# Sphingolipids in fungi: Biosynthesis and key roles in biological processes

Thomas Bergia, Sonia Rippa

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## To cite this version:

Thomas Bergia, Sonia Rippa. Sphingolipids in fungi: Biosynthesis and key roles in biological processes. Fungal Biology Reviews, 2025, 52, pp.100430. 10.1016/j.fbr.2025.100430. hal-05041888

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# Sphingolipids in fungi: Biosynthesis and key roles in biological processes

Thomas Bergia, Sonia Rippa*

Unité Génie Enzymatique et Cellulaire, CNRS, UMR 7025, Alliance Sorbonne Université, Université de technologie de Compègne, Compègne, France

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## ARTICLE INFO

**Keywords:**

Abiotic stress

Biosynthesis pathway

Fungi

Membrane lipids

Sphingolipids

Sphingolipid inhibitors

## ABSTRACT

Sphingolipids are complex lipids involved in the global cell physiology. They are significant components of the plasma membrane, contributing to the organization of lipids and anchoring of proteins. In all organisms, the biosynthesis pathway of sphingolipids begins in the endoplasmic reticulum and ends in the Golgi apparatus, where the biosynthesis of more complex sphingolipids occurs. In recent years, the key roles of sphingolipids in fungal biological processes and in the capacity of fungi to adapt to various environments have become of increasing interest. Sphingolipids are also promising as alternative targets to the emergence of multi-drug resistant pathogenic fungal strains. This review presents an outline of the fungal sphingolipid biosynthesis pathway, and an overview of recent advances in their biological roles.

## 1. Introduction

Lipids are a diverse and ubiquitous group of organic compounds involved in various key biological functions. According to the Lipid Metabolites And Pathway Strategy (Lipid MAPS) consortium, lipids are divided into eight categories: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and polyketides (Fahy et al., 2011). Lipids are major constituents of cell membranes, serve as energy storage sources, and are involved in signaling pathways and cell growth (Fahy et al., 2011; Storck et al., 2018). Taking into account their role in eukaryotic membranes, glycerophospholipids, sphingolipids and sterols are considered as major lipids (Lingwood and Simons, 2010; Yang et al., 2018). Glycerophospholipids are composed of a polar head group attached to a glycerol backbone with up to two fatty acyl chains, and sterols are steroid alcohols. Sphingolipids are complex lipids composed of a sphingoid base also known as Long-Chain Base (LCB) (Fig. 1).

During sphingolipid biosynthesis (as detailed below), the LCB is linked to a fatty acid via an amide bond and the whole structure forms a ceramide (Cer). The family of Cers consists of closely related molecules with different LCBs, number of insaturations, hydroxylation positions, fatty acid chain lengths, and possibly a methylation (Mota Fernandes et al., 2018; Hannun and Obeid, 2011, Table 1). Cers can be linked to different head groups, resulting in different complex sphingolipids (Fig. 1). The head group of Cers can be a phosphate (ceramide-1-phosphate). In fungal acidic glycosphingolipids, it can be an inositol phosphate (InositolPhosphorylCeramide (IPC)), a mannosylated

inositol phosphate (MannosylInositolPhosphorylCeramide (MIPC)), 2 mannosylated inositol phosphates (Mannosyl(InositolPhosphoryl)₂Ceramide (M(IP)₂C)) (Ishino et al., 2022; Marquès et al., 2018). The class of GlycosylInositolPhosphorylCeramides (GIPCs) gathers sphingolipids IPCs, MIPCs and M(IP)₂Cs (Buré et al., 2014). In the neutral glycosphingolipids, the head group of Cers is a mono hexose (monohexosylceramide, cerebroside) like a glucose (GlucosylCeramide, GlcCer), a galactose (GalactosylCeramide, GalCer) (Barreto-Bergter et al., 2004), or a multi-hexose group (neurosporaside) (Costantino et al., 2011) (Fig. 1).

Sphingolipid composition varies depending on the fungal species and on the study conducted. It is noteworthy that the existing information on sphingolipid content comes mainly from yeasts. In different isolates of Candida auris, it has been shown that the class of Cers represents the main sphingolipids, followed by GlcCers, LCBs, IPCs (Ali et al., 2024). In Cryptococcus grubii and Cryptococcus gattii, the LCB content ranges between 1.2 and 2.6 % of the total sphingolipids. The Cer structures represent 49–80 %, the IPC content ranges between 2.4 and 4.4 %, and the GlcCer content ranges between 16 and 45 % (Singh et al., 2017). In the filamentous fungus Mortiella alpina, Cers species also constitute the major part of sphingolipids (80 %), followed by GlcCer and LCB species (Wang et al., 2011). Ejsing et al. (2009) reported that M(IP)₂Cs are predominant in Saccharomyces cerevisiae compared to MIPCs or IPCs. However, Nakase et al. (2010) reported that IPCs are the major complex sphingolipids in this yeast. Klose et al. (2012) have shown that complex sphingolipids represent about 6–10 % of the lipids in S. cerevisiae, with IPCs being predominant. In Candida albicans, MIPCs are the major

<footnote>* Corresponding author.
E-mail address: sonia.rippa@utc.fr (S. Rippa).</footnote>
complex sphingolipids compared to IPCs and M(IP)₂C (Prasad and Singh, 2020).

A substantial body of research has demonstrated the pivotal function of sphingolipids in various biological processes within the context of fungal cells. These lipids constitute integral components of cellular membranes, play a crucial role in cell division and cell death processes, and are implicated in the response to diverse external stresses, including antifungal drugs. This review begins with a description of the existing knowledge of the fungal sphingolipid biosynthetic pathway and then provides a comprehensive account of recent advances in their biological functions.

## 2. Sphingolipid biosynthesis

### 2.1. Sphingolipid precursors and ceramide biosynthesis in fungi

Intermediates and final products of the fungal sphingolipid biosynthesis pathway are given in Table 1. As in animals and plants, fungal sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of L-serine and palmitoyl-CoA and leads to the 3-ketoDi-HydroSphingosine (3-ketoDHS, 3-ketosphinganine) (Fig. 2). This step is catalyzed by the Serine Palmitoyl-Transferase (SPT) protein complex (Barreto-Bergter et al., 2004; Pinto et al., 1992; Zhu et al., 2023). The uptake of exogenous serine is the main source of de novo sphingolipid biosynthesis in S. cerevisiae (Esch et al., 2020). In fungi, as in other species, this biosynthesis step can be inhibited by SPT inhibitors (lipoxamycin, myriocin, simplifungin, sphingofungins, valsafungins and viridiofungins) (Table 2). The 3-ketoDHS is reduced by the 3-ketosphinganine reductase leading to DiHydroSphingosine (DHS) (Beeler et al., 1998). DHS is a sphingoid backbone, also known as sphinganine, which is an LCB. In contrast to mammalian cells, where DHS is the most important LCB, a higher diversity of LCBs is found in fungi (Zhu et al., 2023). DHS can be hydroxylated on C4 by the dihydrospingosine C4-hydroxylase, resulting in the formation of PHYtoSphingosine (PHS), another LCB. The transfer of acyl groups on PHS, catalyzed by the Cer synthase protein complex, leads to the synthesis of PhytoCeramides (PhCers, Cers B). DHS is also acetylated, leading to DihydroCeramides (DhCers, Cers A) (Guillas et al., 2001; Schorling et al., 2001). The acylation reaction involves the transfer of non-hydroxylated Very Long Chain Fatty Acids (VLCFAs) (Oh et al., 1997; Toke and Martin, 1996). In contrast to humans, fungal Cer synthases are capable to attach an extensive variety of fatty acids to their LCBs (Zhu et al., 2023). This step can be inhibited by Cer synthase inhibitors (australifungin or

fumonisins) (Table 2). In addition, PhCers are synthesized by the hydroxylation of DhCers at the C4 position of the sphingosine backbone (Grilley et al., 1998; Haak et al., 1997). Then, a fatty acyl 2-hydroxylase leads to the biosynthesis of 2-OH-DhCers (hydroxyDhCers, Cers B') from DhCers and to the biosynthesis of 2-OH-PhCers (hydroxyPhCers, Cers C) from PhCers in S. cerevisiae (Marquès et al., 2018). The enzyme responsible for the hydroxylation of Cers C remains unidentified; however Ccc2, a P-type copper-transporting ATPase, has been implicated in the synthesis of 2,x-OH-PhCers (Cers D) in S. cerevisiae (Beeler et al., 1997; Marquès et al., 2018).

