The sterol C-24 methyltransferase encoding gene, erg6, is essential for viability of Aspergillus species.

Triazoles, the most widely used class of antifungal drugs, inhibit the biosynthesis of ergosterol, a crucial component of the fungal plasma membrane. Inhibition of a separate ergosterol biosynthetic step, catalyzed by the sterol C-24 methyltransferase Erg6, reduces the virulence of pathogenic yeasts, but its effects on filamentous fungal pathogens like Aspergillus fumigatus remain unexplored. Here, we show that the lipid droplet-associated enzyme Erg6 is essential for the viability of A. fumigatus and other Aspergillus species, including A. lentulus, A. terreus, and A. nidulans. Downregulation of erg6 causes loss of sterol-rich membrane domains required for apical extension of hyphae, as well as altered sterol profiles consistent with the Erg6 enzyme functioning upstream of the triazole drug target, Cyp51A/Cyp51B. Unexpectedly, erg6-repressed strains display wild-type susceptibility against the ergosterol-active triazole and polyene antifungals. Finally, we show that erg6 repression results in significant reduction in mortality in a murine model of invasive aspergillosis. Taken together with recent studies, our work supports Erg6 as a potentially pan-fungal drug target.

Azoles activate type I and type II programmed cell death pathways in crop pathogenic fungi.

Triazoles are widely used to control pathogenic fungi. They inhibit the ergosterol biosynthetic pathway, but the precise mechanisms leading to fungicidal activities in many fungal pathogens are poorly understood. Here, we elucidate the mode of action of epoxiconazole and metconazole in the wheat pathogen Zymoseptoria tritici and the rice blast fungus Magnaporthe oryzae. We show that both azoles have fungicidal activity and reduce fluidity, but not integrity, of the plasma membrane. This impairs localisation of Cdc15-like F-BAR proteins, resulting in defective actin ring assembly and incomplete septation. However, mutant studies and pharmacological experiments in vitro and in planta show that azole lethality is due to a combination of reactive oxygen species-induced apoptosis and macroautophagy. Simultaneous inhibition of both programmed cell death pathways abolishes azole-induced cell death. Other classes of ergosterol biosynthesis inhibitors also induce apoptosis and macroautophagy, suggesting that activation of these two cell death pathways is a hallmark of ergosterol synthesis-targeting fungicides. This knowledge will inform future crop protection strategies.

A secondary mechanism of action for triazole antifungals in Aspergillus fumigatus mediated by hmg1.

Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance.

The SET-domain protein CgSet4 negatively regulates antifungal drug resistance via the ergosterol biosynthesis transcriptional regulator CgUpc2a.

Invasive fungal infections, which pose a serious threat to human health, are increasingly associated with a high mortality rate and elevated health care costs, owing to rising resistance to current antifungals and emergence of multidrug-resistant fungal species. Candida glabrata is the second to fourth common cause of Candida bloodstream infections. Its high propensity to acquire resistance toward two mainstream drugs, azoles (inhibit ergosterol biosynthesis) and echinocandins (target cell wall), in clinical settings, and its inherent low azole susceptibility render antifungal therapy unsuccessful in many cases. Here, we demonstrate a pivotal role for the SET {suppressor of variegation 3 to 9 [Su(var)3-9], enhancer of zeste [E(z)], and trithorax (Trx)} domain-containing protein, CgSet4, in azole and echinocandin resistance via negative regulation of multidrug transporter-encoding and ergosterol biosynthesis (ERG) genes through the master transcriptional factors CgPdr1 and CgUpc2A, respectively. RNA-Seq analysis revealed that C. glabrata responds to caspofungin (CSP; echinocandin antifungal) stress by downregulation and upregulation of ERG and cell wall organization genes, respectively. Although CgSet4 acts as a repressor of the ergosterol biosynthesis pathway via CgUPC2A transcriptional downregulation, the CSP-induced ERG gene repression is not dependent on CgSet4, as CgSet4 showed diminished abundance on the CgUPC2A promoter in CSP-treated cells. Furthermore, we show a role for the last three enzymes of the ergosterol biosynthesis pathway, CgErg3, CgErg5, and CgErg4, in antifungal susceptibility and virulence in C. glabrata. Altogether, our results unveil the link between ergosterol biosynthesis and echinocandin resistance and have implications for combination antifungal therapy.

