Transcriptional and Post-Translational Roles of Calcineurin in Cationic Stress and Glycerol Biosynthesis in Cryptococcus neoformans

Abstract

Stress management is an adaptive advantage for survival in adverse environments. Pathogens face this challenge during host colonization, requiring an appropriate stress response to establish infection. The fungal pathogen Cryptococcus neoformans undergoes thermal, oxidative, and osmotic stresses in the environment and animal host. Signaling systems controlled by Ras1, Hog1, and calcineurin respond to high temperatures and osmotic stress. Cationic stress caused by Na+, K+, and Li+ can be overcome with glycerol, the preferred osmolyte. Deleting the glycerol phosphate phosphatase gene (GPP2) prevents cells from accumulating glycerol due to a block in the last step of its biosynthetic pathway. Gpp2 accumulates in a phosphorylated form in a cna1Δ strain, and a physical interaction between Gpp2 and Cna1 was found; moreover, the gpp2Δ strain undergoes slow growth and has attenuated virulence in animal models of infection. We provide biochemical evidence that growth in 1 M NaCl increases glycerol content in the wild type, whereas gpp2Δ, cna1Δ, and cnb1Δ mutants fail to accumulate it. The deletion of cnb1Δ or cna1Δ renders yeast cells sensitive to cationic stress, and the Gfp-Gpp2 protein assumes an abnormal localization. We suggest a mechanism in which calcineurin controls Gpp2 at the post-translational level, affecting its localization and activity, leading to glycerol biosynthesis. Also, we showed the transcriptional profile of glycerol-deficient mutants and established the cationic stress response mediated by calcineurin; among the biological processes differentially expressed are carbon utilization, translation, transmembrane transport, glutathione metabolism, oxidative stress response, and transcription regulation. To our knowledge, this is the first time that this transcriptional profile has been described. These results have implications for pathogen stress adaptability.