Each of the azole resistant strains had a different antifungal susceptibility pattern that correlated with different mutations in the cyp 51A gene. The lack of differences between sterols highlights the fact that the resistance mechanism in those strains was target-dependent and not sterol-dependent, as will be discussed later.
Instead, the sterol composition of single enzyme defective A. Downstream in the pathway, the synthesis of ergosterol involves the transformation of fecosterol, consisting of double-bond rearrangements in the steroid nucleus and in the side chain, isomerization of the double connection in the C-8 to the C-7 followed by the desaturation at C-5 and C, and the reduction of the C The enzymatic sequence of these three last steps could differ between fungal taxa and growth conditions Nes et al.
Single gene deletion of each of the C-5 sterol desaturases Erg3A, Erg3B, and Erg3C revealed a different sterol profile in terms of total amount of ergosterol and sterol composition. Again this phenotype was different depending on the deleted enzyme. This suggests that although A. Ergosterol biosynthetic pathway. Sterols of A. TABLE 1. Three of the most widely used antifungal drugs, triazoles, polyenes, and allylamines, are aimed at ergosterol, and they are either fungicidal but toxic to the host polyenes or fungi static and more vulnerable to resistance triazoles.
The lethality caused by double gene deletion has been shown, although neither gene is itself essential Mellado et al. Moreover, the importance of A. Also, the in vitro and in vivo correlation of azole resistance has been widely documented; with a clear association of resistant A. Resistance acquired through exposure to azoles in the patientis correlated with cyp 51A single mutations Diaz-Guerra et al.
This latter type of resistance mechanism may have evolved in the environment through the exposure of the fungus to azole fungicides used in agriculture Snelders et al. In isolation the tandem bp promoter insertions cannot explain the observed azole cross resistant phenotype Mellado et al. The sterol content of azole resistant A. It is more likely that azole resistance in these variants results from a reduced affinity of drug binding to the Cyp51A enzyme.
In this sense, 3D protein models of Cyp51A in combination with azoles have provided the basis to address how point mutations can affect azole drug resistance Alcazar-Fuoli et al. Regarding the polyene antifungals, the association between A. Secondary resistance to AMB is generally not observed, even in patients whose therapy has failed Moosa et al. Acquired resistance to AMB has been most extensively evaluated in yeasts and is associated with mutations in the ERG 3 gene, which is linked to qualitative and quantitative alterations of membrane lipids and an absence of ergosterol Kelly et al.
However, the deletion of three independent erg 3-like genes in A. Other studies have determined that neither ergosterol content, cell wall composition, or lipid peroxidation levels correlate with heightened A. Terbinafine belongs to the allylamine class of antifungals that inhibit squalene epoxidase Erg1.
Erg1 catalyzes the first oxygenation step in sterol biosynthesis and is suggested to be one of the rate-limiting enzymes in this pathway. Alterations in A. Also, a point mutation, FL, in the squalene epoxidase enzyme was found to confer a strong terbinafine resistance phenotype.
The equivalent mutation was introduced into the homologous gene of A. Since terbinafine is not currently used in the management of IA, the appearance of terbinafine-resistant strains in the clinical setting is unlikely, however, its potential use in combination with other antifungal drugs should be considered.
Global gene expression studies identified that ergosterol biosynthesis is likely to be highly sensitive to environmental perturbations. Among them, adaptation to the host environment when A.
The transcriptome of A. Although the target site of azole activity is well studied, the role of other proteins in the mode of action of these drugs in fungi is poorly understood.
Recently, a critical role for SrbA-mediated regulation of ergosterol biosynthesis and triazole drug interactions in A. SrbA was identified by transcriptional profiling under hypoxia conditions as a regulator of ergosterol biosynthetic genes in response to low oxygen levels. Also, several genes encoding enzymes that require high levels of oxygen were found to be transcriptionally repressed in the absence of SrbA, including the enzymes Erg6, Erg11, Erg24, Erg25, and Erg3.
In addition, the apparent control of cyp 51 transcript levels by SrbA suggests an additional target for drug development. A role for SrbA in the development of triazole resistance has also been suggested, since alterations in this transcription factor or its DNA-binding affinity could transcriptionally initiate changes that could alter target abundance and would lead to triazole resistance Blosser and Cramer, The genome-wide expression response of A.
In conditions of iron limitation, A. The results described above, together with the characterization of functional siderophore deleted mutants, probed the link between fungal ergosterol and siderophore biosynthesis in A. The latter would appear to feed both biochemical pathways Yasmin et al.
