Biofilm-associated metabolism via ERG251 in Candida albicans.

Biofilm formation by the fungal pathogen Candida albicans is the basis for its ability to infect medical devices. The metabolic gene ERG251 has been identified as a target of biofilm transcriptional regulator Efg1, and here we report that ERG251 is required for biofilm formation but not conventional free-living planktonic growth. An erg251Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo catheter infection model. In both in vitro and in vivo biofilm contexts, cell number is reduced and hyphal length is limited. To determine whether the mutant defect is in growth or some other aspect of biofilm development, we examined planktonic cell features in a biofilm-like environment, which was approximated with sealed unshaken cultures. Under those conditions, the erg251Δ/Δ mutation causes defects in growth and hyphal extension. Overexpression in the erg251Δ/Δ mutant of the paralog ERG25, which is normally expressed more weakly than ERG251, partially improves biofilm formation and biofilm hyphal content, as well as growth and hyphal extension in a biofilm-like environment. GC-MS analysis shows that the erg251Δ/Δ mutation causes a defect in ergosterol accumulation when cells are cultivated under biofilm-like conditions, but not under conventional planktonic conditions. Overexpression of ERG25 in the erg251Δ/Δ mutant causes some increase in ergosterol levels. Finally, the hypersensitivity of efg1Δ/Δ mutants to the ergosterol inhibitor fluconazole is reversed by ERG251 overexpression, arguing that reduced ERG251 expression contributes to this efg1Δ/Δ phenotype. Our results indicate that ERG251 is required for biofilm formation because its high expression levels are necessary for ergosterol synthesis in a biofilm-like environment.

Combination therapy of itraconazole and an acylhydrazone derivative (D13) for the treatment of sporotrichosis in cats.

Acylhydrazone (AH) derivatives represent a novel category of anti-fungal medications that exhibit potent activity against Sporothrix sp., both in vitro and in a murine model of sporotrichosis. In this study, we demonstrated the anti-fungal efficacy of the AH derivative D13 [4-bromo-N'-(3,5-dibromo-2-hydroxybenzylidene)-benzohydrazide] against both planktonic cells and biofilms formed by Sporothrix brasiliensis. In a clinical study, the effect of D13 was then tested in combination with itraconazole (ITC), with or without potassium iodide, in 10 cats with sporotrichosis refractory to the treatment of standard of care with ITC. Improvement or total clinical cure was achieved in five cases after 12 weeks of treatment. Minimal abnormal laboratory findings, e.g., elevation of alanine aminotransferase, were observed in four cats during the combination treatment and returned to normal level within a week after the treatment was ended. Although highly encouraging, a larger and randomized controlled study is required to evaluate the effectiveness and the safety of this new and exciting drug combination using ITC and D13 for the treatment of feline sporotrichosis. IMPORTANCE: This paper reports the first veterinary clinical study of an acylhydrazone anti-fungal (D13) combined with itraconazole against a dimorphic fungal infection, sporotrichosis, which is highly endemic in South America in animals and humans. Overall, the results show that the combination treatment was efficacious in ~50% of the infected animals. In addition, D13 was well tolerated during the course of the study. Thus, these results warrant the continuation of the research and development of this new class of anti-fungals.

SAR study of N'-(Salicylidene)heteroarenecarbohydrazides as promising antifungal agents.

Clinically available antifungal drugs have therapeutic limitations due to toxicity, narrow spectrum of activity, and intrinsic or acquired drug resistance. Thus, there is an urgent need for new broad-spectrum antifungal agents with low toxicity and a novel mechanism of action. In this context, we have successfully identified several highly promising lead compounds, i.e., aromatic N'-(salicylidene)carbohydrazides, exhibiting excellent antifungal activities against Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus and several other fungi both in vitro and in vivo. Building upon these highly promising results, 71 novel N'-(salicylidene)heteroarenecarbohydrazides 5 were designed, synthesized and their antifungal activities examined against fungi. Based on the SAR study, four highly promising lead compounds, i.e., 5.6a, 5.6b, 5.7b and 5.13a were identified, which exhibited excellent potency against C. neoformans, C. albicans and A. fumigatus, and displayed impressive time-kill profiles against C. neoformans with exceptionally high selectivity indices (SI ≥ 500). These four lead compounds also showed synergy with clinical antifungal drugs, fluconazole, caspofungin (CS) and amphotericin B against C. neoformans. For the SAR study, we also employed quantitative structure-activity relationship (QSAR) analysis by taking advantage of the accumulated data on a large number of aromatic and heteroaromatic N'-(salicylidene)carbohydrazides, which successfully led to rational design and selection of promising compounds for chemical synthesis and biological evaluation.

