Ergosterol Biosynthesis In Fungi Research Articles

Discovery of Novel Cinnamic Acid Derivatives as Fungicide Candidates.

Structural diversity derivatization from natural products is an important and effective method of discovering novel green pesticides. Cinnamic acids are abundant in plants, and their unparalleled structures endow them with various excellent biological activities. A series of novel cinnamic oxime esters were designed and synthesized to develop high antifungal agrochemicals. The antifungal activity, structure-activity relationship, and action mechanism were systematically studied. Compounds 7i, 7u, 7v, and 7x exhibited satisfactory activity against Gaeumannomyces graminis var. tritici, with inhibition rates of ≥90% at 50 μg/mL. Compounds 7z and 7n demonstrated excellent activities against Valsa mali and Botrytis cinerea, with median effective concentration (EC50) values of 0.71 and 1.41 μg/mL, respectively. Compound 7z exhibited 100% protective and curative activities against apple Valsa canker at 200 μg/mL. The control effects of 7n against gray mold on tomato fruits and leaves were all >96%, exhibiting superior or similar effects to those of the commercial fungicide boscalid. Furthermore, the quantitative structure-activity relationship was established to guide the further design of higher-activity compounds. The preliminary results on the action mechanism revealed that 7n treatment could disrupt the function of the nucleus and mitochondria, leading to reactive oxygen species accumulation and cell membrane damage. Its primary biochemical mechanism may be inhibiting fungal ergosterol biosynthesis. The novel structure, simple synthesis, and excellent activity of cinnamic oxime esters render them promising potential fungicides.

The yeast mRNA-binding protein Cth2 post-transcriptionally modulates ergosterol biosynthesis in response to iron deficiency

Sterol synthesis is an iron-dependent metabolic pathway in eukaryotes. Consequently, fungal ergosterol biosynthesis (ERG) is down-regulated in response to iron deficiency. In this report, we show that, upon iron limitation or overexpression of the iron-regulated mRNA-binding protein Cth2, the yeast Saccharomyces cerevisiae down-regulates the three initial enzymatic steps of ergosterol synthesis (ERG1, ERG7 and ERG11). Mechanistically, we show that Cth2 protein limits the translation and promotes the decrease in the mRNA levels of these specific ERG genes, which contain consensus Cth2-binding sites defined as AU-rich elements (AREs). Thus, expression of CTH2 leads to the accumulation of initial sterol intermediates, such as squalene, and to the drop of ergosterol levels. Changes in CTH2 expression levels disturb the response of yeast cells to stresses related to membrane integrity such as high ethanol and sorbitol concentrations. Therefore, CTH2 should be considered as a critical regulatory factor of ergosterol biosynthesis during iron deficiency.

Controlling antifungal activity with light: Optical regulation of fungal ergosterol biosynthetic pathway with photo-responsive CYP51 inhibitors.

Invasive fungal infections (IFIs) have been associated with high mortality, highlighting the urgent need for developing novel antifungal strategies. Herein the first light-responsive antifungal agents were designed by optical control of fungal ergosterol biosynthesis pathway with photocaged triazole lanosterol 14α-demethylase (CYP51) inhibitors. The photocaged triazoles completely shielded the CYP51 inhibition. The content of ergosterol in fungi before photoactivation and after photoactivation was 4.4% and 83.7%, respectively. Importantly, the shielded antifungal activity (MIC80≥64μg/mL) could be efficiently recovered (MIC80=0.5-8μg/mL) by light irradiation. The new chemical tools enable optical control of fungal growth arrest, morphological conversion and biofilm formation. The ability for high-precision antifungal treatment was validated by invivo models. The light-activated compound A1 was comparable to fluconazole in prolonging survival in Galleria mellonella larvae with a median survival of 14 days and reducing fungal burden in the mouse skin infection model. Overall, this study paves the way for precise regulation of antifungal therapy with improved efficacy and safety.

