Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM.

Invasive fungal infections have significantly increased in the last few decades. Three classes of drugs are commonly used to treat these infections: polyenes, azoles and echinocandins. Unfortunately each of these drugs has drawbacks; polyenes are toxic, resistance against azoles is emerging and echinocandins have narrow spectrum of activity. Thus, the development of new antifungals is urgently needed. In this context, fungal sphingolipids have emerged as a potential target for new antifungals, because their biosynthesis in fungi is structurally different than in mammals. Besides, some fungal sphingolipids play an important role in the regulation of virulence in a variety of fungi. This review aims to highlight the diverse strategies that could be used to block the synthesis or/and function of fungal sphingolipids.

species cause invasive infections in humans. Lipids play an important role in the progression of these infections. Independent studies done by our group and others provide some detail about the functions of these lipids in infections. However, the pathways of biosynthesis and the metabolism of these lipids are not completely understood. To thoroughly understand the physiological role of these lipids, a proper structure and composition analysis of lipids is demanded. In this study, a detailed spectroscopic analysis of lipid extracts from and strains is presented. Sphingolipid profiling by LC-ESI-MS/MS was used to analyze sphingosine, dihydrosphingosine, sphingosine-1-phosphate, dihydrosphingosine-1-phosphate, ceramide, dihydroceramide, glucosylceramide, phytosphingosine, phytosphingosine-1-phosphate, phytoceramide, α-hydroxy phytoceramide, and inositolphosphorylceramide species. A total of 13 sterol species were identified using GC-MS, where ergosterol is the most abundant species. TheP-NMR-based phospholipid analysis identified phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidyl-,-dimethylethanolamine, phosphatidyl--monomethylethanolamine, phosphatidylglycerol, phosphatidic acid, and lysophosphatidylethanolamine. A comparison of lipid profiles among different strains illustrates a marked change in the metabolic flux of these organisms, especially sphingolipid metabolism. These data improve our understanding of the structure, biosynthesis, and metabolism of common lipid groups of and should be useful while studying their functional significance and designing therapeutic interventions.

By employing electrospray ionization tandem mass spectrometry (ESI-MS/MS), the phospholipidomes of eight hemiascomycetous human pathogenic Candida species have been characterized. Over 200 phospholipid molecular species were identified and quantified. There were no large differences among Candida species in phosphoglyceride class composition; however, differences in phosphoglycerides components (i.e., fatty acyl chains) were identified. In contrast, differences in sphingolipid class composition as well as in molecular species were quite evident. The phospholipid compositions of C. albicans, C. glabrata, C. parapsilosis, C. kefyr, C. tropicalis, C. dubliniensis, C. krusei, and C. utilis could be further discriminated by principal component analysis. Notwithstanding that a single strain of each species was analyzed, our data do point to a typical molecular species imprint of Candida strains.

Sphingolipids form of a unique and complex group of bioactive lipids in fungi. Structurally, sphingolipids of fungi are quite diverse with unique differences in the sphingoid backbone, amide linked fatty acyl chain and the polar head group. Two of the most studied and conserved sphingolipid classes in fungi are the glucosyl- or galactosyl-ceramides and the phosphorylinositol containing phytoceramides. Comprehensive structural characterization and quantification of these lipids is largely based on advanced analytical mass spectrometry based lipidomic methods. While separation of complex lipid mixtures is achieved through high performance liquid chromatography, the soft – electrospray ionization tandem mass spectrometry allows a high sensitivity and selectivity of detection. Herein, we present an overview of lipid extraction, chromatographic separation and mass spectrometry employed in qualitative and quantitative sphingolipidomics in fungi.

The action of multidrug efflux pumps in MDR (multidrug resistance) acquisition has been proposed to partially depend on the transport of physiological substrates which may indirectly affect drug partition and transport across cell membranes. In the present study, the PDR18 gene [ORF (open reading frame) YNR070w], encoding a putative PDR (pleiotropic drug resistance) transporter of the ATP-binding cassette superfamily, was found to mediate plasma membrane sterol incorporation in yeast. The physiological role of Pdr18 is demonstrated to affect plasma membrane potential and is proposed to underlie its action as a MDR determinant, conferring resistance to the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid). The action of Pdr18 in yeast tolerance to 2,4-D, which was found to contribute to reduce [14C]2,4-D intracellular accumulation, may be indirect, given the observation that 2,4-D exposure deeply affects the sterol plasma membrane composition, this effect being much stronger in a Δpdr18 background. PDR18 activation under 2,4-D stress is regulated by the transcription factors Nrg1, controlling carbon source availability and the stress response, and, less significantly, Yap1, involved in oxidative stress and MDR, and Pdr3, a key regulator of the yeast PDR network, consistent with a broad role in stress defence. Taken together, the results of the present study suggest that Pdr18 plays a role in plasma membrane sterol incorporation, this physiological trait contributing to an MDR phenotype.

SUMMARY Cryptococcus neoformans (C. neoformans) is estimated to cause about 220,000 new cases every year in patients with AIDS, despite advances in antifungal treatments. C. neoformans possesses a remarkable ability to disseminate through an immunocompromised host, making treatment difficult. Here, we examine the mechanism of survival of C. neoformans under varying host conditions and find a role for ceramide synthase in C. neoformans virulence. This study also provides a detailed lipidomics resource for the fungal lipid research community in addition to discovering a potential target for antifungal therapy.

C8-desaturated and C9-methylated glucosylceramide (GlcCer) is a fungal-specific sphingolipid that plays an important role in the growth and virulence of many species. In this work, we investigated the contribution of Aspergillus nidulans sphingolipid Δ8-desaturase (SdeA), sphingolipid C9-methyltransferases (SmtA/SmtB) and glucosylceramide synthase (GcsA) to fungal phenotypes, sensitivity to Psd1 defensin and Galleria mellonella virulence. We showed that ΔsdeA accumulated C8-saturated and unmethylated GlcCer, while gcsA deletion impaired GlcCer synthesis. Although increased levels of unmethylated GlcCer were observed in smtA and smtB mutants, ΔsmtA and wild-type cells showed a similar 9,Me-GlcCer content, reduced by 50 % in the smtB disruptant. The compromised 9,Me-GlcCer production in the ΔsmtB strain was not accompanied by reduced filamentation or defects in cell polarity. When combined with the smtA deletion, smtB repression significantly increased unmethylated GlcCer levels and compromised filamentous growth. Furthermore, sdeA and gcsA mutants displayed growth defects and raft mislocalization, which were accompanied by reduced neutral lipids levels and attenuated G. mellonella virulence in the ΔgcsA strain. Finally, ΔsdeA and ΔgcsA showed increased resistance to Psd1, suggesting that GlcCer synthesis and fungal sphingoid base structure specificities are relevant not only to differentiation but also to proper recognition by this antifungal defensin.