogens, but the underlying mechanisms among different species are quite distinct. In human opportunistic pathogen A. fumigatus, functions of histone acetylation are yet to be characterized. In this project, we previously identified thirteen genes which encode putative histone acetyltransferases (HATs) in A. fumigatus using bioinformatics analysis. To determine whether HATs affecting A. fumigatus virulence, we employed the Galleria mellonella infection model to screen all thirteen full-deletion mutants of HAT genes. Surprisingly, among these mutants, the depletion of the histone acetyltransferase Elp3 showed a minor growth defect but significantly attenuated in virulence. Based on this finding, we will further investigate the biological functions of histone acetyltransferase Elp3 by site-directed mutagenesis, in vitro and in vivo enzymatic assay, and mouse model in A. fumigatus. Moreover, with the combination of RNA-seq, ChIP-qPCR, acetylation proteomics approaches, we aim to identify the putative target genes and HAT substrates regulated by Elp3, and therefore provide new insights into the regulatory mechanism of A. fumigatus Elp3 for altering gene expression and protein post-translational modification. On the basis of results from this project, we will finally systematically elucidate the relationship between the histone acetyltransferase Elp3 and the virulence of A. fumigatus and pave the fundamental way for better understanding the mechanism of histone acetylation modification regulating the virulence of A. fumigatus and other pathogenic fungi.

深入了解烟曲霉的毒力因子是有效治疗烟曲霉感染的关键。已有证据显示组蛋白乙酰化修饰与动植物病原菌的毒力密切相关，但不同物种间乙酰化修饰的调控机制差异较大。目前对于人类重要条件致病真菌烟曲霉中组蛋白乙酰化修饰的功能还知之甚少。本项目前期运用信息学分析预测出烟曲霉中存在13个编码组蛋白乙酰转移酶的基因，通过大蜡螟模型毒力筛选，发现组蛋白乙酰转移酶Elp3的缺失几乎不影响烟曲霉生长但毒力显著降低。拟进一步通过位点突变、体内外酶活检测、小鼠毒力模型，研究烟曲霉组蛋白乙酰转移酶Elp3的生物学功能。并结合RNA-seq、ChIP-qPCR、乙酰化蛋白质组学等手段，探究Elp3的转录调控途径和靶点蛋白，进而从基因转录和蛋白翻译后修饰两个层面解析具体分子机制和调控网络，最终系统地阐明组蛋白乙酰转移酶Elp3与烟曲霉毒力的关系，为深入认识组蛋白乙酰化修饰调控烟曲霉乃至病原真菌毒力的作用机制奠定理论基础。

烟曲霉（Aspergillus fumigatus）是一种重要的人类条件致病性病原真菌。近年来，随着免疫缺陷和免疫低下人群数量逐年增加，曲霉深部感染患者数量急剧上升，以致曲霉病的死亡率高达30%-95%。世界卫生组织于近日发布了有史以来第一份对人类健康构成巨大威胁真菌病原体清单，烟曲霉属于威胁级别最高的类别。人们已经认识到烟曲霉具有超强的对不良环境的适应能力以及和宿主竞争营养的生存能力，然而影响其致病性的因素和分子机制还远未阐明。本研究通过遗传学、生化与结构生物学等多种手段揭示并阐明了烟曲霉延伸复合体催化亚基Elp3除了组蛋白乙酰化功能以外，主要通过其tRNA修饰功能感知氨基酸营养平衡来调控烟曲霉的生长、生物膜形成和毒力。研究中对烟曲霉中延伸复合体六个亚基进行敲除，发现任意亚基缺失后均导致菌丝生长缓慢，生物膜形成能力降低和毒力显著减弱，表明烟曲霉延伸复合物作为一个整体发挥功能。其次发现催化亚基Elp3的KAT和SAM结构域与其功能紧密相关。研究发现，与其他真菌中Elp3发挥组蛋白乙酰转移酶的功能不同，烟曲霉中Elp3是通过对tRNA摆动位置U34的修饰发挥功能，且对于tRNA的修饰作用直接影响维持氨基酸代谢平衡的重要转录因子CpcA的表达。该研究不仅为深入认识tRNA修饰参与调控烟曲霉乃至其他病原真菌的营养感知和毒力的调控关系奠定基础，同时也为寻找新型药物靶点提供新的思路。