New miconazole-based azoles derived from eugenol show activity against Candida spp. and Cryptococcus gattii by inhibiting the fungal ergosterol biosynthesis

This work describes the design, synthesis and antifungal activity of new imidazoles and 1,2,4-triazoles derived from eugenol and dihydroeugenol. These new compounds were fully characterized by spectroscopy/spectrometric analyses and the imidazoles 9, 10, 13 e 14 showed relevant antifungal activity against Candida sp. and Cryptococcus gattii in the range of 4.6–75.3 μM. Although no compound has shown a broad spectrum of antifungal activity against all evaluated strains, some azoles were more active than either reference drugs employed against specific strains. Eugenol-imidazole 13 was the most promising azole (MIC: 4.6 μM) against Candida albicans being 32 times more potent than miconazole (MIC: 150.2 μM) with no relevant cytotoxicity (selectivity index >28). Notably, dihydroeugenol-imidazole 14 was twice as potent (MIC: 36.4 μM) as miconazole (MIC: 74.9 μM) and more than 5 times more active than fluconazole (MIC: 209.0 μM) against alarming multi-resistant Candida auris. Furthermore, in vitro assays showed that most active compounds 10 and 13 altered the fungal ergosterol biosynthesis, reducing its content as fluconazole does, suggesting the enzyme lanosterol 14α-demethylase (CYP51) as a possible target for these new compounds. Docking studies with CYP51 revealed an interaction between the imidazole ring of the active substances with the heme group, as well as insertion of the chlorinated ring into a hydrophobic cavity at the binding site, consistent with the behavior observed with control drugs miconazole and fluconazole. The increase of azoles-resistant isolates of Candida species and the impact that C. auris has had on hospitals around the world reinforces the importance of discovery of azoles 9, 10, 13 e 14 as new bioactive compounds for further chemical optimization to afford new clinically antifungal agents.

Ipconazole Disrupts Mitochondrial Homeostasis and Alters GABAergic Neuronal Development in Zebrafish

Ipconazole, a demethylation inhibitor of fungal ergosterol biosynthesis, is widely used in modern agriculture for foliar and seed treatment, and is authorized for use in livestock feed. Waste from ipconazole treatment enters rivers and groundwater through disposal and rain, posing potential toxicity to humans and other organisms. Its metabolites remain stable under standard hydrolysis conditions; however, their neurodevelopmental toxicity is unknown. We investigated the potential neurodevelopmental toxicity of ipconazole pesticides in zebrafish (Danio rerio). Our behavioral monitoring demonstrated that the locomotive activity of ipconazole-exposed zebrafish larvae was reduced during early development, even when morphological abnormalities were undetected. Molecular profiling demonstrated that the mitochondrial-specific antioxidants, superoxide dismutases 1 and 2, and the genes essential for mitochondrial genome maintenance and functions were specifically reduced in ipconazole-treated (0.02 μg/mL) embryos, suggesting underlying ipconazole-driven oxidative stress. Consistently, ipconazole treatment substantially reduced hsp70 expression and increased ERK1/2 phosphorylation in a dose-dependent manner. Interrupted gad1b expression confirmed that GABAergic inhibitory neurons were dysregulated at 0.02 μg/mL ipconazole, whereas glutamatergic excitatory and dopaminergic neurons remained unaffected, resulting in an uncoordinated neural network. Additionally, ipconazole-treated (2 μg/mL) embryos exhibited caspase-independent cell death. This suggests that ipconazole has the potential to alter neurodevelopment by dysregulating mitochondrial homeostasis.

Inhibiting C-4 Methyl Sterol Oxidase with Novel Diazaborines to Target Fungal Plant Pathogens.

With resistance to current agricultural fungicides rising, a great need has emerged for new antifungals with unexploited targets. In response, we report a novel series of diazaborines with potent activity against representative fungal plant pathogens. To identify their mode of action, we selected for resistant isolates using the model fungus Saccharomyces cerevisiae. Whole-genome sequencing of independent diazaborine-resistant lineages identified a recurring mutation in ERG25, which encodes a C-4 methyl sterol oxidase required for ergosterol biosynthesis in fungi. Haploinsufficiency and allele-swap experiments provided additional genetic evidence for Erg25 as the most biologically relevant target of our diazaborines. Confirming Erg25 as putative target, sterol profiling of compound-treated yeast revealed marked accumulation of the Erg25 substrate, 4,4-dimethylzymosterol and depletion of both its immediate product, zymosterol, as well as ergosterol. Encouraged by these mechanistic insights, the potential utility of targeting Erg25 with a diazaborine was demonstrated in soybean-rust and grape-rot models of fungal plant disease.