### 2.2. Acidic glycosphingolipid pathway

Cers A, B, B', C and D are the precursors of phosphorylated and glycosylated acidic sphingolipids (Fig. 3). In S. cerevisiae, Cers could be transferred from the endoplasmic reticulum to the Golgi apparatus via vesicular or non-vesicular transport (Funato et al., 2020; Funato and Riezman, 2001). Cers are then linked to a phosphorylated inositol at the C1 hydroxyl group by a phosphodiester bond. This reaction is catalyzed by the InositolPhosphorylCeramide (IPC) synthase and produces IPCs (Nagiec et al., 1997; Sato et al., 2009). This step can be inhibited by IPC inhibitors (Aureobasidin A (AbA), khafrefungin, pleofungins and rustmicin) (Table 2). It should be noted that AbA, khafrefungin, rustmicin are non-toxic to animal cells due to the absence of IPC synthase (Vicente et al., 2003; Zhu et al., 2023). As in the case of plants, further glycosylation of IPCs by glycosyltransferases leads to GIPCs in fungi. Subsequently, IPCs are mannosylated by the action of IPC mannosyl transferase to produce Mannose-Inositol-PhosphorylCeramides (MIPCs) (Beeler et al., 1994, 1997). Next, Mannose-DiInositol-PhosphorylCeramides (M(IP)₂Cs) are synthesized by the action of inositolphosphotransferase (Dickson et al., 1997). GIPCs are structural homologs of gangliosides in the animal kingdom, but gangliosides contain neither phosphate nor inositol (Gronnier et al., 2016; Takahashi et al., 2009). GIPCs are distributed widely among fungal species of the two phyla Ascomycota and Basidiomycota (Guimarães et al., 2014; Takahashi et al., 2009). In *Aspergillus fumigatus*, an N-acetylglucosaminyltransferase has been found to be involved in the initiation of zwitterionic GIP biosynthesis (Engel et al., 2015).

### 2.3. Neutral glycosphingolipid pathway

Neutral glycosphingolipids are synthesized from DhCers (Fig. 4). These are desaturated at the Δ4 position of the sphingoid backbone by

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Chemical structure of fungal sphingolipids adapted from Mota Fernandes et al. (2018). Black: sphingoid base (Long chain Base, LCB). Red: fatty acid chain. Blue: head group composition. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
the action of DhCer Δ4-desaturase, resulting in the production of Δ4-ceramides (Ternes et al., 2002). Subsequent desaturation of Δ4-ceramides on the sphingoid backbone is catalyzed by Cer Δ8-desaturase, resulting in the formation of Δ4,Δ8-ceramides (Takakuwa et al., 2002). The sphingolipid C9-methyltransferase then catalyzes the addition of a methyl group, specifically at the C9 position of the sphingoid base, leading to Δ4,Δ8-9-me-ceramides (Ternes et al., 2006; Zhu et al., 2023). The transport of Δ4,Δ8-9-me-ceramides to the Golgi apparatus can occur via both non-vesicular and vesicular mechanisms. Subsequent to this, Δ4,Δ8-9-methyl-ceramides are converted into GlcCers or GalCers, categorized as two distinct classes of monohexosylceramides. The

biosynthesis of GlcCers occurs by the action of a GlcCer synthase, which catalyzes the transfer of glucose moiety from the UDP-glucose (Barreto-Bergter et al., 2004; Leipelt et al., 2001). This step can be inhibited by GlcCer synthase inhibitors (as D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol, PPMP) (Table 2). The biosynthesis of GalCers is catalyzed by the Cer galactosyltransferase. However, the gene encoding a galactosyl transferase has only been identified in two species, Neurospora crassa and Magnaporthe grisea (Jiang et al., 2021). All fungi studied so far contain GlcCers or GalCers, and most species likely contain both types of glycosphingolipids. S. cerevisiae does not produce GlcCers (Warnecke and Heinz, 2003).

<table><caption>**Table 1**

Chemical structures of fungal sphingolipids and precursors. Chemical structures are adapted from Mota Fernandes et al. (2018) and Marqués et al. (2018).</caption><thead><tr><th>Class</th><th>Name</th><th>Chemical structure</th></tr></thead><tbody><tr><td>Sphingolipid precursor</td><td>3-ketodihydrosphingosine (3-ketoDHS, 3-ketosphinganine)</td><td></td></tr><tr><td rowspan="2">Long chain bases (LCBs)</td><td>Dihydrosphingosine (DHS, sphinganine)</td><td></td></tr><tr><td>Phytosphingosine (PHS)</td><td></td></tr><tr><td rowspan="5">Ceramides (Cers)</td><td>Dihydroceramide (DhCer, Cer A)</td><td></td></tr><tr><td>2-OH-Dihydroceramide (HydroxyDhCer, Cer B')</td><td></td></tr><tr><td>Δ4-ceramide</td><td></td></tr><tr><td>Δ4,Δ8-9-me-ceramide</td><td></td></tr><tr><td>Phytoceramide (PhCer, Cer B)</td><td></td></tr><tr><td rowspan="2">Neutral glycosphingolipids</td><td>2-OH-Phytoceramide (HydroxyPhCer, Cer C)</td><td></td></tr><tr><td>2,x-OH-Phytoceramide (Cer D)</td><td></td></tr><tr><td rowspan="2">Acidic glycosphingolipids</td><td>Δ4,Δ8-9-me-ceramide<br>R: Glucose (Glucosylceramide, GlcCer)<br>R: Galactose (Galactosylceramide, GalCer)</td><td></td></tr><tr><td>InositolPhosphorylCeramide (IPC)<br>R: Mannose-inositol (MIPC)<br>R: Inositol-P-mannose-inositol-P (M(IP)<sub>2</sub>C)<br>R: GlcNα1-2 (GIPC)<br>R: Manα1-2 (GIPC)<br>R: Manα1-6 (GIPC)</td><td></td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Sphingolipid precursors and ceramide biosynthesis steps in fungal cells. Black: lipids. Brown arrows indicate each enzymatic step in endoplasmic reticulum. Green: sphingolipid inhibitors. SPT: Serine Palmitoyl Transferase. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

**3. Biological roles of sphingolipids in fungi**

Knowledge of the sphingolipid biosynthetic pathway, the use of
mutants and inhibitors, has allowed a deeper understanding of the role
of sphingolipids in fungal cell biology and stress tolerance.

**3.1. Sphingolipid role in membranes, protein localization and vacuolar morphology**

Several sphingolipid classes are present in the fungal plasma mem-
brane with some discrepancies observed between species. In the model
yeast *S. cerevisiae*, IPCs, MIPCs and M(IP)₂Cs are highly present
(Hechtberger et al., 1994; Patton and Lester, 1991), with M(IP)₂Cs being
the most abundant sphingolipids (Dickson et al., 1997; Leber et al.,
1997). Furthermore, a significant quantity of Cers has been highlighted
(Schneiter et al., 1999). IPCs and MIPCs are also found in the plasma
membrane of *C. albicans*, with IPCs being the major compounds
(Urbanek et al., 2022). In the filamentous fungus *N. crassa* plasma
membrane, GlcCer species are in majority, in comparison to Cers species
(Huber et al., 2019).

