Calcineurin regulatory network control of Aspergillus fumigatus hyphal septation

由烟曲霉菌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 烟曲霉菌生长调控及致病机理研究。我们建立了钙调神经磷酸酶(CN)作为一种关键的磷酸酶 烟曲霉菌丝生长、隔膜和侵袭性疾病所必需的。我们是第一个将 真菌CN复合体和结构导向的抑制剂策略在动物模型中显示了靶向CN的有效性。 然而，CN如何调控生长和发病机制在很大程度上尚不清楚。我们演示了 CN复合体由催化(CNAA)和调节(Cna B)亚基组成，动态定位于Active 生长点，菌丝顶端和隔膜，很可能在这些活跃的点上与关键效应器相互作用来调节 分隔和菌丝伸展。我们证实，隔膜和菌丝伸展的缺陷不是由 通过CN依赖的转录因子CrzA或主要的CN结合蛋白CbpA。使用突变 方法，我们发现CN通过短线性底物在隔膜与这些未知的效应器结合 结合基序(PxIxIT/LxVP)和CN在隔膜的错误定位导致异常隔膜和发育迟缓 增长，表明CN的直接监管作用。我们最新的CN蛋白质组学和磷蛋白质组学方法 发现了几个与菌丝生长相关的和隔膜相关的蛋白(SAP)作为潜在的CN效应因子。AS 顺理成章的下一步，我们现在将CN的主要功能定义为协调生长和毒力的磷酸酶 通过它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基因、结构和新的 强大的蛋白质组学数据来定义CN特异性控制。我们的中心假设是CN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的菌丝生长 隔膜，从而控制侵袭性疾病。在目标1中，我们将确定菌丝上的关键CN信号效应器 通过双分子荧光互补和亲和力分析。效应器的本地化研究 在CN抑制/条件表达/删除背景中，将确认其对功能的CN依赖。 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 在效应器中预测的CN结合基序，以确认CN-效应器的相互作用。效应器突变体将是 筛选菌丝生长和间隔缺陷，以关联CN依赖的效应器调节。在目标2中，我们 将利用正交法和LC-2对优先排序的SAP进行体内磷酸化分析 MS/MS分析以深入了解CN中各自SAP中磷酸化的特定残基 禁止/删除背景。最后，CN依赖的隔膜效应器对隔膜、隔孔的贡献 封闭性、菌丝伸长和致病力将使用基因缺失的迭代方法进行验证，有针对性的 突变、体外生长筛选和小鼠模型验证。这项研究将首次确定和 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.