Ergosterol Biosynthesis and Regulation Impact the Antifungal Resistance and Virulence of Candida spp.

Abstract

Ergosterol is a key fungal sterol that is mainly found in the plasma membrane and is responsible for the proper membrane structure, rigidity, permeability and activity of membrane proteins. Ergosterol plays a crucial role in the ability of fungi to adapt to environmental stresses. The biosynthesis of ergosterol is also intimately connected with the antifungal resistance and virulence of pathogenic fungi. The most common etiological agents of life-threatening fungal infections are yeasts belonging to the genus Candida. The antifungal agents mostly used to treat Candida spp. infections are azoles, which act as competitive inhibitors of sterol demethylase, a key enzyme in the fungal ergosterol biosynthetic pathway. Although most studies on ergosterol biosynthesis, its regulation and the uptake of sterols are from the baker’s yeast Saccharomyces cerevisiae, the study of ergosterol biosynthesis and its relationship to antifungal drug resistance and virulence in pathogenic fungi is of utmost importance. The increasing antifungal drug resistance of Candida spp. and the limited armamentarium of antimycotics pose a challenge in the development of new therapeutic approaches. This review summarizes the available data on ergosterol biosynthesis and related phenomena in Candida albicans and non-albicans Candida species (Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida auris) with special emphasis on C. albicans and C. glabrata as the most common etiological agents of systemic candidiasis.

1. Introduction

Sterols are essential constituents of eukaryotic membranes responsible for membrane integrity, fluidity and permeability [1,2,3,4]. They are mainly present in the plasma membrane and contribute to the proper distribution and activity of proteins, which are often located in specific microdomains of the plasma membrane [5,6,7].

Sterol biosynthetic pathways in fungi contain phylum-specific branches with distinct end products. Although ergosterol is the dominant sterol in Ascomycota and Basidiomycota, fungi also produce different types of sterols such as 24-methyl-cholesterol in Entomophthorales, 24-ethyl-cholesterol in Glomeromycota, brassicasterol in Taphrinales or cholesterol in Pneumocystis [8].

The presence of ergosterol in the plasma membrane contributes to fungal resistance to diverse environmental stresses, e.g., high temperature, osmotic stress, oxidative stress, cell wall stress, low pH or ethanol [9,10,11]. Ergosterol is also involved in the regulation of the yeast cell cycle, a function known as “sparking” [12]. The most common antifungal drugs used in medicine and agriculture target ergosterol or its biosynthesis (azoles, polyenes or allylamines) [13,14]. Moreover, the antifungal resistance and virulence of pathogenic fungi are closely intertwined with the ergosterol biosynthesis and the sterol composition of the plasma membrane [15,16].

Candida spp. are among the most common causes of fungal infections in humans, ranging from mild oral and vaginal infections to severe invasive disease [17,18,19]. The majority of invasive infections are caused by Candida albicans, followed by Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei, collectively known as non-albicans Candida (NAC) yeasts. Based on geographic location, C. albicans, C. glabrata and C. parapsilosis, represent the three major pathogenic Candida species, which are relatively distant from each other phylogenetically, morphologically and genetically [19,20]. In recent years, a new multidrug-resistant yeast Candida auris has emerged [19]. Fluconazole and other triazoles are among the most commonly used antifungal agents for the treatment of infections with Candida spp. Currently, only two other classes of antifungals (polyenes and echinocandins) are available for the treatment of invasive fungal infections. In recent decades, resistance to all three antifungal classes has been on the rise, especially in NAC yeast species [17,21].

In this review, we briefly describe the ergosterol biosynthetic pathway and highlight specific aspects of ergosterol biosynthesis in Candida spp. with special emphasis on its regulation and relevance for antifungal resistance and virulence. The focus is on C. albicans and C. glabrata as the leading pathogens of invasive candidiasis worldwide.

2. Sterol Biosynthesis

The ergosterol molecule is composed of four rigid rings. It contains a hydrophilic hydroxyl group at carbon 3 and a hydrophobic aliphatic tail at carbon 17 [22]. The synthesis of an ergosterol molecule requires a lot of energy (at least 24 molecules of ATP), the presence of oxygen and two cofactors, NADPH and iron [23].

The biosynthesis of ergosterol can be divided into three distinct modules, each of which generates typical end products. The reactions of the first and second module of sterol synthesis are localized in the mitochondria and vacuoles, respectively. The first module leads to the production of mevalonate, the second module results in the formation of farnesyl pyrophosphate (FPP). FPP is also required for the metabolism of dolichol, heme, ubiquinone or prenylated proteins. The third module involves a series of successive reactions generating different sterol structures. In contrast to the previous modules, the final part of ergosterol biosynthesis occurs in the endoplasmic reticulum [23,24,25]. Differences in the enzymatic make-up of sterol biosynthesis in mammals, plants or fungi lead to the creation of cholesterol, phytosterols or ergosterol [3,23]. Ergosterol biosynthesis in Candida spp. resembles the pathway described in Saccharomyces cerevisiae [26]. The third module of ergosterol biosynthesis (Figure 1) begins with the production of squalene, continues with its cyclization to lanosterol and leads to the formation of several intermediates: 4,4-dimethylcholesta-8,14,24-trienol, 4,4-dimethylzymosterol, zymosterol, fecosterol, episterol, ergosta-5,7,24(28)-trienol, ergosta-5,7,22,24(28)-tetraenol and finally ergosterol is formed [11,24,25]. The disruption of individual enzymatic reactions in the third module leads to the accumulation of a mixture of different ergosterol precursors. Due to the low substrate specificity of the enzymes in the final steps of ergosterol biosynthesis, the enzymes can accept a wide range of sterols with similar structures [25]. The list of enzymes involved in the third module of ergosterol synthesis, their corresponding genes and their counterparts in Candida spp. are summarized in

Table 1. Genes involved in the third module of sterol biosynthesis, encoded enzymes and their counterparts in C. albicans, C. glabrata and C. parapsilosis.

