Hsp90 is a molecular chaperone for many signal transducers and may influence evolution by releasing previously silent genetic variation in response to environmental change. In fungi separated by approximately 800 million years of evolution, Hsp90 potentiated the evolution of drug resistance in a different way, by enabling new mutations to have immediate phenotypic consequences. Resistance was abrogated by Hsp90 inhibitors and by febrile temperatures, suggesting new therapeutic strategies and a clinical benefit of fever. During selection in a human host, drug resistance that was initially Hsp90-dependent evolved toward independence. Thus, Hsp90 can act in diverse ways to couple environmental contingency to the emergence and fixation of new traits.

SUMMARY Pathogenic fungi have become a leading cause of human mortality due to the increasing frequency of fungal infections in immunocompromised populations and the limited armamentarium of clinically useful antifungal drugs. Candida albicans , Cryptococcus neoformans , and Aspergillus fumigatus are the leading causes of opportunistic fungal infections. In these diverse pathogenic fungi, complex signal transduction cascades are critical for sensing environmental changes and mediating appropriate cellular responses. For C. albicans , several environmental cues regulate a morphogenetic switch from yeast to filamentous growth, a reversible transition important for virulence. Many of the signaling cascades regulating morphogenesis are also required for cells to adapt and survive the cellular stresses imposed by antifungal drugs. Many of these signaling networks are conserved in C. neoformans and A. fumigatus , which undergo distinct morphogenetic programs during specific phases of their life cycles. Furthermore, the key mechanisms of fungal drug resistance, including alterations of the drug target, overexpression of drug efflux transporters, and alteration of cellular stress responses, are conserved between these species. This review focuses on the circuitry regulating fungal morphogenesis and drug resistance and the impact of these pathways on virulence. Although the three human-pathogenic fungi highlighted in this review are those most frequently encountered in the clinic, they represent a minute fraction of fungal diversity. Exploration of the conservation and divergence of core signal transduction pathways across C. albicans , C. neoformans , and A. fumigatus provides a foundation for the study of a broader diversity of pathogenic fungi and a platform for the development of new therapeutic strategies for fungal disease.

Candida albicans is the leading fungal pathogen of humans, causing life-threatening disease in immunocompromised individuals. Treatment of candidiasis is hampered by the limited number of antifungal drugs whose efficacy is compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. We previously established that the molecular chaperone Hsp90, which regulates the form and function of diverse client proteins, potentiates resistance to the azoles in C. albicans and in the model yeast Saccharomyces cerevisiae. Genetic studies in S. cerevisiae revealed that Hsp90's role in azole resistance is to enable crucial cellular responses to the membrane stress exerted by azoles via the client protein calcineurin. Here, we demonstrate that Hsp90 governs cellular circuitry required for resistance to the only new class of antifungals to reach the clinic in decades, the echinocandins, which inhibit biosynthesis of a critical component of the fungal cell wall. Pharmacological or genetic impairment of Hsp90 function reduced tolerance of C. albicans laboratory strains and resistance of clinical isolates to the echinocandins and created a fungicidal combination. Compromising calcineurin function phenocopied compromising Hsp90 function. We established that calcineurin is an Hsp90 client protein in C. albicans: reciprocal co-immunoprecipitation validated physical interaction; Hsp90 inhibition blocked calcineurin activation; and calcineurin levels were depleted upon genetic reduction of Hsp90. The downstream effector of calcineurin, Crz1, played a partial role in mediating calcineurin-dependent stress responses activated by echinocandins. Hsp90's role in echinocandin resistance has therapeutic potential given that genetic compromise of C. albicans HSP90 expression enhanced the efficacy of an echinocandin in a murine model of disseminated candidiasis. Our results identify the first Hsp90 client protein in C. albicans, establish an entirely new role for Hsp90 in mediating resistance to echinocandins, and demonstrate that targeting Hsp90 provides a promising therapeutic strategy for the treatment of life-threatening fungal disease.