Also, in hypoxic conditions SrbA was found to activate siderophore-mediated iron uptake in response to hypoxia and iron starvation in part by transcriptional activation of another transcription factor, HapX Blatzer et al. Transcriptional regulation of ergosterol biosynthesis in A. Erg6 has a role in the synthesis of secondary sterols ethyl sterols , and its down-regulation suggests that A.
However, different results were found for the A. These results may indicate the existence of differential responses for overcoming the distinct stresses imposed by these drugs. In addition, genes encoding for Erg24, Erg25, and Erg3 were differentially expressed when A. Collectively, the above observations suggest that ergosterol biosynthesis is prone to perturbation by environmental conditions, and in a manner dependent upon Erg6, Erg11, Erg24, Erg25, and Erg3 functions, thereby highlighting these gene functions as alternative or synergic inhibitors of the pathway.
An important finding in A. These sterols can also be final metabolites of the sterol pathway and they are predominantly found in higher plants, though absent in mammalian cells, which cannot alkylate the C of sterols. In plants C ethyl sterols have multiple roles to play in growth and development, however, few reports exist on the detection of ethyl sterols in fungi, and their role in A.
The sterol profile analysis of A. Genome wide studies are providing us with a very useful platform from which to dissect the linkages between cellular sterol biosynthesis and other cell functions or metabolic pathways. Such new findings might be further explored for novel drug development or for possible combinatorial therapeutic strategies to fight invasive fungal diseases.
Among them, genes encoding enzymes involved in cellular stress, cell wall synthesis, and transport have been found to be differentially expressed under antifungal exposure or underhost imposed stresses. To conclude, A. Biosynthetic control under different environmental circumstances is observed, demonstrating that Aspergillus has alternatives to overcome severe drawbacks.
Therefore, the precise knowledge of this complex biosynthetic pathway could facilitate the future development of novel and more selective antifungal drugs in order to improve efficacy and to minimize drug resistance.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Albarrag, A. Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: GC, YC, and GC single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter. Agents Chemother. Alcazar-Fuoli, L. Agents 38, — Probing the role of point mutations in the cyp51A gene from Aspergillus fumigatus in the model yeast Saccharomyces cerevisiae.
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Biochim Biophys Acta — Luo X, Su P, Zhang W Advances in microalgae-derived phytosterols for functional food and pharmaceutical applications. Mar Drugs 13 7 — World J Microbiol Biotechnol 34 4 PLoS Genet 10 1 :e Nature — As mentioned above, ergosterol biosynthesis depends on oxygen and heme as cofactors for critical enzymes of the pathway Figure 1.
During aerobic growth, heme is properly synthesized and available for binding to Hap1 protein. Rox1 and Mot3 regulatory factors act synergistically to achieve stringent repression of target genes [ 89 , 90 ]. As a general result, under aerobic conditions, ERG genes display basal levels necessary for ergosterol biosynthesis, whereas the hypoxic-responsive genes are fully repressed.
Conversely, a decrease in oxygen bioavailability causes a drop in heme and ergosterol levels that triggers the activation of signaling pathways that induce the expression of both hypoxic and ERG genes [ 94 ].
The up-regulation of oxygen-dependent enzymes within the ergosterol biosynthesis pathway upon oxygen limitation could be considered as a cellular strategy to compensate for their decrease in activity. Oxygen depletion also enhances the expression of the oxygen-dependent fatty acid desaturase Ole1 through the Mga2 transcriptional factor [ 97 ]. Importantly, Hap1 seems to shift from an activator to a transcriptional repressor when oxygen availability decreases [ 98 ].
However, conflicting results have been observed for the effect of Hap1 on the expression of ERG genes during anaerobic conditions [ 75 , 96 , 99 , ].
These discrepancies could be due to the use of yeast strains expressing different Hap1 factors, or being cultivated in media with different oxygen, ergosterol or fatty acid availability [ 29 , 96 , ]. Many aspects of their regulation are still unknown due to the crosstalk between different environmental signals and the opposite regulatory effects triggered by Hap1, Rox1, Mot3, Upc2 and Ecm22 regulatory factors on a large number of ERG genes.
Several genome-wide expression studies based on microarray and RNA-Seq analyses under low oxygen conditions have reflected that the pattern of expression differs among ERG genes, although some general trends can be extracted [ 94 , 96 , 98 , , , , ]. ERG genes involved in the first two modules of the ergosterol biosynthesis pathway seem to be down-regulated e. As expected, the number of ERG genes with altered expression increases with the severity of oxygen restriction, and genes encoding for oxygen-dependent enzymes are up-regulated e.
