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.

R21AI163301

2021-06-01

2023-05-31