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.

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.

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.

Identification of Antifungal Compounds against Multidrug-Resistant Candida auris Utilizing a High-Throughput Drug-Repurposing Screen.

Candida auris is an emerging fatal fungal infection that has resulted in several outbreaks in hospitals and care facilities. Current treatment options are limited by the development of drug resistance. Identification of new pharmaceuticals to combat these drug-resistant infections will thus be required to overcome this unmet medical need. We have established a bioluminescent ATP-based assay to identify new compounds and potential drug combinations showing effective growth inhibition against multiple strains of multidrug-resistant Candida auris The assay is robust and suitable for assessing large compound collections by high-throughput screening (HTS). Utilizing this assay, we conducted a screen of 4,314 approved drugs and pharmacologically active compounds that yielded 25 compounds, including 6 novel anti-Candida auris compounds and 13 sets of potential two-drug combinations. Among the drug combinations, the serine palmitoyltransferase inhibitor myriocin demonstrated a combinational effect with flucytosine against all tested isolates during screening. This combinational effect was confirmed in 13 clinical isolates of Candida auris.

The Future of Antifungal Drug Therapy: Novel Compounds and Targets.

Fungal infections are a universal problem and are routinely associated with high morbidity and mortality rates in immunocompromised patients. Existing therapies comprise five different classes of antifungal agents, four of which target the synthesis of ergosterol and cell wall glucans. However, the currently available antifungals have many limitations, including poor oral bioavailability, narrow therapeutic indices, and emerging drug resistance resulting from their use, thus making it essential to investigate the development of novel drugs which can overcome these limitations and add to the antifungal armamentarium. Advances have been made in antifungal drug discovery research and development over the past few years as evidenced by the presence of several new compounds currently in various stages of development. In the following minireview, we provide a comprehensive summary of compounds aimed at one or more novel molecular targets. We also briefly describe potential pathways relevant for fungal pathogenesis that can be considered for drug development in the near future.

Fungal sphingolipids: role in the regulation of virulence and potential as targets for future antifungal therapies.

INTRODUCTION: The antifungal therapy currently available includes three major classes of drugs: polyenes, azoles and echinocandins. However, the clinical use of these compounds faces several challenges: while polyenes are toxic to the host, antifungal resistance to azoles and echinocandins has been reported. AREAS COVERED: Fungal sphingolipids (SL) play a pivotal role in growth, morphogenesis and virulence. In addition, fungi possess unique enzymes involved in SL synthesis, leading to the production of lipids which are absent or differ structurally from the mammalian counterparts. In this review, we address the enzymatic reactions involved in the SL synthesis and their relevance to the fungal pathogenesis, highlighting their potential as targets for novel drugs and the inhibitors described so far. EXPERT OPINION: The pharmacological inhibition of fungal serine palmitoyltransferase depends on the development of specific drugs, as myriocin also targets the mammalian enzyme. Inhibitors of ceramide synthase might constitute potent antifungals, by depleting the pool of complex SL and leading to the accumulation of the toxic intermediates. Acylhydrazones and aureobasidin A, which inhibit GlcCer and IPC synthesis, are not toxic to the host and effectively treat invasive mycoses, emerging as promising new classes of antifungal drugs.