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.

Filamentous and dimorphic fungi cause invasive mycoses associated with high mortality rates. Among the fungal determinants involved in the establishment of infection, glycosphingolipids (GSLs) have gained increased interest in the last few decades. GSLs are ubiquitous membrane components that have been isolated from both filamentous and dimorphic species and play a crucial role in polarized growth as well as hypha-to-yeast transition. In fungi, two major classes of GSLs are found: neutral and acidic GSLs. Neutral GSLs comprise glucosylceramide and galactosylceramide, which utilize Δ4-Δ8-9-methyl-sphingadienine as a sphingoid base, linked to a C16–18 fatty acid chain, forming ceramide, and to a sugar residue, such as glucose or galactose. In contrast, acidic GSLs include glycosylinositol phosphorylceramides (GIPCs), composed of phytosphingosine attached to a long or very long fatty acid chain (C18–26) and to diverse and complex glycan groups via an inositol-phosphate linker. GIPCs are absent in mammalian cells, while fungal glucosylceramide and galactosylceramide are present but diverge structurally from their counterparts. Therefore, these compounds and their biosynthetic pathways represent potential targets for the development of selective therapeutic strategies. In this minireview, we discuss the enzymatic steps involved in the production of fungal GSLs, analyze their structure, and address the role of the currently characterized genes in the biology and pathogenesis of filamentous and dimorphic fungi.

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 indexes, 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 review we provide a comprehensive summary of compounds aimed at novel molecular target(s). We also briefly describe potential pathways, relevant for fungal pathogenesis, that can be considered for drug development in the near future.

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. Identifying 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. Utilizing this assay, we conducted a screen of 4,314 approved drugs and pharmacologically active compounds which 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 deleterious effects of human-induced climate change have long been predicted. However, the imminent emergence and spread of new diseases, including fungal infections through the rise of thermotolerant strains, is still neglected, despite being a potential consequence of global warming. Thermotolerance is a remarkable virulence attribute of the mold Aspergillus fumigatus. Under high-temperature stress, opportunistic fungal pathogens deploy an adaptive mechanism known as heat shock (HS) response controlled by heat shock transcription factors (HSFs). In eukaryotes, HSFs regulate the expression of several heat shock proteins (HSPs), such as the chaperone Hsp90, which is part of the cellular program for heat adaptation and a direct target of HSFs. We recently observed that the perturbation in cell wall integrity (CWI) causes concomitant susceptibility to elevated temperatures in A. fumigatus, although the mechanisms underpinning the HS response and CWI cross talking are not elucidated. Here, we aim at further deciphering the interplay between HS and CWI. Our results show that cell wall ultrastructure is severely modified when A. fumigatus is exposed to HS. We identify the transcription factor HsfA as essential for A. fumigatus viability, thermotolerance, and CWI. Indeed, HS and cell wall stress trigger the coordinated expression of both hsfA and hsp90. Furthermore, the CWI signaling pathway components PkcA and MpkA were shown to be important for HsfA and Hsp90 expression in the A. fumigatus biofilms. Lastly, RNA-sequencing confirmed that hsfA regulates the expression of genes related to the HS response, cell wall biosynthesis and remodeling, and lipid homeostasis. Our studies collectively demonstrate the connection between the HS and the CWI pathway, with HsfA playing a crucial role in this cross-pathway regulation, reinforcing the importance of the cell wall in A. fumigatus thermophily.