Ergosterol Production Research Articles

A Comprehensive in vitro and in silico Assessment on Inhibition of CYP51B and Ergosterol Biosynthesis by Eugenol in Rhizopus oryzae

Mucormycosis, also known as Zygomycosis, is a disease caused by invasive fungi, predominantly Rhizopus species belonging to the Order of Mucorales. Seeing from the chemistry perspective, heterocyclic compounds with an "azole" moiety are widely employed as antifungal agent for minimising the effect of mucormycosis as a prescribed treatment. These azoles serve as non-competitive inhibitors of fungal CYP51B by predominantly binding to its heme moiety, rendering its inhibition. However, long-term usage and abuse of azoles as antifungal medicines has resulted in drug resistance among certain fungal pathogens. Hence, there is an unmet need to find alternative therapeutic compounds. In present study, we used various in vitro tests to investigate the antifungal activity of eugenol against R. oryzae/R. arrhizus, including ergosterol quantification to test inhibition of ergosterol production mediated antifungal action. The minimum inhibitory concentration (MIC) value obtained for eugenol was 512μg/ml with reduced ergosterol concentration of 77.11 ± 3.25% at MIC/2 concentration. Further, the molecular interactions of eugenol with fungal CYP51B were meticulously studied making use of proteomics in silico study including molecular docking and molecular dynamics simulations that showed eugenol to be strongly interacting with heme in an identical fashion to that shown by azole drugs (in this case, clotrimazole was evaluated). This is the first of a kind study showing the simulation study of eugenol with CYP51B of fungi. This inhibition results in ergosterol synthesis and is also studied and compared with keeping clotrimazole as a reference.

Effect of Farnesol in Trichoderma Physiology and in Fungal-Plant Interaction.

Farnesol is an isoprenoid intermediate in the mevalonate (MVA) pathway and is produced by the dephosphorylation of farnesyl diphosphate. Farnesol plays a central role in cell growth and differentiation, controls production of ubiquinone and ergosterol, and participates in the regulation of filamentation and biofilm formation. Despite these important functions, studies of farnesol in filamentous fungi are limited, and information on its effects on antifungal and/or biocontrol activity is scarce. In the present article, we identified the Trichoderma harzianum gene dpp1, encoding a diacylglycerol pyrophosphatase that catalyzes production of farnesol from farnesol diphosphate. We analyzed the function of dpp1 to address the importance of farnesol in Trichoderma physiology and ecology. Overexpression of dpp1 in T. harzianum caused an expected increase in farnesol production as well as a marked change in squalene and ergosterol levels, but overexpression did not affect antifungal activity. In interaction with plants, a dpp1-overexpressing transformant acted as a sensitizing agent in that it up-regulated expression of plant defense salicylate-related genes in the presence of a fungal plant pathogen. In addition, toxicity of farnesol on Trichoderma and plants was examined. Finally, a phylogenetic study of dpp1 was performed to understand its evolutionary history as a primary metabolite gene. This article represents a step forward in the acquisition of knowledge on the role of farnesol in fungal physiology and in fungus-environment interactions.

Antifungal activities of metconazole against the emerging wheat pathogen Fusarium pseudograminearum

Fusarium crown rot of wheat is a serious fungal disease that occurs worldwide. The disease has been emerging in the major wheat-growing areas in China since 2010. Fusarium pseudogramineaum is the predominant causative pathogen of crown rot of wheat in China. The 14α-demethylation inhibitor (DMI) fungicide metconazole has been shown to be effective against Fusarium spp., but little is known about its specific activity against F. pseudogramineaum. Metconazole exhibited strong antifungal activities against all thirty-nine F. pseudogramineaum strains collected from the major wheat-growing areas in China. Metconazole inhibited mycelial growth and conidial germ tube elongation of F. pseudograminearum. Metconazole treatment significantly reduced the production of major toxins and the expression levels of toxin biosynthesis genes. Genome-wide transcriptional profiling of F. pseudograminearum in response to metconazole indicated that the expression of genes involved in ergosterol biosynthesis, including fungicide target genes (cyp51 genes), was significantly induced by metconazole. Nine ATP-binding cassette (ABC) transporter-encoding genes were significantly expressed in response to metconazole treatment. Reduced ergosterol production and antioxidant enzyme activities were observed after metconazole treatment. Greenhouse experiments indicated a significant reduction in crown rot occurrence in wheat after seed treatment with metconazole. This study evaluated the potential of metconazole to manage wheat crown rot and provides information to understand its antifungal activities and mechanism of action against F. pseudograminearum.