The biological functions of sphingolipids in plant pathogenic fungi.

Sphingolipids are critically significant in a range of biological processes in animals, plants, and fungi. In mammalian cells, they serve as vital components of the plasma membrane (PM) in maintaining its structure, tension, and fluidity. They also play a key role in a wide variety of biological processes, such as intracellular signal transduction, cell polarization, differentiation, and migration. In plants, sphingolipids are important for cell development and for cell response to environmental stresses. In pathogenic fungi, sphingolipids are crucial for the initiation and the development of infection processes afflicting humans. However, our knowledge on the metabolism and function of the sphingolipid metabolic pathway of pathogenic fungi affecting plants is still very limited. In this review, we discuss recent developments on sphingolipid pathways of plant pathogenic fungi, highlighting their uniqueness and similarity with plants and animals. In addition, we discuss recent advances in the research and development of fungal-targeted inhibitors of the sphingolipid pathway, to gain insights on how we can better control the infection process occurring in plants to prevent or/and to treat fungal infections in crops.

Role of sphingolipids in the host-pathogen interaction.

Fungal pathogens have been under the spotlight as their expanding geographic range combined with their potential harm to vulnerable populations turns them into increasingly threats to public health. Therefore, it is ultimately important to unveil the mechanisms associated with their infection process for further new treatment discovery. With this purpose, sphingolipid-based research has gained attention over the last years as these molecules have key properties that can regulate fungal pathogenicity. Here we discuss some of these properties as well as their role in fungal diseases, focusing on the subgroup of glycosphingolipids, as they represent promising molecules for drug discovery and for the development of fungal vaccines.

The Heat Shock Transcription Factor HsfA Plays a Role in Membrane Lipids Biosynthesis Connecting Thermotolerance and Unsaturated Fatty Acid Metabolism in Aspergillus fumigatus.

Thermotolerance is a remarkable virulence attribute of Aspergillus fumigatus, but the consequences of heat shock (HS) to the cell membrane of this fungus are unknown, although this structure is one of the first to detect changes in ambient temperature that imposes on the cell a prompt adaptative response. Under high-temperature stress, fungi trigger the HS response controlled by heat shock transcription factors, such as HsfA, which regulates the expression of heat shock proteins. In yeast, smaller amounts of phospholipids with unsaturated fatty acid (FA) chains are synthesized in response to HS, directly affecting plasma membrane composition. The addition of double bonds in saturated FA is catalyzed by Δ9-fatty acid desaturases, whose expression is temperature-modulated. However, the relationship between HS and saturated/unsaturated FA balance in membrane lipids of A. fumigatus in response to HS has not been investigated. Here, we found that HsfA responds to plasma membrane stress and has a role in sphingolipid and phospholipid unsaturated biosynthesis. In addition, we studied the A. fumigatus Δ9-fatty acid desaturase sdeA and discovered that this gene is essential and required for unsaturated FA biosynthesis, although it did not directly affect the total levels of phospholipids and sphingolipids. sdeA depletion significantly sensitizes mature A. fumigatus biofilms to caspofungin. Also, we demonstrate that hsfA controls sdeA expression, while SdeA and Hsp90 physically interact. Our results suggest that HsfA is required for the adaptation of the fungal plasma membrane to HS and point out a sharp relationship between thermotolerance and FA metabolism in A. fumigatus. IMPORTANCE Aspergillus fumigatus causes invasive pulmonary aspergillosis, a life-threatening infection accounting for high mortality rates in immunocompromised patients. The ability of this organism to grow at elevated temperatures is long recognized as an essential attribute for this mold to cause disease. A. fumigatus responds to heat stress by activating heat shock transcription factors and chaperones to orchestrate cellular responses that protect the fungus against damage caused by heat. Concomitantly, the cell membrane must adapt to heat and maintain physical and chemical properties such as the balance between saturated/unsaturated fatty acids. However, how A. fumigatus connects these two physiological responses is unclear. Here, we explain that HsfA affects the synthesis of complex membrane lipids such as phospholipids and sphingolipids and controls the enzyme SdeA, which produces monounsaturated fatty acids, raw material for membrane lipids. These findings suggest that forced dysregulation of saturated/unsaturated fatty acid balance might represent novel strategies for antifungal therapy.