Gene	C. albicans Gene ID	C. glabrata Gene ID	C. parapsilosis Gene ID	Predicted/Confirmed Function
ERG9	C2_08610W_A [37]	CAGL0M07095g [38]	CPAR2_406760 *	squalene synthase
ERG1	C1_08590C_A [39]	CAGL0D05940g [40]	CPAR2_210480 *	squalene epoxidase
ERG7	C2_02460W_A [37]	CAGL0J10824g [41]	CPAR2_301800 *	lanosterol synthase
ERG11	C5_00660C_A [32]	CAGL0E04334g [29]	CPAR2_303740 [42]	lanosterol 14α-demethylase
ERG24	C2_09400C_A [43]	CAGL0I02970g *	CPAR2_405900 *	C-14 sterol reductase
ERG25	CR_02370W_A [44]	CAGL0K04477g [41]	CPAR2_801410 *	C-4 methyl sterol oxidase
ERG26	C4_06270C_A [45]	CAGL0G00594g [41]	CPAR2_302110 *	C-3 sterol dehydrogenase
ERG27	CR_01140C_A [46]	CAGL0M11506g [41]	CPAR2_801560 *	3-keto sterol reductase
ERG28	C2_01090C_A *	CAGL0J02684g *	CPAR2_213750 *	scaffold activity
ERG6	C3_02150C_A [30]	CAGL0H04653g [33]	CPAR2_405010 *	C-24 sterol methyltransferase
ERG2	C1_00800C_A [47]	CAGL0L10714g [48]	CPAR2_201490 *	C-8 sterol isomerase
ERG3	C1_04770C_A [31]	CAGL0F01793g [29]	CPAR2_105550 [49]	C-5 sterol desaturase
ERG5	C7_02840C_A [36]	CAGL0M07656g *	CPAR2_703970 *	C-22 sterol desaturase
ERG4	C3_00760W_A [50]	CAGL0A00429g *	CPAR2_502980 *	C-24 sterol reductase

* Systematic name of the orthologous genes from Candida Genome Database [51].

.

However, it is worth mentioning that specific steps of the ergosterol biosynthetic pathway have also been described in other fungal species. The four-ring triterpene lanosterol, is a common sterol intermediate of various fungal species [52,53]. In yeasts, the main reactions form a zymosterol branch, shortly described above. In filamentous fungi, however, an eburicol branch of ergosterol biosynthesis is found. In the zymosterol branch, repeated demethylation of lanosterol leads to the formation of zymosterol, and subsequently, zymosterol is methylated by C-24 sterol methyltransferase (ERG6) at the C-24 position to form fecosterol. In the eburicol branch, eburicol is first formed by the methylation of lanosterol by C-24 sterol methyltransferase at the C-24 position, and then the C-14 and C-4 methyl groups are removed from lanosterol. Finally, fecosterol is synthesized as the final product of the enzymatic reactions of both branches [52].

The characterization of the C-24 sterol methyltransferase in Mucor lusitanicus pointed to at least four alternative pathways for the biosynthesis of sterols in this organism [54]. In contrast to filamentous fungi, there is no direct experimental evidence for alternative ergosterol production pathways in yeasts. The presence of alternative ergosterol biosynthesis routes may potentially lead to failure of antifungal therapy. In addition, ergosterol biosynthesis in yeasts can be redirected following the inhibition of lanosterol 14α-demethylase (ERG11). Inhibition of lanosterol 14α-demethylase, either by drugs or due to a mutation, leads to ergosterol depletion and conversion of lanosterol into toxic 14α-methylergosta-8,24(28)-dienol. The accumulation of this diol is considered to be the main cause of the antifungal effect of azoles [27,55]. Re-routing the ergosterol biosynthesis of Candida to the production of toxic sterols could improve the efficacy of antifungal therapy. However, in yeast cells with ERG6 or ERG3 mutation/gene disruption, alternative sterols accumulate that are able to support cell growth [11,13]. This can lead to persistent or recurrent infections in immunocompromised patients and due to the narrow spectrum of antifungals, to an increased mortality rate in these patients. Alternatively, higher doses of antifungals (e. g., amphotericin B, which is known for its nephrotoxicity) may be required, which can be a significant challenge in patients with a severely debilitated immune system. An alternative sterol biosynthesis pathway is shown in Figure 2.

3. Transcription Factors Involved in Ergosterol Biosynthesis Regulation in Candida spp.

Inhibition of ergosterol biosynthesis either by mutations in ERG genes or by antifungal drugs leads to increased expression of several ergosterol biosynthesis genes in Candida yeast cells [28,57,58,59,60]. Upregulation of the ERG11 gene, which encodes a key enzyme of the fungal ergosterol biosynthetic pathway, was observed in the presence of azoles in C. albicans, C. glabrata, C. tropicalis, C. krusei and C. parapsilosis, indicating a conserved response to sterol biosynthesis inhibitors in pathogenic yeasts [57,61].

The major transcriptional regulators of ergosterol biosynthesis in Candida spp. are homologs of the zinc cluster transcription factors Upc2p and Ecm22p of S. cerevisiae [62,63]. Ergosterol biosynthesis in S. cerevisiae is also controlled by the products of the ROX1, MOT3 and HOG1 genes [64].

The N-terminus of Upc2p contains a nuclear localization signal followed by a DNA-binding domain. The C-terminus is a regulatory domain, which contains a sterol-binding domain. Binding of ergosterol negatively affects the transcriptional activation of the UPC2 gene. Upc2p activates the expression of ERG genes by binding to the sterol-regulatory element (SRE) in the promoters of the regulated genes [65,66]. Tan et al. [67] reported a key role of the molecular chaperone Hsp90p in Upc2p activation and showed that in the presence of enough ergosterol, Upc2p is associated with Hsp90p and retains its inactive cytosolic form. Ergosterol dissociation from Upc2p induces a conformational change of its C-terminus, Hsp90p is released from Upc2p, and the transcription factor can be transported into the nucleus [67].

