解码新生隐球菌赖氨酸乙酰化影响真菌毒性的分子机制和调控网络

Fungal pathogens are major threats to human health. Cryptococcus neoformans is the leading cause of human death among all fungal pathogens, leading 650,000 deaths per year globally. While current anti-fungal therapies are ineffective, new drug development is hampered by the lack of knowledge of novel molecular targets in fungal pathogenicity. In this study, we discovered that deacetylase inhibitors are potent anti-fungal agents in both in vitro and in animal infection models. Our data demonstrated that deacetylase inhibitors are able to block the expression of genes of many essential virulence factors and dampen fungal proliferation during the early stage of pulmonary infection. However, the molecular mechanism of the effect of deacetylase inhibitors in fungal virulence remains unknown. In our study, we propose to systematically analyze the correlation between post-translational protein acetylation and Cryptococcus neoformans pathogenicity, underlining the importance of acetylation as a novel modulator of fungal virulence. To decipher our hypothesis, we employed acetylome analysis and generated the first database of acetylation network in Cryptococcus neoformans. We detected over 1400 acetylated proteins and more than 3500 acetylated lysine sites. Using bioinformatic analysis, we have identified novel fungal virulence factors, whose activities are regulated by acetylated lysine residues. On the other hand, we generated and screened the deacetylase knockout library, identified two important deacetylases that positively regulate Cryptococcus virulence in the host. We will employ proteomics, acetylomics, molecular biology, and bioinformatic analysis to decipher their functions in modulating fungal virulence. Our study describes a novel regulation of virulence in Cryptococcus neoformans, and provides potential drug targets for anti-fungal therapy development.

病原真菌对人类造成了极大的威胁，每年造成超过200万人死亡。新生隐球菌在病原性真菌引起的死亡人数中居首位，每年导致65万人死亡。对于其致病机制的深入了解是推动药物靶点研发的关键。申请人前期研究发现，乙酰化抑制剂可以有效抑制真菌毒性因子的表达，并且可以大幅度降低宿主中的菌落数量。然而，目前对乙酰化调控隐球菌毒性的分子机制一无所知。本项目以赖氨酸乙酰化(Kac)参与调控新生隐球菌毒性的分子机制为研究方向。首先通过乙酰化蛋白组学方法，构建新生隐球菌全蛋白赖氨酸乙酰化位点图谱，分析Kac调控毒性的可能途径。并以核心毒性因子做为切入点解析Kac修饰决定毒性发挥的关键分子机制。其次，通过系统筛选新生隐球菌脱乙酰化基酶敲除菌株，找出调控毒性的关键性基酶，利用组学、分子生物学、生物信息学等手段，深度解码Kac调控毒性的分子机制。本项目以一个崭新的角度理解真菌毒性的调控途径，为寻求新的药物靶点提供理论依据。

真菌感染是人类最难控制的疾病之一，每年感染真菌疾病的人数高达3亿人，死亡人数达160万，其中新型隐球菌与白色念珠菌、烟曲霉菌并称为人类三大致病真菌，具有高致病性和高致死率。近期研究表明，乙酰化类药物具有抗真菌的作用，可影响致病真菌的生长、毒力、耐药性和应激信号反应等，因此我们通过比较乙酰组学分析人类病原体新型隐球菌、白色念珠菌和烟曲霉菌与非致病真菌发酵酵母的乙酰化修饰的进化异同，发现不同真菌的乙酰化修饰进化高度动态，但在核心生物学过程如翻译过程、TCA循环过程、呼吸过程、核糖体组分、以及组蛋白修饰等进化守恒；发现致病真菌享有与发酵酵母明显不同的进化保守motif，该motif与致病真菌的毒力因子的乙酰化motif具有显著相关性。同时我们证实，真菌转录延伸因子Tef1活性受Kac调控，脱乙酰基酶Dac2、Dac4可直接参与Tef1乙酰化修饰调节。另外转录因子Lzr4蛋白存在乙酰化-泛素化的交互作用共同调节真菌的耐药性。质谱数据显示Lzr4蛋白存在多个乙酰化和泛素化位点，其中Lzr4K97是重要的耐药调控位点，缺失后可使菌株具有唑类药物的耐受性；E3 连接酶Cdc4通过调节Lzr4K113和Lzr4K441位点调控Lzr4的泛素-蛋白酶体过程，Lzr4R97促进该过程的发生。与此同时，我们通过多组学比较分析的方法，整合了宿主应答隐球菌感染时转录组、蛋白质组和乙酰化修饰组学调控网络，全面分析了宿主应答隐球菌感染的分子机制，通过器官比较乙酰化修饰组学分析，我们发现脑、肺组织应答隐球菌感染的乙酰化修饰调控的存在很大差异，暗示了中枢神经系统和其他组织之间的免疫差异，为破译不同器官的免疫应答调控机制提供了新的思路。