Targeting Sterylglucosidase A to Treat Aspergillus fumigatus Infections.

Invasive fungal infections are a leading cause of death in immunocompromised patients. Current therapies have several limitations, and innovative antifungal agents are critically needed. Previously, we identified the fungus-specific enzyme sterylglucosidase as essential for pathogenesis and virulence of Cryptococcus neoformans and Aspergillus fumigatus (Af) in murine models of mycoses. Here, we developed Af sterylglucosidase A (SglA) as a therapeutic target. We identified two selective inhibitors of SglA with distinct chemical scaffolds that bind in the active site of SglA. Both inhibitors induce sterylglucoside accumulation and delay filamentation in Af and increase survival in a murine model of pulmonary aspergillosis. Structure-activity relationship (SAR) studies identified a more potent derivative that enhances both in vitro phenotypes and in vivo survival. These findings support sterylglucosidase inhibition as a promising antifungal approach with broad-spectrum potential. IMPORTANCE Invasive fungal infections are a leading cause of death in immunocompromised patients. Aspergillus fumigatus is a fungus ubiquitously found in the environment that, upon inhalation, causes both acute and chronic illnesses in at-risk individuals. A. fumigatus is recognized as one of the critical fungal pathogens for which a substantive treatment breakthrough is urgently needed. Here, we studied a fungus-specific enzyme, sterylglucosidase A (SglA), as a therapeutic target. We identified selective inhibitors of SglA that induce accumulation of sterylglucosides and delay filamentation in A. fumigatus and increase survival in a murine model of pulmonary aspergillosis. We determined the structure of SglA, predicted the binding poses of these inhibitors through docking analysis, and identified a more efficacious derivative with a limited SAR study. These results open several exciting avenues for the research and development of a new class of antifungal agents targeting sterylglucosidases.

Host populations, challenges, and commercialization of cryptococcal vaccines.

Vaccines are one of the most effective public health tools to prevent and manage infectious diseases. Since the first clinical use of vaccines in the late 18th century, many vaccines have been successfully developed to combat bacterial and viral infections, including the most recent Coronavirus Disease 2019 (COVID-19) pandemic. However, there remains no vaccine that is clinically available to treat or prevent invasive fungal diseases, including cryptococcal meningoencephalitis. This fungal disease is uniformly fatal without treatment and has a global mortality rate of over 70%. Despite a dire need for an effective cryptococcal vaccine, there are many scientific and economic challenges to overcome prior to making it a reality. Here, we discuss some of these challenges as well as steps that the community is taking for commercialization of effective cryptococcal vaccines.

Sterylglucosides in Fungi.

Sterylglucosides (SGs) are sterol conjugates widely distributed in nature. Although their universal presence in all living organisms suggests the importance of this kind of glycolipids, they are yet poorly understood. The glycosylation of sterols confers a more hydrophilic character, modifying biophysical properties of cell membranes and altering immunogenicity of the cells. In fungi, SGs regulate different cell pathways to help overcome oxygen and pH challenges, as well as help to accomplish cell recycling and other membrane functions. At the same time, the level of these lipids is highly controlled, especially in wild-type fungi. In addition, modulating SGs metabolism is becoming a novel tool for vaccine and antifungal development. In the present review, we bring together multiple observations to emphasize the underestimated importance of SGs for fungal cell functions.

Vaccine protection by Cryptococcus neoformans Δsgl1 is mediated by γδ T cells via TLR2 signaling.