It is imperative to acknowledge the pivotal function of sphingolipids within the context of plasma membranes, a role that arises from their participation in the process of lipid domain formation. These domains, which include lipid rafts and sphingolipid-enriched microdomains, are distinguished by their distinct lipid compositions and the specific types of associated proteins (Fig. 5).

Lipid rafts are defined as compacted regions of condensed bilayers, which are enriched in sterols tightly packed with sphingolipids (Mollinedo, 2012). They have multiple functions including cell signaling, polarity and endocytosis (Wachtler and Balasubramanian, 2006). According to Nakase et al. (2010), the absence of the major complex sphingolipids MIPCs, in the yeast *Schizosaccharomyces pombe* *IMT1*, *IMT2* and *IMT3* triple deficient mutant (depleted in IPC mannosyl transferase), causes a defect in sterol localization. Neutral glycosphingolipids, especially methylated ones, also play a role. Indeed, a raft mislocalization is observed in ceramide Δ8-desaturase and GlcCer

synthase deficient mutants of the filamentous fungus *Aspergillus nidulans* (Mota Fernandes et al., 2016). Additionally, total membranes isolated from *Cryptococcus neoformans* in which GlcCers are present but not methylated, are leaky and lipids extracted from these membranes do not form rafts *in vitro* compared to the lipids extracted from wild-type membranes (Singh et al., 2012). In the filamentous fungus *Scedospo-rium boydii*, treated with myriocin (SPT inhibitor), a decrease in PHS, DhCers, αOH-Cers, GlcCers and some IPCs is observed. This is associated with a lack of rafts along the plasma membrane. In the same fungus, a treatment with PPMP (GlcCer synthase inhibitor) induces an accumulation of α-OH-Δ8-9Me-Cers and a decrease of αOH-Cers, GlcCers and some IPCs. In this case, lipid rafts are randomly accumulated along the membrane (Rollin-Pinheiro et al., 2019).

Fungal plasma membranes also contain sphingolipid-enriched domains. These are not ergosterol-enriched lipid rafts, but gel domains composed mainly of sphingolipids (Aresta-Branco et al., 2011) (Fig. 5). They could be important diffusion barriers for lipids and proteins and would contribute to the greater immobility or characteristically slower diffusion of proteins in the plasma membrane. Sphingolipid-enriched domains may also play a crucial role in maintaining the trans-membrane proton gradient and ion homeostasis, or stabilizing the plasma membrane when the cell wall is damaged or removed (Santos et al., 2020).

Lipid domains remain a complex concept because they can also differ in the associated proteins. In *S. cerevisiae*, Can1p (*H*⁺-arginine symporter) and Pma1p (plasma membrane *H*⁺-ATPase) are compartmentalized into 2 types of domains within the plasma membrane: Membrane Compartment of Can1p (MCC), with a punctate pattern, and membrane compartment of Pma1p, with a network-like pattern (Malínská et al., 2003). It is suggested that the MCC is enriched in ergosterol (Grossmann et al., 2007) and that the membrane compartment of Pma1p is enriched in sphingolipids (*van 't Klooster et al., 2020*). In *S. cerevisiae* lacking M (IP)₂Cs by the deletion of inositolphosphotranferase 1, there is a clear alteration in Pma1p membrane distribution, but no change in Can1p distribution. Cells have more rigid and compact sphingolipid-enriched domains, while the dielectric properties, particularly those modulated
<table><caption>**Table 2**
Effects of sphingolipid biosynthesis inhibitors on fungi.</caption><thead><tr><th>Compounds</th><th>Organisms</th><th>Effects</th><th>References</th></tr></thead><tbody><tr><td><strong>Serine Palmitoyl-Transferase (SPT) inhibitors</strong><br>Lipoxamycin</td><td>C. albicans,<br>C. guillermondii,<br>C. parapsilosis,<br>C. pseudotropicalis,<br>C. tropicalis,<br>C. neoformans,<br>S. cerevisiae<br>C. albicans</td><td>Growth inhibition</td><td>Mandala et al. (1994)</td></tr><tr><td><strong>Myriocin</strong></td><td>A. fumigatus<br>A. corymbifera,<br>C. albicans,<br>C. glabrata,<br>C. parapsilosis,<br>C. neoformans,<br>R. microsporus,<br>R. oryzae,<br>R. pusillus,<br>S. cerevisiae<br>S. boydii<br>C. auris<br>S. turcica</td><td>Biofilm biomass reduction, growth inhibition<br>Biofilm biomass reduction<br>Growth inhibition</td><td>Lattif et al. (2011);<br>De Melo et al. (2013)<br>Perdoni et al. (2015)<br>Ishijima et al. (2016)</td></tr><tr><td><strong>Simplifungin</strong></td><td>C. albicans,<br>C. parapsilosis,<br>C. neoformans,<br>S. cerevisiae<br>A. flavus,<br>A. fumigatus,<br>C. albicans,<br>C. guillermondii<br>C. parapsilosis,<br>C. pseudotropicalis,<br>C. tropicalis,<br>C. neoformans,<br>S. cerevisiae<br>C. albicans</td><td>Reduced growth and biofilm formation, impaired<br>germination process<br>Growth defects<br>Abnormal appressoria development, conidial<br>development inhibition<br>Growth inhibition</td><td>Rollin-Pinheiro et al. (2019)<br>Stieber et al. (2024)<br>Li et al. (2024)<br>Ishijima et al. (2016)</td></tr><tr><td><strong>Sphingofungins</strong></td><td>A. fumigatus,<br>A. parapsilosis,<br>R. oryzae,<br>R. microsporus,<br>R. pusillus<br>C. albicans</td><td>Growth inhibition</td><td>Horn et al. (1992);<br>VanMiddlesworth et al. (1992);<br>Mandala et al. (1994)</td></tr><tr><td><strong>Valsafungins</strong></td><td>C. albicans<br>C. parapsilosis,<br>R. oryzae,<br>R. microsporus,<br>R. pusillus<br>C. albicans</td><td>Growth inhibition</td><td>Ishijima et al. (2016)</td></tr><tr><td><strong>Viridiofungins</strong><br>Ceramide synthase inhibitors<br>Australifungin</td><td>A. fumigatus,<br>C. albicans,<br>C. guillermondii,<br>C. parapsilosis,<br>C. pseudotropicalis,<br>C. tropicalis,<br>C. neoformans,<br>S. cerevisiae<br>S. cerevisiae<br>Candida albicans<br>Candida spp.<br>B. cinerea</td><td>Growth inhibition<br>Growth inhibition</td><td>Mandala et al. (1997a)<br>Mandala et al. (1995)</td></tr><tr><td><strong>Fumonisins</strong><br>Inositol Phosphoryl Ceramide (IPC) synthase inhibitors<br>Aureobasidin A (AbA)</td><td>C. albicans,<br>C. grabata,<br>C. tropicalis,<br>C. neoformans,<br>S. cerevisiae<br>Candida albicans<br>Candida spp.<br>B. cinerea</td><td>Biofilm biomass reduction<br>Filamentation and biofilm development inhibition<br>Growth inhibition, delayed conidial germination<br>initiation, restricted elongation of germ tube<br>Anti-biofilm effect<br>Growth defect<br>Growth inhibition</td><td>Lattif et al. (2011)<br>Tan and Tay (2012)<br>Wang et al. (2015)<br>Munusamy et al. (2018)<br>Fukuda et al. (2023)<br>Mandala et al. (1997b)</td></tr><tr><td><strong>Khafrefungin</strong></td><td>C. albicans,<br>C. neoformans,<br>S. cerevisiae<br>A. fumigatus,<br>C. albicans,<br>C. grabata,<br>C. parapsilosis,<br>C. tropicalis,<br>C. neoformans</td><td>Growth inhibition</td><td>Yano et al. (2007)</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 2 (continued)**</caption><thead><tr><th>Compounds</th><th>Organisms</th><th>Effects</th><th>References</th></tr></thead><tbody><tr><td rowspan="5">Rustmicin</td><td>C. neoformans,<br>S. cerevisiae</td><td rowspan="3">Growth inhibition</td><td rowspan="3">Harris et al. (1998)</td></tr><tr><td>C. albicans,<br>C. tropicalis,<br>C. neoformans</td></tr><tr><td>C. albicans,<br>C. glabrata,<br>C. krusei,<br>C. parapsilosis,<br>C. pseudotropicalis,<br>C. tropicalis,<br>S. cerevisiae</td></tr><tr><td>C. albicans,<br>C. grabata,<br>C. parapsilosis,<br>C. tropicalis,<br>C. neoformans</td><td>Growth inhibition</td><td>Mandala et al. (1998)</td></tr><tr><td>C. neoformans</td><td>Growth inhibition</td><td>Yano et al. (2007)</td></tr><tr><td>GlucosylCeramide (GlcCer) synthase inhibitors</td><td></td><td></td><td></td></tr><tr><td>D-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (P4)</td><td>A. fumigatus</td><td>Growth arrest after polarity establishment</td><td>Levery et al. (2002)</td></tr><tr><td>D-threo-3',4'-ethylenedioxy-P4</td><td>A. nidulans</td><td>Halted growth after germ tube emergence</td><td>Levery et al. (2002)</td></tr><tr><td>D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP)</td><td>A. fumigatus</td><td>Halted growth before germ tube emergence</td><td>Levery et al. (2002)</td></tr><tr><td>N'--(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM)</td><td>S. boydii</td><td>Growth and biofilm reduction</td><td>Rollin-Pinheiro et al. (2019)</td></tr><tr><td></td><td>A. fumigatus,<br>B. dermatitis,<br>C. krusei,<br>C. glabrata,<br>C. parapsilosis,<br>C. guilliermondii,<br>Coccidioides spp.,<br>C. gattii,<br>C. neoformans,<br>H. capsulatum,<br>P. variotii,<br>R. oryzae</td><td>Growth inhibition</td><td>Mor et al. (2015)</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Acidic glycosphingolipid biosynthesis pathway in fungal cells. Black: lipids. Dark red arrows indicate each enzymatic step in the Golgi apparatus. Green: IPC (InositolPhosphorylCeramide) inhibitors. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

