Proteomic Profiling of the Pathogenic Fungus Cryptococcus neoformans upon Regulation of the cyclic-AMP/Protein Kinase A Signaling Pathway (2015)
The pathogenic yeast Cryptococcus neoformans causes life-threatening meningoencephalitis in immunocompromised individuals. The ability of C. neoformans to cause disease depends on the elaboration of virulence factors including a polysaccharide capsule, melanin deposition in the cell wall, the ability to grow at 37°C, and the secretion of extracellular enzymes. The cyclic-AMP/Protein Kinase A (PKA) signal transduction pathway is a key regulator of virulence in C. neoformans and may also regulate the trafficking of virulence factors. The influence of PKA1 expression on the intracellular and extracellular proteomes and identification of Pka1 phosphorylation targets using phosphoproteomics have not been investigated for C. neoformans. In our current study, I performed quantitative proteomics using a galactose-inducible/glucose-repressible expression strain of the PKA1 gene to identify regulated proteins in the secretome and proteome. During investigation of the secretome, five proteins showed changes in extracellular abundance upon Pka1 induction. These included the Cig1 and Aph1 proteins with known roles in virulence, as well as an α-amylase, a glyoxal oxidase, and a novel protein. Targeted proteomics of these Pka1-regulated proteins allowed us to identify the secreted proteins in biological samples suggesting their potential as biomarkers of infection. During investigation of the intracellular proteome, I identified a broad and conserved influence by PKA1. Furthermore, an analysis of protein-ptotein interactions emphasized the impact of PKA activity on several clusters of proteins involving translation and the ribosome, the proteasome, and diverse metabolic processes. Lastly, a phosphoproteomic study identified six potential targets of Pka1 phosphorylation including the master iron regulator, Cir1. Construction of site-directed mutants showed that Pka1 phosphorylation of Cir1 impacted the production ofcapsule and melanin, cell size, and the ability to grow under low iron conditions. Overall, thedata presented in this thesis have contributed a better understanding of the broad and conserved influence of Pka1 on cellular regulation and secretion in C. neoformans, and the discovery of potential biomarkers may facilitate the monitoring of disease progression. Additionally, the identification of new Pka1 phosphorylation targets present opportunities for the development of a molecular understanding of the regulation of virulence as well as novel therapeutic strategies for treatment of cryptococcosis.