Calcineurin regulatory network control of Aspergillus fumigatus hyphal septation

烟曲霉引起的侵袭性曲霉病是免疫功能低下患者的主要感染性杀手。 开发有效的抗真菌疗法的一个重大障碍是缺乏关于真菌的知识。 烟曲霉生长和发病机制的调节。我们确定钙调神经磷酸酶 (CN) 作为一种关键的磷酸酶 烟曲霉菌丝生长、分隔和侵袭性疾病所需的。我们是第一个结晶的 真菌 CN 复合物和结构引导抑制剂策略表明，靶向 CN 在动物模型中有效。 然而，CN 到底如何调节生长和发病机制在很大程度上尚不清楚。我们证明了 CN 复合物由催化 (CnaA) 和调节 (CnaB) 亚基组成，动态定位于活性 生长点、菌丝尖端和隔膜，可能与这些活性点的关键效应器相互作用以调节 分隔和菌丝延伸。我们证实分隔和菌丝延伸的缺陷不是介导的 通过 CN 依赖性转录因子 CrzA 或主要 CN 结合蛋白 CbpA。使用突变 方法，我们证明 CN 通过短线性底物与隔膜处的这些未知效应器结合 结合基序 (PxIxIT/LxVP) 和 CN 从隔膜错误定位导致异常隔膜和发育不良 增长，表明 CN 的直接监管作用。我们最近的 CN 蛋白质组学和磷酸化蛋白质组学方法 发现了几种作为潜在 CN 效应子的菌丝生长相关蛋白和隔膜相关蛋白 (SAP)。作为 合乎逻辑的下一步，我们现在将 CN 的主要功能定义为磷酸酶，以协调生长和毒力 通过与这些效应器的相互作用。我们的总体目标是利用我们强大的遗传、结构和新能力 强大的蛋白质组数据来定义 CN 特异性控制。我们的中心假设是 CN 精心策划了入侵 通过磷酸化-去磷酸化结合和调节关键效应蛋白来促进菌丝生长 隔膜，从而控制侵袭性疾病。在目标 1 中，我们将识别菌丝中的关键 CN 信号传导效应器 通过双分子荧光互补和亲和力测定进行隔膜。效应器的定位研究 在 CN 抑制/条件表达/删除背景中将确认其功能的 CN 依赖性。 CN-效应子结合的分子模型和分子动力学模拟将指导我们的靶向突变 效应器中预测的 CN 结合基序以确认 CN 效应器相互作用。效应突变体将是 筛选菌丝生长和隔膜缺陷，以关联 CN 依赖性效应调节。在目标 2 中，我们 将利用正交方法和 LC-对优先 SAP 进行体内磷酸化分析 MS/MS 分析可深入了解 CN 中相应 SAP 中磷酸化的特定残基 抑制/删除背景。最后，CN依赖性隔膜效应器对隔膜、隔膜孔的贡献 密封、菌丝延伸和毒力将使用基因删除、靶向的迭代方法进行验证 突变、体外生长筛选和小鼠模型验证。这项研究将首次确定并 定义控制真菌生长和毒力的新型隔膜效应器的 CN 信号网络。

Invasive aspergillosis due to Aspergillus fumigatus is a leading infectious killer of immunocompromised patients. A significant barrier to developing effective antifungal therapeutics is the lack of knowledge regarding the regulation of A. fumigatus growth and pathogenesis. We established calcineurin (CN) as a critical phosphatase required for A. fumigatus hyphal growth, septation, and invasive disease. We were the first to crystallize the fungal CN complex, and structure-guided inhibitor strategies showed targeting CN effective in animal models. However, exactly how CN regulates growth and pathogenesis is largely undefined. We demonstrated that the CN complex, comprised of catalytic (CnaA) and regulatory (CnaB) subunits, is dynamically localized at active points of growth, the hyphal tip and septum, likely interacting with key effectors at these active points to regulate septation and hyphal extension. We confirmed that defects in septation and hyphal extension are not mediated via the CN-dependent transcription factor CrzA or the major CN-binding protein CbpA. Using mutational approaches, we showed that CN binds to these unknown effectors at the septum via short linear substrate binding motifs (PxIxIT/LxVP) and CN mislocalization from the septum leads to aberrant septation and stunted growth, indicating CN’s direct regulatory role. Our recent CN proteomic and phosphoproteomic approaches uncovered several hyphal growth-related and septum-associated proteins (SAPs) as potential CN effectors. As a logical next step, we will now define CN’s main function as a phosphatase to orchestrate growth and virulence via its interaction with these effectors. Our overall objective is to leverage our strong genetic, structural and new robust proteomic data to define CN specific control. Our central hypothesis is that CN orchestrates invasive hyphal growth by binding and regulating key effector proteins through phosphorylation-dephosphorylation at the septum, thereby governing invasive disease. In Aim 1, we will identify key CN signaling effectors at the hyphal septum by bimolecular fluorescence complementation and affinity assays. Localization studies of the effectors in CN inhibited/conditional expression/deletion backgrounds will confirm their CN-dependency for function. Molecular modeling of CN-effector binding and molecular dynamics simulations will guide our targeted mutations of predicted CN-binding motifs in the effectors to confirm CN-effector interactions. Effector mutants will be screened for hyphal growth and septation defects to correlate CN-dependent effector regulation. In Aim 2, we will perform in vivo phosphorylation analyses of the prioritized SAPs by utilizing orthogonal approaches and LC- MS/MS analysis to gain in-depth insight into specific residues phosphorylated in the respective SAPs in CN inhibited/deletion backgrounds. Finally, contributions of CN-dependent septal effectors to septation, septal pore sealing, hyphal extension, and virulence will be validated using an iterative approach of genetic deletion, targeted mutations, in vitro growth screening, and murine model validation. This study will, for the first time, identify and define the CN signaling network of novel septal effectors controlling fungal growth and virulence.