Friday, November 22, 2024
11/22/2024
Invasive aspergillosis (IA), caused by the fungus Aspergillus fumigatus, is a leading infectious cause of death in immunocompromised patients. A significant barrier to deciphering the mechanisms of pathogenesis and developing effective antifungals is a lack of understanding of the regulation of A. fumigatus growth leading to invasive disease. Cellular recycling mediated via autophagy-associated proteins is a key catabolic pathway and critical to invasive fungal growth and virulence in the well-known nutrient-limited host environment. Protein kinase A (PKA) is vital for growth and virulence of numerous fungal pathogens. However, the underlying basis for its regulation of pathogenesis remains poorly understood in any species. Our A. fumigatus PKA-dependent whole proteome and phosphoproteome studies employing advanced mass spectroscopic (MS) approaches identified numerous previously undefined PKA-regulated proteins in catabolic pathways. Preliminary characterization of two such novel direct PKA target proteins, Atg24 and Not4, demonstrated their requirement for A. fumigatus virulence and also revealed functional importance of PKA-dependent phosphorylation of specific target sites. We have identified and prioritized more than 30 autophagy-associated proteins as likely novel PKA-regulated effectors, and will now characterize the top 6 priority proteins with regard to their roles in fungal growth and pathogenesis, as well as their regulation by PKA. Our overall objective is to leverage our robust PKA whole proteomic and phosphoproteomic data to define fungal-specific mechanisms of PKA control over autophagy function and validate these PKA-regulated effectors’ novel contributions to fungal virulence. In Aim 1, we will define PKA control over autophagy by validating PKA-dependent phosphorylation of prioritized phosphoregulated effectors via MS analysis and expression-regulated effectors via qRT-PCR. We will characterize the role of each PKA effector in fungal growth and stress responses through deletion and phosphosite mutagenesis approaches. We will also perform structural elucidation of PKA-effector phosphoregulation and function using molecular modeling and molecular dynamics simulations. In Aim 2, we will validate the roles of PKA-regulated autophagy proteins in virulence and host-pathogen interaction using our murine model of invasive aspergillosis to assess their impact on mortality, fungal burden and host lung tissue damage via histological analysis. We will also assess the impact of PKA-regulated proteins on both the pathogen and host response to infection by expression profiling of A. fumigatus autophagy marker genes, as well as simultaneous examination of the host immune cytokine response during infection with wild-type and mutant strains. PKA regulates catabolic processes critical to fungal growth, yet an understanding of PKA regulation of autophagy pathways is lacking in any organism. We will define novel PKA-dependent regulation of newly- identified fungal-specific autophagy-associated proteins and provide critical insight into future exploitation of PKA-dependent control over A. fumigatus disease.
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