by ergosterol, remain essentially unchanged. This supports that M (IP₂)Cs are important components of the sphingolipid-enriched domains but not of the ergosterol-enriched domains (Bento-Oliveira et al., 2020). The integral plasma membrane tetraspan protein Nce102 is also a

structurally and functionally important component of the MCC. A study of *S. cerevisiae* and *C. albicans* shows an increase in Nce102 level in response to increased sphingolipid demand. Internalization of Nce102 into the vacuolar membrane is observed when excess sphingolipid precursors are supplied (Zahumenský et al., 2022).

Membrane domains also allow for the incorporation of other proteins (Fig. 5). Thus, GPI-anchored proteins, known to play critical roles in various cellular processes, such as cell signaling and cell adhesion, have been described to be found in lipid rafts and possibly in sphingolipid-enriched domains (Aresta-Branco et al., 2011; Saha et al., 2016). Similarly, in *S. cerevisiae*, myriocin inhibits trafficking of the methionine transporter Mup1 to the plasma membrane. This can be restored by adding PHS to the culture medium, confirming a sphingolipid requirement for Mup1 activity (Hepowit et al., 2021).

Sphingolipids can also disrupt plasma membrane properties and enzyme functions. In *S. pombe*, Csn1, a membrane protein that functions as an inositol phosphosphingolipid phospholipase C (Isc1 phospholipase), hydrolyzes complex sphingolipids to produce Cers (Feoktistova et al., 2001). In Csn1p mutants, increased plasma membrane MIPCs disrupt the intrafacial distribution of ergosterol, the properties of the plasma membrane, and the functions of N-glycosylated cell wall assembly enzymes (Willet et al., 2023).

The metabolism of sphingolipids and precursors may also control the membrane contact sites of vacuoles by tethering proteins (Fig. 5). For example, the tethering protein Ymr160w is a component of vacuole contact sites with the nuclear endoplasmic reticulum, the mitochondria and the peroxisomes. In *S. cerevisiae*, Ymr160w-mediated contact sites are enhanced when sphingolipid levels are decreased by myriocin or AbA (Bisinski et al., 2022). In addition, in tricalbin-deleted *S. cerevisiae* cells (tricalbins being tethering proteins at the endoplasmic reticulum-plasma membrane and endoplasmic reticulum-Golgi contact sites) PHS accumulates and induces vacuole division. Removing the
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Neutral glycosphingolipid biosynthesis pathway in fungal cells. Black: lipids. Brown arrows indicate enzymatic steps in the endoplasmic reticulum. Dark red arrows indicate enzymatic steps in the Golgi apparatus. Green: sphingolipid inhibitors. DhCer: DihydroCeramide. GlcCer: GlucosylCeramide. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Biological roles of sphingolipids in fungi. MCC: Membrane Compartment of Can1p. MCP: Membrane Compartment of Pma1p. GPI: GlycosylPhosphatidylInositol. UPR: Unfolded Protein Response.</figcaption></figure>

nucleus-vacuole junction restores vacuolar morphology, supporting that the PHS transport across the nucleus-vacuole junction induces vacuolar division (Hanaoka et al., 2023).

Finally, sphingolipids have been shown to be involved in vacuole formation and fusion. Indeed, early stationary phase vacuoles in *S. cerevisiae* are characterized by an increase in putative raft components as complex sphingolipids, including IPCs with longer and more hydroxylated chains (Kim and Budin, 2024). Furthermore, when *S. pombe* MIPC mannosyltransferase-deficient mutants are subjected to hypotonic

stress conditions, vacuoles fail to fuse normally. This result suggests that MIPCs are required for normal vacuole morphology and for the fusion process (Nakase et al., 2010).

### 3.2. Sphingolipid role in cell growth, conidiation and death

Sphingolipid biosynthesis is critical for fungal growth (Figs. 5 and 6). Indeed, inhibitors of SPT, Cer synthase, IPC synthase or GlcCer synthase cause growth inhibition in a wide range of fungi (Table 2). Mycelial
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Sphingolipid roles on growth, conidiation and germination in fungi. DhCers: DihidroCeramides. PhCers: PhytoCeramides. GlcCers: GlucosylCeramide. IPC: InositolPhosphorylCeramide. Black dotted arrows: hidden intermediates in the sphingolipid biosynthesis pathway. Squares: sphingolipids. Blue and orange arrows: GlcCers and acidic glycosphingolipids respectively, biological implications. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

growth assays have also shown that the deletion of genes involved in Cer biosynthesis, results in significantly reduced hyphal growth on *Fusarium graminearum* (Wang et al., 2023). A cell separation defect is also observed in a strain of *S. cerevisiae*, lacking acidic glycosphingolipids (Epstein et al., 2012). Nevertheless, the addition of exogenous sphingolipids can inhibit the yeast growth. For example, the LCB PHS has a negative effect on the growth of *S. cerevisiae* (Chung et al., 2001). Cers are also proposed to inhibit cell growth by arresting the cell cycle in the G₁ phase in *S. cerevisiae* (Nickels and Broach, 1996). This indicates that maintaining an equilibrium in different sphingolipid biosynthesis is capital for yeast growth.