C. albicans possess a single homolog of the S. cerevisiae UPC2 and ECM22 genes named CaUPC2, which encodes a central transcriptional regulator of ergosterol biosynthesis in C. albicans [68,69,70]. CaUPC2 gene deletion leads to an inability to upregulate ergosterol biosynthesis genes, leading to reduced ergosterol levels in cells [68,69,71]. On the contrary, gain-of-function mutations in the CaUPC2 gene lead to increased expression of CaERG11 [70,72] and an increased cellular ergosterol content [72].

The biosynthesis of ergosterol in C. glabrata is also regulated by homologs of the S. cerevisiae UPC2 and ECM22 genes [73,74]. A study by Nagi et al. [73] identified two homologous genes in C. glabrata designated as CgUPC2A and CgUPC2B. The induction of ERG genes by lovastatin or serum was severely impaired in strains lacking the CgUPC2A gene [73]. Deletion of the CgUPC2A gene also leads to the downregulation of several genes involved in ergosterol biosynthesis and a decreased content of ergosterol in C. glabrata cells [74]. The obtained results showed that the transcriptional regulator CgUpc2Ap plays a crucial role in the transcriptional regulation of ergosterol biosynthesis in C. glabrata [73,74].

However, further studies on Candida spp. revealed, in addition to Upc2p, the presence of other transcriptional factors contributing to the regulation of ergosterol biosynthesis. The transcription factor CaNdt80p binds directly to the promoters of several ERG genes in C. albicans, including CaERG3, CaERG4, CaERG6, CaERG7, CaERG11, CaERG24 and CaERG25. CaNdt80p is also required for the transcriptional activation of ERG genes in response to fluconazole treatment [75]. Another transcription factor with a suggested role in the regulation of ergosterol biosynthesis in C. albicans is CaEfg1p, a regulator of the morphogenetic transformation from yeast into hyphae [76]. The CaERG3 and CaERG11 genes are negatively or positively regulated by CaEfg1p, respectively [76,77]. Moreover, the Caefg1Δ deletion mutant showed increased susceptibility to drugs targeting ergosterol or its biosynthesis [76]. Zheng et al. [78] showed that deletion of the CaTOP2 gene leads to the upregulation of CaERG11 gene expression and increased ergosterol biosynthesis. A recent study pointed out the role of the CaRpn4p transcription factor in C. albicans ergosterol biosynthesis [79]. Yau et al. [79] reported that the function of CaRpn4p is required for mitigating the inhibition of ergosterol biosynthesis by fluconazole. ERG genes are strongly upregulated in the C. albicans Cazap1Δ deletion mutant, suggesting that CaZap1p is a negative regulator of ERG genes in C. albicans [80].

Ollinger et al. [81] showed that CgROX1 gene deletion in the Cgupc2AΔ mutant strain leads to a significant increase in the cellular ergosterol content. The product of the CgROX1 gene is thus a negative regulator of ERG genes in C. glabrata. The authors also predict the existence of an additional positive regulator of ERG genes in C. glabrata, which is not yet known [81]. Pais et al. [82] showed that the transcription factor CgRpn4p plays a role in the regulation of several ergosterol biosynthesis genes (CgERG1, CgERG2, CgERG3 and CgERG11) in C. glabrata in the presence of fluconazole. Deletion of the CgRPN4 gene leads to a significant decrease in the ergosterol content upon early fluconazole exposure. CgRpn4p also binds directly to the CgERG11 gene promoter through the TTGCAAA binding motif [82]. CgZap1p, a transcription factor normally involved in the C. glabrata adaptation to zinc deficiency, negatively affects the expression of the CgERG3 and CgERG5 genes in the presence of fluconazole. Fluconazole treatment caused a more pronounced depletion of ergosterol in the Cgzap1Δ deletion mutant (which exhibits increased fluconazole sensitivity) compared with that in the parental strain [83].

In addition, the SAGA complex is proposed to be involved in the co-regulation of ergosterol biosynthesis [84,85]. The SAGA complex is an evolutionarily conserved transcriptional coactivator that regulates gene expression through its histone acetyltransferase and deubiquitinase activities, recognition of specific histone modifications and interactions with transcription factors [86]. Disruption of the CgADA2 gene, one of the components of the SAGA complex, leads to pleiotropic phenotypes in C. glabrata.

The Cgada2Δ deletion mutant showed increased susceptibility to amphotericin B, an ergosterol-binding antifungal agent, pointing to changes in the sterol composition of the Cgada2Δ strain [87]. Another study [88] showed that CgSet4p acts as a repressor of ERG genes via transcriptional downregulation of the CgUPC2A gene. CgSET4 encodes a stress-responsive and chromatin-associated protein. CgSET4 gene deletion resulted in increased expression of genes involved in the late steps of ergosterol biosynthesis and an elevated cellular ergosterol content. RNA-sequencing of CgSET1, which encodes histone H3K4 methyltransferase, revealed that it plays a role in azole-induced expression of all genes involved in the late ergosterol biosynthesis pathway [89]. A schematic representation of the regulation of ERG genes in Candida spp. is shown in Figure 3.

Histone modifications, such as acetylation or methylation, also influence resistance to antifungal drug in Candida spp. [90,91,92]. In C. glabrata, disruption of the CgRPH1 gene, which encodes a putative histone demethylase, leads to increased susceptibility to fluconazole and decreased basal expression of CgPDR1 (the main transcription factor in yeasts multidrug resistance) and CgCDR1 (encoding the efflux pump) [92]. Moreover, inhibition of CgGcn5p with histone acetyltransferase activity minimized the emergence of CgPDR1 gain-of-function mutations in C. glabrata, suggesting the importance of histone modifications in both the maintenance and acquisition of azole resistance [90]. These findings point to the connection between the ergosterol biosynthesis regulation and chromatin modifications and show that chromatin modifications are important for azole tolerance in Candida spp.

