Calcineurin regulatory network control of Aspergillus fumigatus hyphal septation

由烟曲霉引起的侵袭性曲霉病是免疫功能低下患者的主要传染性杀手。 开发有效的抗真菌治疗的一个重要障碍是缺乏关于抗真菌药物的知识。 调节A。烟曲霉生长和致病性。我们建立了钙调神经磷酸酶（CN）作为一个关键的磷酸酶 需要A。烟曲霉菌丝生长、分隔和侵入性疾病。我们是第一个将 真菌CN复合物，和结构导向的抑制剂策略显示靶向CN在动物模型中有效。 然而，CN究竟如何调节生长和发病机制在很大程度上是不确定的。我们证明了 CN复合物由催化亚基（CnaA）和调节亚基（CnaB）组成，动态定位于活性位点， 生长点，菌丝尖端和隔膜，可能与这些活性点的关键效应物相互作用，以调节 分隔和菌丝延伸。我们证实，在分隔和菌丝延伸的缺陷是不介导的 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，我们发现CN通过短的线性底物结合到这些未知的效应器上 结合基序（PxIxIT/LxVP）和CN从隔膜的错误定位导致异常分隔和发育不良 增长，表明CN的直接调节作用。我们最近的CN蛋白质组学和磷酸化蛋白质组学方法 揭示了几个菌丝生长相关和隔膜相关蛋白（SAP）作为潜在的CN效应。作为 合乎逻辑的下一步是，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 确定CN特异性对照的稳健蛋白质组学数据。我们的中心假设是CN编排了侵入性的 菌丝生长通过结合和调节关键效应蛋白通过磷酸化-去磷酸化在 隔膜，从而控制侵入性疾病。在目标1中，我们将确定菌丝中的关键CN信号效应子 通过双分子荧光互补和亲和测定来鉴定隔膜。效应器的定位研究 在CN中，抑制的/条件性表达/缺失背景将证实其功能的CN依赖性。 CN-效应子结合的分子建模和分子动力学模拟将指导我们的靶向突变 的预测CN结合基序的效应，以确认CN效应相互作用。效应突变体将 筛选菌丝生长和分隔缺陷以关联CN依赖性效应调控。在目标2中， 将通过利用正交方法和LC-1对优先的SAP进行体内磷酸化分析。 MS/MS分析，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依赖性隔效应物对分隔、隔孔和细胞分裂的作用。 密封，菌丝延伸和毒力将使用遗传缺失的迭代方法进行验证， 突变、体外生长筛选和鼠模型验证。这项研究将首次确定和 定义控制真菌生长和毒力的新型间隔效应子的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.