The involvement of GlcCers in fungal growth has been particularly well studied (Fig. 6). For instance, loss of a methyl group on GlcCers in *C. neoformans* mutants causes a growth arrest (Singh et al., 2012). As *C. neoformans* cells divide, the amount of GlcCers in the cell wall increases, particularly at the bud neck. Addition of GlcCer antibodies to cell cultures inhibits cell budding and cell growth (Rodrigues et al., 2000). In addition, the absence of Gcs1, which is responsible for GlcCer biosynthesis (Fig. 4) in *C. neoformans* mutants, causes a growth defect in vitro (Rittershaus et al., 2006). In filamentous fungi, GlcCers have been shown to be involved in hyphal growth. Thus, in *A. nidulans*, the LCB Δ8 desaturation and C9 methylation of GlcCers are important for growth. Indeed, mutants of a Δ8-desaturase and C9-methyltransferases, accumulating C8-saturated and unmethylated GlcCers, display a growth defect (Mota Fernandes et al., 2016). Finally, when *S. boydii* is treated with myriocin or with PPMP, a decrease in cell density is observed. Specifically, myriocin induces short germ tubes and PPMP induces longer germ tubes than myriocin but hyphae fail to mature (Rollin-Pinheiro et al., 2019).

Ceramides and complex sphingolipids are involved in fungal growth in different ways in different species. In *S. pombe*, Lag1 and Lac1 Cer

synthases could be responsible for producing different types of Cers. The Lac1 protein would have a preference for producing long-chain PhCers, whereas Lag1 would synthesize long-chain DhCers. Lac1, but not Lag1, is specifically required for proper control of cell growth (Flor-Parra et al., 2022). On the contrary, the absence of Lag1 in *C. albicans* causes severe defects in the growth and hyphal morphogenesis. Analysis of Cers and sphingolipid compositions shows that Lag1 predominantly synthesizes Cers involved in the generation of inositol-containing sphingolipids, suggesting the role of acidic glycosphingolipids in cell growth (Cheon et al., 2012). The homolog of Lag1 in *A. nidulans*, BarA, also appears to produce a specialized pool of Cers that is particularly important for hyphal tip organization (Li et al., 2006).

Studies show that complex sphingolipids are also required for conidiation, spore formation and germination (Fig. 5). When the protein kinase YpkA, a regulator of sphingolipid production, is deleted in *A. fumigatus*, a decrease in DhCers, Cers, GlcCers and an increase in IPCs are observed. This results in a poor growth and a lack of conidiation (Fabri et al., 2019). Similarly, the deletion of enzymes involved in the GlcCer biosynthesis in *N. crassa* and *Magnaporthe oryzae* affects growth and conidiation (Huber et al., 2019; Wang et al., 2024). Likewise, monoclonal antibodies used against GlcCers block the differentiation of *Colletotrichum gloeosporioides* conidia into mycelia (Da Silva et al., 2004) and significantly reduce the growth of *Fonsecaea pedrosoi* (Nimrichter et al., 2004). In addition, delayed initiation of conidial germination and restricted germ tube elongation are observed in *Botrytis cinerea* with AbA (Wang et al., 2015). Finally, studies on an IPC synthase mutant in *Aspergillus oryzae* suggest that IPCs enhance mycelial growth, spore formation, and development, but reduce the accumulation of organic compounds (Ge et al., 2022).

Sphingolipids are also known to be involved in cell death (Fig. 5). The LCBs PHS and DHS have been suggested to be involved in the
induction of apoptosis in *A. nidulans*. They induce a rapid DNA condensation independent of mitosis, extensive DNA fragmentation and phosphatidylserine exposure, all common morphological features characteristic of apoptosis (Cheng et al., 2003). More recently, sphingosine has been shown to have a direct effect on mitochondria, leading to the release of cytochrome C as a hallmark of apoptosis on *Candida glabrata* and *A. fumigatus* (Arani et al., 2022). Likewise, in *Pichia pastoris* cells, Cers and sphingosine increase during the decline phase and may be associated with cell apoptosis (Liu et al., 2022; Yu et al., 2022). In *S. pombe* Ccs1-3 mutants, an accumulation of MIPCs is observed and is associated with cell wall defect, leading to growth arrest, cytoplasm compression, and cell death (Willet et al., 2023).

### 3.3. Sphingolipid role in the unfolded protein response

The Unfolded Protein Response (UPR) is a signal transduction pathway that senses the fidelity of protein folding in the lumen of endoplasmic reticulum. It monitors the abnormal accumulation of unfolded or misfolded proteins that induces cellular stress (Hetz et al., 2020). Several studies show that Cers regulate the UPR. For example, high DhCer and PhCer levels and an UPR failure are observed in a *sec14-1*<sup>ts</sup> (thermosensitive allele of the structural gene for the major yeast phosphatidylinositol transfer protein) and *tlg2* (Tlg2 target membrane-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) deleted yeast mutant. In this mutant, when *KES1* (encoding a sterol-binding protein) is deleted, or in the presence of a ceramidase, a decrease in DhCer and PhCer levels is observed and the UPR is reactivated. In the same mutant, when *SIT4* (gene encoding the major catalytic subunit of Cer-activated protein phosphatase) is deleted, the UPR activity is restored. This result shows that Cers can exert their effect through Sit4 protein. Thus, elevation of endogenous Cers represses the UPR through increased Sit4 activity in the *sec14-1*<sup>ts</sup> and *tlg2* deleted mutant (Mousley et al., 2008). In addition, recent work suggests that the UPR can also sense various defects within the endoplasmic reticulum membrane. In *S. cerevisiae*, the loss of Isc1 phospholipase catabolizing complex Cers, causes UPR induction which requires VLCFA-containing PhCers. It demonstrates the intersection between ceramide metabolism, membrane homeostasis, and the UPR (Rajakumar et al., 2024).

## 4. Sphingolipid roles in adaptation to stress

### 4.1. Oxidative stress

As reported in Table 3, both Cers and glycosphingolipids have been shown to play a role in the susceptibility of fungal cells to oxidative stress. In the case of *S. cerevisiae*, the Isc1 phospholipase is known to generate the bioactive sphingolipid class of Cers. Loss of this enzyme results in increased sensitivity to hydrogen peroxide, leading to a shortened chronological lifespan and increased apoptotic cell death. It is then shown that Isc1 phospholipase plays an important role in the regulation of cellular redox homeostasis in *S. cerevisiae* (Almeida et al., 2008). Interestingly, a study in *S. cerevisiae* has shown that the upregulation of M(IP)₂C biosynthesis also induces an oxidative stress hypersensitivity and a shortened chronological lifespan. Conversely, *S. cerevisiae* strains lacking M(IP)₂Cs exhibit increased resistance to oxidative stress under hydrogen peroxide and have a prolonged chronological lifespan (Aerts et al., 2006). To investigate the role of the GlCer synthesis pathway in the oxidative stress response in *N. crassa*, Huber et al. (2019) exposed mutant strains to increasing concentrations of reactive oxygen species generators. The results demonstrate that different intermediates of the GlCer synthesis pathway regulate the sensitivity to superoxide and hydroxyl radicals, but not to organic hydroperoxides. In another study, Wang et al. (2024) have found that the *M. oryzae DES1* (encoding the DhCer Δ4-desaturase) deleted mutant is more resistant to hydrogen peroxide than the wild type. This result supports the regulatory role of neutral glycosphingolipid pathway in the

oxidative stress sensitivity.