Detoxification and adaptation mechanisms of Trichoderma atroviride to antifungal agents

Abstract Filamentous fungi are crucial for recycling of organic material in nature. In natural habitats, they cope with many stress factors and therefore their adaptation ability to various conditions is very high. Trichoderma sp., fungi used in agriculture as biocontrol agent, are exposed to a variety of toxic molecules including pesticides and fungicides. They have to fight with toxic molecules using stress adaptation mechanisms known as the stress response. Adaptation of fungi to stress, especially to chemical stress, is not well studied in environmental fungal strains. Moreover, the adaptation process presents a risk of resistance mechanism induction to antifungal agents. Such resistant strains could be spread in the environment. This work aims to contribute to the knowledge of the adaptation process spread throughout the fungal kingdom. Transcriptional response of ABC transporters, the main detoxification efflux pumps of subfamily B and G in presence of antifungal agents, is shown. On the other hand, as azoles are the most commonly used antifungal structures in clinical practice and agriculture, changes in important fungal ergosterol biosynthesis genes as a result of their exposure to various azoles structure are highlighted.

Synthesis, Biological Evaluation, and Structure-Activity Relationships of 4-Aminopiperidines as Novel Antifungal Agents Targeting Ergosterol Biosynthesis.

The aliphatic heterocycles piperidine and morpholine are core structures of well-known antifungals such as fenpropidin and fenpropimorph, commonly used as agrofungicides, and the related morpholine amorolfine is approved for the treatment of dermal mycoses in humans. Inspired by these lead structures, we describe here the synthesis and biological evaluation of 4-aminopiperidines as a novel chemotype of antifungals with remarkable antifungal activity. A library of more than 30 4-aminopiperidines was synthesized, starting from N-substituted 4-piperidone derivatives by reductive amination with appropriate amines using sodium triacetoxyborohydride. Antifungal activity was determined on the model strain Yarrowia lipolytica, and some compounds showed interesting growth-inhibiting activity. These compounds were tested on 20 clinically relevant fungal isolates (Aspergillus spp., Candida spp., Mucormycetes) by standardized microbroth dilution assays. Two of the six compounds, 1-benzyl-N-dodecylpiperidin-4-amine and N-dodecyl-1-phenethylpiperidin-4-amine, were identified as promising candidates for further development based on their in vitro antifungal activity against Candida spp. and Aspergillus spp. Antifungal activity was determined for 18 Aspergillus spp. and 19 Candida spp., and their impact on ergosterol and cholesterol biosynthesis was determined. Toxicity was determined on HL-60, HUVEC, and MCF10A cells, and in the alternative in vivo model Galleria mellonella. Analysis of sterol patterns after incubation gave valuable insights into the putative molecular mechanism of action, indicating inhibition of the enzymes sterol C14-reductase and sterol C8-isomerase in fungal ergosterol biosynthesis.

Double-stranded RNA targeting fungal ergosterol biosynthesis pathway controls Botrytis cinerea and postharvest grey mould.

SummaryPathogenic fungi cause major postharvest losses. During storage and ripening, fruit becomes highly susceptible to fungi that cause postharvest disease. Fungicides are effective treatments to limit disease. However, due to increased public concern for their possible side effects, there is a need to develop new strategies to control postharvest fungal pathogens. Botrytis cinerea, a common postharvest pathogen, was shown to uptake small double‐stranded RNA (dsRNA) molecules from the host plant. Such dsRNA can regulate gene expression through the RNA interference system. This work aimed to develop a synthetic dsRNA simultaneously targeting three essential transcripts active in the fungal ergosterol biosynthesis pathway (dsRNA‐ERG). Our results show initial uptake of dsRNA in the emergence zone of the germination tube that spreads throughout the fungus and results in down‐regulation of all three targeted transcripts. Application of dsRNA‐ERG decreased B. cinerea germination and growth in in vitro conditions and various fruits, leading to reduce grey‐mould decay. The inhibition of growth or decay was reversed by the addition of ergosterol. While dual treatment with dsRNA‐ERG and ergosterol‐inhibitor fungicide reduced by 100‐fold the required amount of fungicide to achieve the same protection rate. The application of dsRNA‐ERG induced systemic protection as shown by decreased decay development at inoculation points distant from the treatment point in tomato and pepper fruits. Overall, this study suggests that dsRNA‐ERG can effectively control B. cinerea growth and grey‐mould development suggesting its efficacy as a future method for postharvest control of fungal pathogens.

