Defining the Aspergillus fumigatus Calcineurin-Dependent Regulatory Network through Whole Phosphoproteome Analysis

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杀手。一个 开发有效的抗真菌疗法的主要障碍是缺乏对规则的理解 对烟曲霉菌丝生长和病害的影响。我们已经确定烟曲霉菌需要钙调神经磷酸酶 生长和毒力。我们还表明，钙调神经磷酸酶动态地定位在活跃的生长点，即 菌丝顶端和隔膜，在这些活性点处需要钙调神经磷酸酶活性来调节 菌丝生长和致病力。钙调神经磷酸酶(CNAA)作为一种磷酸酶作用于底物去磷酸化 并通过转录因子CrzA。然而，与我们的cnaa缺失株相比，我们的ΔcrzA株 表现出最小的菌丝生长缺陷，令人惊讶的是，CrzA没有定位在活跃的生长点。 广泛的定向突变筛选显示，钙调神经磷酸酶底物结合PxIxIT基序，以及一个 新颖的FxDxF基序，是CNAA定位不可缺少的。这些新的发现表明，主要影响 钙调神经磷酸酶介导的生长和毒力可能是由于钙调神经磷酸酶与尚未确定的 这些关键增长点的关键影响因素。作为合乎逻辑的下一步，我们现在试图定义钙调神经磷酸酶的主要 作为一种磷酸酶，通过与这些未知效应器的相互作用来协调生长和毒力。 我们的总体目标是确定控制烟曲霉菌丝生长的钙调神经磷酸酶下游效应物。 和致命性。我们的假设是，钙调神经磷酸酶作为一种关键的磷酸酶，与磷酸化的 效应器使它们去磷酸化以控制生长和毒力。要定义这些交互和 调节促进菌丝生长和毒力的效应物的机制，我们将利用整体超强 敏感的蛋白质组学策略来定义完整的CNAA磷酸蛋白质组。 在目标1中，我们将定义烟曲霉菌的天然蛋白质组和依赖钙调神经磷酸酶的蛋白质组。 使用两种独立的定量全球磷酸蛋白质组学方法与定量分析 天然蛋白质组。这将定义野生型菌株中去磷酸化的下游效应底物， 由于有磷酸酶活性，但在钙调神经磷酸酶缺陷突变体中仍有磷酸化。我们会 然后使用多反应监测来测量蛋白质表达趋势以及验证效应器 来自磷酸盐蛋白质组。在目标2中，我们将确定已识别的CNAA效应器的优先顺序，以验证它们在 钙调神经磷酸酶通过迭代方法在生长和疾病中介导下游效应。我们将使用基因 缺失(单个、多个)、体外生长筛选、靶向突变和小鼠模型验证 毒性缺陷。这一多方面的方法将确定关键的依赖磷酸化的下游 钙调神经磷酸酶的效应者，并开发了真菌特异性控制菌丝生长和毒力的模型。我们会 生成由病原体中的钙调神经磷酸酶调节的先前未描述的下游效应器的地图，该图 将对更广泛的钙调神经磷酸酶、真菌生长和发病机制研究产生重要的生物学影响。

Invasive aspergillosis due to Aspergillus fumigatus is a leading killer in immunocompromised patients. A significant barrier to developing effective antifungal therapeutics is the lack of understanding of the regulation of A. fumigatus hyphal growth and disease. We have established that calcineurin is required for A. fumigatus growth and virulence. We also showed that calcineurin is dynamically localized at active points of growth, the hyphal tip and septum, and that calcineurin phosphatase activity is required at these active points to regulate hyphal growth and virulence. Calcineurin (CnaA) functions as a phosphatase to dephosphorylate substrates and through the transcription factor, CrzA. However, in contrast to our CnaA deletion strain, our ΔcrzA strain exhibited a minimal hyphal growth defect and, surprisingly, CrzA did not localize at the active points of growth. An extensive targeted mutagenesis screen revealed that the calcineurin substrate binding PxIxIT motif, and a novel FxDxF motif, are indispensable for CnaA localization. These new findings indicate that the major effects on calcineurin-mediated growth and virulence are likely due to calcineurin interactions with as yet undefined key effectors at these critical growth points. As a logical next step, we now seek to define calcineurin’s main function as a phosphatase to orchestrate growth and virulence via its interaction with these unknown effectors. Our overall goal is to identify the calcineurin downstream effectors that control A. fumigatus hyphal growth and virulence. Our hypothesis is that calcineurin, as a critical phosphatase, interacts with phosphorylated effectors to dephosphorylate them to control growth and virulence. To define these interactions and the mechanism of regulation of effectors facilitating hyphal growth and virulence, we will utilize holistic ultra- sensitive proteomic strategies to define the complete CnaA phosphoproteome. In Aim 1, we will define the A. fumigatus native proteome and calcineurin-dependent phosphoproteome using two independent quantitative global phosphoproteomic approaches paired with quantitative analysis of the native proteome. This will define downstream effector substrates dephosphorylated in the wild-type strain, due to phosphatase activity, but still phosphorylated in the calcineurin phosphatase-deficient mutant. We will then employ Multiple Reaction Monitoring to measure protein expression trends as well as validate effectors from the phosphoproteome. In Aim 2, we will prioritize the identified CnaA effectors to validate their role in calcineurin-mediated downstream effects in growth and disease via an iterative approach. We will use genetic deletion (single, multiple), in vitro growth screening, targeted mutations, and a murine model validation for virulence defects. This multi-faceted approach will identify key phosphorylation-dependent downstream effectors of calcineurin and develop a model for fungal-specific control of hyphal growth and virulence. We will generate a map of previously undescribed downstream effectors regulated by calcineurin in a pathogen, which will have important biologic ramifications for broader calcineurin, fungal growth, and pathogenesis studies.