The anti-cancer efficacy of a novel phenothiazine derivative is independent of dopamine and serotonin receptor inhibition.

Introduction: An attractive, yet unrealized, goal in cancer therapy is repurposing psychiatric drugs that can readily penetrate the blood-brain barrier for the treatment of primary brain tumors and brain metastases. Phenothiazines (PTZs) have demonstrated anti-cancer properties through a variety of mechanisms. However, it remains unclear whether these effects are entirely separate from their activity as dopamine and serotonin receptor (DR/5-HTR) antagonists. Methods: In this study, we evaluated the anti-cancer efficacy of a novel PTZ analog, CWHM-974, that was shown to be 100-1000-fold less potent against DR/5-HTR than its analog fluphenazine (FLU). Results: CWHM-974 was more potent than FLU against a panel of cancer cell lines, thus clearly demonstrating that its anti-cancer effects were independent of DR/5-HTR signaling. Our results further suggested that calmodulin (CaM) binding may be necessary, but not sufficient, to explain the anti-cancer effects of CWHM-974. While both FLU and CWHM-974 induced apoptosis, they induced distinct effects on the cell cycle (G0/G1 and mitotic arrest respectively) suggesting that they may have differential effects on CaM-binding proteins involved in cell cycle regulation. Discussion: Altogether, our findings indicated that the anti-cancer efficacy of the CWHM-974 is separable from DR/5-HTR antagonism. Thus, reducing the toxicity associated with phenothiazines related to DR/5-HTR antagonism may improve the potential to repurpose this class of drugs to treat brain tumors and/or brain metastasis.

Bacterial structural genomics target enabled by a recently discovered potent fungal acetyl-CoA synthetase inhibitor.

The compound ethyl-adenosyl monophosphate ester (ethyl-AMP) has been shown to effectively inhibit acetyl-CoA synthetase (ACS) enzymes and to facilitate the crystallization of fungal ACS enzymes in various contexts. In this study, the addition of ethyl-AMP to a bacterial ACS from Legionella pneumophila resulted in the determination of a co-crystal structure of this previously elusive structural genomics target. The dual functionality of ethyl-AMP in both inhibiting ACS enzymes and promoting crystallization establishes its significance as a valuable resource for advancing structural investigations of this class of proteins.

The Mystery of Candida albicans Hyphal Morphogenesis in the Macrophage Phagolysosome.

C. albicans transitions between budding yeast and filamentous hyphal forms in a process that is tightly associated with its virulence. This transition also occurs after the fungus has been phagocytosed by macrophages. A number of somewhat discordant models have been proposed for the environmental characteristics of the phagolysosome that induce this transition. H. B. Wilson and M. C. Lorenz (Infect Immun 91:e00087-23, 2023, https://doi.org/10.1128/iai.00087-23) revisited these models and found that none of them explained morphogenesis in the macrophage.

Candida albicans Oropharyngeal Infection Is an Exception to Iron-Based Nutritional Immunity.