Surprisingly, the function of CgUpc2Ap extends beyond the regulation of ergosterol biosynthesis [93,94]. Recent results of Vu et al. [93] point to the link between ergosterol biosynthesis and CgPdr1p-mediated drug efflux in C. glabrata. Inhibition of the ergosterol biosynthetic pathway leads to the activation of CgPdr1p by the transcription factor CgUpc2Ap, which has been shown to bind directly to the promoters of the CgPDR1 and CgCDR1 genes [93].

In C. parapsilosis, the products of the CpUPC2 and CpNDT80 genes are also involved in the positive regulation of ERG gene expression. Disruption of the CpUPC2 gene resulted in reduced ERG gene expression [49].

Deletion of the UPC2 gene increased susceptibility of yeast cells to azoles and other drugs targeting sterol biosynthesis (terbinafine, fenpropimorph and lovastatin) in various Candida spp. [49,68,69,71,73,74].

Ergosterol biosynthesis can also be modulated by iron concentration and the presence of oxygen [95,96]. Several ERG enzymes (Erg11p, Erg25p, Erg3p and Erg5p) require iron as a cofactor. Therefore, iron availability directly influences the activity of these enzymes and the flux through ergosterol biosynthesis. Reduced iron availability can lead to a decrease in the ergosterol concentration in Candida cells and impaired virulence. Iron deprivation leads to a decreased membrane ergosterol content, a significant increase in membrane fluidity and enhanced passive diffusion of drugs in Candida cells. The expression of ERG11, ERG1, ERG2 and ERG25 genes was considerably downregulated under iron-limited conditions. On the other hand, upregulated expression of the ERG3 gene was observed in iron-deprived cells [95,97]. A strain of C. glabrata lacking the heme-binding protein CgDap1p displayed growth defects under iron-limited conditions, decreased production of ergosterol, increased accumulation of 14α-methyl sterols, including lanosterol and squalene, and increased azole susceptibility. Hosogaya et al. [96] proposed that CgDap1p post-transcriptionally modifies CgErg11p in C. glabrata.

Hypoxia induces the expression of the main transcriptional regulator Upc2p and genes involved in ergosterol biosynthesis in C. albicans, C. glabrata and C. parapsilosis [60,98,99,100]. In a previous study, MacPherson et al. [69] showed that an Caupc2Δ deletion strain is unable to grow under anaerobic conditions. The transcriptional factor CgUpc2Ap is essential for the growth of C. glabrata under hypoxic conditions [81]. Hypoxia also induces the import of exogenous sterol. Sterols are imported into yeast cells via plasma membrane transporters belonging to the ABC family and are regulated by Upc2p [60].

4. Sterol Profiles of C. albicans and C. glabrata Ergosterol Biosynthesis Mutants

Several laboratories have studied the ergosterol biosynthesis pathway in C. albicans and C. glabrata. Deletion of ERG genes usually leads to the absence of ergosterol in the mutant cells. The loss of function of enzymes involved in ergosterol biosynthesis leads to the accumulation of various ergosterol precursors [29,30,31,41]. ERG genes are categorized as essential and non-essential. Various sterol molecules can be accumulated as a result of the deletion of non-essential ERG genes and incorporated into the plasma membrane to ensure some degree of the critical functions of the plasma membrane. Similar to S. cerevisiae, the first enzyme of the ergosterol biosynthesis module, the squalene synthase encoded by the ERG9 gene, has been shown to be essential in C. glabrata in vitro. However, the absence of squalene synthase did not block the growth of C. glabrata in mice, probably due to the ability of the host cholesterol to complement the defect in the ergosterol pathway [38]. C. albicans CaERG9, CaERG7 (lanosterol synthase) and C. glabrata CgERG7 genes were also found to be essential [37,41].

ERG1 (squalene epoxidase) seems to be essential for both C. albicans [39] and C. glabrata [40]. Functional characterization of the Caerg1Δ deletion mutant revealed that the absence of squalene epoxidase activity leads to an accumulation of squalene and a complete lack of ergosterol [39]. Inactivation of the CgERG1 gene in C. glabrata by transposon mutagenesis also led to an increase in the cellular amount of squalene and a lower ergosterol content [40]. The null mutant of S. cerevisiae Scerg1Δ is unable to grow aerobically but is viable under anaerobic conditions in the presence of exogenous ergosterol [101].

ERG11, encoding lanosterol 14α-demethylase, is usually considered essential [32,37]. The accumulation of 14α-methylergosta-8,24(28)-dienol in cells with a mutation in ERG11 is thought to be the main reason for the lethality of erg11Δ mutants. It is also proposed that ERG11 deletion is only possible in the presence of non-functional ERG3 gene [31,32] or in media supplemented with ergosterol under anaerobic conditions [29]. However, there are few reports demonstrating viable erg11Δ deletion mutants in C. albicans and C. glabrata, even in the absence of CaERG3/CgERG3 gene mutation [5,29,31,32]. The observed differences in the viability of erg11Δ deletion mutants could be due to a different susceptibility of the yeast cells to the lethal effect of the diol formed. Bard et al. [32] suggested that C. albicans can sequester toxic diols in an esterified form. In contrast to in vitro conditions, a conditional mutant of C. glabrata in the CgERG11 gene was viable with no growth defect observed in mice [102]. The sterol intermediates that accumulate in C. albicans cells lacking lanosterol 14α-demethylase activity are mainly lanosterol, obtusifoliol, 24-methylene lanosterol, 14α-methylfecosterol, 4,14-dimethylzymosterol and 14α-methylergosta-8,24(28)-dienol [5,31,32]. Sterol analysis of the C. glabrata Cgerg11Δ deletion mutant confirmed the accumulation of 14α-methylsterols (lanosterol and obtusifoliol) as major sterols and a minor amount of 4, 14-dimethylzymosterol [29].

