Friday, November 22, 2024
11/22/2024
Invasive aspergillosis due to Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients. The mortality rate is a staggering 40-60%, worsened by emerging resistance to azole antifungals. Despite this, there is a significant knowledge gap surrounding the effectors governing A. fumigatus antifungal response and resistance. We established calcineurin (CN) as a critical phosphatase required for A. fumigatus growth, invasive disease, antifungal response and resistance. However, how CN regulates these processes is largely undefined. We demonstrated CN-dependent transcriptional control over antifungal response via its transcription factor, CrzA. Our CN proteomic and phosphoproteomic approaches uncovered several cell wall and membrane-related proteins as potential CN effectors. We also defined unique changes in the CN proteome in response to azole and echinocandin antifungals, and detected CN-dependent alterations in cell wall β-glucan and membrane ergosterol and lipid profiles. We were also the first to crystallize the fungal CN complex, and our structure-guided inhibitor strategies showed targeting CN is effective in animal models. Our objective is to leverage our novel genetic, proteomic and lipidomic data to define CN control over antifungal response and resistance. Our central hypothesis is that CN orchestrates parallel transcriptional and phosphoregulatory control of key effectors in cell wall and membrane organization, thereby specifically governing antifungal response and drug resistance. In Aim 1, we will identify key CN effectors for cell wall and membrane integrity and antifungal response by bimolecular fluorescence complementation and affinity assays. CN effector localization studies in CN inhibited/deletion backgrounds will confirm CN-dependency in response to antifungals. Molecular modeling of CN-effector binding and molecular dynamics simulations will guide targeted mutations of predicted CN-binding motifs in effectors. These integrated approaches will elucidate the unknown role played by CN in antifungal response through binding and regulation of key effectors in cell wall and membrane synthesis. In Aim 2, we will interrogate CN transcriptional control of effectors by examining expression in the background of CN and CN- dependent transcription factor inhibition/deletions, including mutations in promoter CDRE sequences. We will perform LC-MS/MS of phosphorylation sites in CN effectors in wild-type vs. CN-inhibited strains under antifungal stress. Phosphomutations in CN effectors will assess the requirement of CN-dependent dephosphorylation at specific residues for function in cell wall and membrane stress responses. In Aim 3, we will genetically characterize contributions of prioritized CN effectors to resistance in azole- and echinocandin-resistant strains using an iterative approach of genetic deletion, targeted mutations, and murine model validation. We will also be the first to examine the emerging azole-resistant A. calidoustus species genetically and in animal models to fully define CN effectors’ specific roles in drug response and disease. This study will, for the first time, define the mechanism of CN control over antifungal response and resistance through cell wall and membrane effectors.
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