Different hypotheses have been proposed to explain these observations. The increase in the expression of oxygen-using enzymes may be taking place to maintain the flux of ergosterol formation. However, the pattern of ERG5 expression contradicts this hypothesis, since it is down-regulated. Another possibility is that only the expression of particular ERG genes is enhanced to prevent the accumulation of toxic sterol intermediates.
The up-regulation of the latter module of the ergosterol biosynthesis pathway under severe low oxygen conditions could favor the rapid production of ergosterol upon reoxygenation [ 94 , , ]. In accordance with this hypothesis, some of the genes that are most strongly induced upon reoxygenation are involved in the two first modules of ergosterol synthesis [ ].
This could be important during anaerobic-to-aerobic transitions, since de novo sterol synthesis is required for the induction of respiratory genes [ ]. Further studies are necessary to fully elucidate the regulation of ERG genes upon oxygen limitation. As already mentioned, yeast cells activate sterol import under anaerobic conditions. By using a hyperactive upc allele, the cell wall mannoprotein Dan1 was identified as a facilitator of sterol influx, in addition to Aus1 and Pdr11 [ 73 ].
When the levels of oxygen are appropriate normoxia , Mot3 represses AUS1 and PDR11 expression [ ], whereas Upc2 allows their up-regulation upon oxygen depletion [ 73 ]. As well as the upc mutation, the constitutively active mutant of Ecm22 imports sterols under normoxia, but the underlying mechanisms are poorly characterized [ ].
The response and adaptation to hyperosmotic stress is mostly governed by the high osmolarity glycerol HOG pathway and its terminal signaling MAPK Hog1 reviewed in [ ]. Upon osmostresses such as high extracellular salt concentrations, Hog1 induces the transcription of MOT3 [ ], leading to a transient increase in Mot3 protein abundance that facilitates its association to particular ERG promoters [ 7 ].
Consistent with this, the expression of the hyperactive upc allele increases ergosterol content and renders cells highly salt sensitive, whereas inhibition of ergosterol synthesis with azole drugs is beneficial for salt-stress tolerance [ 7 ]. As already indicated, ergosterol biosynthesis depends on iron in four steps, which are catalyzed by enzymes that contain a cofactor in the form of heme Erg5, Erg11 and its regulator Dap1 or oxo-diiron Erg25 and Erg3 centers Figure 1.
Consequently, iron deficiency reduces the metabolic flux through the sterol pathway, leading to a decrease in ergosterol and zymosterol levels, and the accumulation of squalene and lanosterol [ 9 ], which are the substrates of Erg1 and Erg11, respectively. Erg11 function is likely to decrease due to the drop in heme levels that occurs when iron is scarce, and the subsequent lanosterol accumulation may inhibit Erg1 [ 9 , 65 ].
Further studies have indicated that the heme-binding domain of the cytochrome b 5 related protein Dap1 is required for the activity of the cytochrome P enzyme Erg11 as well as for growth in iron-deficient conditions [ ].
Although it has been proposed that Dap1 increases Erg11 protein abundance in a heme-dependent manner, the regulation of Erg11 by Dap1 has not been fully deciphered [ , ].
In any case, under iron deprivation, the loss of Dap1 is rescued by ERG11 overexpression but not by increasing heme biosynthesis [ ]. In addition to Dap1, mutants in other components of ergosterol biosynthesis, such as in the essential genes ERG25 and ERG29 , also lead to growth defects in low iron and respiratory conditions, respectively, due to impaired ergosterol production [ , ].
A recent study has revealed that, similarly to Erg25, Erg29 participates in the methyl sterol oxidase step of ergosterol biosynthesis [ 13 ]. Defects in this step lead to the accumulation of toxic intermediates of the methyl sterol oxidase reaction that increase mitochondrial oxidation and affect the stability of the yeast frataxin homolog Ffh1, which is implicated in mitochondrial iron metabolism [ 13 ].
As a consequence, erg29 mutants exhibit defects in iron-sulfur cluster assembly and mitochondrial iron accumulation [ 13 , ]. These results emphasize the multiple connections between iron metabolism and ergosterol biosynthesis. Genome-wide expression studies have shown that the expression of ERG genes is altered in response to iron deficiency [ , ]. Consistent with this, the protein levels of both Erg1 and Erg11 decrease in response to iron depletion [ 9 ].
However, the abundance of other Erg proteins, such as Erg6 and Erg25, is kept roughly constant or even increases, which is the case of Erg3 [ 9 ]. In this sense, a recent global kinetic study of gene expression during the progress of iron deficiency has shown that the pattern of expression differs among ERG genes and depends on the severity of the depletion [ ].