Potential dual inhibition of SE and CYP51 by eugenol conferring inhibition of Candida albicans: Computationally curated study with experimental validation

Ergosterol is the key sterol component in the cell membrane of fungi including moulds and yeasts. Any decrease in the levels of ergosterol in the cell membrane of fungi render them venerable to cell membrane damage and even its death. Majority of antifungal drug targets the key enzymes involved in ergosterol biosynthesis pathway. The biochemical pathway for the synthesis of Ergosterol is a complex one, though the reactions carried by Squalene Epoxidase (SE) and 14α-demethylase (CYP51- a member of Cytochrome P450 family) serves to the key rate limiting reactions that can impact the overall production of Ergosterol. Allylamines class of antifungal drug target SE while Azoles target the CYP51. Currently advancement in the drug development is focused to introduce newer drugs that can simultaneously inhibit both this rate limiting enzymes. However, natural compounds established to possess antifungal activity but the major loophole about their understanding lies in the fact that their mode of action are severely unstudied. One such well-established antifungal natural phytochemical is Eugenol, and in current manuscript we investigated its efficacy to interact with both, SE and CYP51 of Candida albicans using molecular Docking, Free energy change calculations and Molecular Dynamics (MD) simulation, showing promising outcomes. For experimental studies, terbinafine, clotrimazole and eugenol showed 4 μg/ml, 2 μg/ml, and 512 μg/ml MIC90 values, respectively against C. albicans and also showed reduction in Ergosterol production at sub-MIC levels. The obtained result indicates the involvement of eugenol in the inhibition of enzymes require in the ergosterol biosynthesis pathway.

Optimization of ultrasound-assisted production of ergosterol from Penicillium brevicompactum by Taguchi statistical method.

Ergosterol as a primary metabolite and precursor of vitamin D2, is the most plentiful mycosterols in fungal cell membrane. Process optimization to increase the yield and productivity of biological products is a topic of interest. Ultrasonic waves have many applications in biotechnology, like cell disruption, and enhancement of primary and secondary metabolites production. This study disclosed an optimal condition for ultrasound-assisted production (UAP) of ergosterol from Penicillium brevicompactum MUCL 19,011 using L9 Taguchi statistical method. The intensity (IS), time of sonication (TS), treatment frequency (TF), and number of days of treatment (DT) were allocated to study the effects of ultrasound on ergosterol production. The results were analyzed using Minitab version 19. The maximum ergosterol, 11mg/g cell dry weight (CDW), was produced on the tenth day while all factors were at a low level. The days of treatment with a contribution of 45.48% was the most significant factor for ergosterol production. For the first time, this study revealed the positive effect of ultrasound on the production of ergosterol. Ergosterol production increased 73% (4.63mg/g CDW) after process optimization. Finally, a mathematical model of ultrasound factors with a regression coefficient of R2 = 0.978 was obtained for the ergosterol production during ultrasound treatment.

