Mitochondrial Activity and Cyr1 Are Key Regulators of Ras1 Activation of C. albicans Virulence Pathways

Abstract

Candida albicans is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of C. albicans morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in C. albicans and similar effects were observed in other Candida species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate different environmental and intracellular signals, including metabolic status, to decide the fate of cellular morphology.

Author Summary

Candida albicans is a successful fungal commensal and pathogen of humans. It is a polymorphic organism and the ability to switch from yeast to hyphal growth is associated with the commensal-to-pathogen switch. Previous research identified the Ras1-cAMP-protein kinase A pathway as a key regulator of hyphal growth. Here, we report that mitochondrial activity plays a key role in Ras1 activation, as respiratory inhibition decreased Ras1 activity and Ras1-dependent filamentation. We found that intracellular ATP modulates Ras1 activity through a pathway involving the GTPase activating protein Ira2 and the adenylate cyclase Cyr1. Based on our data the canonical Ras1 signaling model in C. albicans needs to be restructured in such a way that Cyr1 is no longer placed downstream of Ras1 but rather in a major signaling node with Ras1 and Ira2. Our studies suggest that the energy status of the cell is the most important signal involved in the decision of C. albicans to undergo the yeast-to-hyphae switch or express genes associated with the hyphal morphology as low intracellular ATP or associated cues override several hypha-inducing signals. Future studies will show if this knowledge can be used to develop therapies that would favor benign host- Candida interactions by promoting low Ras1 activity.