Emergence of Antifungal Azole Resistance in the Fungal Strains of Tinea corporis, Tinea capitis, Tinea cruris and Tinea pedis from the Locality of Southern Punjab, Pakistan

Background: Dermatophytes are the most common group of fungi causing fungal infections all over the world. They are classified into three main groups Trichophyton, Microsporum and Epidermophyton. Among these, Trichophyton has the highest prevalence rate (70-90%) as compared to the others. The global emergence of fungal infections is varied due to the socio-economic conditions throughout the world. Developing countries, like Pakistan, are facing an increase in the number of dermatophytoses, including frequent relapses and treatment failures.
 Objectives: The study have been conducted to identify the emerging fungal species, the role of commonly available antifungals such as azoles including voriconazole, ketoconazole, fluconazole and amphotericin B were used to determine the drug resistance among these species.
 Methodology: Nine groups of dermatophytes and non-dermatophyte fungi isolated from the patients of tinea corporis, tinea cruris, tinea capitis and tinea pedis infections were analyzed for Phenotypic diversity, antifungal susceptibility and strains identification, was performed by cultural characteristics and microscopy.
 Results: Nine groups of isolated fungal strains were identified as Trichophyton interdigitale, Trichophyton mentagrophyte, Trichophyton rubrum amongst dermatophytes class and Aspergillus terreus, Aspergillus verisocolor, Aspergillus niger, Acretonium sordidulum and Acremonium sclerotigenum of non-dermatophytes class.
 Conclusion: The study revealed Trichophytone interdigitale group as more frequent dermatophytes. Whereas, among the antifungal drugs, fluconazole that targets the Erg 1 gene of ergosterol biosynthesis in fungi is less effective most common antifungal drugs available locally.

Anethum graveolens Essential Oil Encapsulation in Chitosan Nanomatrix: Investigations on In Vitro Release Behavior, Organoleptic Attributes, and Efficacy as Potential Delivery Vehicles Against Biodeterioration of Rice (Oryza sativa L.)

The study deals with first time report on encapsulation of chemically characterized Anethum graveolens essential oil within chitosan nanomatrix (Nm-AGEO) using ionic gelation technique to enhance the antimicrobial, antiaflatoxigenic, antioxidant, and in situ efficacy against stored rice biodeterioration. GC-MS analysis of AGEO revealed dill apiol (33.79%), carvone (27.19%), and limonene (13.76%) as major components. Nm-AGEO characterization through scanning electron microscopy (SEM), X-ray diffractometry (XRD), and Fourier transform infrared spectroscopy (FT-IR) confirmed successful encapsulation of AGEO within chitosan as an encapsulant. Biphasic and sustained release pattern reflected controlled volatilization of bioactives, helpful in shelf-life extension of stored food commodities. Nm-AGEO caused significant impairment in fungal ergosterol biosynthesis and enhanced leakage of vital ions indicating destabilization in plasma membrane integrity. Inhibition of methylglyoxal (aflatoxin inducer) biosynthesis by Nm-AGEO confirmed novel antiaflatoxigenic mechanism of action, suggesting its future exploitation for development of aflatoxin-resistant rice varieties through green transgenics. Nm-AGEO induced impairment in antioxidant defense enzymes (SOD, CAT) and non-enzymatic defense biomolecules GSH and GSSG revealing biochemical mechanism of action. In silico modeling of carvone and limonene with Omt-A and Ver-1 genes suggested molecular mechanism of aflatoxin inhibition. Treatment of rice samples with Nm-AGEO caused significant protection from aflatoxin B1 contamination and lipid peroxidation without altering organoleptic properties. Moreover, favorable safety profile for mammalian system and non-phytotoxic nature of chitosan-fabricated AGEO nanoemulsion-based delivery system recommend attention of food industries for its formulation as potential green preservative.