Ergosterol production at elevated temperatures by Upc2-overexpressing Kluyveromyces marxianus using Jerusalem artichoke tubers as feedstock

Ergosterol is an important precursor in the pharmaceutical industry for the production of numerous drugs. In this study, Kluyveromyces marxianus that showed more potential for ergosterol production than some other yeasts was reported. The effects of transcription factors UPC2, MOT3, and ROX1 of K. marxianus on ergosterol synthesis were explored, and a Upc2-overexpressing strain produced 1.78 times more ergosterol (167.33 mg/L) than the wild-type strain (60.04 mg/L). A total of 239.98 mg/L ergosterol was produced when glucose was replaced with fructose to limit ethanol production. Enhanced aeration increased ergosterol titer from 63.09 mg/L to 128.46 mg/L at 42 °C. The ergosterol titer reached 304.37 mg/L in a shake flask at 37 °C, or 1124.38 and 948.32 mg/L at 37 °C and 42 °C, respectively, in a 5 L bioreactor, using Jerusalem artichoke tubers as the sole carbon source. This study establishes a platform for ergosterol biosynthesis using inexpensive materials.

Determination of Optimum Conditions for Ergosterol Production by Saccharomyces cerevisiae

Ergosterol is an important provitamin in the present-day of industrial biotechnology. Seven yeast strains were obtained from the market of Basrah city and subjected to screening for their ergosterol production ability using liquid-state fermentation, the cultural conditions, and nutritional requirements for optimal production of ergosterol by Saccharomyces cerevisiae under laboratory conditions were determined. Y.6 is the best isolate of the yeast that produced ergosterol. It was identified as S. cerevisiae, with a similarity rate of 97% by using the Vitek2 device, this S. cerevisiae (Y.6) was further subjected to optimization conditions. The results showed that the best medium for production was yeast extract peptone dextrose broth. The effect of two cheaper carbon sources, molasses and date juice were investigated. Maximum ergosterol (0.55% ) was produced using a medium containing date juice, with a replacement ratio of 75%, an incubation time of 72 hours, pH 5, at a temperature of 30 °C, and an inoculation volume of 4 ml. The percentage of ergosterol was (0.47, 0.55, 0.74, 0.66, 0.68 and 0.78 %), respectively.

Mode of action of nanochitin whisker against Fusarium pseudograminearum

Nanochitin whisker (NC) is an advanced nanobiomaterial with novel physicochemical and biological properties. Fusarium pseudograminearum (Fpg) is an important pathogenic fungus causing wheat crown rot disease. This study explored the mode of action of NC against Fpg as a target microorganism. The effects of different treatments and concentrations of NC on the fungal growth and conidial germination were investigated by in vitro bioassay. The impacts of NC on cell structure integrity, membrane permeability, pathogenesis related key enzymes activity, and the mycotoxin production were examined by electron microscopy, fluorescence spectroscopy, IR spectroscopy, conductometry, and spectrophotometry, respectively. The results showed that NC significantly reduced hyphal growth, and the spore germination rate of Fpg declined by 33.0 % and 23.2 % when Fpg was treated with 30 and 300 μg/mL of NC, respectively. NC vigorously influenced structural stability of cell wall by destroying dextran structure, and strongly stimulated ergosterol production altering membrane integrity of the fungus. It reduced the activities of enzymes related to energy-supply like nicotinamide adenine dinucleotide oxidase and succinate dehydrogenase remarkably. The production of fungal mycotoxin deoxynivalenol was also decreased by NC. These findings provide an important basis for fully understanding the mechanism of nanobiomaterial in plant fungal disease control.

Triazole Derivatives Target 14α-Demethylase (LDM) Enzyme in Candida albicans Causing Ergosterol Biosynthesis Inhibition.