We previously reported that administration of Cryptococcus neoformans Δsgl1 mutant vaccine, accumulating sterylglucosides (SGs) and having normal capsule (GXM), protects mice from a subsequent infection even during CD4+ T cells deficiency, a condition commonly associated with cryptococcosis. Here, we studied the immune mechanism that confers host protection during CD4+T deficiency. Mice receiving Δsgl1 vaccine produce IFNγ and IL-17A during CD4+ T (or CD8+ T) deficiency, and protection was lost when either cytokine was neutralized. IFNγ and/or IL-17A are produced by γδ T cells, and mice lacking these cells are no longer protected. Interestingly, ex vivo γδ T cells are highly stimulated in producing IFNγ and/or IL-17A by Δsgl1 vaccine, but this production was significantly decreased when cells were incubated with C. neoformans Δcap59/Δsgl1 mutant, accumulating SGs but lacking GXM. GXM modulates toll-like receptors (TLRs), including TLR2. Importantly, neither Δsgl1 nor Δcap59/Δsgl1 stimulate IFNγ or IL-17A production by ex vivo γδ T cells from TLR2-/- mice. Finally, TLR2-/- animals do not produce IL-17A in response to Δsgl1 vaccine and were no longer protected from WT challenge. Our results suggest that SGs may act as adjuvants for GXM to stimulate γδ T cells in producing IFNγ and IL-17A via TLR2, a mechanism that is still preserved upon CD4+ T deficiency.

Vaccination with Live or Heat-Killed Aspergillus fumigatus ΔsglA Conidia Fully Protects Immunocompromised Mice from Invasive Aspergillosis.

Aspergillus fumigatus causes invasive aspergillosis (IA) in immunocompromised patients, resulting in high mortality rates. Currently, no vaccine formulations to promote immune protection in at-risk individuals have been developed. In this work, we deleted the sterylglucosidase-encoding gene, sglA, in Aspergillus fumigatus and investigated its role in fungal virulence and host vaccine protection. The ΔsglA mutant accumulated sterylglucosides (SGs), newly studied immunomodulatory glycolipids, and exhibited reduced hyphal growth and altered compositions of cell wall polysaccharides. Interestingly, the ΔsglA mutant was avirulent in two murine models of IA and was fully eliminated from the lungs. Both corticosteroid-induced immunosuppressed and cyclophosphamide-induced leukopenic mice vaccinated with live or heat-killed ΔsglA conidia were fully protected against a lethal wild-type A. fumigatus challenge. These results highlight the potential of SG-accumulating strains as safe and promising vaccine formulations against invasive fungal infections. IMPORTANCE Infections by Aspergillus fumigatus occur by the inhalation of environmental fungal spores called conidia. We found that live mutant conidia accumulating glycolipids named sterylglucosides are not able to cause disease when injected into the lung. Interestingly, these animals are now protected against a secondary challenge with live wild-type conidia. Remarkably, protection against a secondary challenge persists even with vaccination with heat-killed mutant conidia. These results will significantly advance the field of the research and development of a safe fungal vaccine for protection against the environmental fungus A. fumigatus.

Microdomain Protein Nce102 Is a Local Sensor of Plasma Membrane Sphingolipid Balance.

Sphingolipids are essential building blocks of eukaryotic membranes and important signaling molecules that are regulated tightly in response to environmental and physiological inputs. While their biosynthetic pathway has been well-described, the mechanisms that facilitate the perception of sphingolipid levels at the plasma membrane remain to be uncovered. In Saccharomyces cerevisiae, the Nce102 protein has been proposed to function as a sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that the production of Nce102 increases following sphingolipid biosynthesis inhibition and that Nce102 is internalized when excess sphingolipid precursors are supplied. This finding suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. The physiological role of Nce102 in the regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of hydroxylated complex sphingolipids in response to heat stress in the nce102Δ deletion mutant. We also demonstrate that Nce102 behaves analogously in the widespread human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species. IMPORTANCE Microorganisms are challenged constantly by their rapidly changing environment. To survive, they have developed diverse mechanisms to quickly perceive stressful situations and adapt to them appropriately. The primary site of both stress sensing and adaptation is the plasma membrane. We identified the yeast protein Nce102 as a marker of local sphingolipid levels and fluidity in the plasma membrane. Nce102 is an important structural and functional component of the membrane compartment Can1 (MCC), a plasma membrane microdomain stabilized by a large cytosolic hemitubular protein scaffold, the eisosome. The MCC/eisosomes are widely conserved among fungi and unicellular algae. To determine if Nce102 carries out similar functions in other organisms, we analyzed the human fungal pathogen Candida albicans and found that Nce102 responds to sphingolipid levels also in this organism, which has potential applications for the development of novel therapeutic approaches. The presented study represents a valuable model for how organisms regulate plasma membrane sphingolipids.