Candida albicans is a commensal of the human gastrointestinal tract and a common cause of human fungal disease, including mucosal infections, such as oropharyngeal candidiasis and disseminated infections of the bloodstream and deep organs. We directly compared the in vivo transcriptional profile of C. albicans during oral infection and disseminated infection of the kidney to identify niche specific features. Overall, 97 genes were differentially expressed between the 2 infection sites. Virulence-associated genes, such as hyphae-specific transcripts, were expressed similarly in the 2 sites. Genes expressed during growth in a poor carbon source (ACS1 and PCK1) were upregulated in oral tissue relative to kidney. Most strikingly, C. albicans in oral tissue shows the transcriptional hallmarks of an iron replete state while in the kidney it is in the expected iron starved state. Interestingly, C. albicans expresses genes associated with a low zinc environment in both niches. Consistent with these expression data, strains lacking transcription factors that regulate iron responsive genes (SEF1, HAP5) have no effect on virulence in a mouse model of oral candidiasis. During microbial infection, the host sequesters iron, zinc, and other metal nutrients to suppress growth of the pathogen in a process called nutritional immunity. Our results indicate that C. albicans is subject to iron and zinc nutritional immunity during disseminated infection but not to iron nutritional immunity during oral infection. IMPORTANCE Nutritional immunity is a response by which infected host tissue sequesters nutrients, such as iron, to prevent the microbe from efficiently replicating. Microbial pathogens subjected to iron nutritional immunity express specific genes to compensate for low iron availability. By comparing the gene expression profiles of the common human fungal pathogen Candida albicans in 2 infection sites, we found that C. albicans infecting the kidney has the transcriptional profile of iron starvation. By contrast, the C. albicans expression profile during oropharyngeal infection indicates the fungus is not iron starved. Two transcription factors that activate the transcriptional response to iron starvation are not required for C. albicans virulence during oral infection but are required for disseminated infection of the kidney. Thus, our results indicate that C. albicans is subject to nutritional iron immunity during disseminated infection but not during oropharyngeal infection, and highlight niche specific differences in the host-Candida albicans interaction.

Candida albicans oropharyngeal infection is an exception to iron-based nutritional immunity.

Candida albicans is a commensal of the human gastrointestinal tract and one of the most causes of human fungal disease, including mucosal infections such as oropharyngeal candidiasis and disseminated infections of the bloodstream and deep organs. We directly compared the in vivo transcriptional profile of C. albicans during oral infection and disseminated infection of the kidney to identify niche specific features. Although the expression of a set of environmentally responsive genes were correlated in the two infection sites (Pearson R 2 , 0.6), XXX genes were differentially expressed. Virulence associated genes such as hyphae-specific transcripts were expressed similarly in the two sites. Genes expressed during growth in a poor carbon source ( ACS1 and PCK1 ) were upregulated in oral tissue relative to kidney. Most strikingly, C. albicans in oral tissue shows the transcriptional hallmarks of an iron-replete state while in the kidney it is in the expected iron starved state. Interestingly, C. albicans expresses genes associated with a low zinc environment in both niches. Consistent with these expression data, deletion of two transcription factors that activate iron uptake genes ( SEF1 , HAP5 ) have no effect on virulence in a mouse model of oral candidiasis. During microbial infection, the host sequesters iron and other metal nutrients to suppress growth of the pathogen in a process called nutritional immunity. Our results indicate that C. albicans is subject to iron and zinc nutritional immunity during disseminated infection but is exempted from iron nutritional immunity during oral infection.

Candida albicans Filamentation Does Not Require the cAMP-PKA Pathway In Vivo.

Candida albicans is one of the most prevalent human fungal pathogens. Its ability to transition between budding yeast and filamentous morphological forms (pseudohyphae and hyphae) is tightly associated with its pathogenesis. Based on in vitro studies, the cAMP-protein kinase A (PKA) pathway is a key regulator of C. albicans morphogenesis. Using an intravital imaging approach, we investigated the role of the cAMP-PKA pathway during infection. Consistent with their roles in vitro, the downstream effectors of the cAMP-PKA pathway Efg1 and Nrg1 function, respectively, as an activator and a repressor of in vivo filamentation. Surprisingly, strains lacking the adenylyl cyclase, CYR1, showed only slightly reduced filamentation in vivo despite being completely unable to filament in RPMI + 10% serum at 37°C. Consistent with these findings, deletion of the catalytic subunits of PKA (Tpk1 and Tpk2), either singly or in combination, generated strains that also filamented in vivo but not in vitro. In vivo transcription profiling of C. albicans isolated from both ear and kidney tissue showed that the expression of a set of 184 environmentally responsive genes correlated well with in vitro filamentation (R2, 0.62 to 0.68) genes. This concordance suggests that the in vivo and in vitro transcriptional responses are similar but that the upstream regulatory mechanisms are distinct. As such, these data emphatically emphasize that C. albicans filamentation is a complex phenotype that occurs in different environments through an intricate network of distinct regulatory mechanisms. IMPORTANCE The fungus Candida albicans causes a wide range of disease in humans from common diaper rash to life-threatening infections in patients with compromised immune systems. As such, the mechanisms for its ability to cause disease are of wide interest. An intensely studied virulence property of C. albicans is its ability to switch from a round yeast form to filament-like forms (hyphae and pseudohyphae). Surprisingly, we have found that a key signaling pathway that regulates this transition in vitro, the protein kinase A pathway, is not required for filamentation during infection of the host. Our work not only demonstrates that the regulation of filamentation depends upon the specific environment C. albicans inhabits but also underscores the importance of studying these mechanisms during infection.