Coordinated Regulation of Membrane Homeostasis and Drug Accumulation by Novel Kinase STK-17 in Response to Antifungal Azole Treatment.

The emergence of antifungal resistance, especially to the most widely used azole class of ergosterol biosynthesis inhibitors, makes fungal infections difficult to treat in clinics and agriculture. When exposed to azoles, fungi can make adaptive responses to alleviate azole toxicity and produce azole tolerance. However, except for azole efflux pumps and ergosterol biosynthesis genes, the role of most azole responsive genes in azole resistance is unknown. In this study, STK-17, whose transcription is upregulated by azoles, was characterized as a novel kinase that is required for azole resistance. Deletion or dysfunction of STK-17 led to azole hypersensitivity in Neurospora crassa and to other ergosterol biosynthesis inhibitors such as amorolfine, terbinafine, and amphotericin B, but not fatty acid and ceramide biosynthesis inhibitors. STK-17 was also required for oxidative stress resistance, but this was not connected to azole resistance. RNA-seq results showed that stk-17 deletion affected the basal expression and the response to ketoconazole of some membrane protein genes, indicating functional association of STK-17 with the membrane. Notably, deletion of stk-17 affected the normal response to azoles of erg genes, including the azole target-encoding gene erg11, and erg2, erg6, and erg24, and led to abnormal accumulation of sterols in the presence of azoles. HPLC-MS/MS analysis revealed increased intracellular azole accumulation in the stk-17 mutant, possibly due to enhanced azole influx and reduced azole efflux that was independent of the major efflux pump CDR4. Importantly, STK-17 was widely distributed and functionally conserved among fungi, thus providing a potential antifungal target. IMPORTANCE Antifungal resistance is increasing worldwide, especially to the most widely used azole class of ergosterol biosynthesis inhibitors, making control of fungal infections more challenging. A lot of effort has been expended in elucidating the mechanism of azole resistance and revealing potential antifungal targets. In this study, by analyzing azole-responsive genes in Neurospora crassa, we discovered STK-17, a novel kinase, that is required for azole resistance in several types of fungi. It has a role in regulating membrane homeostasis, responses to azole by ergosterol biosynthesis genes and azole accumulation, thus, deepening our understanding on the mechanism of azole stress response. Additionally, STK-17 is conserved among fungi and plays important roles in fungal development and stress resistance. Kinase inhibitors are broadly used for treating diseases, and our study pinpoints a potential drug target for antifungal development.

K143R Amino Acid Substitution in 14-α-Demethylase (Erg11p) Changes Plasma Membrane and Cell Wall Structure of Candida albicans.

The opportunistic pathogen Candida albicans is responsible for life-threating infections in immunocompromised individuals. Azoles and polyenes are two of the most commonly used antifungals and target the ergosterol biosynthesis pathway or ergosterol itself. A limited number of clinically employed antifungals correspond to the development of resistance mechanisms. One resistance mechanism observed in clinical isolates of azole-resistant C. albicans is the introduction of point mutations in the ERG11 gene, which encodes a key enzyme (lanosterol 14-α-demethylase) on the ergosterol biosynthesis pathway. Here, we demonstrate that a point mutation K143R in ERG11 (C. albicans ERG11K143R/K143R) contributes not only to azole resistance, but causes increased gene expression. Overexpression of ERG11 results in increased ergosterol content and a significant reduction in plasma membrane fluidity. Simultaneously, the same point mutation caused cell wall remodeling. This could be facilitated by the unmasking of chitin and ß-glucan on the fungal cell surface, which can lead to recognition of the highly immunogenic ß-glucan, triggering a stronger immunological reaction. For the first time, we report that a frequently occurring azole-resistance strategy makes C. albicans less susceptible to azole treatment while, at the same time, affects its cell wall architecture, potentially leading to exposure of the pathogen to a more effective host immune response.

