DataSheet_1_Identification and Phenotypic Characterization of Hsp90 Phosphorylation Sites That Modulate Virulence Traits in the Major Human Fungal Pathogen Candida albicans.docx

The highly conserved, ubiquitous molecular chaperone Hsp90 is a key regulator of cellular proteostasis and environmental stress responses. In human pathogenic fungi, which kill more than 1.6 million patients each year worldwide, Hsp90 governs cellular morphogenesis, drug resistance, and virulence. Yet, our understanding of the regulatory mechanisms governing fungal Hsp90 function remains sparse. Post-translational modifications are powerful components of nature’s toolbox to regulate protein abundance and function. Phosphorylation in particular is critical in many cellular signaling pathways and errant phosphorylation can have dire consequences for the cell. In the case of Hsp90, phosphorylation affects its stability and governs its interactions with co-chaperones and clients. Thereby modulating the cell’s ability to cope with environmental stress. Candida albicans, one of the leading human fungal pathogens, causes ~750,000 life-threatening invasive infections worldwide with unacceptably high mortality rates. Yet, it remains unknown if and how Hsp90 phosphorylation affects C. albicans virulence traits. Here, we show that phosphorylation of Hsp90 is critical for expression of virulence traits. We combined proteomics, molecular evolution analyses and structural modeling with molecular biology to characterize the role of Hsp90 phosphorylation in this non-model pathogen. We demonstrated that phosphorylation negatively affects key virulence traits, such as the thermal stress response, morphogenesis, and drug susceptibility. Our results provide the first record of a specific Hsp90 phosphorylation site acting as modulator of fungal virulence. Post-translational modifications of Hsp90 could prove valuable in future exploitations as antifungal drug targets.

高度保守且普遍存在的分子伴侣Hsp90在维持细胞蛋白质稳态和对环境应激反应的调控中发挥着关键作用。在每年全球导致超过160万患者死亡的致病真菌中，Hsp90控制着细胞的形态发生、耐药性和致病力。然而，我们对调控真菌Hsp90功能的调控机制的理解仍然十分有限。翻译后修饰是自然界调节蛋白质丰度和功能的强大工具之一。特别是磷酸化在许多细胞信号通路中至关重要，错误的磷酸化可能会对细胞产生灾难性的后果。在Hsp90的情况下，磷酸化影响其稳定性并调控其与共伴侣和客户的相互作用，从而调节细胞应对环境应激的能力。白色念珠菌，作为主要的人体真菌病原体之一，全球每年导致约75万起危及生命的侵袭性感染，死亡率极高。然而，目前尚不清楚Hsp90的磷酸化是否以及如何影响C. albicans的致病性特征。在本研究中，我们表明Hsp90的磷酸化对于致病性特征的表达至关重要。我们结合蛋白质组学、分子进化分析和结构建模以及分子生物学技术，以表征Hsp90磷酸化在非模式病原体中的作用。我们证明磷酸化对关键致病性特征，如热应激反应、形态发生和药物敏感性产生负面影响。我们的研究首次记录了特定的Hsp90磷酸化位点作为真菌致病力的调节剂。Hsp90的翻译后修饰在未来作为抗真菌药物靶点的探索中可能具有重大价值。