Candida albicans is the most dominant and prevalent cause of fungal infections in humans. Azoles are considered as first-line drugs for the treatment of these infections. However, their prolonged and insistent use has led to multidrug resistance and treatment failures. To overcome this, modification or derivatization of the azole ring has led to the development of new and effective antifungal molecules. In a previous study, we reported on the development of new triazole-based molecules as potential antifungal agents against Candida auris. In this study, the most potent molecules from the previous study were docked and simulated with lanosterol 14-alpha demethylase enzyme. These compounds were further evaluated for in vitro susceptibility testing against C. albicans. In silico results revealed favorable structural dynamics of the compounds, implying that the compounds would be able to effectively bind to the target enzyme, which was further manifested by the strong interaction of the test compounds with the amino acid residues of the target enzyme. In vitro studies targeting quantification of ergosterol content revealed that pta1 was the most active compound and inhibited ergosterol production by >90% in both drug-susceptible and resistant C. albicans isolates. Furthermore, RT-qPCR results revealed downregulation of ERG11 gene when C. albicans cells were treated with the test compound, which aligns with the decreased ergosterol content. In addition, the active triazole derivatives were also found to be potent inhibitors of biofilm formation. Both in silico and in vitro results indicate that these triazole derivatives have the potential to be taken to the next level of antifungal drug development.

Evaluation of quinoxaline derivatives as potential ergosterol biosynthesis inhibitors: design, synthesis, ADMET, molecular docking studies, and antifungal activities

Because of the increased infection incidence and the advent of medication resistance, fungi have become a severe medical problem. Ergosterol is a structural component of the fungal cell membrane, and its synthetases (14α-demethylase (CYP51)) are thought to be the key to inhibiting ergosterol synthesis. Based on the active site (CYP51) analysis, we created a series of quinoxaline derivatives in this study. Following that, these target compounds were produced and tested for antifungal efficacy. The majority of the compounds show significant antifungal activity against a variety of Candida species. Compounds 9, 11, 17, 20, and 21 have antifungal properties that are similar to the positive control medicines and have considerable inhibitory effects, with MIC50 values ranging from 0.78 to 3.12 μg/mL. Compound 9 has the strongest antifungal activity against all Candida species, outperforming Ketoconazole and Fluconazole, and was thought to be the series' most active compound. As a result, the action mechanism for the most powerful blows was investigated further. They can alter ergosterol synthesis by blocking the activity of the 14α-demethylase (CYP51) target, according to preliminary mechanisms. According to this study, compounds 9, 11, 17, 20, and 21 have an efficient effect on the ergosterol production pathway. Moreover, the cytotoxicity of the most promising compounds was investigated, revealing the importance of 9, 11, 17, 20, and 21 hits as prospective anticandidal therapeutics. Furthermore, the ADMET characteristics, as well as the created binding models, will be valuable for further lead optimization.

Steroidal Antimetabolites Protect Mice against Trypanosoma brucei.

Trypanosoma brucei, the causative agent for human African trypanosomiasis, is an emerging ergosterol-dependent parasite that produces chokepoint enzymes, sterol methyltransferases (SMT), not synthesized in their animal hosts that can regulate cell viability. Here, we report the lethal effects of two recently described natural product antimetabolites that disrupt Acanthamoeba sterol methylation and growth, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT) that can equally target T. brucei. We found that CHT/ERGT inhibited cell growth in vitro, yielding EC50 values in the low nanomolar range with washout experiments showing cidal activity against the bloodstream form, consistent with their predicted mode of suicide inhibition on SMT activity and ergosterol production. Antimetabolite treatment generated altered T. brucei cell morphology and death rapidly within hours. Notably, in vivo ERGT/CHT protected mice infected with T. brucei, doubling their survival time following daily treatment for 8–10 days at 50 mg/kg or 100 mg/kg. The current study demonstrates a new class of lead antibiotics, in the form of common fungal sterols, for antitrypanosomal drug development.

