Phosphoproteomic Identification of Aspergillus Fumigatus Virulence-associated Protein Kinase A Downstream Effectors

Invasive aspergillosis (IA) caused by Aspergillus fumigatus is among the leading infectious causes of deathin immunocompromised patients. Protein kinase A (PKA) signaling is a critical pathway required for the virulenceof several important fungal pathogens, including A. fumigatus. However, little is known regarding the specifictargets of PKA in any organism. Furthermore, the majority of mechanistic studies relating to the pathway in fungihave been limited to non-pathogenic model yeasts. Unraveling the PKA signaling pathway should thereforeprovide key insight into the mechanisms of fungal pathogenesis. We have recently identified novel regulatorymechanisms of PKA activity in A. fumigatus via phosphorylation of the catalytic and regulatory subunits. Wehave also defined the PKA interactome via mass spectroscopic analysis that identified numerous potential targetproteins. These include both known and uncharacterized transcriptional regulators, which hint at diverse andnovel roles for PKA signaling in this species. These findings underscore the contrast between PKA regulatorymechanisms in filamentous fungi and yeasts, and also signify that important undefined variations exist in theactivation of downstream effectors governing virulence and disease establishment. We hypothesize that PKAacts as one master regulator of fungal pathogenic processes through the activation of specific effector proteinsthat promote growth and virulence. Our overall goal is to identify proteins that significantly contribute to thevirulence of A. fumigatus in a PKA-dependent manner. To identify these effectors and better understand themolecular mechanism of A. fumigatus pathogenesis, we will utilize a high-resolution proteomics-based approachto now define the full PKA-dependent phosphoproteome. Following identification, the PKA-dependent effectorswill be screened for their contributions to virulence using genetic, in vitro, and animal model strategies. In Aim1, we will define the A. fumigatus native proteome and PKA-dependent phosphoproteome using two independentquantitative global proteomic approaches. Effectors will be defined as those phosphorylated in a wild-type strainbut dephosphorylated in a PKA-deficient mutant strain under basal or stress-inducing conditions. Comparison ofspecific protein levels between the native proteomes of the wild-type and mutant strains will be used to validatechanges in phosphorylation versus changes in protein expression. In Aim 2, we will identify PKA-dependenteffectors with virulence contributions. Following prioritization via a bioinformatic and proteomic scoring system,we will use an iterative approach of genetic deletion, in vitro growth screening, and a robust murine model of IAfor validation of virulence defects. This strategy will enable the comprehensive, high-resolution identification ofPKA-dependent effectors specifically contributing to the growth and virulence of A. fumigatus. Cataloguing themost complete set of PKA targets for any fungal species to date, as well as the identification of specific sites ofphosphorylation, will greatly increase our understanding of the fundamental pathobiology of A. fumigatus andpotentially facilitate future studies toward novel targeting strategies.