Developmental transitions between single-cell yeast and multicellular filaments underpin virulence of diverse fungal pathogens. For the leading human fungal pathogen Candida albicans, filamentation is thought to be required for immune cell escape via induction of an inflammatory programmed cell death. Here we perform a genome-scale analysis of C. albicans morphogenesis and identify 102 negative morphogenetic regulators and 872 positive regulators, highlighting key roles for ergosterol biosynthesis and N-linked glycosylation. We demonstrate that C. albicans filamentation is not required for escape from host immune cells; instead, macrophage pyroptosis is driven by fungal cell-wall remodelling and exposure of glycosylated proteins in response to the macrophage phagosome. The capacity of killed, previously phagocytized cells to drive macrophage lysis is also observed with the distantly related fungal pathogen Cryptococcus neoformans. This study provides a global view of morphogenetic circuitry governing a key virulence trait, and illuminates a new mechanism by which fungi trigger host cell death.

Reversible phosphorylation is a fundamental regulatory mechanism, intricately coordinated by kinases and phosphatases, two classes of enzymes widely disrupted in human disease. To better understand the functions of the relatively understudied phosphatases, we have used complementary affinity purification and proximity-based interaction proteomics approaches to generate a physical interactome for 140 human proteins harboring phosphatase catalytic domains. We identified 1,335 high-confidence interactions (1,104 previously unreported), implicating these phosphatases in the regulation of a variety of cellular processes. Systematic phenotypic profiling of phosphatase catalytic and regulatory subunits revealed that phosphatases from every evolutionary family impinge on mitosis. Using clues from the interactome, we have uncovered unsuspected roles for DUSP19 in mitotic exit, CDC14A in regulating microtubule integrity, PTPRF in mitotic retraction fiber integrity, and DUSP23 in centriole duplication. The functional phosphatase interactome further provides a rich resource for ascribing functions for this important class of enzymes.

The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection.

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.

The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen Candida albicans is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of C. albicans HSF1 induces morphogenesis in the absence of external cues. HSF1 depletion compromises Hsp90 function, thereby driving filamentation. HSF1 overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.