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

Abstract

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.

Keywords: Candida albicans; inflammasome; pyroptosis.

FIG 1

Caspase-1/11-dependent cell death occurs in the initial stages of C. albicans infection. WT and congenic casp1/11^−/− mice were infected with C. albicans SC5314 by tail vein infection. Animals were sacrificed at 24 (A) and 48 (B) h postinfection, and kidneys were harvested for histology and fungal burden. Sections from kidneys with similar fungal burdens in the two mouse backgrounds were stained with PAS stain and by TUNEL. TUNEL staining at 24 h is predominantly caspase 1/11 dependent, while caspase-1/11-independent TUNEL staining is observed at 48 h.

FIG 2

Genome-wide screen of S. cerevisiae Σ1278 background nonessential deletion mutant collection for pyroptosis-deficient filamentous strains. (A) Screening strategy and funnel. (B) Representative raw data with upper and lower cutoffs (±2 SDs) depicted as dashed lines. Two illustrative hits are indicated by colored diamonds. (C) Representative single strain confirmation of decreased LDH release from J774 murine macrophages. Percent LDH release is relative to chemically lysed cells. Bars indicate means, and error bars are standard deviations for two to three biological replicates with triplicate technical replicates. (D) Venn diagram of gene set members with roles in polyene resistance, effects on vacuole morphology, functions in lipid droplet homeostasis, and consensus motifs for Upc2, a transcription factor involved in ergosterol biosynthesis and hypoxic response.

FIG 3

Disruption of yeast ergosterol homeostasis decreases pyroptosis. (A) Cryptococcus neoformans cap59Δ acapsular mutant was treated with the indicated concentrations of fluconazole overnight in YPD. The yeast cells were harvested and cocultured with J774 cells for 24 h prior to assay for cell lysis using LDH release. The bars indicate means, and error bars indicate standard deviations for two to three independent experiments performed in triplicate. Treated and untreated groups were analyzed using ordinary one-way ANOVA. P < 0.05 was considered statistically significant. NS, not significant. (B) WT, dan1Δ, and vps1Δ S. cerevisiae strains harboring plasmids expressing the sterol-binding domain–GFP fusion protein were exposed to J774 cells. Nonphagocytosed cells were removed by washing, and the macrophages were imaged by confocal microscopy using both DIC optics and fluorescence channels. Arrows indicate the circumferential localization of the reporter to the plasma membrane in WT cells, while arrowheads indicate exclusively cytoplasmic localization for the dan1Δ mutant. (C) WT and dan1Δ cells containing either empty vector or MSB2 expressed from a 2μ plasmid were exposed to macrophages. Percent LDH release is relative to chemically lysed cells. Bars indicate means, and error bars are standard deviations for two to three biological replicates with triplicate technical replicates. (D) The dan1Δ strain containing both the ergosterol reporter and pMSB2 was phagocytosed by J774 macrophages and analyzed as described for panel A.

FIG 4

Specific O-mannosyltransferase isoforms are required for C. albicans-induced pyroptosis. (A and B) J774 cells were exposed to C. albicans MSB2 and msb2ΔΔ strains (A) and the indicated mannosyltransferase PMT mutants (B) before being processed for LDH release. The bars for both panels indicate means, and error bars are the standard deviations. Asterisks indicate statistically significant differences between strains by two-sided, unpaired Student’s t test (A) or one-way ANOVA (B) with the limit of significance set at P < 0.05. (C) Gram-stained images of PMT4 complemented strain and pmt4ΔΔ mutants undergoing filamentation within J774 cells.

FIG 5

Ergosterol-containing liposomes trigger J774 cell pyroptosis. J774 cells were exposed to liposomes containing either nonsterol lipid or ergosterol. LDH release was measured and is expressed relative to chemically induced lysis. Bars indicate means, and error bars are standard deviations for two to three biological replicates with triplicate technical replicates. The differences between control and ergosterol-containing liposomes were significant (P < 0.05, one-way ANOVA) at all concentrations.