Yck2 links mitochondrial function and pH homeostasis to cell wall remodeling and antifungal susceptibility in Cryptococcus neoformans

Abstract

Invasive fungal infections are on the rise due to climate change, antifungal resistance, and increased usage of immunomodulating therapies. The antifungal arsenal is limited in number and in efficacy necessitating novel therapeutics. Deletion of the gene encoding the fungal yeast casein kinase, YCK2, has pleiotropic effects, impacting morphology, drug resistance, metabolism, cell wall, and virulence. Yck2 has recently been shown to be druggable, however the mechanisms by which Yck2 elicits its impacts are unknown. We show that C. neoformans Yck2 is a regulator of cell wall masking, thermotolerance, general stress, and drug resistance. Proximity labeling of Yck2-interacting proteins revealed that Yck2 interacts with proteins that function in the mitochondria as well as the essential plasma membrane H+ ATPase, Pma1. A role for Yck2 in mitochondrial regulation was supported by sensitivity to mitochondrial inhibitors and increased mitochondrial ROS production in the absence of Yck2, and presence of Yck2 in mitochondrial fractions. Using pHluorin-expressing cells we also found that intracellular pH is elevated in cells lacking Yck2 supporting a role in regulating Pma1 activity. Both mitochondria and cellular pH may contribute to cellular signaling and may explain the multitude of phenotypes associated with the yck2Δ mutant. Our results begin to identify the mechanisms by which Yck2 contributes to fungal cellular homeostasis which may support efforts to optimize Yck2 inhibitors. Author Summary Cryptococcus neoformans is a pathogenic fungus that causes ∼20% of AIDS-related mortality. Current antifungals are associated with resistance, high-cost, and/or ineffectiveness necessitating the need for novel therapies. We found that the fungal casein kinase, Yck2, contributes to cell wall remodeling, thermotolerance, drug resistance, and stress response, all of which are important for fungal pathogenesis. Our work revealed that Yck2 modulates mitochondrial function and regulates the intracellular pH, both of which may have cascading effects on the cell. Recent work has shown that fungal Yck2 can be successfully targeted for inhibition. Our data begins to elucidate how Yck2 is functioning in the cell, supporting efforts to develop Yck2 inhibitors as antifungals.