Analysis of the Protein Kinase A-Regulated Proteome of Cryptococcus neoformans Identifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation

Abstract

The opportunistic fungal pathogenCryptococcus neoformanscauses life-threatening meningitis in immunocompro-mised individuals. The expression of virulence factors, including capsule and melanin, is in part regulated by the cyclic-AMP/protein kinase A (cAMP/PKA) signal transduction pathway. In this study, we investigated the influence of PKA on the composi-tion of the intracellular proteome to obtain a comprehensive understanding of the regulation that underpins virulence. Throughquantitative proteomics, enrichment and bioinformatic analyses, and an interactome study, we uncovered a pattern of PKA reg-ulation for proteins associated with translation, the proteasome, metabolism, amino acid biosynthesis, and virulence-relatedfunctions. PKA regulation of the ubiquitin-proteasome pathway inC. neoformansshowed a striking parallel with connectionsbetween PKA and protein degradation in chronic neurodegenerative disorders and other human diseases. Further investigationof proteasome function with the inhibitor bortezomib revealed an impact on capsule production as well as hypersusceptibilityfor strains with altered expression or activity of PKA. Parallel studies with tunicamycin also linked endoplasmic reticulum stresswith capsule production and PKA. Taken together, the data suggest a model whereby expression of PKA regulatory and catalyticsubunits and the activation of PKA influence proteostasis and the function of the endoplasmic reticulum to control the elabora-tion of the polysaccharide capsule. Overall, this study revealed both broad and conserved influences of the cAMP/PKA pathwayon the proteome and identified proteostasis as a potential therapeutic target for the treatment of cryptococcosis.