Structural Characterization of the Reaction and Substrate Specificity Mechanisms of Pathogenic Fungal Acetyl-CoA Synthetases.

Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.

Derivatives of the Antimalarial Drug Mefloquine Are Broad-Spectrum Antifungal Molecules with Activity against Drug-Resistant Clinical Isolates.

The antifungal pharmacopeia is critically small, particularly in light of the recent emergence of multidrug-resistant pathogens, such as Candida auris Here, we report that derivatives of the antimalarial drug mefloquine have broad-spectrum antifungal activity against pathogenic yeasts and molds. In addition, the mefloquine derivatives have activity against clinical isolates that are resistant to one or more of the three classes of antifungal drugs currently used to treat invasive fungal infections, indicating that they have a novel mechanism of action. Importantly, the in vitro toxicity profiles obtained using human cell lines indicated that the toxicity profiles of the mefloquine derivatives are very similar to those of the parent mefloquine, despite being up to 64-fold more active against fungal cells. In addition to direct antifungal activity, subinhibitory concentrations of the mefloquine derivatives inhibited the expression of virulence traits, including filamentation in Candida albicans and capsule formation/melanization in Cryptococcus neoformans Mode/mechanism-of-action experiments indicated that the mefloquine derivatives interfere with both mitochondrial and vacuolar function as part of a multitarget mechanism of action. The broad-spectrum scope of activity, blood-brain barrier penetration, and large number of previously synthesized analogs available combine to support the further optimization and development of the antifungal activity of this general class of drug-like molecules.

Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus.

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.

Repurposing Estrogen Receptor Antagonists for the Treatment of Infectious Disease.

The concept of repurposing previously approved medications to the treatment of new indications by taking advantage of off-target effects has gained traction in recent years, particularly in areas of medicine that do not offer large profits to pharmaceutical firms. As infectious disease discovery research has declined among large pharmaceutical companies, the potential payoff of repurposing has become attractive. From these efforts, the triphenylethylene class of selective estrogen receptor modulators related to tamoxifen has shown activity against a wide range of medically important human pathogens, including bacteria, fungi, parasites, and viruses. Because it has activity against many pathogens affecting people in resource-limited areas of the world, TAM and related drugs may be particularly useful. Here, we review the in vitro, in vivo, and mechanistic studies of the anti-infective activity of tamoxifen, toremifene, clomiphene, and their analogs. We also discuss the pharmacologic properties of this privileged scaffold and its potential utility in treating infectious diseases.

A Genome-Wide Screen of Deletion Mutants in the Filamentous Saccharomyces cerevisiae Background Identifies Ergosterol as a Direct Trigger of Macrophage Pyroptosis.