Loss-of-Function ROX1 Mutations Suppress the Fluconazole Susceptibility of upc2AΔ Mutation in Candida glabrata, Implicating Additional Positive Regulators of Ergosterol Biosynthesis.

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress-induced transcription factors such as Upc2 and Upc2A as well as homologs of sterol response element binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2AΔ deletion mutant. This led to the identification of loss-of-function mutations in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2AΔ mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels of ergosterol and decreased levels of the toxic sterol 14α methyl-ergosta-8,24(28)-dien-3ß, 6α-diol, relative to the upc2AΔ mutant. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata. IMPORTANCE Candida glabrata is one of the most important human fungal pathogens and has reduced susceptibility to azole-class inhibitors of ergosterol biosynthesis. Although ergosterol is the target of two of the three classes of antifungal drugs, relatively little is known about the regulation of this critical cellular pathway. Sterols are both essential components of the eukaryotic plasma membrane and potential toxins; therefore, sterol homeostasis is critical for cell function. Here, we identified two new negative regulators in C. glabrata of ergosterol (ERG) biosynthesis gene expression. Our results also indicate that in addition to Upc2A, the only known activator of ERG genes, additional positive regulators of this pathway must exist.

The Effects of Tormentic Acid and Extracts from Callistemon citrinus on Candida albicans and Candida tropicalis Growth and Inhibition of Ergosterol Biosynthesis in Candida albicans.

Candida albicans and Candida tropicalis are the leading causes of human fungal infections worldwide. There is an increase in resistance of Candida pathogens to existing antifungal drugs leading to a need to find new sources of antifungal agents. Tormentic acid has been isolated from different plants including Callistemon citrinus and has been found to possess antimicrobial properties, including antifungal activity. The study aimed to determine the effects of tormentic and extracts from C. citrinus on C. albicans and C. tropicalis and a possible mode of action. The extracts and tormentic acid were screened for antifungal activity using the broth microdilution method. The growth of both species was inhibited by the extracts, and C. albicans was more susceptible to the extract compared to C. tropicalis. The growth of C. albicans was inhibited by 80% at 100 µg/ml of both the DCM: methanol extract and the ethanol: water extract. Tormentic acid reduced the growth of C. albicans by 72% at 100 µg/ml. The effects of the extracts and tormentic acid on ergosterol content in C. albicans were determined using a UV/Vis scanning spectrophotometer. At concentrations of tormentic acid of 25 µg/ml, 50 µg/ml, 100 µg/ml, and 200 µg/ml, the content of ergosterol was decreased by 22%, 36%, 48%, and 78%, respectively. Similarly, the DCM: methanol extract at 100 µg/ml and 200 µg/ml decreased the content by 78% and 88%, respectively. A dose-dependent decrease in ergosterol content was observed in cells exposed to miconazole with a 25 µg/ml concentration causing a 100% decrease in ergosterol content. Therefore, tormentic acid inhibits the synthesis of ergosterol in C. albicans. Modifications of the structure of tormentic acid to increase its antifungal potency may be explored in further studies.

Effect of the essential oils of Satureja montana L., Myristica fragrans H. and Cymbopogon flexuosus S. on mycotoxin-producing Aspergillus flavus and Aspergillus ochraceus antifungal properties of essential oils.

Essential oils can be a useful alternative to the use of synthetic fungicides because they have biological potential and are relatively safe for food and agricultural products. The objectives of the present study were to evaluate the antifungal and antimycotoxigenic activities of the essential oils from Satureja montana L., Myristica fragrans H. and Cymbopogon flexuosus S. against Aspergillus flavus and Aspergillus ochraceus, as well as their effects on ergosterol synthesis and membrane morphology. The antifungal potential was evaluated by mycelial growth analysis and scanning electron microscopy. Fungicidal effects against A. flavus, with MFC of 0.98, 15.62 and 0.98 µL/mL, respectively, were observed for the essential oils from S. montana, M. fragrans and C. flexuosus. Aspergillus ochraceus did not grow in the presence of concentrations of 3.91, 15.62 and 0.98 µL/mL of the essential oils from S. montana, M. fragrans and C. flexuosus, respectively. The essential oils significantly inhibited the production of ochratoxin A by the fungus A. ochraceus. The essential oils also inhibited the production of aflatoxin B1 and aflatoxin B2. The biosynthesis of ergosterol was inhibited by the applied treatments. Biological activity in the fungal cell membrane was observed in the presence of essential oils, given that deleterious effects on the morphologies of the fungi were detected. The essential oils under study are promising as food preservatives because they significantly inhibit toxigenic fungi that contaminate food. In addition, the essential oils hindered the biosynthesis of mycotoxins.