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-β-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo.

Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans

Ergosterol is the most important membrane sterol in fungal cells and a component not found in the membranes of human cells. We identified the ERG6 gene in the AIDS-associated fungal pathogen, Cryptococcus neoformans, encoding the sterol C-24 methyltransferase of fungal ergosterol biosynthesis. In this work, we have explored its relationship with high-temperature growth and virulence of C. neoformans by the construction of a loss-of-function mutant. In contrast to other genes involved in ergosterol biosynthesis, C. neoformans ERG6 is not essential for growth under permissive conditions in vitro. However, the erg6 mutant displayed impaired thermotolerance and increased susceptibility to osmotic and oxidative stress, as well as to different antifungal drugs. Total lipid analysis demonstrated a decrease in the erg6Δ strain membrane ergosterol content. In addition, this mutant strain was avirulent in an invertebrate model of C. neoformans infection. C. neoformans Erg6 was cyto-localized in the endoplasmic reticulum and Golgi complex. Our results demonstrate that Erg6 is crucial for growth at high temperature and virulence, likely due to its effects on C. neoformans membrane integrity and dynamics. These pathogen-focused investigations into ergosterol biosynthetic pathway components reinforce the multiple roles of ergosterol in the response of diverse fungal species to alterations in the environment, especially that of the infected host. These studies open perspectives to understand the participation of ergosterol in mechanism of resistance to azole and polyene drugs. Observed synergistic growth defects with co-inhibition of Erg6 and other components of the ergosterol biosynthesis pathway suggests novel approaches to treatment in human fungal infections.

Plant-Derived Isoquinoline Alkaloids that Target Ergosterol Biosynthesis Discovered by Using a Novel Antifungal Screening Tool

Background: 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. Methods: 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. Findings: 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. Interpretation: 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-β-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo. Funding Statement: This work was supported by Faculty Research Grant, Hong Kong Baptist University (FRG 2/17-18/103), the Hong Kong Baptist University (HKBU) Interdisciplinary Research Matching Scheme (RC-IRMS/15-16/02),NSFC funding 31670360, 81973293, U1702286 and Natural Science Foundation of SZU 860-000002110131. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Female guinea pigs were used for the in vivo antifungal study, which was approved by the laboratory animal ethics committee of Shenzhen University, China [Approval number: SYXK (Yue) 2014-0140].

Azole-induced cell wall carbohydrate patches kill Aspergillus fumigatus

Azole antifungals inhibit the fungal ergosterol biosynthesis pathway, resulting in either growth inhibition or killing of the pathogen, depending on the species. Here we report that azoles have an initial growth-inhibitory (fungistatic) activity against the pathogen Aspergillus fumigatus that can be separated from the succeeding fungicidal effects. At a later stage, the cell wall salvage system is induced. This correlates with successive cell integrity loss and death of hyphal compartments. Time-lapse fluorescence microscopy reveals excessive synthesis of cell wall carbohydrates at defined spots along the hyphae, leading to formation of membrane invaginations and eventually rupture of the plasma membrane. Inhibition of β-1,3-glucan synthesis reduces the formation of cell wall carbohydrate patches and delays cell integrity failure and fungal death. We propose that azole antifungals exert their fungicidal activity by triggering synthesis of cell wall carbohydrate patches that penetrate the plasma membrane, thereby killing the fungus. The elucidated mechanism may be potentially exploited as a novel approach for azole susceptibility testing.

Green Ultrasound versus Conventional Synthesis and Characterization of Specific Task Pyridinium Ionic Liquid Hydrazones Tethering Fluorinated Counter Anions: Novel Inhibitors of Fungal Ergosterol Biosynthesis.

