Abstract：
Cryptococcus neoformans is a life-threatening fungal pathogen that is a causative agent for pulmonary infection and meningoencephalitis in both immunocompetent and immunodeficient individuals. Recent studies have elucidated the important function of the target of rapamycin (TOR) signaling pathway in the modulation of C. neoformans virulence factor production and pathogenicity in animal infection models. Herein, we discovered that Ypk1, a critical component of the TOR signaling pathway, acts as a critical modulator in fungal pathogenicity through post-translational modifications (PTMs). Mass spectrometry analysis revealed that Ypk1 is subject to protein acetylation at lysines 315 and 502, and both sites are located within kinase functional domains. Inhibition of the C. neoformans TOR pathway by rapamycin activates the deacetylation process for Ypk1. The YPK1Q strain, a hyper-acetylation of Ypk1, exhibited increased sensitivity to rapamycin, decreased capsule formation ability, reduced starvation tolerance, and diminished fungal pathogenicity, indicating that deacetylation of Ypk1 is crucial for responding to stress. Deacetylase inhibition assays have shown that sirtuin family proteins are critical to the Ypk1 deacetylation mechanism. After screening deacetylase mutants, we found that Dac1 and Dac7 directly interact with Ypk1 to facilitate the deacetylation modification process via a protein-protein interaction. These findings provide new insights into the molecular basis for regulating the TORC-Ypk1 axis and demonstrate an important function of protein acetylation in modulating fungal pathogenicity.
OBJECTIVE: Cryptococcus neoformans is an important opportunistic fungal pathogen in humans. While there are currently few effective antifungal treatments, the absence of novel molecular targets in fungal pathogenicity hinders the development of new drugs. There is increasing evidence that protein post-translational modifications (PTMs) can modulate the pathogenicity of fungi. In this study, we discovered that the pathogenicity of C. neoformans was significantly impacted by the dynamic acetylation changes of Ypk1, the immediate downstream target of the TOR complex. We discovered that Ypk1 is acetylated at lysines 315 and 502, both of which are within kinase functional domains. Deacetylation of Ypk1 is necessary for formation of the capsule structure, the response to the TOR pathway inhibitor rapamycin, nutrient utilization, and host infection. We also demonstrate that the sirtuin protein family is involved in the Ypk1 deacetylation mechanism. We anticipate that the sirtuin-Ypk1 regulation axis could be used as a potential target for the development of antifungal medications.
OBJECTIVE: Cryptococcus neoformans is an important opportunistic fungal pathogen in humans. While there are currently few effective antifungal treatments, the absence of novel molecular targets in fungal pathogenicity hinders the development of new drugs. There is increasing evidence that protein post-translational modifications (PTMs) can modulate the pathogenicity of fungi. In this study, we discovered that the pathogenicity of C. neoformans was significantly impacted by the dynamic acetylation changes of Ypk1, the immediate downstream target of the TOR complex. We discovered that Ypk1 is acetylated at lysines 315 and 502, both of which are within kinase functional domains. Deacetylation of Ypk1 is necessary for formation of the capsule structure, the response to the TOR pathway inhibitor rapamycin, nutrient utilization, and host infection. We also demonstrate that the sirtuin protein family is involved in the Ypk1 deacetylation mechanism. We anticipate that the sirtuin-Ypk1 regulation axis could be used as a potential target for the development of antifungal medications.
摘要：
新生隐球菌是一种威胁生命的真菌病原体，是免疫活性和免疫缺陷个体中肺部感染和脑膜脑炎的病原体。最近的研究已经阐明了雷帕霉素（TOR）信号通路的靶在动物感染模型中调节新生梭菌毒力因子的产生和致病性的重要功能。在这里，我们发现Ypk1是TOR信号通路的关键组成部分，通过翻译后修饰（PTM）充当真菌致病性的关键调节剂。质谱分析显示Ypk1在赖氨酸315和502处经历蛋白质乙酰化,并且两个位点都位于激酶功能结构域内。雷帕霉素对新生梭菌TOR途径的抑制激活了Ypk1的脱乙酰化过程。YPK1Q菌株，Ypk1的超乙酰化表现出对雷帕霉素的敏感性增加，胶囊形成能力下降，饥饿耐受性降低，和减少真菌的致病性，表明Ypk1的脱乙酰作用对于响应胁迫至关重要。脱乙酰酶抑制试验表明，沉默调节蛋白家族蛋白对Ypk1脱乙酰机制至关重要。筛选脱乙酰酶突变体后，我们发现Dac1和Dac7直接与Ypk1相互作用，通过蛋白质-蛋白质相互作用促进脱乙酰修饰过程。这些发现为调节TORC-Ypk1轴的分子基础提供了新的见解，并证明了蛋白质乙酰化在调节真菌致病性中的重要功能。
目的：新生隐球菌是人类重要的机会性真菌病原体。虽然目前很少有有效的抗真菌治疗方法，真菌致病性中缺乏新的分子靶标阻碍了新药的开发。越来越多的证据表明，蛋白质翻译后修饰（PTM）可以调节真菌的致病性。在这项研究中,我们发现,新生梭状芽孢杆菌的致病性受到TOR复合物的直接下游靶标Ypk1的动态乙酰化变化的显著影响.我们发现Ypk1在赖氨酸315和502处被乙酰化,这两者都在激酶功能结构域内。Ypk1的脱乙酰是形成胶囊结构所必需的，对TOR通路抑制剂雷帕霉素的反应，养分利用率，和宿主感染。我们还证明sirtuin蛋白家族参与Ypk1脱乙酰化机制。我们预计sirtuin-Ypk1调节轴可用作开发抗真菌药物的潜在靶标。
目的：新生隐球菌是人类重要的机会性真菌病原体。虽然目前很少有有效的抗真菌治疗方法，真菌致病性中缺乏新的分子靶标阻碍了新药的开发。越来越多的证据表明，蛋白质翻译后修饰（PTM）可以调节真菌的致病性。在这项研究中,我们发现,新生梭状芽孢杆菌的致病性受到TOR复合物的直接下游靶标Ypk1的动态乙酰化变化的显著影响.我们发现Ypk1在赖氨酸315和502处被乙酰化,这两者都在激酶功能结构域内。Ypk1的脱乙酰是形成胶囊结构所必需的，对TOR通路抑制剂雷帕霉素的反应，养分利用率，和宿主感染。我们还证明sirtuin蛋白家族参与Ypk1脱乙酰化机制。我们预计sirtuin-Ypk1调节轴可用作开发抗真菌药物的潜在靶标。