The transcription factor Cas5 suppresses hyphal morphogenesis during yeast-form growth in Candida albicans.

Candida albicans is an opportunistic human pathogen that exists as yeast, hyphal or pseudohyphal forms depending on pH, nutrients, and temperature. The morphological transition from yeast to hyphae, which is required for the complete virulence of C. albicans, is controlled by many transcription factors that activate or repress hypha-specific genes. The C. albicans transcriptional factor Cas5, a key regulator of genes involved in cell wall integrity, affects the susceptibility of C. albicans to fluconazole, an inhibitor of ergosterol synthesis. In this study, we found that deletion of CAS5 in C. albicans decreased the expression levels of a set of ergosterol biosynthesis genes, such as ERG2, ERG3, ERG5, ERG6, ERG11, and ERG24, resulting in the accumulation of lanosterol and zymosterol, which are intermediate metabolites in the ergosterol biosynthesis pathway. Interestingly, it was observed that the cas5Δ/Δ mutant could not maintain the yeast form under non-hypha-inducing conditions, while the CAS5-overexpressing cells could not form hyphae under hypha-inducing conditions. Consistent with these observations, the cas5Δ/Δ mutant highly expressed hypha-specific genes, ALS3, ECE1, and HWP1, under non-hypha-inducing conditions. In addition, CAS5 transcription was significantly downregulated immediately after hyphal initiation in the wild-type strain. Furthermore, the cas5Δ/Δ mutant reduced the transcription of NRG1, which encodes a major repressor of hyphal morphogenesis, while Cas5 overexpression increased the transcription of NRG1 under hypha-inducing conditions. Collectively, this study suggests the potential role of Cas5 as a repressor of hypha-specific genes during yeast-form growth of C. albicans.

Mevalonate kinase of Leishmania donovani protects parasite against oxidative stress by modulating ergosterol biosynthesis.

Leishmaniasis comprises of a wide variety of diseases, caused by protozoan parasite belonging to the genus Leishmania. Leishmania parasites undergo different types of stress during their lifetime and have developed strategies to overcome this damage. Identifying the mechanistic approach used by the parasite in dealing with the stress is of immense importance for unfolding the survival strategy adopted by the parasite. Mevalonate kinase (MVK) is an important regulatory factor in the mevalonate pathway in both bacteria and eukaryotes. In this study, we explored the role of Leishmania donovani mevalonate kinase (LdMVK) in parasite survival under stress condition. Hydrogen peroxide (H2O2) and menadione, the two known oxidants were used to carry out the experiments. The MVK expression was found to be up regulated ∼2.1 fold and ∼2.3 fold under oxidative stress condition and under the effect of anti-Leishmania drug, AmBisome respectively. The cell viability declined under the effect of MVK inhibitor viz: vanadyl sulfate (VS). The level of intracellular ROS was also found to be increased under the effect of MVK inhibitor. To confirm the findings, LdMVK over expression (LdMVK OE) and LdMVK knockdown (LdMVK KD) parasites were generated. The level of ergosterol, an important component of plasma membrane in L. donovani, was observed and found to be reduced by nearly 60 % in LdMVK KD parasite and increased by nearly 30 % in LdMVK OE parasites as compared to wild type. However, the ergosterol content was found to be elevated under oxidative stress. Furthermore, LdMVK was also found to be associated with maintaining the plasma membrane integrity and also in preventing the peroxidation of cellular lipids when exposed to oxidative stress. The above data clearly suggests that MVK has a vital role in protecting the parasite from oxidative stress. These findings may also explore the contribution of LdMVK in drug unresponsiveness which may help in future rational drug designing for leishmaniasis.

