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

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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 ?crzA 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.

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