Interplay between acetylation and ubiquitination of Isw1 confers multidrug resistance in Cryptococcus neoformans

Cryptococcus neoformans poses a great threat to human communities, given that it quickly becomes resistant to available antifungal drugs. Herein, a conserved chromatin remodeler, Isw1, is shown to function as a master transcriptional modulator of genes responsible for multidrug resistance. It reciprocally controls drug resistance, and cells with disrupted ISW1 demonstrate profound resistance to fluconazole, ketoconazole, 5-fluorocytosine and 5-fluorouracil. Mass spectrometry reveals that Isw1 is both acetylated and ubiquitinated. These two protein posttranslational modifications confer an interplay regulation mechanism that controls Isw1 protein degradation via a ubiquitin-mediated proteasome and, consequently, C. neoformans resistance to drugs. Functional mutagenesis analysis of acetylation and ubiquitination sites reveals that the acetylation status of the lysine 97 residue on Isw1 coordinates its ubiquitination processes at lysines 113 and 441 by modulating the protein interactions between Isw1 and Cdc4, an E3 ligase. Clinical C. neoformans isolates overexpressing the undegradable ISW1 mutant demonstrate impaired drug-resistant phenotypes. Collectively, our studies reveal a sophisticated acetylation-Isw1-ubiquintation regulation axis that controls multidrug resistance in fungal pathogens. Overall design: performed paired-end RNA-seq of Cryptococcus neoformans H99s as Control group and isw1? strains as in 50ml YPD broth at OD600=0.8, respectively in biological triplicates.

新型隐球菌（Cryptococcus neoformans）对人类社群构成严重威胁，因其可快速对现有抗真菌药物产生耐药性。本研究证实，保守型染色质重塑因子Isw1可作为多药耐药相关基因的核心转录调控因子。其对药物耐药性具有双向调控作用：ISW1基因敲除的菌株对氟康唑、酮康唑、5-氟胞嘧啶及5-氟尿嘧啶均表现出显著耐药性。质谱分析法显示，Isw1同时存在乙酰化与泛素化修饰。这两种蛋白质翻译后修饰构成了一套交互调控机制，通过泛素介导的蛋白酶体通路调控Isw1的蛋白降解，进而影响新型隐球菌的药物耐药性。对乙酰化与泛素化位点的功能诱变分析表明，Isw1第97位赖氨酸残基的乙酰化状态可通过调节Isw1与E3泛素连接酶（E3 ligase）Cdc4的蛋白相互作用，调控其第113位与第441位赖氨酸残基的泛素化过程。过表达不可降解ISW1突变体的临床新型隐球菌分离株，其耐药表型受到显著削弱。综上，本研究揭示了一条调控真菌病原体多药耐药性的精密乙酰化-Isw1-泛素化调控轴。
实验整体设计：分别以培养于50ml YPD液体培养基、OD600值为0.8的新型隐球菌H99株作为对照组，以同等条件培养的ISW1基因缺失菌株为实验组，每组进行三次生物学重复的双端RNA测序。