Phagocytic cells such as macrophages play an important role in the host defense mechanisms mounted in response to the common human fungal pathogen Candida albicansIn vitro, C. albicans triggers macrophage NLRP3-Casp1/11-mediated pyroptosis, an inflammatory programmed cell death pathway. Here, we provide evidence that Casp1/11-dependent pyroptosis occurs in the kidney of infected mice during the early stages of infection. We have also used a genome-wide screen of nonessential Σ1278b Saccharomyces cerevisiae genes to identify genes required for yeast-triggered macrophage pyroptosis. The set of genes identified by this screen was enriched for those with functions in lipid and sterol homeostasis and trafficking. These observations led us to discover that cell surface localization and/or total levels of ergosterol correlate with the ability of S. cerevisiae, C. albicans, and Cryptococcus neoformans to trigger pyroptosis. Since the mammalian sterol cholesterol triggers NLRP3-mediated pyroptosis, we hypothesized that ergosterol may also do so. Consistent with that hypothesis, ergosterol-containing liposomes but not ergosterol-free liposomes induce pyroptosis. Cell wall mannoproteins directly bind ergosterol, and we found that Dan1, an ergosterol receptor mannoprotein, as well as specific mannosyltransferases, is required for pyroptosis, suggesting that cell wall-associated ergosterol may mediate the process. Taken together, these data indicate that ergosterol, like mammalian cholesterol, plays a direct role in yeast-mediated pyroptosis.IMPORTANCE Innate immune cells such as macrophages are key components of the host response to the human fungal pathogen Candida albicans Macrophages undergo pyroptosis, an inflammatory, programmed cell death, in response to some species of pathogenic yeast. Prior to the work described in this report, yeast-triggered pyroptosis has been observed only in vitro; here, we show that pyroptosis occurs in the initial stages of murine kidney infection, suggesting that it plays an important role in the initial response of the innate immune system to invasive yeast infection. We also show that a key component of the fungal plasma membrane, ergosterol, directly triggers pyroptosis. Ergosterol is also present in the fungal cell wall, most likely associated with mannoproteins, and is increased in hyphal cells compared to yeast cells. Our data indicate that specific mannoproteins are required for pyroptosis. This is consistent with a potential mechanism whereby ergosterol present in the outer mannoprotein layer of the cell wall is accessible to the macrophage-mediated process. Taken together, our data provide the first evidence that ergosterol plays a direct role in the host-pathogen interactions of fungi.

The Celecoxib Derivative AR-12 Has Broad-Spectrum Antifungal Activity In Vitro and Improves the Activity of Fluconazole in a Murine Model of Cryptococcosis.

Only one new class of antifungal drugs has been introduced into clinical practice in the last 30 years, and thus the identification of small molecules with novel mechanisms of action is an important goal of current anti-infective research. Here, we describe the characterization of the spectrum of in vitro activity and in vivo activity of AR-12, a celecoxib derivative which has been tested in a phase I clinical trial as an anticancer agent. AR-12 inhibits fungal acetyl coenzyme A (acetyl-CoA) synthetase in vitro and is fungicidal at concentrations similar to those achieved in human plasma. AR-12 has a broad spectrum of activity, including activity against yeasts (e.g., Candida albicans, non-albicans Candida spp., Cryptococcus neoformans), molds (e.g., Fusarium, Mucor), and dimorphic fungi (Blastomyces, Histoplasma, and Coccidioides) with MICs of 2 to 4 µg/ml. AR-12 is also active against azole- and echinocandin-resistant Candida isolates, and subinhibitory AR-12 concentrations increase the susceptibility of fluconazole- and echinocandin-resistant Candida isolates. Finally, AR-12 also increases the activity of fluconazole in a murine model of cryptococcosis. Taken together, these data indicate that AR-12 represents a promising class of small molecules with broad-spectrum antifungal activity.

Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase.

AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target.

Structure-activity relationships for the antifungal activity of selective estrogen receptor antagonists related to tamoxifen.

Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM) tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1) the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2) an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3) electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

Repurposing the antihistamine terfenadine for antimicrobial activity against Staphylococcus aureus.