Targeting fungal membrane homeostasis with imidazopyrazoindoles impairs azole resistance and biofilm formation.

Fungal infections cause more than 1.5 million deaths annually. With an increase in immune-deficient susceptible populations and the emergence of antifungal drug resistance, there is an urgent need for novel strategies to combat these life-threatening infections. Here, we use a combinatorial screening approach to identify an imidazopyrazoindole, NPD827, that synergizes with fluconazole against azole-sensitive and -resistant isolates of Candida albicans. NPD827 interacts with sterols, resulting in profound effects on fungal membrane homeostasis and induction of membrane-associated stress responses. The compound impairs virulence in a Caenorhabditis elegans model of candidiasis, blocks C. albicans filamentation in vitro, and prevents biofilm formation in a rat model of catheter infection by C. albicans. Collectively, this work identifies an imidazopyrazoindole scaffold with a non-protein-targeted mode of action that re-sensitizes the leading human fungal pathogen, C. albicans, to azole antifungals.

Three Models of Vaccination Strategies Against Cryptococcosis in Immunocompromised Hosts Using Heat-Killed Cryptococcus neoformans Δsgl1.

Vaccines are one of the greatest medical accomplishments to date, yet no fungal vaccines are currently available in humans mainly because opportunistic mycoses generally occur during immunodeficiencies necessary for vaccine protection. In previous studies, a live, attenuated Cryptococcus neoformans Δsgl1 mutant accumulating sterylglucosides was found to be avirulent and protected mice from a subsequent lethal infection even in absence of CD4+ T cells, a condition most associated with cryptococcosis (e.g., HIV). Here, we tested three strategies of vaccination against cryptococcosis. First, in our preventative model, protection was achieved even after a 3-fold increase of the vaccination window. Second, because live C. neoformans Δsgl1-vaccinated mice challenged more than once with WT strain had a significant decrease in lung fungal burden, we tested C. neoformans Δsgl1 as an immunotherapeutic. We found that therapeutic administrations of HK C. neoformans Δsgl1 post WT challenge significantly improves the lung fungal burden. Similarly, therapeutic administration of HK C. neoformans Δsgl1 post WT challenge resulted in 100% or 70% survival depending on the time of vaccine administration, suggesting that HK Δsgl1 is a robust immunotherapeutic option. Third, we investigated a novel model of vaccination in preventing reactivation from lung granuloma using C. neoformans Δgcs1. Remarkably, we show that administration of HK Δsgl1 prevents mice from reactivating Δgcs1 upon inducing immunosuppression with corticosteroids or by depleting CD4+ T cells. Our results suggest that HK Δsgl1 represents a clinically relevant, efficacious vaccine that confers robust host protection in three models of vaccination against cryptococcosis even during CD4-deficiency.

Extracellular Vesicles Regulate Biofilm Formation and Yeast-to-Hypha Differentiation in Candida albicans.

In this study, we investigated the influence of fungal extracellular vesicles (EVs) during biofilm formation and morphogenesis in Candida albicans. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that C. albicans EVs inhibited biofilm formation in vitro. By time-lapse microscopy and SEM, we showed that C. albicans EV treatment stopped filamentation and promoted pseudohyphae formation with multiple budding sites. The ability of C. albicans EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. C. albicans EVs from distinct strains inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 h. EVs from S. cerevisiae and H. capsulatum modestly reduced morphogenesis, and the effect was evident after 24 h of incubation. The inhibitory activity of C. albicans EVs on phase transition was promoted by a combination of lipid compounds, which were identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, C. albicans EVs were also able to reverse filamentation. Finally, C. albicans cells treated with C. albicans EVs for 24 h lost their capacity to penetrate agar and were avirulent when inoculated into Galleria mellonella. Our results indicate that fungal EVs can regulate yeast-to-hypha differentiation, thereby inhibiting biofilm formation and attenuating virulence. IMPORTANCE The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on biofilm formation and virulence. Morphogenesis is controlled by a diversity of stimuli, including osmotic stress, pH, starvation, presence of serum, and microbial components, among others. Apart from external inducers, C. albicans also produces autoregulatory substances. Farnesol and tyrosol are examples of quorum-sensing molecules (QSM) released by C. albicans to regulate yeast-to-hypha conversion. Here, we demonstrate that fungal EVs are messengers impacting biofilm formation, morphogenesis, and virulence in C. albicans. The major players exported in C. albicans EVs included sesquiterpenes, diterpenes, and fatty acids. The understanding of how C. albicans cells communicate to regulate physiology and pathogenesis can lead to novel therapeutic tools to combat candidiasis.