### 4.2. pH variations

Sphingolipid composition has also been shown to be involved in the adaptation of fungal cells to pH changes (Table 3). For example, *S. cerevisiae* strains lacking sphingolipids are unable to grow at low pH (Patton and Lester, 1991). Lipidomic profiling of the yeasts *S. cerevisiae* and *Zygosaccharomyces bailii* treated with acetic acid, has revealed a general increase of Cers, IPCs, MIPCs and M(IP)₂Cs (Lindberg et al., 2013). Likewise, the acidification of the cytosol in *Fusarium oxysporum* leads to an increase in the LCB sphingolipid DHS (Fernandes et al., 2023). Furthermore, *S. cerevisiae* lacks the ability to synthesize GlCers but adapts to alkaline conditions after exposure to GlCers from *Aspergillus kawachii* grown on koji cereal (Sawada et al., 2015).

The role of sphingolipids in plasma membrane composition seems to explain their involvement in pH variation adaptation. Indeed, a study in *S. cerevisiae* suggests that the accumulation of IPCs (due to a defect in MIPC biosynthesis) is detrimental to yeast under low pH conditions (pH 2.5-4.0) and that downregulation of IPC levels is one of the adaptation mechanisms to low pH. However, low pH hypersensitivity is suppressed by altering plasma membrane ergosterol homeostasis, suggesting a functional relationship between complex sphingolipids and ergosterol under low pH conditions (Otsu et al., 2020). More specifically, neutral and acidic pH cause changes in the membrane composition and increase the sphingolipid content in *Sodiomyces tronii*, an alkaliphilic fungus that grows at the optimal pH of 9.2 (Bondarenko et al., 2017). The change in the sphingolipid membrane composition induced by a pH variation has also been observed in the acidophilic fungus *Sistotrema brinkmannii*. The optimal growth pH of this fungus is about 3.0-4.0. The composition of membrane lipids at pH 2.6 does not differ much from the optimal conditions, while in the near-neutral region there is a twofold increase in sphingolipids. This indicates the adaptive value of sphingolipids for the protection of cell membranes in *S. brinkmannii* (Ianutsevich et al., 2023).

More specifically, in *C. neoformans*, growing at neutral/alkaline pH but not at acidic pH, very long chain PhCers are significantly increased at low pH. Two enzymes of the sphingolipid pathway, IPC synthase and Isc1 phospholipase, are critical for promoting fungal growth at acidic pH. They have been shown to regulate the plasma membrane H⁺-ATPase Pma1 by distinct mechanisms: Isc1 through PhCer C26 and Ipc1 through complex sphingolipids or/and diacylglycerol (Garcia et al., 2008). Furthermore, an accumulation of DHS and GlCer species is observed in *C. neoformans* when exposed to an acidic environment, accompanied by a higher abundance of PHS, some long-chain α-OH PhCers and IPCs. This finding suggests that *C. neoformans* alters the metabolism of several sphingolipid species, with these changes depending on specific Cer isoforms, to generate sufficient amounts of the resulting complex sphingolipids (Munshi et al., 2018).

### 4.3. Temperature variations

Research has demonstrated the importance of sphingolipids in maintaining cellular viability during temperature fluctuations (Table 3). For instance, sphingolipid-deficient strains of *S. cerevisiae* are unable to withstand heat shock (Patton et al., 1992). In addition, it has been observed in several cases that sphingolipid levels vary with temperature changes. For example, an increase in membrane sphingolipid content has been observed in *Rhizomucor tauricus* and *Myceliophytora thermophila* following a heat shock. This is accompanied by modifications in the amounts of different phospholipids and an increase in sterol content (Ianutsevich et al., 2016). Specifically, a temperature shift from 24 °C to 39 °C leads to a multi-fold increase in Cers as well as a transient increase in DHS and PHS. *De Novo* Cer biosynthesis involving Cer synthase, but not sphingolipid degradation, appears to be the underlying mechanism responsible for the heat-induced Cer elevation (Wells et al., 1998). In *A. oryzae*, the expression level of the gene *AoAUR1*, which encodes the
<table><caption>**Table 3**
Sphingolipid roles in fungal adaptation to stress and interplay with antifungal drugs.</caption><thead><tr><th colspan="5">Oxidative stress</th></tr><tr><th>Treatment</th><th>Species</th><th>Effect</th><th>References</th></tr></thead><tbody><tr><td rowspan="5">H<sub>2</sub>O<sub>2</sub></td><td>S. cerevisiae: M(IP)<sub>2</sub>C upregulation</td><td rowspan="2">Fungal sensitivity to H<sub>2</sub>O<sub>2</sub></td><td>Aerts et al. (2006)</td></tr><tr><td>S. cerevisiae: Isc1 lacking</td><td>Almeida et al. (2008)</td></tr><tr><td>N. crassa: ΔDES1, ΔDES2, ΔSMT</td><td rowspan="4">Fungal resistance to H<sub>2</sub>O<sub>2</sub></td><td>Huber et al. (2019)</td></tr><tr><td>S. cerevisiae: M(IP)<sub>2</sub>C lacking</td><td>Aerts et al. (2006)</td></tr><tr><td>N. crassa: ΔLAC with accumulated MIPC</td><td>Huber et al. (2019)</td></tr><tr><td>M. oryzae: ΔMODES1</td><td>Wang et al. (2024)</td></tr><tr><td rowspan="3">Menadione<br/>Tert-<br/>butylhydroperoxide</td><td>N. crassa: ΔDES1, ΔDES2, ΔSMT</td><td>Fungal sensitivity to tert-butylhydroperoxide</td><td>Huber et al. (2019)</td></tr><tr><td>N. crassa: ΔDES1, ΔDES2, ΔSMT; ΔGCS; ΔLAC with accumulated MIPC</td><td>Fungal resistance to menadione and tert-butylhydroperoxide</td><td>Huber et al. (2019)</td></tr><tr><td></td><td></td><td></td></tr><tr><th colspan="5">pH variations</th></tr><tr><th>Treatment</th><th>Species</th><th>Effect</th><th>References</th></tr><tr><td rowspan="6">Low pH</td><td>S. cerevisiae: ΔLCB1</td><td rowspan="2">Fungal sensitivity to low pH</td><td>Patton and Lester (1991)</td></tr><tr><td>S. cerevisiae: IPC accumulation</td><td>Otsu et al. (2020)</td></tr><tr><td>C. neoformans</td><td rowspan="4">Increasing content of different sphingolipid classes</td><td>Garcia et al. (2008); Munshi et al. (2018)</td></tr><tr><td>Z. bailii</td><td>Lindberg et al. (2013)</td></tr><tr><td>S. tronii</td><td>Bondarenko et al. (2017)</td></tr><tr><td>S. cerevisiae</td><td>Otsu et al. (2020)</td></tr><tr><td rowspan="4">Neutral/alkalin pH</td><td>F. oxysporum</td><td>Fernandes et al. (2023)</td></tr><tr><td>A. kawachii treated with GlcCer</td><td>Sawada et al. (2015)</td></tr><tr><td>S. tronii</td><td>Bondarenko et al. (2017)</td></tr><tr><td>S. brinkmannii</td><td>Ianutsevich et al. (2023)</td></tr><tr><th colspan="5">Temperature variations</th></tr><tr><th>Treatment</th><th>Species</th><th>Effect</th><th>References</th></tr><tr><td rowspan="2">High temperatures</td><td>S. cerevisiae</td><td rowspan="6">Increasing content of different sphingolipid classes</td><td>Jenkins et al. (1997)</td></tr><tr><td>S. cerevisiae</td><td>Wells et al. (1998)</td></tr><tr><td>M. thermophila</td><td>Anutsevich et al. (2016)</td></tr><tr><td>Cryptococcus, Saccharomyces and Wickerkamomyces genera</td><td>Rezanka et al. (2018)</td></tr><tr><td>S. cerevisiae</td><td>Randez-Gil et al. (2020)</td></tr><tr><td>S. lacrymans</td><td>Senik et al. (2021)</td></tr><tr><td rowspan="4">Low temperatures</td><td>A. oryzae</td><td>AoAUR1 downregulation</td><td>Ge et al. (2022)</td></tr><tr><td>Cryptococcus, Saccharomyces and Wickerkamomyces genera</td><td>Increasing content of different sphingolipid classes</td><td>Rezanka et al. (2018)</td></tr><tr><td>K. lactis</td><td>Decreasing content of different sphingolipid classes</td><td>Tanji et al. (2004)</td></tr><tr><td>Cryptococcus, Saccharomyces and Wickerkamomyces genera</td><td>Rezanka et al. (2018)</td></tr><tr><th colspan="5">Nutrient deprivation</th></tr><tr><th>Treatment</th><th>Species</th><th>Effect</th><th>References</th></tr><tr><td>Amino acid or glucose<br/>starvation</td><td>S. cerevisiae ΔCSG2</td><td>Disturbing of Aur1 stability, accumulation of PHS.</td><td>Liu et al. (2023)</td></tr><tr><td>Iron starvation</td><td>S. cerevisiae: appropriate levels of sphingolipids mediated by TORC2-Ypk1</td><td>Aft1 nuclear localization</td><td>Montellà-Manuel et al. (2023)</td></tr><tr><td rowspan="2">Nitrogen-limiting condition<br/>or starvation</td><td>S. pombe</td><td>Impairing of the sorting of the amino acid transporter from the plasma membrane to the vacuole</td><td>Nakase et al. (2010)</td></tr><tr><td>S. cerevisiae TORC1 inhibition</td><td>Complex sphingolipid synthesis</td><td>Shimobayashi et al. (2013)</td></tr><tr><th colspan="5">Hypoxia</th></tr><tr><th>Treatment</th><th>Species</th><th>Effect</th><th>References</th></tr><tr><td rowspan="2">Hypoxia</td><td>C. albicans</td><td>Increasing sphingolipid content</td><td>Burgain et al. (2020)</td></tr><tr><td>P. pastoris</td><td>Increasing Cers carrying methylated moieties, decreasing di- and tri- unsaturated LCB moieties, reduction of C42 species and favoring C44 species of inositol containing sphingolipids</td><td>Adelantado et al. (2017)</td></tr><tr><th colspan="5">NaCl stress</th></tr><tr><th>Treatment</th><th>Species</th><th>Effect</th><th>References</th></tr><tr><td rowspan="4">NaCl</td><td>S. cerevisiae ELO2 overexpression</td><td>Increasing of IPC, MIPC and M(IP)<sub>2</sub>C species</td><td>Zhu et al. (2020)</td></tr><tr><td>M. oryzae ΔMODES1</td><td>Higher growth rates than the wild type or the complemented strain</td><td>Wang et al. (2024)</td></tr><tr><td>S. boydii + myriocin</td><td>Fungal susceptibility to NaCl</td><td>Pinto et al. (1992)</td></tr><tr><td>S. boydii + PPMP</td><td>No NaCl susceptibility alteration</td><td>Pinto et al. (1992)</td></tr><tr><th colspan="5">Drought stress</th></tr><tr><th>Treatment</th><th>Species</th><th>Effect</th><th>References</th></tr><tr><td>Drought</td><td>C. geophilum</td><td>Upregulated genes encoding sphingosine hydroxylase or ceramidase</td><td>Li et al. (2023)</td></tr><tr><th colspan="5">Interplay of sphingolipids with antifungal drugs</th></tr><tr><th>Treatment</th><th>Species</th><th>Effect</th><th>References</th></tr><tr><td rowspan="2">Fluconazole</td><td>C. albicans + myriocin</td><td>Efflux pump mislocalization, enhanced fluconazole activity</td><td>Wang et al. (2022)</td></tr><tr><td>S. cerevisiae + AbA</td><td>Enhanced fluconazole activity</td><td>Rollin-Pinheiro et al. (2023)</td></tr></tbody><tfoot><tr><td colspan="4" style="text-align: right;">(continued on next page)</td></tr></tfoot></table>
<table><caption>**Table 3 (continued)**</caption><caption>Interplay of sphingolipids with antifungal drugs</caption><thead><tr><th>Treatment</th><th>Species</th><th>Effect</th><th>References</th></tr></thead><tbody><tr><td rowspan="2">Amphotericin B</td><td>C. neoformans + AbA</td><td>Increasing amphotericin B effect</td><td>Chen et al. (2024)</td></tr><tr><td>C. auris + myriocin</td><td>Increasing susceptibility to amphotericin B</td><td>Stieber et al. (2024)</td></tr></tbody></table>