Staphylococcus aureus is a rapidly growing health threat in the U.S., with resistance to several commonly prescribed treatments. A high-throughput screen identified the antihistamine terfenadine to possess, previously unreported, antimicrobial activity against S. aureus and other Gram-positive bacteria. In an effort to repurpose this drug, structure-activity relationship studies yielded 84 terfenadine-based analogues with several modifications providing increased activity versus S. aureus and other bacterial pathogens, including Mycobacterium tuberculosis. Mechanism of action studies revealed these compounds to exert their antibacterial effects, at least in part, through inhibition of the bacterial type II topoisomerases. This scaffold suffers from hERG liabilities which were not remedied through this round of optimization; however, given the overall improvement in activity of the set, terfenadine-based analogues provide a novel structural class of antimicrobial compounds with potential for further characterization as part of the continuing process to meet the current need for new antibiotics.

Efg1 directly regulates ACE2 expression to mediate cross talk between the cAMP/PKA and RAM pathways during Candida albicans morphogenesis.

The cyclic AMP/protein kinase A (cAMP/PKA) and regulation of Ace2 and morphogenesis (RAM) pathways are important regulators of the yeast-to-hypha transition in Candida albicans that interact genetically during this process. To further understand this interaction, we have characterized the expression of ACE2 during morphogenesis. In normoxic, planktonic conditions, ACE2 expression is very low in stationary-phase cells at both the mRNA and protein levels. Upon shifting to Spider medium, ACE2/Ace2p levels increase. Although Ace2 is not absolutely required for hypha formation, ace2Δ/Δ mutants show delayed hypha formation in Spider medium (but not others) and morphological changes to the hyphal tip and lateral yeast. We also show that Efg1 directly binds the promoter of Ace2 in stationary phase, and ACE2 levels are increased in strains lacking Efg1 and the protein kinase A proteins Tpk1 and Tpk2, indicating that the PKA pathway directly regulates ACE2 expression. ACE2 expression is positively regulated by Tec1 and Brg1, which bind the promoters of ACE2 in hyphal cells but not in the yeast phase. Under embedded conditions, Efg1 is dispensable for filamentation and Ace2 is required. We have found that ACE2 expression is much higher in embedded cells than in planktonic cells, providing a potential rationale for this observation. Taken together, our observations indicate that the PKA pathway directly regulates the RAM pathway under specific conditions and are consistent with a model where the two pathways carry out similar functions that depend on the specific environmental context.

Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF hand proteins and synergize with fluconazole in vivo.

UNLABELLED: Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene. Tamoxifen and toremifene are fungicidal and synergize with fluconazole and amphotericin B in vitro. In a mouse model of disseminated cryptococcosis, tamoxifen at concentrations achievable in humans combines with fluconazole to decrease brain burden by ~1 log10. In addition, these drugs inhibit the growth of Cryptococcus neoformans within macrophages, a niche not accessible by current antifungal drugs. Toremifene and tamoxifen directly bind to the essential EF hand protein calmodulin, as determined by thermal shift assays with purified C. neoformans calmodulin (Cam1), prevent Cam1 from binding to its well-characterized substrate calcineurin (Cna1), and block Cna1 activation. In whole cells, toremifene and tamoxifen block the calcineurin-dependent nuclear localization of the transcription factor Crz1. A large-scale chemical genetic screen with a library of C. neoformans deletion mutants identified a second EF hand-containing protein, which we have named calmodulin-like protein 1 (CNAG_05655), as a potential target, and further analysis showed that toremifene directly binds Cml1 and modulates its ability to bind and activate Cna1. Importantly, tamoxifen analogs (idoxifene and methylene-idoxifene) with increased calmodulin antagonism display improved anti-cryptococcal activity, indicating that calmodulin inhibition can be used to guide a systematic optimization of the anti-cryptococcal activity of the triphenylethylene scaffold. IMPORTANCE: Worldwide, cryptococcosis affects approximately 1 million people annually and kills more HIV/AIDS patients per year than tuberculosis. The gold standard therapy for cryptococcosis is amphotericin B plus 5-flucytosine, but this regimen is not readily available in regions where resources are limited and where the burden of disease is highest. Herein, we show that molecules related to the breast cancer drug tamoxifen are fungicidal for Cryptococcus and display a number of pharmacological properties desirable for an anti-cryptococcal drug, including synergistic fungicidal activity with fluconazole in vitro and in vivo, oral bioavailability, and activity within macrophages. We have also demonstrated that this class of molecules targets calmodulin as part of their mechanism of action and that tamoxifen analogs with increased calmodulin antagonism have improved anti-cryptococcal activity. Taken together, these results indicate that tamoxifen is a pharmacologically attractive scaffold for the development of new anti-cryptococcal drugs and provide a mechanistic basis for its further optimization.