Invasive fungal infections are a leading cause of mortality among immunocompromised individuals. Treatment is notoriously difficult with the limited armamentarium of antifungal drugs, whose efficacy is compromised by host toxicity, a limited activity spectrum, or the emergence of drug resistance. We previously established that the molecular chaperone Hsp90 enables the emergence and maintenance of fungal drug resistance. For the most prevalent fungal pathogen of humans, Candida albicans, Hsp90 mediates resistance to azoles, which inhibit ergosterol biosynthesis and are the most widely deployed antifungals in the clinic. For the emerging opportunistic pathogen Aspergillus terreus, Hsp90 is required for basal resistance to echinocandins, which inhibit ␤(1, 3)-glucan synthesis and are the only new class of antifungals to reach the clinic in decades. Here, we explore the therapeutic potential of Hsp90 inhibitors in fungal disease using a tractable host-model system, larvae of the greater wax moth Galleria mellonella, and a murine model of disseminated disease. Combination therapy with Hsp90 inhibitors that are well tolerated in humans and an azole rescued larvae from lethal C. albicans infections. Combination therapy with an Hsp90 inhibitor and an echinocandin rescued larvae from infections with the most lethal mold, Aspergillus fumigatus. In a murine model of disseminated candidiasis, genetic compromise of C. albicans HSP90 expression enhanced the therapeutic efficacy of an azole. Thus, harnessing Hsp90 provides a much-needed strategy for improving the treatment of fungal disease because it enhances the efficacy of existing antifungals, blocks the emergence of drug resistance, and exerts broadspectrum activity against diverse fungal pathogens.antifungal ͉ Aspergillus fumigatus ͉ azole ͉ Candida albicans ͉ drug resistance

Antifungal therapy is a central component of patient management for acute and chronic mycoses. Yet, treatment choices are restricted because of the sparse number of antifungal drug classes. Clinical management of fungal diseases is further compromised by the emergence of antifungal drug resistance, which eliminates available drug classes as treatment options. Once considered a rare occurrence, antifungal drug resistance is on the rise in many high-risk medical centers. Most concerning is the evolution of multidrug-resistant organisms refractory to several different classes of antifungal agents, especially among common Candida species. The mechanisms responsible are mostly shared by both resistant strains displaying inherently reduced susceptibility and those acquiring resistance during therapy. The molecular mechanisms include altered drug affinity and target abundance, reduced intracellular drug levels caused by efflux pumps, and formation of biofilms. New insights into genetic factors regulating these mechanisms, as well as cellular factors important for stress adaptation, provide a foundation to better understand the emergence of antifungal drug resistance.

Summary Background Hsp90 is an environmentally contingent molecular chaperone that influences the form and function of diverse regulators of cellular signaling. Hsp90 potentiates the evolution of fungal drug resistance by enabling crucial cellular stress responses. Here we demonstrate that in the leading fungal pathogen of humans, Candida albicans, Hsp90 governs cellular circuitry required not only for drug resistance but also for the key morphogenetic transition from yeast to filamentous growth that is crucial for virulence. This transition is normally regulated by environmental cues, such as exposure to serum, that are contingent upon elevated temperature to induce morphogenesis. The basis for this temperature dependence has remained enigmatic. Results We show that compromising Hsp90 function pharmacologically or genetically induces a transition from yeast to filamentous growth in the absence of external cues. Elevated temperature relieves Hsp90-mediated repression of the morphogenetic program. Hsp90 regulates morphogenetic circuitry by repressing Ras1-PKA signaling. Modest Hsp90 compromise enhances the phenotypic effects of activated Ras1 signaling while deletion of positive regulators of the Ras1-PKA cascade blocks the morphogenetic response to Hsp90 inhibition. Consistent with the requirement for morphogenetic flexibility for virulence, depletion of C. albicans Hsp90 attenuates virulence in a murine model of systemic disease. Conclusions Hsp90 governs the integration of environmental cues with cellular signaling to orchestrate fungal morphogenesis and virulence, suggesting new therapeutic strategies for life-threatening infectious disease. Hsp90’s capacity to govern a key developmental program in response to temperature change provides a new mechanism that complements the elegant repertoire that organisms utilize to sense temperature.

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.

Fungal infections are a major contributor to infectious disease-related deaths across the globe. Candida species are among the most common causes of invasive mycotic disease, with Candida albicans reigning as the leading cause of invasive candidiasis. Given that fungi are eukaryotes like their human host, the number of unique molecular targets that can be exploited for antifungal development remains limited. Currently, there are only three major classes of drugs approved for the treatment of invasive mycoses, and the efficacy of these agents is compromised by the development of drug resistance in pathogen populations. Notably, the emergence of additional drug-resistant species, such as Candida auris and Candida glabrata, further threatens the limited armamentarium of antifungals available to treat these serious infections. Here, we describe our current arsenal of antifungals and elaborate on the resistance mechanisms Candida species possess that render them recalcitrant to therapeutic intervention. Finally, we highlight some of the most promising therapeutic strategies that may help combat antifungal resistance, including combination therapy, targeting fungal-virulence traits, and modulating host immunity. Overall, a thorough understanding of the mechanistic principles governing antifungal drug resistance is fundamental for the development of novel therapeutics to combat current and emerging fungal threats.