Identification of calcineurin-binding proteins in A. fumigatus septum formation

描述（由申请人提供）：由烟曲霉引起的侵袭性曲霉病（IA）已经超过侵袭性念珠菌病，成为免疫功能低下患者最常见的真菌死亡原因。美国传染病学会强调烟曲霉是迫切需要实质性治疗突破的六种病原体之一。尽管发病率和死亡率不断上升，烟曲霉发病机制的基本决定因素尚不清楚。我们的长期目标是了解烟曲霉生长和疾病的分子控制。鼻中隔是一种基本的生物结构，对许多真菌的生长很重要，是理想的治疗靶点。烟曲霉菌丝产生区隔推进并侵入宿主组织。我们已经证明，钙调神经磷酸酶稳定地定位于烟曲霉间隔，遗传或药理学抑制钙调神经磷酸酶阻止菌丝生长并破坏分离。尽管它在生长中起着关键作用，但尚未有研究关注菌丝间隔以了解其发病机制，并且钙调磷酸酶介导的间隔形成控制尚不清楚。我们的中心假设是，钙调磷酸酶与烟曲霉中隔的其他蛋白质复合物调节中隔的形成和菌丝的延伸。本应用程序的目的是确定在菌丝间隔处与钙调磷酸酶结合的蛋白质。我们的方法将分为两个具体目标：1)通过钙调神经磷酸酶A和B的下拉测定，然后进行LC/MS/MS分析，鉴定烟曲霉中的钙调神经磷酸酶结合蛋白。钙调磷酸酶A和钙调磷酸酶B高度集中在隔膜上，表明它们之间有很强的相互作用，这一作用不同于钙调磷酸酶的已知功能。我们将确定钙调磷酸酶结合在隔膜开始了解生长调节的机制。2)验证室间隔定位和假定的钙调磷酸酶结合蛋白的钙调磷酸酶依赖性。我们将在野生型烟曲霉中对这些新发现的蛋白进行优先排序和定位，以验证它们的细胞位置和功能。为了表征已鉴定的钙调磷酸酶结合间隔蛋白的钙调磷酸酶依赖性，我们将利用我们独特的钙调磷酸酶缺失菌株。这些结果将有一个

DESCRIPTION (provided by applicant): Invasive aspergillosis (IA) caused by the fungus Aspergillus fumigatus has surpassed invasive candidiasis as the most frequent fungal cause of death in immunocompromised patients. The Infectious Diseases Society of America highlighted A. fumigatus as one of only six pathogens for which a substantive treatment breakthrough is urgently needed. Despite an increasing incidence of disease and mortality, the basic determinants underlying A. fumigatus pathogenesis are not well understood. Our long-term goal is to understand the molecular control of A. fumigatus growth and disease. The septum is a fundamental biologic structure important for the growth of many fungi and an ideal therapeutic target. A. fumigatus hyphae produce compartmentalizing septa to advance and invade host tissue. We have shown that calcineurin stably localizes to the A. fumigatus septum and genetic or pharmacologic inhibition of calcineurin blocks hyphal growth and disrupts septation. Despite its pivotal role in growth, no studies have focused on the hyphal septum to understand pathogenesis, and calcineurin-mediated control of septum formation is not understood. Our central hypothesis is that calcineurin complexes with other proteins at the A. fumigatus septum to regulate septum formation and hyphal extension. The objective of this application is to define those proteins that bind to calcineurin at the hyphal septum. Our approach will be divided into two specific aims: 1) Identify calcineurin- binding proteins in A. fumigatus through calcineurin A and B pulldown assays followed by LC/MS/MS analysis. Calcineurin A and B are highly concentrated at the septum, arguing for a strong interaction, a role different than calcineurin's known function as a phosphatase. We will identify what calcineurin binds at the septum to begin to understand the mechanism of growth regulation. 2) Validate septal localization and calcineurin-dependency of putative calcineurin-binding protein. We will prioritize and localize those newly identified proteins in wild-type A. fumigatus to verify their cellular location and function. To characterize the calcineurin-dependency of the identified calcineurin-binding septal proteins, we will utilize our unique set of calcineurin deletion strains. These results will have a significant impact in understanding underlying growth mechanisms, focusing on the novel theory that calcineurin directly regulates septum formation to orchestrate hyphal growth and disease. Our approach to define the proteins interacting with calcineurin at the septum will begin to unravel the larger molecular basis of A. fumigatus pathogenesis. This strategy will move us toward our long- term goal of developing novel IA therapeutics by identifying exploitable fungal-specific targets to block septum formation and growth, leading to a substantial impact on IA mortality. PUBLIC HEALTH RELEVANCE: Invasive fungal infection caused by Aspergillus fumigatus is a leading cause of death for patients with lowered immune systems. Despite this high mortality, the fundamental mechanism of how A. fumigatus grows and invades is not understood and this knowledge is critical to improving diagnosis and treatment for this deadly disease. The septum is crucial for A. fumigatus growth and disease and we have shown that the calcineurin signaling pathway controls normal septum formation and could be targeted for newer treatments.