Candida albicans triggers NLRP3-mediated pyroptosis in macrophages.

Pyroptosis is an inflammasome-mediated programmed cell death pathway triggered in macrophages by a variety of stimuli, including intracellular bacterial pathogens. Activation of pyroptosis leads to the secretion of interleukin-1ß (IL-1ß) and pore-mediated cell lysis. Although not considered an intracellular pathogen, Candida albicans is able to kill and, thereby, escape from macrophages. Here, we show that C. albicans-infected bone marrow-derived macrophages (BMDM) and murine J774 macrophages undergo pyroptotic cell death that is suppressed by glycine and pharmacologic inhibition of caspase-1. Infection of BMDM harvested from mice lacking components of the inflammasome revealed that pyroptosis was dependent on caspase-1, ASC, and NLRP3 and independent of NLRC4. In contrast to its role during intracellular bacterial infection, pyroptosis does not restrict C. albicans replication. Nonfilamentous Candida spp. did not trigger pyroptosis, while Candida krusei, which forms pseudohyphae in macrophages, triggered much lower levels than did C. albicans. Interestingly, a Saccharomyces cerevisiae strain from the filamentous background Σ1278 also triggered low, but significant, levels of pyroptosis. We have found that deletion of the transcription factor UPC2 decreases pyroptosis but has little effect on filamentation in the macrophage. In addition, a gain-of-function mutant of UPC2 induces higher levels of pyroptosis than does a matched control strain. Taken together, these data are most consistent with a model in which filamentation is necessary but not sufficient to trigger NLRP3 inflammasome-mediated pyroptosis. This is the first example of a fungal pathogen triggering pyroptosis and indicates that C. albicans-mediated macrophage damage is not solely due to hypha-induced physical disruption of cellular integrity.

Cryptococcus neoformans phosphoinositide-dependent kinase 1 (PDK1) ortholog is required for stress tolerance and survival in murine phagocytes.

Cryptococcus neoformans PKH2-01 and PKH2-02 are orthologous to mammalian PDK1 kinase genes. Although orthologs of these kinases have been extensively studied in S. cerevisiae, little is known about their function in pathogenic fungi. In this study, we show that PKH2-02 but not PKH2-01 is required for C. neoformans to tolerate cell wall, oxidative, nitrosative, and antifungal drug stress. Deletion of PKH2-02 leads to decreased basal levels of Pkc1 activity and, consequently, reduced activation of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway in response to cell wall, oxidative, and nitrosative stress. PKH2-02 function also is required for tolerance of fluconazole and amphotericin B, two important drugs for the treatment of cryptococcosis. Furthermore, OSU-03012, an inhibitor of human PDK1, is synergistic and fungicidal in combination with fluconazole. Using a Galleria mellonella model of low-temperature cryptococcosis, we found that PKH2-02 is also required for virulence in a temperature-independent manner. Consistent with the hypersensitivity of the pkh2-02Δ mutant to oxidative and nitrosative stress, this mutant shows decreased survival in murine phagocytes compared to that of wild-type (WT) cells. In addition, we show that deletion of PKH2-02 affects the interaction between C. neoformans and phagocytes by decreasing its ability to suppress production of tumor necrosis factor alpha (TNF-α) and reactive oxygen species. Taken together, our studies demonstrate that Pkh2-02-mediated signaling in C. neoformans is crucial for stress tolerance, host-pathogen interactions, and both temperature-dependent and -independent virulence.