In contrast to S. cerevisiae, the CaERG24 gene (C-14 sterol reductase) is not essential in C. albicans [43]. The deletion of the CaERG24 gene in C. albicans leads to the production of ignosterol and a minor amount of ergosta-8,14,22-trienol, which was only detected in the C. albicans Caerg24Δ deletion mutant [43].

The genes ERG25 (C-4 methylsterol oxidase), ERG26 (C-3-sterol dehydrogenase) and ERG27 (3-keto-sterol reductase) are essential in both S. cerevisiae and C. albicans [44,45,46]. The CgERG25 and CgERG26 genes of C. glabrata also appear to be essential for cell growth [41]. Candida spp. with a disturbed function of the C-4 methylsterol oxidase produce a significantly elevated amount of 4, 4-dimethylzymosterol [41,44]. Cells lacking the ERG27 gene exhibit a complete loss of both 3-keto-sterol reductase (ERG27) and lanosterol synthase (ERG7) activities, pointing to the interaction of both enzymes [46,103]. As a result, the C. albicans Caerg27Δ mutant, similar to S. cerevisiae, accumulates non-sterol intermediates like squalene, squalene epoxide and squalene dioxide [46].

Based on the viability of deletion mutants in the genes CaERG2 (C-8 sterol isomerase) [47], CaERG6 (C-24 sterol methyltransferase) [30] or CaERG3 (C-5 sterol desaturase) [31] in C. albicans and CgERG3 [29] and CgERG6 [33] in C. glabrata, these genes are considered non-essential, similar to S. cerevisiae [9,10,11].

C. albicans and C. glabrata erg6Δ deletion mutants accumulate sterols without C-24 methylation in the side chain [30,33], similar to the S. cerevisiae Scerg6Δ deletion mutant [104]. In accordance with the role of C-24 sterol methyltransferase in ergosterol biosynthesis, the predominant sterols in erg6Δ mutants are zymosterol, cholesta-5,7,24-trienol, cholesta-7,24-dienol and cholesta-5,7,22,24-tetraenol [30,33]. Interestingly, the Cgerg6Δ deletion mutant is not completely devoid of ergosterol [33]. Abrogation of C. lusitaniae ClERG6 gene function resulted in a 50% reduction in the ergosterol content in the mutant compared to the wild type [56]. Based on these observations, we propose that ergosterol biosynthesis in some Candida spp. occurs via an alternative pathway that is responsible for the synthesis of a minor amount of ergosterol. The presence of an alternative C-24 sterol methyltransferase in the genome of some Candida yeasts may resemble the situation in the genus Leishmania [105,106]. Leishmania major has two C-24 sterol methyltransferase orthologs identified as a tandem repeat on chromosome 36, namely SMT80 and SMT90. Since both SMTs are required for the synthesis of ergostane-based sterols, only SMT80 of L. major has a significant effect on cellular sterol composition [105]. However, the presence of alternative genes for ergosterol biosynthesis in Candida cells remains speculative.

Deletion of the C. albicans CaERG2 gene results in the accumulation of ergosta-5,8,22-trienol, fecosterol and ergosta-8-enol in the mutant cells [28].

Several studies have focused on the function of the CaERG3 gene, which encodes the C-5 sterol desaturase [27,31,34,35,55]. The C. albicans Caerg3Δ deletion mutant with diminished C-5-sterol desaturase activity accumulates several ergosterol precursors: ergosta-7,22-dienol, ergosta-7-enol, as well as fecosterol and episterol [27,31,34,35]. The Cgerg3Δ deletion mutant of C. glabrata mainly accumulates ergosta-7,22-dienol and to a lesser extent, ergosta-8,22-dienol and ergosta-7-enol [29]. A recent study by Luna-Tapia et al. [55] demonstrated that C. albicans CaERG3 gene deletion can be at least partially complemented by homologous sequences of the C. glabrata or C. auris ERG3 genes. Despite the ability of ERG3 gene orthologs to restore ergosterol biosynthesis in C. albicans cells, these findings point to the species-specific properties of C-5 sterol desaturase in different Candida spp. [55].

The absence of ergosterol and the accumulation of various sterol precursors have profound effects on yeast physiology and plasma membrane properties.

5. Ergosterol Influence on Membranes and Organelles of Candida spp.

Ergosterol is the predominant sterol in the yeast plasma membrane and influences its biophysical properties such as permeability, fluidity and stability [1]. However, ergosterol is also present in other membranous organelles, such as mitochondria [107]. The lipid composition (mainly sterols and sphingolipids) of the plasma membrane is also responsible for the proper integration and activity of membrane proteins, including multidrug transporters [5,6,108,109].

Cdr1p belongs to the ABC (ATP-binding cassette) family of transporters that use the energy of ATP for substrate transport across the plasma membrane. The increased efflux activity of ABC transporters is considered to be the most common azole resistance mechanism in Candida spp. [13]. Alterations in ergosterol levels may affect the proper folding, assembly or surface localization of efflux pumps. Disruption of plasma membrane organization caused by ergosterol depletion and accumulation of its precursors is likely responsible for inadequate integration of CaCdr1p into the plasma membrane and reduced efflux activity of the pump [5,6,39,110,111]. Suchodolski et al. [5] showed that in the C. albicans Caerg11Δ deletion mutant, the CaCdr1p efflux pump is mislocalized to the vacuole. However, CaMdr1p, which belongs to the MFS (major facilitator superfamily) transporters, did not show any sorting or activity defects when expressed in S. cerevisiae ergosterol deletion mutants. This fact points to different lipid specificities of the multidrug transporters in C. albicans [111].

The lack of ergosterol in the plasma membrane of the Caerg11Δ deletion mutant is also responsible for the mislocalization of the CaPma1p plasma membrane H+-ATPase to the vacuole, leading to its decreased activity [5,6].