Therefore, we hypothesize that additional regulatory factors, including Hap1, Upc2, Ecm22 and others may directly or indirectly respond to iron limitation to control ERG gene expression and ergosterol synthesis.
The yeast S. In recent years, ergosterol biosynthesis has been greatly studied in S. The balanced regulation of all the enzymes in the ergosterol biosynthesis pathway is an essential determinant of the efficiency of sterol synthesis and, therefore, of optimal growth and adaptation to environmental cues. Moreover, one of the main approaches to overproduce sterols of high-value for food and pharmaceutical industries includes yeast genetic modification.
However, changes in ergosterol biosynthesis lead to pleiotropic defects that limit cellular proliferation and adaptation to stresses. Because of these reasons, the study of ergosterol biosynthesis regulation may provide new ideas for enhancing sterol production and the adaptation of yeast cell factories to the environment.
Furthermore, chemicals could be developed to increase the production of specific sterols during yeast fermentation. For instance, the treatment with terbinafine, which targets the squalene epoxidase Erg1, results in the accumulation of squalene, which can be used for the synthesis of terpenes.
In this sense, the genetic alteration of sterol metabolism could reduce the concentration of these expensive chemicals, drastically lowering production costs. The ergosterol biosynthetic pathway constitutes one of the main targets for antifungal agents in health and agriculture. Nevertheless, the currently available drugs have been related to emerging resistance by fungal pathogens, significant side effects and toxicity reviewed in [ ].
The detailed study of the molecular mechanisms that contribute to the regulation, synthesis and transport of sterols in S.
Ergosterol regulation by particular metabolites and environmental cues is still far from being understood in all its complexity.
Future lines of investigation should include the identification of factors that regulate the subcellular localization and function of Erg proteins and the structural and functional characterization of the fungal-specific zinc-finger regulatory proteins that control the expression of ERG genes. Conceptualization, S. Both authors have read and agreed to the published version of the manuscript.
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
National Center for Biotechnology Information , U. Journal List Genes Basel v. Genes Basel. Published online Jul Author information Article notes Copyright and License information Disclaimer. Received Jun 26; Accepted Jul This article has been cited by other articles in PMC. Abstract Ergosterol is an essential component of fungal cell membranes that determines the fluidity, permeability and activity of membrane-associated proteins.
Keywords: ergosterol, sterol biosynthesis, sterol regulation, yeast, Saccharomyces cerevisiae , oxygen, iron. Introduction Sterols are essential components of eukaryotic cellular membranes that maintain membrane structural integrity, fluidity and permeability. Ergosterol Synthesis, Uptake and Detoxification in S. Ergosterol Biosynthesis in S. Open in a separate window. Figure 1. Sterol Acquisition and Transport in S. Sterol Detoxification Yeast cells can overproduce ergosterol or sterol intermediates, which can be incorporated into cellular membranes to some extent, to modulate their physicochemical properties.
Regulation of Ergosterol Biosynthesis To alter the ergosterol composition of lipid bilayers and, consequently, to be able to properly adapt to particular environmental stresses, yeast cells have evolved different regulatory mechanisms that tightly control sterol levels. Subcellular Localization of Ergosterol Biosynthesis Enzymes Multiple enzymes that participate in the late stage of ergosterol biosynthesis are transmembrane-containing proteins that associate into a functional complex, denoted the ergosome, to facilitate catalysis [ 53 ].
Transcriptional Regulation 3. Transcriptional Regulation by Sterols The expression of many enzymes within the ergosterol biosynthesis pathway is regulated at the transcriptional level by sterol abundance through the action of transcriptional factors that bind to 7-base pair DNA motifs, known as sterol regulatory elements SREs , located in the promoter of their corresponding genes reviewed in [ 26 ].
Figure 2. Figure 3. Transcriptional Regulation by Oxygen As mentioned above, ergosterol biosynthesis depends on oxygen and heme as cofactors for critical enzymes of the pathway Figure 1. Regulation by Iron Bioavailability As already indicated, ergosterol biosynthesis depends on iron in four steps, which are catalyzed by enzymes that contain a cofactor in the form of heme Erg5, Erg11 and its regulator Dap1 or oxo-diiron Erg25 and Erg3 centers Figure 1.
Conclusions The yeast S. Author Contributions Conceptualization, S. Conflicts of Interest The authors declare no conflict of interest. References 1. Maxfield F. Role of cholesterol and lipid organization in disease. Tarkowska D. Plant ecdysteroids: Plant sterols with intriguing distributions, biological effects and relations to plant hormones.
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