Repurposing vilanterol as a novel potential antifungal for Candida albicans: In-silico & in-vitro approach

Candida albicans infects immune-compromised persons, and its resistance to antifungal drugs has become a significant health concern, which causes a higher mortality rate worldwide. This study aims to find novel antifungals against Candida albicans using a computational drug repurposing approach targeting lanosterol 14-alpha demethylase and experimental testing of the drugs. We used the virtual docking method for initial drug screening. We did minimum inhibitory concentration (MIC), and disc diffusion assay for antifungal determination, and the fungicidal property was detected using the fluorescent microscopy method. Further, we carried out Erg11p drug interaction studies by molecular dynamics simulations and binding free energy analysis. Docking results identified bimatoprost and vilanterol as novel drug hits for C. albicans. In-vitro studies confirmed that vilanterol possesses an active antifungal activity (MIC- 500 µg/ml). The fluorescent microscopy study revealed that vilanterol disrupts the cell membrane of C. albicans at a tested concentration of 600 µg/ml. Molecular dynamics simulations and structural decomposition analysis confirmed the durability of the Erg11p-vilanterol complex's interaction. Our study concludes that vilanterol has a novel potential fungicidal activity against C. albicans. On the other hand, we hypothesize it may alter the ergosterol production by binding lanosterol 14α-demethylase. Previous clinical studies have proved that vilanterol oral doses of up to 500 μg were safe in humans. Our study shows that vilanterol may be a new drug class for treating C. albicans. Therefore, we report that vilanterol could be repurposed possibly against C. albicans infections.

Formulation and Charaterization of Antifungal Drug Microemulsion Hydrogel for Topical Drug Delivery

Clotrimazole is a broad-spectrum antimycotic drug, Candida albicans as well as other fungal infections are typically treated with this drug. “Clinical clotrimazole seems to be a topical therapy treating tineapedis (athlete's foot), vulvovaginal candidiasis, and oropharyngeal candidiasis.” That is a synthetic azole antimycotic. It inhibits fungal growth by decreasing the production of ergosterol. Clotrimazole has become a medication of interest for a variety of illnesses, including sickle cell anaemia, malaria, and some cancers, in addition to its antimycotic properties. It's been mixed with other molecules as well, to create clotrimazole compounds with enhanced pharmacological effectiveness, including such metals. Numerous novel pharmaceutical formulations enabling variable releases are also being developed. Clotrimazole is a well-tolerated, little-adverse drug, however certain immune compromised individuals are developing treatment resistance. Clotrimazole's pharmaceutical chemistry, use, and pharmacology are discussed in this study.

Regulated targeting of the monotopic hairpin membrane protein Erg1 requires the GET pathway.

The guided entry of tail-anchored proteins (GET) pathway targets C-terminally anchored transmembrane proteins and protects cells from lipotoxicity. Here, we reveal perturbed ergosterol production in ∆get3 cells and demonstrate the sensitivity of GET pathway mutants to the sterol synthesis inhibiting drug terbinafine. Our data uncover a key enzyme of sterol synthesis, the hairpin membrane protein squalene monooxygenase (Erg1), as a non-canonical GET pathway client, thus rationalizing the lipotoxicity phenotypes of GET pathway mutants. Get3 recognizes the hairpin targeting element of Erg1 via its classical client-binding pocket. Intriguingly, we find that the GET pathway is especially important for the acute upregulation of Erg1 induced by low sterol conditions. We further identify several other proteins anchored to the endoplasmic reticulum (ER) membrane exclusively via a hairpin as putative clients of the GET pathway. Our findings emphasize the necessity of dedicated targeting pathways for high-efficiency targeting of particular clients during dynamic cellular adaptation and highlight hairpin proteins as a potential novel class of GET clients.

Recent progress in strategies for steroid production in yeasts.

As essential structural molecules of fungal cell membrane, ergosterol is not only an important component of fungal growth and stress-resistance but also a key precursor for manufacturing steroid drugs of pharmaceutical or agricultural significance. So far, ergosterol biosynthesis in yeast has been elucidated elaborately, and efforts have been made to increase ergosterol production through regulation of ergosterol metabolism and storage. Furthermore, the same intermediates shared by yeasts and animals or plants make the construction of heterologous sterol pathways in yeast a promising approach to synthesize valuable steroids, such as phytosteroids and animal steroid hormones. During these challenging processes, several obstacles have arisen and been combated with great endeavors. This paper reviews recent research progress of yeast metabolic engineering for improving the production of ergosterol and heterologous steroids. The remaining tactics are also discussed.