In a recent study by Okamoto et al. [41], it was shown that the CgERG25 gene may play an important role in stabilizing sterol-rich lipid domains in the plasma membrane of C. glabrata. The correct plasma membrane localization of the CgAus1p sterol importer depends on the presence of the CgERG25 gene [41]. CgAus1p is important for antifungal azole resistance in C. glabrata [112,113].

Changes in the sterol composition also influence the biophysical properties of the plasma membrane. A study by Suchodolski et al. [5] showed that the deletion of the C. albicans CaERG11 gene leads to reduced plasma membrane fluidity and decreased plasma membrane potential. The changes in plasma membrane potential could be the result of decreased activity of the CaPma1p plasma membrane H+-ATPase [5]. Reduced plasma membrane fluidity has also been described in miconazole-treated C. albicans cells [114]. The absence of the CgERG6 gene also impairs the integrity of the C. glabrata plasma membrane. Disruption of the C. glabrata CgERG6 gene leads to plasma membrane hyperpolarization and reduced membrane fluidity [33].

Membrane fluidity, which is strongly influenced by sterol composition, can affect the passive diffusion of molecules in yeast cells [115]. Diminished squalene epoxidation leads to increased rhodamine 6G or [3H] fluconazole intracellular accumulation in both C. glabrata and C. albicans [39,40]. Increased passive diffusion, as a result of leakage in the plasma membrane could contribute to the increased susceptibility of erg deletion mutants to antifungal drugs [108,115].

The lack of cellular ergosterol also affects the function of vacuoles [5,47] and mitochondria [5]. Defects in vacuolar fusion and reduced intracellular ATP levels have been found in the Caerg11Δ deletion strain [5]. Changes in vacuole morphology and shared stress phenotypes with vacuole-deficient mutants were also observed in the C. albicans–Caerg24Δ and Caerg2Δ deletion mutants [47].

The studies of various erg mutants of C. albicans and C. glabrata revealed a close relationship between intact ergosterol biosynthesis and antifungal drug susceptibility [5,29,31,39,40]. C. albicans Caerg1Δ mutant exhibits increased susceptibility to azoles, terbinafine, cycloheximide and, surprisingly, also to nystatin and amphotericin B [39]. The absence of the CgERG1 gene rendered the C. glabrata cells more susceptible to azoles and terbinafine. A slight decrease in amphotericin B susceptibility was observed [40]. Disruption of the ERG11 gene resulted in antifungal azole and amphotericin B resistance in both C. albicans and C. glabrata [5,29,31]. On the other hand, the Caerg11Δ deletion strain exhibits increased susceptibility to metabolic inhibitors, such as cycloheximide, fluphenazine, brefeldin A and hygromycin B [31]. The intact C. albicans CaERG11 gene is also required for growth in the presence of cell wall- and plasma membrane-damaging agents [116].

Although the Caerg24Δ strain shows significantly higher susceptibility to terbinafine [43,47] and various metabolic inhibitors (cycloheximide, cerulenin, fluphenazine and brefeldin A) the C. albicans cells exhibit slight resistance to azoles, nystatin and also to caspofungin, a cell wall biosynthesis inhibitor [43,47].

Deletion of the CaERG6 gene in C. albicans resulted in hypersusceptibility to a variety of metabolic inhibitors, namely fluphenazine, cycloheximide, cerulenin, brefeldin A, terbinafine and morpholines [30]. Surprisingly, no increase in susceptibility to azoles was observed [30]. This fact is in stark contrast to the observations in S. cerevisiae and C. glabrata erg6Δ deletion mutants, which showed decreased susceptibility to azoles as a result of ERG6 gene deletion [11,33,117]. The lack of CgErg6p enzyme activity influences also C. glabrata susceptibility to cell wall integrity inhibitors and calcineurin signaling [33]. Susceptibility to fluconazole, terbinafine and fenpropimorph has been reported for the C. lusitaniae Clerg6Δ deletion strain [56]. A common feature of C. albicans, C. lusitaniae and C. glabrata ERG6 gene deletion mutants is polyene resistance [30,33,56].

A C. albicans strain lacking the CaERG2 gene shows increased susceptibility to fenpropimorph but not to amorolfine, tridemorph or terbinafine. On the other hand, the Caerg2Δ mutant is resistant both to amphotericin B and caspofungin [47].

Disruption of the CaERG3 gene leads to reduced susceptibility to azoles in C. albicans [31,118,119], but the cells remain susceptible to metabolic inhibitors such as cycloheximide, fluphenazine, brefeldin A and hygromycin B [31]. Surprisingly, no resistance to polyenes was observed in the Caerg3Δ deletion mutant [31]. Despite the antifungal azole resistance observed in vitro, a mutation in the CaERG3 gene, which causes inactivation of C-5 sterol desaturase, did not lead to fluconazole resistance in vivo [34]. Disruption of C. parapsilosis CpERG3 in both susceptible and resistant clinical isolates resulted in a loss of sterol desaturase activity and, simultaneously, a highly azole-resistant phenotype [120]. In contrast to previous observations, the cells of Cgerg3Δ and C. glabrata are not resistant to azoles [29]. The observed differences in the susceptibility of erg6Δ and erg3Δ deletion mutants of various yeast species to antifungal compounds point to possible minor differences in ergosterol biosynthesis of different yeast species.

A C. albicans mutant lacking the CaERG5 gene encoding C-22 sterol desaturase exhibits sensitivity to geldanamycin, a pharmacological Hsp90p inhibitor. It is hypothesized that the CaERG5 gene is a genetic interactor of CaHSP90 [121].

Taken together, changes in the susceptibility of erg deletion mutants to various drugs are thought to be due to altered efflux pump activities, enhanced passive diffusion and rearrangements of sterol metabolism [5,11,27,31].

6. Ergosterol Biosynthesis and Antifungal Resistance of Candida spp. Clinical Isolates

Changes in ergosterol biosynthesis are frequently observed in clinical isolates of Candida spp. that are resistant to antifungal azoles or polyenes [58,59,122,123,124].

