Mapping the Hsp90 Genetic Network Reveals Ergosterol Biosynthesis and Phosphatidylinositol-4-Kinase Signaling as Core Circuitry Governing Cellular Stress

摘要

Candida albicans is a leading human fungal pathogen that causes life-threatening systemic infections. A key regulator of C . albicans stress response, drug resistance, morphogenesis, and virulence is the molecular chaperone Hsp90. Targeting Hsp90 provides a powerful strategy to treat fungal infections, however, the therapeutic utility of current inhibitors is compromised by toxicity due to inhibition of host Hsp90. To identify components of the Hsp90-dependent circuitry governing virulence and drug resistance that are sufficiently divergent for selective targeting in the pathogen, we pioneered chemical genomic profiling of the Hsp90 genetic network in C . albicans . Here, we screen mutant collections covering ~10% of the genome for hypersensitivity to Hsp90 inhibition in multiple environmental conditions. We identify 158 HSP90 chemical genetic interactors, most of which are important for growth only in specific environments. We discovered that the sterol C-22 desaturase gene ERG5 and the phosphatidylinositol-4-kinase (PI4K) gene STT4 are HSP90 genetic interactors under multiple conditions, suggesting a function upstream of Hsp90. By systematic analysis of the ergosterol biosynthetic cascade, we demonstrate that defects in ergosterol biosynthesis induce cellular stress that overwhelms Hsp90’s functional capacity. By analysis of the phosphatidylinositol pathway, we demonstrate that there is a genetic interaction between the PI4K Stt4 and Hsp90. We also establish that Stt4 is required for normal actin polarization through regulation of Wal1, and suggest a model in which defects in actin remodeling induces stress that creates a cellular demand for Hsp90 that exceeds its functional capacity. Consistent with this model, actin inhibitors are synergistic with Hsp90 inhibitors. We highlight new connections between Hsp90 and virulence traits, demonstrating that Erg5 and Stt4 enable activation of macrophage pyroptosis. This work uncovers novel circuitry regulating Hsp90 functional capacity and new effectors governing drug resistance, morphogenesis and virulence, revealing new targets for antifungal drug development. Author Summary Hsp90 is an essential and conserved molecular chaperone that is required for the folding and function of a wide range of client proteins, especially those involved in signaling and stress responses. In the human fungal pathogen Candida albicans , Hsp90 governs drug resistance, morphogenesis, and virulence. In our previous analysis, we developed a chemical genomic approach to map the HSP90 chemical genetic network in C . albicans , an organism for which classical genetic approaches are hampered by the lack of a complete sexual cycle. Here, we confirm the environmental contingency of the Hsp90 genetic network in C . albicans , identify novel genetic interactions, and demonstrate new circuitry that regulates Hsp90 functional capacity in the cell. In the context of treatment of infectious disease, the challenge in avoiding host toxicity and achieving specificity lies in either the development of pathogen-selective Hsp90 inhibitors or identifying pathogen-specific components of the circuitry through which Hsp90 governs virulence. This work provides insight into such circuitry in the leading fungal pathogen of humans.