Cryptococcal Meningitis Reported With Fingolimod Treatment: Case Series.

BACKGROUND AND OBJECTIVES: To describe the characteristics of patients with MS reporting cryptococcal meningitis (CM) while treated with fingolimod. METHODS: The Novartis safety database was searched for cases with CM between January 26, 2006, and February 28, 2020. The reporting rate of CM was estimated based on the case reports received and exposure to fingolimod in the postmarketing setting during the relevant period. RESULTS: A total of 60 case reports of CM were identified, mostly from the United States. The median age was 48 years, and 51.8% were women. Most of the patients had recovered or were recovering at the time of final report. A fatal outcome occurred in 13 cases. During the study period, the rate of CM in patients with MS receiving fingolimod was estimated to be 8 per 100,000 patient-years (95% CI: 6.0; 10.0). The incidence of CM seemed to increase with duration of treatment; however, this relationship remains uncertain due to wide CIs and missing data. DISCUSSION: The causal relationship between fingolimod treatment and CM is not yet fully understood. The CM mortality rate in fingolimod-treated patients is similar to that reported in HIV-negative patients. Vigilance for signs and symptoms of CM in patients receiving fingolimod, particularly the new onset of headaches and altered mental status, is essential. Early diagnosis and treatment are critical to reducing CM-associated mortality.

Sphingolipid depletion suppresses UPR activation and promotes galactose hypersensitivity in yeast models of classic galactosemia.

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations on the GALT gene, which encodes the Leloir pathway enzyme galactose-1-phosphate uridyltransferase. Previous studies have shown that the endoplasmic reticulum unfolded protein response (UPR) is relevant to galactosemia, but the molecular mechanism behind the endoplasmic reticulum stress that triggers this response remains elusive. In the present work, we show that the activation of the UPR in yeast models of galactosemia does not depend on the binding of unfolded proteins to the ER stress sensor protein Ire1p since the protein domain responsible for unfolded protein binding to Ire1p is not necessary for UPR activation. Interestingly, myriocin - an inhibitor of the de novo sphingolipid synthesis pathway - inhibits UPR activation and causes galactose hypersensitivity in these models, indicating that myriocin-mediated sphingolipid depletion impairs yeast adaptation to galactose toxicity. Supporting the interpretation that the effects observed after myriocin treatment were due to a reduction in sphingolipid levels, the addition of phytosphingosine to the culture medium reverses all myriocin effects tested. Surprisingly, constitutively active UPR signaling did not prevent myriocin-induced galactose hypersensitivity suggesting multiple roles for sphingolipids in the adaptation of yeast cells to galactose toxicity. Therefore, we conclude that sphingolipid homeostasis has an important role in UPR activation and cellular adaptation in yeast models of galactosemia, highlighting the possible role of lipid metabolism in the pathophysiology of this disease.

Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores by aiding in clearance but can also potentially serve as a niche for their dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high-order plasma membrane domains enriched in cholesterol and sphingomyelin [SM]) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages' ability to phagocytose C. neoformans. We found that cholesterol or SM depletion resulted in significantly deficient immunoglobulin G (IgG)-mediated phagocytosis of fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency, whereas a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable SM (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis, whereas the raft-inhibiting conformation (cis-AzoSM) significantly but reversibly blunted phagocytosis. We observed that the effect on phagocytosis may be facilitated by Fcγ receptor (FcγR) function, whereby IgG immune complexes crosslink to FcγRIII, resulting in tyrosine phosphorylation of FcR γ-subunit (FcRγ), an important accessory protein in the FcγR signaling cascade. Correspondingly, cholesterol or SM depletion resulted in decreased FcRγ phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcRγ phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating FcγRIII-mediated phagocytosis of C. neoformans.

Structure and inhibition of Cryptococcus neoformans sterylglucosidase to develop antifungal agents.

Pathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3ß-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3ß-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1's substrate specificity and enable the rational design of antifungal agents targeting Sgl1.