Artemisinin elevates ergosterol levels of Candida albicans to synergise with amphotericin B against oral candidiasis.

Oral candidiasis, especially caused by Candida albicans, is the most common fungal infection of the oral cavity. The increase in drug resistance and lack of new antifungal agents call for new strategies of antifungal treatment. This study repurposed artemisinin (Art) as a potentiator to the polyene amphotericin B (AmB) and characterised their synergistic mechanism against C. albicans and oral candidiasis. The synergistic antifungal activity between Art and AmB was identified by the checkerboard and recovery plate assays according to the fractional inhibitory concentration index (FICI). Art showed no antifungal activity even at >200 mg/L. However, it significantly reduced AmB dosages against the wild-type strain and 75 clinical isolates of C. albicans (FICI ≤ 0.5). Art significantly upregulated expression of genes from the ergosterol biosynthesis pathway (ERG1, ERG3, ERG9 and ERG11), as shown by RT-qPCR, and elevated the ergosterol content of Candida cells. Increased ergosterol content significantly enhanced binding between fungal cells and the polyene agent, resulting in sensitisation of C. albicans to AmB. Drug combinations of Art and AmB showed synergistic activity against oral mucosal infection in vivo by reducing the epithelial infection area, fungal burden and inflammatory infiltrates in murine oropharyngeal candidiasis. These findings indicate a novel synergistic antifungal drug combination and a new Art mechanism of action, suggesting that drug repurposing is a clinically practical means of antifungal drug development and treatment of oral candidiasis.

Identification of Thiazoyl Guanidine Derivatives as Novel Antifungal Agents Inhibiting Ergosterol Biosynthesis for Treatment of Invasive Fungal Infections.

Invasive fungal infections (IFIs) are fatal infections, but treatment options are limited. The clinical efficacies of existing drugs are unsatisfactory because of side effects, drug-drug interaction, unfavorable pharmacokinetic profiles, and emerging drug-resistant fungi. Therefore, the development of antifungal drugs with a new mechanism is an urgent issue. Herein, we report novel aryl guanidine antifungal agents, which inhibit a novel target enzyme in the ergosterol biosynthesis pathway. Structure-activity relationship development and property optimization by reducing lipophilicity led to the discovery of 6h, which showed potent antifungal activity against Aspergillus fumigatus in the presence of serum, improved metabolic stability, and PK properties. In the murine systemic A. fumigatus infection model, 6h exhibited antifungal efficacy equivalent to voriconazole (1e). Furthermore, owing to the inhibition of a novel target in the ergosterol biosynthesis pathway, 6h showed antifungal activity against azole-resistant A. fumigatus.

The transcriptome analysis on urea response mechanism in the process of ergosterol synthesis by Cordyceps cicadae.

Nitrogen source is required for the growth of Cordyceps cicadae and involved in the regulation of metabolite synthesis. In order to further investigate the regulatory effects of nitrogen sources on the ergosterol synthesis by C. cicadae. We first confirmed that urea could significantly increase the ergosterol synthesis. The transcriptome analysis showed that compared with biomass cultured in the control fermentation medium (CFM), 1340 differentially expressed genes (DEGs) were obtained by Gene Ontology (GO) annotation, and 312 DEGs were obtained by Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation from the biomass cultured in CFM + CO(NH2)2. Urea up-regulated D-3-phosphoglycerate dehydrogenase gene transcription level and down-regulated enolase and L-serine/L-threonine ammonialyase gene transcription level, increased serine synthesis, allosterically activate pyruvate kinase, to promote the synthesis of pyruvate and CH3CO ~ SCOA, the primer of ergosterol; Urea increase the genes transcription related with ergosterol synthesis by up-regulating the steroid regulatory element binding protein gene transcription levels. The transcriptome results were provided by those of qRT-PCR. Collectively, our finding provided valuable insights into the regulatory effect of nitrogen source on the ergosterol synthesis by C. cicadae.