Novel culture chamber to evaluate in vitro plant-microbe volatile interactions: Effects of Trichoderma harzianum volatiles on wheat plantlets

The field of plant-microbe interactions mediated by Biogenic Volatile Organic Compounds (BVOCs) still faces several limitations due to the lack of reliable equipment. We present a novel device designed to evaluate in vitro plant-microbe volatile interactions, the plant-microbe VOC Chamber. It was tested by evaluating the effects exerted on wheat development by volatiles from three Trichoderma harzianum strains, a wild type and two genetically modified strains; one expressing the tri5 gene, which leads to the synthesis and emission of the volatile trichodiene, and the other by silencing the erg1 gene, impairing ergosterol production. The wild type and the erg1-silenced strain enhanced fresh weight and length of the aerial part, but reduced root dry weight. Interestingly, no differences were found between them. Conversely, the tri5-transformant strain reduced root and aerial growth compared to the control and the other strains. No differences were observed regarding chlorophyll fluorescence quantum yield and leaf chlorophyll content, suggesting that the released BVOCs do not interfere with photosynthesis. The plant-microbe VOC Chamber proved to be a simple and reliable method to evaluate the in vitro effects of microbial BVOCs on plant development, perfect for the screening of microorganisms with interesting volatile traits.

Evaluation of parental and transgenerational effects of clotrimazole in Daphnia magna — A multi-parametric approach

Azole antifungals inhibit the cytochrome P450 complex, decreasing the production of ergosterol in fungi, and compromising the biosynthesis of ecdysteroids in crustaceans, which are hormones regulating reproduction and ecdysis. The azole antifungal clotrimazole (CLO) raises environmental concerns due its toxicity. This work evaluated the effects on the number of moults, feeding rate, growth, reproduction, transgenerational reproductive effects on two different generations (F0, parental generation; and F1, organisms born from F0), and energetic balance in Daphnia magna. Neonates (<24 h) were exposed to sublethal concentrations (0, 2.7, and 3.4 mg/L) of CLO, to assess its effects on the moulting process. Neonates were also exposed to environmentally realistic concentrations of CLO (0, 30, 150, 750, and 3750 ng/L) for 24 and 96 h, to assess adverse effects on their feeding behaviour. Effects on energy reserves (fatty acids, glycogen, and protein levels) were also measured in animals exposed to CLO. A reproduction test was carried out to evaluate the amount and size of neonates from F0 and F1 generations. CLO exposure decreased the number of moults, and the size of organisms, but did not alter the feeding pattern of 5 days old individuals. However, neonates (<24 h) exposed to CLO had a significant decrease in their feeding pattern. CLO decreased the fatty acids content in exposed animals, but did not change glycogen and protein. CLO also decreased the size of adult daphnids from the third brood, born from animals exposed in F0; in F1 animals, the size of neonates from the third brood was decreased. This study evidenced the toxic effects caused by CLO on growth, feeding and reproduction of D. magna. Nevertheless, it is not possible to conclude whether the effects are due to the inhibition of cytochrome P450 enzymes, or to unspecific effects caused by general toxic stress and decreased nutrition.

Air temperature more than drought duration affects litter decomposition under flow intermittency

