Transcriptome and Phosphoproteomics analysis spreadsheets

Adenylyl cyclase (AC) is the central regulator of cAMP signalling in fungal pathogens, yet fungal ACs uniquely contain a protein phosphatase 2C (PP2C) domain whose function remains unclear. Here, we characterise the PP2C domain of the AC Cac1 in the human pathogen <i>Cryptococcus neoformans</i>. Structural and biochemical analyses show that Cac1-PP2C adopts a non-canonical scaffold but functions as a Ser/Thr-specific metalloenzyme. Deletion of the PP2C or AC catalytic (ACC) domain demonstrated that PP2C is required for full Cac1 activity, influencing melanin and capsule synthesis, sexual differentiation, titan cell formation, and cell wall integrity. Notably, the PP2C-null mutant induced Th2-biased immunity and extensive pulmonary damage yet failed to cause mortality in mice. Integrated transcriptomic and phosphoproteomic analyses further revealed shared and domain-specific signalling outputs mediated by PP2C and ACC. This study provides the first comprehensive characterisation of a fungal AC-linked PP2C domain and defines its pivotal role in <i>C. neoformans</i> pathobiology.

腺苷酸环化酶（Adenylyl cyclase, AC）是真菌病原体中环腺苷酸（cAMP）信号通路的核心调控因子，然而真菌AC独特地携带一个功能尚未阐明的蛋白磷酸酶2C（protein phosphatase 2C, PP2C）结构域。本研究针对人类病原体新型隐球菌（Cryptococcus neoformans）中AC同源蛋白Cac1的PP2C结构域展开功能表征。结构生物学与生化分析结果显示，Cac1-PP2C采用非经典的支架结构，但作为丝氨酸/苏氨酸特异性金属酶发挥功能。通过敲除PP2C结构域或AC催化（ACC）结构域的实验证实，PP2C是Cac1发挥完全活性所必需的，其可影响黑色素与荚膜合成、有性分化、泰坦细胞形成以及细胞壁完整性。值得注意的是，PP2C缺失突变体能够诱导Th2偏倚型免疫应答并引发广泛的肺部损伤，但无法导致小鼠死亡。整合转录组学与磷酸化蛋白质组学分析进一步揭示了PP2C与ACC所介导的共有及结构域特异性信号传导输出。本研究首次对真菌AC关联的PP2C结构域开展了全面的功能表征，并明确了其在新型隐球菌（C. neoformans）致病生物学中的关键作用。