IPC synthase, is down-regulated at 22 °C and 42 °C compared to 30 °C, which is the optimal growth temperature. Inhibition of *AoAUR1* increases the tolerance of *A. oryzae* to temperature stress and significantly decreases the level of IPCs and increases the contents of DhCers and GalCers. Thus, the downregulation of *AoAUR1* expression could be one of the mechanisms that enables *A. oryzae* to grow at low and high temperatures (Ge et al., 2022). Moreover, a study on different yeast strains belonging to the genera *Cryptococcus*, *Saccharomyces*, *Schizosaccharomyces* and *Whickerharmomyces*, grown at different temperatures (up to 40 °C) shows that C18-LCB molecular species are associated with low growth temperatures. A higher proportion of C20-LCB molecular species is produced at higher temperatures. The relative content of IPC increases with increasing temperature, while the content of M(IP)₂C decreases (Rezanka et al., 2018). In *S. cerevisiae*, an increase in C20 PHS and C20 DHS sphingoid bases is also observed early after heat stress, followed by an increase in two Cers. This indicates that sphingolipids are involved in the heat stress adaptation of yeast. This adaptation could be structural or sphingolipids could be second messengers or signal transducers as described in mammalian systems (Jenkins et al., 1997). Recently, an adaptive laboratory evolution in the presence of myriocin has been used to remodel the lipid profile of a strain of *S. cerevisiae*. Interestingly, the higher sphingolipid content correlates with the greater improvement in fitness at 40 °C compared to the parental strain. More specifically, the strain synthesizes sphingolipids with a shorter chain length (Randez-Gil et al., 2020).

Regarding the role of the neutral glycosphingolipids in adaptation to temperature changes, in the yeast *Kluyveromyces lactis*, cerebroside levels are significantly lower at 15 °C than in cells grown at 25 °C or 35 °C. More specifically, the 9-methyl branched chain in yeast cerebroside molecules appears to be essential for maintaining sufficient membrane fluidity for a low temperature environment (Tanji et al., 2004). Interestingly, in the basidiomycete fungus *Serpula lacrymans*, an increase of the freezing resistance is observed after pre-cultivation of mycelia at elevated temperatures. Lipid analysis revealed an increase in phosphatidylcholine and sphingolipid glyceroceramides. Adaptation to elevated temperature could lead to the predominance of membrane lipids (Senik et al., 2021).