Stream intermittency — periodic sequences of water flow cessation and resumption — occurs throughout the year, across seasons. Even though temperature is a known regulator of litter decomposition in both terrestrial and aquatic environments, comparative experiments on drought durations at distinct temperatures on microbial-mediated decomposition in streams experiencing intermittency are still lacking. Here, three drought temperatures (5, 15 and 25 °C) and two durations (short: 2.5 weeks; long: 5 weeks) were applied in a microcosm study to oak leaf discs colonized in a reference stream; mass loss and associated microbial parameters (fungal biomass, microbial activity, and sporulation rates) were evaluated following re-submersion for 2 weeks. Higher mass loss was found at 15 °C than 25 °C. A prolongation of the drought exposure period had no effect on mass loss, suggesting an early (≤ 2.5 weeks) inhibitor effect of drought on microbial-mediated leaf degradation. Fungal biomass was highest at 25 °C following a short drought, and decreased with a longer drought period at both 15 °C and 25 °C. Microbial activity was not affected by either drought duration or temperature. Sporulation rates and fungal diversity were significantly reduced by the longer drought period; in the short treatment, maximum values were found at 15 °C. In contrast to longer droughts, aquatic fungal communities during short dry periods seem to invest in energetically-expensive physiological responses to desiccation (e.g., ergosterol production) promoting biomass accrual at the expense of mass loss and reproductive output. Under more severe desiccation (higher duration and temperature), the lower diversity of fungal communities seem to result in negative legacy effects for fungal growth and reproductive capacity after flow resumption. These results suggest that native riparian vegetation, through its ability to regulate temperature in streams, may be critical in protecting freshwaters from intensified severity of drought periods in streams experiencing intermittency.

Azole-Resistant Alleles of ERG11 in Candida glabrata Trigger Activation of the Pdr1 and Upc2A Transcription Factors.

Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol α-14 demethylase enzyme, which is referred to as Erg11. Inhibition of Erg11 ultimately leads to a reduction in ergosterol production, an essential fungal membrane sterol. Many Candida species, such as Candida albicans, develop mutations in this enzyme which reduces the azole binding affinity and results in increased resistance. Candida glabrata is also a pathogenic yeast that has low intrinsic susceptibility to azole drugs and easily develops elevated resistance. In C. glabrata, these azole resistant mutations typically cause hyperactivity of the Pdr1 transcription factor and rarely lie within the ERG11 gene. Here, we generated C. glabrata ERG11 mutations that were analogous to azole resistance alleles from C. albicans ERG11. Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. Resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. Crucially, the presence of the DM ERG11 allele was sufficient to activate the Pdr1 transcription factor in the absence of azole drugs. Our data indicate that azole resistance linked to changes in ERG11 activity can involve cellular effects beyond an alteration in this key azole target enzyme. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata.

UHPLC-HRMS-Based Untargeted Lipidomics Reveal Mechanism of Antifungal Activity of Carvacrol against Aspergillus flavus.

Aspergillus flavus is a common contaminant in grain, oil and their products. Its metabolite aflatoxin B1 (AFB1) has been proved to be highly carcinogenic. Therefore, it is of great importance to find possible antifungal substances to inhibit the growth and toxin production of Aspergillus flavus. Carvacrol (CV) was reported as a potent antifungal monoterpene derived from plants. In this paper, the antifungal effects and mechanism of CV on Aspergillus flavus were investigated. CV was shown good inhibition on the growth of Aspergillus flavus and the production of AFB1. CV used in concentrations ranging from 0, 50, 100 and 200 μg/mL inhibited the germination of spores, mycelia growth and AFB1 production dose-dependently. To explore the antifungal mechanism of CV on Aspergillus flavus, we also detected the ergosterol content of Aspergillus flavus mycelia, employed Scanning Electron Microscopy (SEM) to observe mycelia morphology and utilized Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-HRMS) to explore the lipidome profiles of Aspergillus flavus. The results showed that the production of ergosterol of mycelia was reduced as the CV treatment concentration increased. SEM photographs demonstrated a rough surface and a reduction in the thickness of hyphae in Aspergillus flavus treated with CV (200 µg/mL). In positive ion mode, 21 lipids of Aspergillus flavus mycelium were downregulated, and 11 lipids were upregulated after treatment with 200-µg/mL CV. In negative ion mode, nine lipids of Aspergillus flavus mycelium were downregulated, and seven lipids upregulated after treatment with 200-µg/mL CV. In addition, the analysis of different lipid metabolic pathways between the control and 200-µg/mL CV-treated groups demonstrated that glycerophospholipid metabolism was the most enriched pathway related to CV treatment.