The antifungal effect of azoles, the most widely used drugs for the treatment of Candida spp. infections, is based on the inhibition of ergosterol biosynthesis and the accumulation of ergosterol precursors [13,19]. Mutations in the ERG11 gene contribute to azole resistance in clinical isolates of C. albicans, C. parapsilosis and C. tropicalis [42,50,124,125,126]. On the other hand, CaERG11 gene overexpression is a common mechanism leading to azole resistance in C. albicans clinical isolates [50,127,128]. However, mutations or overexpression of the CgERG11 gene do not appear to be an important mechanism of azole resistance in C. glabrata [129]. C. auris, first isolated in 2009, has evolved into a highly multidrug resistant pathogen. Azole resistance in C. auris has been associated with substitution mutations in the ERG11 gene [130,131,132,133,134,135].

Overexpression of the CauERG11 gene, as a consequence of chromosome duplication or increased copy number variations also corresponds to antifungal azole resistance in C. auris [132,136,137,138].

The antifungal effect of polyenes is the result of physical interaction between the ergosterol molecule and the polyene, which leads to the formation of pores in the plasma membrane and the leakage of cell contents. Accumulated ergosterol precursors are believed to have a lower affinity to polyenes [13,14]. The absence of ergosterol renders the yeast cells resistant for polyenes [48,58,59]. In contrast to the laboratory strain of C. glabrata deleted in the CgERG6 gene, clinical isolates carrying substitution mutations in the CgERG6 gene showed increased susceptibility to antifungal azoles [58,59]. The widespread molecular mechanism of resistance to amphotericin B in C. auris isolates involves the loss of ERG6 gene functions [139,140]. The cross-resistance of C. auris to amphotericin B and fluconazole has emerged as a result of point mutations in the ERG3 and ERG11 genes [136].

A substitution mutation in C. glabrata CgERG2 leads to the accumulation of ergosta-type sterols, like fecosterol, ergosta-5,8,22-trienol or ergosta-8-enol in clinical isolates. The absence of ergosterol also leads to concomitant resistance of the cells to amphotericin B [48,141]. The C. kefyr is recognized as an emerging fungal pathogen in intensive care units [142]. Candida kefyr CkERG2 gene is a major target conferring resistance to amphotericin B.

Mutations in the CaERG3 gene and inactivation of desaturase activity have been observed in some C. albicans azole-resistant clinical isolates [27,123]. Although the loss of the CaERG3 gene enables C. albicans to grow in the presence of azoles in the laboratory, the contribution of the loss of C-5 sterol desaturase activity to azole resistance in the clinical setting is less certain [118]. It is proposed that fitness defects, such as decreased virulence, may disfavor the selection of strains with a mutation in CaERG3 in patients [35]. A point mutation in the C. parapsilosis CpERG3 gene, causing complete loss of C-5 sterol desaturase activity, results in azole resistance and upregulation of ERG genes [49,120]. Azole-resistant C. glabrata clinical isolates usually do not contain mutations in the CgERG3 gene [143].

The role of the C. albicans CaERG5 and CaERG4 genes in the resistance of clinical isolates to azoles and amphotericin B has also been assessed [36,50]. Clinical isolates of C. albicans harboring mutation in the CaERG5 gene do not accumulate ergosterol and contain more than 80% of ergosta-5,7-dienol [36]. The intact ergosterol biosynthesis regulator Upc2p is also required for the azole resistance in Candida spp. clinical isolates [74,144,145].

In addition, exogenous sterol uptake, like serum cholesterol, can replace ergosterol biosynthesis in C. glabrata growing in host tissues. It is believed that cholesterol can replace ergosterol in the plasma membrane and partially restore the crucial functions of ergosterol in the plasma membrane (fluidity, permeability and activity of membrane bound enzymes/transporters). It is hypothesized that the compensation of azole-induced ergosterol loss by cholesterol contributes to azole resistance in C. glabrata clinical isolates [40,60,112]. In accordance with this, loss of the CgAUS1 gene in C. glabrata sensitized the pathogen to azoles [60].

7. Ergosterol Biosynthesis Modulations Affect the Virulence of Candida spp.

Biofilm formation is one of the most important virulence factors in Candida spp. Biofilm is associated with increased resistance to antifungals and chronic and recurrent infections [146]. Some studies point to the fact that ergosterol biosynthesis influences biofilm formation in C. albicans [147,148]. In addition, yeast cells associated with biofilm contain a lower concentration of ergosterol in the plasma membrane compared to planktonic cells [149]. The lower concentration of ergosterol in the plasma membrane of biofilm cells may limit the efficacy of antifungal drugs targeting ergosterol [150].

Several studies have shown transcriptional changes in genes involved in ergosterol biosynthesis in biofilms formed by diverse Candida spp. [98,151,152,153]. Up-regulation of different ERG genes has been reported in biofilm cells of C. albicans [152] and C. parapsilosis [98]. C. dubliniensis biofilm cells upregulate the genes CdERG25 and CdERG3 after treatment with fluconazole [151]. Moreover, C. tropicalis biofilm resistance to voriconazole seems to be associated with ERG genes overexpression and alterations in the cell membrane sterol content [153]. It was proposed that the increased expression of ERG genes could be the consequence of hypoxic conditions within the biofilm [98,152].

The ability to adhere to host cells is another important virulence factor of Candida spp. and is mediated by the cell wall-bound adhesins. Adherence to biotic and abiotic surfaces is also one of the first steps in biofilm formation. It has been suggested that an altered ergosterol content may influence the cell wall composition and the amount or availability of adhesins on the C. albicans cell surface [147,154,155]. The hydrophobicity of the cell surface (CSH) also has an effect on the invasiveness and adherence of pathogenic cells [147,156].