## 4.4. *Nutrient deprivation and hypoxia*

LCBs or complex sphingolipids, such as MIPCs play a role in nutrient-limiting conditions (Table 3). For example, in *S. cerevisiae*, when TORC1 (Target Of Rapamycin Complex 1), a conserved serine/threonine kinase that controls cell growth in response to nutrients, is inhibited by nitrogen starvation, the synthesis of complex sphingolipids downstream of SPT is stimulated. This indicates that the complex sphingolipid synthesis upon TORC1 inhibition is a physiological response to nitrogen starvation (Shimobayashi et al., 2013). Similarly, in *S. pombe* under nitrogen-limiting conditions, MIPCs could be required for endocytosis of a plasma membrane-localized amino acid transporter. Indeed, sorting of the transporter from the plasma membrane to the vacuole is severely impaired in a MIPC deficient mutant. In addition, lipid rafts are unstable. Thus, MIPCs are required for lipid-raft-mediated endocytosis of plasma membrane transporter under nitrogen-limiting conditions (Nakase et al., 2010). Besides, in *S. cerevisiae*, Aft1 is a transcription factor involved in iron homeostasis and controls the expression of high-affinity iron uptake genes. It has been shown that TORC2 (Target Of Rapamycin Complex 2)-Ypk1 (AGC family protein kinase) signaling

pathway and the biosynthesis of sphingolipids, such as IPCs and precursors LCBs and Cers, are required for Aft1 nuclear localization. When TORC2-Ypk1 is impaired, the deficiency of Aft1 nuclear localization and the impaired transcriptional response in the absence of iron, are partially suppressed by exogenous addition of the LCB DHS. Thus, the sphingolipid pathway mediated by TORC2/Ypk1 proteins are important in the cellular response to iron deprivation (Montellà-Manuel et al., 2023). Finally, the deletion of the calcium release channel Csg2, under amino acid or glucose starvation conditions in *S. cerevisiae*, increases calcium levels in the endoplasmic reticulum. This disturbs the stability of IPC synthase and leads to the accumulation of the bioactive sphingolipid PHS, which specifically and completely blocks autophagy and induces loss of starvation resistance. PHS may represent a nutrient signal that provides growth information to TORC1, leading to TORC1 activation and subsequent suppression of autophagy (Liu et al., 2023).

With respect to hypoxia, a low level of oxygen in *C. albicans* leads to an increase in several lipid classes, including sphingolipids. This is due to a dynamic reprogramming of many fundamental metabolic pathways, such as glycolysis (Burgain et al., 2020). Furthermore, in a recombinant strain of *P. pastoris*, the sphingolipid pathway is highly up-regulated under hypoxic conditions. In particular, an increase in Cers with methylated moieties is observed, while the amount of Cers with di- and tri-unsaturated LCB moieties decreases. Hypoxia also affects inositol-containing sphingolipids, reducing the relative amounts of C42 species and favoring the content of C44 species. In addition, reduced amounts of ergosterol and inositol are observed. Taken together, these results suggest a potential mechanism by which cells adapt to a state of ergosterol and inositol deprivation under hypoxic conditions, namely through an increase in sphingolipid species with longer fatty acid chains (Adelantado et al., 2017).

## 4.5. *Osmotic and drought stresses*

An involvement of sphingolipids has also been shown in fungal osmotic stress tolerance (Table 3). Indeed, myriocin treatment in *S. boydii* results in cells with punctual membrane alterations that are more susceptible to 5 % NaCl, indicating that the reduction of the sphingolipid content leads to a more fragile plasma membrane. On the contrary, PPMP (inhibitor of the GlcCer synthesis), which also affects membrane integrity, does not alter NaCl sensitivity. These results suggest that each inhibitor acts differently on the susceptibility of the plasma membrane to NaCl stress (Rollin-Pinheiro et al., 2019). In addition, an increase in cell survival rate is observed at 1.0 M NaCl in the ELO2 (involved in the biosynthesis of VLCFAs) overexpressing strain of *S. cerevisiae*. An analysis of complex sphingolipids has revealed that the levels of IPC t18:0/26:0, MIPC t18:0/22:0(2OH), d18:0/22:0, d20:0/24:0, M(IP)₂C d20:0/26:0, t18:0/26:0(2OH), and d20:0/26:0(2OH) are higher. This suggests that complex sphingolipids with longer acyl chains, may enhance membrane integrity and raft structure to promote osmotic stress tolerance (Zhu et al., 2020).

Some research also reports a negative role for the sphingolipid pathway in salt stress adaptation. For instance, it has been reported that the DhCer Δ4-desaturase 1 mutant of *M. oryzae* has a higher growth rate than the wild type and the complemented strain under osmotic stress (0.7 M NaCl). The phosphorylation level of Osm1 remains elevated in the mutant after treatment. The Osm1 MAPK pathway has been identified as a critical mediator of hyperosmotic stress adaptation in *M. oryzae*. Taken together, these observations suggest that DhCer Δ4-desaturase 1
has a regulatory effect on hyperosmotic stress resistance in *M. oryzae* (Wang et al., 2024).

A recent paper, on the ectomycorrhizal fungus *Cenococcum geophilum*, also reports an involvement of sphingolipids in drought stress. Indeed, drought-tolerant strains respond to polyethylene glycol-induced drought stress, by upregulating genes encoding enzymes of the sphingolipid metabolic pathway, such as sphingosine hydroxylase and ceramidase. Therefore, the sphingolipid metabolism is an important pathway for *C. geophilum* to alleviate drought stress (Li et al., 2023).

### 4.6. Interplay of sphingolipids with antifungal drugs

Recently, sphingolipids have also been shown to be involved in sensitivity to antifungal drugs targeting ergosterol. For example, in *C. albicans*, myriocin enhances the activity of the ergosterol biosynthesis inhibitor fluconazole. This synergistic effect occurs by blocking the membrane localization of the efflux pump Cdr1, which is involved in the detoxification of fluconazole (Wang et al., 2022). Similarly, in *S. cerevisiae* expressing the *C. albicans* efflux pump Cdr2, homologous to Cdr1, the IPC synthase inhibitor AbA enhances the activity of fluconazole (Rollin-Pinheiro et al., 2023). Pasrija et al. (2008) have shown, in sphingolipid and ergosterol biosynthesis mutants, that Cdr1 is selectively recruited to lipid rafts. Fluconazole sensitivity is then linked to the localization of the efflux pumps in rafts via sphingolipids.

In addition, sublethal concentrations of myriocin increase the susceptibility of *C. auris* to amphotericin B (Stieber et al., 2024). Amphotericin B is an antifungal compound that binds to membrane ergosterol (Laniado-Laborín and Cabrales-Vargas, 2009). Similarly, the inhibition of the IPC synthase by AbA increases the effect of amphotericin B on *C. neoformans*. This is due to a better affinity of IPC than amphotericin B for ergosterol, leading to a competition (Chen et al., 2024). Therefore, this new knowledge could improve the efficacy of antifungal treatments against drug resistant pathogens.

## 5. Conclusion

In conclusion, sphingolipids are highly diverse compounds. Complex glycosphingolipids are key components in fungal cell physiology due to their role in the plasma membrane, particularly in domain formation, and LCB/Cers are particularly involved in cell signaling. Sphingolipids influence various biological processes in fungi such as protein folding, protein anchoring and activity, vacuolar morphology, cell division, cell growth and death. They also have pivotal roles in cell adaptation to stress. Knowledge of sphingolipids will not only improve the understanding of fungal physiology, which will help to optimize future industrial applications, but will also help to find new potential targets for antifungal therapies in the context of emerging drug resistance. The synergistic antifungal activity observed between sphingolipid and sterol inhibitors are of interest for antifungal treatments. This could be applied to animal medicine and phytoprotection strategies. In addition, metabolic processes can be conserved in eukaryotic cells, even if the metabolism of differentiated cells grouped in dedicated organs is very different. Knowledge of the sphingolipid metabolism in fungi, especially in the model yeast *S. cerevisiae*, will provide insights into other eukaryotic metabolisms, including human metabolic disorders (e.g., the recent use of a yeast model of human galactosemia, suggests a role for the sphingolipid metabolism and the UPR in this disease). Finally, genome editing, the expansion of lipidomic databases, and the creation of new mathematical models of fungal sphingolipid metabolism, which could be used to assess the effects of stress, may allow to better study and/or predict the role of sphingolipids in all species.

## Funding

The pHD thesis of Thomas Bergia was funded by the French ministry of higher education and research (Ministère de l'Enseignement

Supérieur et de la Recherche, MESR).

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for this journal and was not involved in the editorial review or the decision to publish this article.

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