Isavuconazole nonwildtype Aspergillus fumigatus isolates from a global surveillance study display alterations in multiple genes involved in the ergosterol biosynthesis pathway not previously associated with resistance to other azoles.

OBJECTIVES: We evaluated 35 azole nonwildtype Aspergillus fumigatus isolates that were collected during 2017-2018 using whole genome sequencing (WGS) to detect alterations in the genes involved in the ergosterol biosynthesis pathway as well as other genes associated with azole resistance. METHODS: Among 297 A fumigatus isolates collected worldwide, 36 isolates displayed nonwildtype MIC values to isavuconazole, itraconazole, or voriconazole when tested by the CLSI reference broth microdilution method. Isolates were submitted to WGS and results were compared to 2 azolewildtype isolates. RESULTS: Among the 35 sequenced isolates (1 failed to produce quality sequences), 29 were nonwildtype to isavuconazole, 16 were nonwildtype to itraconazole, and 9 were nonwildtype to voriconazole (CLSI M59Ed2 criteria). A total of 9 isolates carried Cyp51A TR34/L98H alterations (8 from Italy and 1 from Belgium) and had nonwildtype MIC values for ≥2 azoles. A Cyp51B Q42L mutation was detected in 3 isolates, 1 nonwildtype voriconazole and 2 nonwildtype isavuconazole isolates. Three isolates harboured multiple mutations in Cyp51A (F46Y, M172V, E427K ± N248T, and D255E), including 1 isolate with the Cyp51B Q42L mutation. Mutations causing frameshifts, early termination, and duplications were observed among several genes and were more prevalent in isavuconazole nonwildtype isolates (66.7%) than in the isolates that were nonwildtype to 1 or 2 other azoles (22.2%). Nine isolates harboured frameshift mutations in a ERG25 homologue that is usually associated with changes in other genes and should be further evaluated. CONCLUSIONS: Cyp51A L98H/TR34 was the most common alteration observed among the azole nonwildtype A fumigatus isolates from a large surveillance study; however, only isolates that were nonwildtype to isavuconazole had alterations in multiple analysed genes. These isolates deserve further evaluation.

iTRAQ-based proteomic analysis of Paracoccidioides brasiliensis in response to hypoxia.

Aerobic organisms require oxygen for energy. In the course of the infection, adaptation to hypoxia is crucial for survival of human pathogenic fungi. Members of the Paracoccidioides complex face decreased oxygen tensions during the life cycle stages. In Paracoccidioides brasiliensis proteomic responses to hypoxia have not been investigated and the regulation of the adaptive process is still unknown, and this approach allowed the identification of 216 differentially expressed proteins in hypoxia using iTRAQ-labelling. Data suggest that P. brasiliensis reprograms its metabolism when submitted to hypoxia. The fungus reduces its basal metabolism and general transport proteins. Energy and general metabolism were more representative and up regulated. Glucose is apparently directed towards glycolysis or the production of cell wall polymers. Plasma membrane/cell wall are modulated by increasing ergosterol and glucan, respectively. In addition, molecules such as ethanol and acetate are produced by this fungus indicating that alternative carbon sources probably are activated to obtain energy. Also, detoxification mechanisms are activated. The results were compared with label free proteomics data from Paracoccidioides lutzii. Biochemical pathways involved with acetyl-CoA, pyruvate and ergosterol synthesis were up-regulated in both fungi. On the other hand, proteins from TCA, transcription, protein fate/degradation, cellular transport, signal transduction and cell defense/virulence processes presented different profiles between species. Particularly, proteins related to methylcitrate cycle and those involved with acetate and ethanol synthesis were increased in P. brasiliensis proteome, whereas GABA shunt were accumulated only in P. lutzii. The results emphasize metabolic adaptation processes for distinct Paracoccidioides species.

Plant-derived isoquinoline alkaloids that target ergosterol biosynthesis discovered by using a novel antifungal screening tool.