The morphological transition of C. albicans from the yeast form to hyphae is an important trait in the invasion, dissemination and damage of host tissue and is considered to be one of the key virulence factors in C. albicans cells [147,157]. Several studies have clearly shown that the sterol content in yeast cells has an impact on the proper development of hyphae. The presence of ergosterol is essential for the polarization of plasma membrane and thus the development of hyphae, as ergosterol is accumulated at the hyphal tips. Changes in ergosterol levels may prevent hyphae formation and host tissue invasion [27,43,55,147,158].

Deletion of genes involved in ergosterol biosynthesis (

Table 2. Ergosterol biosynthesis modulations affect the virulence of Candida spp.

Candida Species	Gene Involved in Ergosterol Biosynthesis	Gene Alteration	Alteration of Function	Reference
C. albicans	CaERG11	deletion	Impaired biofilm formation/elevated CSH */impairment of filamentation Impairment of oral candidiasis Increased susceptibility to oxidative stress Inability to grow in the presence of L. fermentum	[116,147,166,167]
	CaERG11	overexpression	Elevated CSH *	[156]
	CaERG24	deletion	Inability to form germ tubes	[43,47]
	CaERG6	overexpression	Restoration of biofilm formation and virulence in G. mellonella	[148]
	CaERG3	deletion	Impairment of filamentation and oral candidiasis	[27,34,55,166]
	CaERG3	substitution mutation (L193R) or deletion mutation (Δ366–378)	Compromised virulence	[159]
	CaERG1	deletion	Impairment of filamentation	[39]
	CaERG2	deletion	Impairment of filamentation	[47]
C. glabrata	CgERG11	deletion	Increased susceptibility to oxidative stress and neutrophils	[168]
	CgERG6	deletion	Increased susceptibility to oxidative stress	[169]
	CgAUS1	deletion	Compromised virulence	[113]

* Cell surface hydrofobicity.

) affects the virulence of Candida spp. as has been shown in mouse models of candidiasis [27,43,116,159,160]. It is also proposed that induction of sterol transport may compensate for the shortage of endogenous ergosterol biosynthesis in yeast pathogens [38,102]. Various authors suggested that cholesterol uptake from serum could support the yeast growth during bloodstream infections and explain the decreased azole susceptibility observed in clinical settings [60,112,113,161,162,163,164]. Sterol uptake has also been found to be involved in the C. glabrata virulence. The absence of the sterol importer encoded by the CgAUS1 gene resulted in a significantly decreased fungal burden in the kidneys [113]. C. glabrata, which contains only a single homolog of the S. cerevisiae sterol transporter, CgAus1p, can import sterols under both aerobic and anaerobic conditions [60,107,112,113,162]. On the other hand, the genome of C. albicans does not contain putative orthologs of S. cerevisiae sterol transporters but is able to grow under anaerobic conditions without supplementation with exogenous ergosterol as an adaptation to the anaerobic environment in the gastrointestinal tract [107,165]. However, C. albicans is able to import sterols under aerobic conditions during the post-exponential growth phase [107]. This fact indicates that different Candida spp. may acquire their pathogenic potential in different ways.

Nagi et al. [113] showed that serum-induced CgAus1p expression and sterol uptake in C. glabrata can be explained by the presence of iron-chelating molecules in the serum. Since ergosterol biosynthesis depends on the presence of iron, the sterol scavenging mechanisms can compensate for decreased ergosterol synthesis during infections caused by low levels of iron in the host environment. These observations point to the importance of iron homeostasis for virulence and ergosterol biosynthesis in pathogenic fungi [113].

During infection, Candida cells are engulfed by host phagocytic cells and exposed to an oxidative burst [170,171]. According to some studies, ergosterol plays a role in oxidative stress tolerance in Candida yeasts [116,168,169]. The CaERG11 gene seems to play a key role in the adaptation to oxidative stress imposed by hydrogen peroxide through the modulation of CaCAT1 gene expression in C. albicans [116].

Candida spp. colonize host mucosal surfaces, including the genital, urinary, respiratory and gastrointestinal tracts as well as the oral cavity. Candida spp. share niches with Lactobacillus spp. in the gastrointestinal and vaginal tract [167]. Recent studies have shown that Lactobacillus spp. can prevent the overgrowth of Candida yeast through ergosterol depletion [167]. The addition of ergosterol improved C. glabrata growth in the co-culture. Moreover, the C. glabrata Cgerg11Δ deletion mutant was unable to grow in the presence of Limosilactobacillus fermentum [167]. Similar results were obtained also in the study of Mailänder-Sánchez et al. [172]. Coculture of C. albicans with Lactobacillus rhamnosus GG suppressed ergosterol biosynthesis and decreased the yeast cellular content of ergosterol. Interestingly, Oliveira et al. [173] showed that the presence of L. rhamnosus ATCC 7469 increased the susceptibility of C. albicans to fluconazole. Lactobacillus spp. thus seem to be a promising option for the treatment or prevention of vulvovaginal candidiasis [174].

8. Conclusions

Ergosterol plays a crucial role in the physiology, antifungal resistance and pathogenesis of Candida spp. In recent years, different aspects of ergosterol biosynthesis, regulation and uptake have been deciphered in great details mainly in C. albicans and C. glabrata yeasts. However, similar research is scarce in other important NAC yeasts species. Little is known about the role of ergosterol biosynthesis, regulation and uptake in multidrug resistant species of C. parapsilosis or C. auris. Further research of ergosterol biosynthesis in Candida spp. could broaden our knowledge on various aspects of antifungal drug resistance and virulence of these fungal pathogens. In addition, knowledge of the precise physiological role of different enzymes involved in ergosterol biosynthesis in Candida spp. could support our efforts to find new targets for the development of new antifungal drugs. Of particular importance are genes encoding enzymes involved in late ergosterol biosynthesis that are not found in humans. Unfortunately, systemic infections caused by drug-resistant Candida spp. isolates and new emerging pathogenic species within the genus have increased in recent decades. We believe that the study of ergosterol biosynthesis as an essential component yeast physiology could be instrumental in overcoming this trend.