The ergosterol pathway is a prime antifungal target as it is required for fungal survival, yet is not involved in human homeostasis. Methods to study the ergosterol pathway, however, are often time-consuming. The minimum inhibitory concentration (MIC) assay is a simple research tool that determines the lowest concentration at which a novel antimicrobial is active in vitro with limited scope to determine the mechanism of action for a drug. In this study, we show that by adding hydrogen peroxide, an oxidative stressor, or glutathione (GSH), an antioxidant, to modify a commonly performed MIC assay allowed us to screen selectively for new antifungal drugs that target ergosterol biosynthesis in fungi. A human pathogen and dermatophyte, Microsporum gypseum, was used as a test organism. When exposed to ergosterol targeting drugs, the hydrogen peroxide treatment significantly decreased fungal survival by reducing ergosterol in the cell wall, whereas GSH increased survival of M. gypseum. Further, by performing a series of experiments with M. gypseum and Trichophyton rubrum, it was determined that the oxidative stress from hydrogen peroxide causes cell death at different developmental stages based on fungal species. These findings allow us to describe a simple, high-throughput method for simultaneously screening new antifungal drugs for activity and effects on the ergosterol pathway. By using this tool, two isoquinoline alkaloids were discovered to be potent inhibitors of ergosterol biosynthesis in vitro by reducing the amount of ergosterol without affecting the expression of 1,3-ß-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo.

The Effect of Honokiol on Ergosterol Biosynthesis and Vacuole Function in Candida albicans.

Ergosterol, an essential constituent of membrane lipids of yeast, is distributed in both the cell membrane and intracellular endomembrane components such as vacuoles. Honokiol, a major polyphenol isolated from Magnolia officinalis, has been shown to inhibit the growth of Candida albicans. Here, we assessed the effect of honokiol on ergosterol biosynthesis and vacuole function in C. albicans. Honokiol could decrease the ergosterol content and upregulate the expression of genes related with the ergosterol biosynthesis pathway. The exogenous supply of ergosterol attenuated the toxicity of honokiol against C. albicans. Honokiol treatment could induce cytosolic acidification by blocking the activity of the plasma membrane Pma1p H+-ATPase. Furthermore, honokiol caused abnormalities in vacuole morphology and function. Concomitant ergosterol feeding to some extent restored the vacuolar morphology and the function of acidification in cells treated by honokiol. Honokiol also disrupted the intracellular calcium homeostasis. Amiodarone attenuated the antifungal effects of honokiol against C. albicans, probably due to the activation of the calcineurin signaling pathway which is involved in honokiol tolerance. In conclusion, this study demonstrated that honokiol could inhibit ergosterol biosynthesis and decrease Pma 1p H+-ATPase activity, which resulted in the abnormal pH in vacuole and cytosol.

Glutamine metabolism modulates azole susceptibility in Trypanosoma cruzi amastigotes.

The mechanisms underlying resistance of the Chagas disease parasite, Trypanosoma cruzi, to current therapies are not well understood, including the role of metabolic heterogeneity. We found that limiting exogenous glutamine protects actively dividing amastigotes from ergosterol biosynthesis inhibitors (azoles), independent of parasite growth rate. The antiparasitic properties of azoles are derived from inhibition of lanosterol 14α-demethylase (CYP51) in the endogenous sterol synthesis pathway. We find that carbons from 13C-glutamine feed into amastigote sterols and into metabolic intermediates that accumulate upon CYP51 inhibition. Incorporation of 13C-glutamine into endogenously synthesized sterols is increased with BPTES treatment, an inhibitor of host glutamine metabolism that sensitizes amastigotes to azoles. Similarly, amastigotes are re-sensitized to azoles following addition of metabolites upstream of CYP51, raising the possibility that flux through the sterol synthesis pathway is a determinant of sensitivity to azoles and highlighting the potential role for metabolic heterogeneity in recalcitrant T. cruzi infection.

The Capture of a Disabled Proteasome Identifies Erg25 as a Substrate for Endoplasmic Reticulum Associated Degradation.

Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin-proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques-including MS-have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.