A series of specific task ionic liquids (ILs) based on a pyridiniumhydrazone scaffold in combination with hexafluorophosphate (PF6−), tetrafluoroboron (BF4−) and/or trifluoroacetate (CF3COO−) counter anion, were designed and characterized by IR, NMR and mass spectrometry. The reactions were conducted under both conventional and green ultrasound procedures. The antifungal potential of the synthesized compounds 2–25 was investigated against 40 strains of Candida (four standard and 36 clinical isolates). Minimum inhibitory concentrations (MIC90) of the synthesized compounds were in the range of 62.5–2000 μg/mL for both standard and oral Candida isolates. MIC90 results showed that the synthesized 1-(2-(4-chlorophenyl)-2-oxoethyl)-4-(2-(4-fluorobenzylidene)hydrazinecarbonyl)-pyridin-1-ium hexafluorophosphate (11) was found to be most effective, followed by 4-(2-(4-fluorobenzylidene)hydrazinecarbonyl)-1-(2-(4-nitrophenyl)-2-oxoethyl)-pyridin-1-ium hexafluorophosphate (14) and 1-(2-ethoxy-2-oxoethyl)-4-(2-(4-fluorobenzylidene)hydrazinecarbonyl)pyridin-1-ium hexafluorophosphate (8). All the Candida isolates showed marked sensitivity towards the synthesized compounds. Ergosterol content was drastically reduced by more active synthesized compounds, and agreed well with MIC90 values. Confocal scanning laser microscopy (CLSM) results showed that the red colored fluorescent dye enters the test agent treated cells, which confirms cell wall and cell membrane damage. The microscopy results obtained suggested membrane-located targets for the action of these synthesized compounds. It appears that the test compounds might be interacting with ergosterol in the fungal cell membranes, decreasing the membrane ergosterol content and ultimately leading to membrane disruption as visible in confocal results. The present study indicates that these synthesized compounds show significant antifungal activity against Candida which forms the basis to carry out further in vivo experiments before their clinical use.

Accumulation of Azole Drugs in the Fungal Plant Pathogen Magnaporthe oryzae Is the Result of Facilitated Diffusion Influx.

Magnaporthe oryzae is an agricultural mold that causes disease in rice, resulting in devastating crop losses. Since rice is a world-wide staple food crop, infection by M. oryzae poses a serious global food security threat. Fungicides, including azole antifungals, are used to prevent and combat M. oryzae plant infections. The target of azoles is CYP51, an enzyme localized on the endoplasmic reticulum (ER) and required for fungal ergosterol biosynthesis. However, many basic drug-pathogen interactions, such as how the azole gets past the fungal cell wall and plasma membrane, and is transported to the ER, are not understood. In addition, reduced intracellular accumulation of antifungals has consistently been observed as a drug resistance mechanism in many fungal species. Studying the basic biology of drug-pathogen interactions may elucidate uncharacterized mechanisms of drug resistance and susceptibility in M. oryzae and potentially other related fungal pathogens. We characterized intracellular accumulation of azole drugs in M. oryzae using a radioactively labeled fluconazole uptake assay to gain insight on whether azoles enter the cell by passive diffusion, active transport, or facilitated diffusion. We show that azole accumulation is not ATP-dependent, nor does it rely on a pH-dependent process. Instead there is evidence for azole drug uptake in M. oryzae by a facilitated diffusion mechanism. The uptake system is specific for azole or azole-like compounds and can be modulated depending on cell phase and growth media. In addition, we found that co-treatment of M. oryzae with ‘repurposed’ clorgyline and radio-labeled fluconazole prevented energy-dependent efflux of fluconazole, resulting in an increased intracellular concentration of fluconazole in the fungal cell.

α-Bisabolol inhibits Aspergillus fumigatus Af239 growth via affecting microsomal ∆24-sterol methyltransferase as a crucial enzyme in ergosterol biosynthesis pathway

Finding new compounds with antifungal properties is an important task due to the side effects of common antifungal drugs and emerging antifungal resistance in fungal strains. ∆24-sterol methyltransferase (24-SMT) is a crucial enzyme that plays important roles in fungal ergosterol biosynthesis pathway and is not found in humans. In the present study, the effects of α-bisabolol on Aspergillus fumigatus Af239 growth and ergosterol synthesis on the base of 24-SMT enzyme activity were studied; in addition, the expression of erg6, the gene encoded 24-SMT, was considered. To our knowledge, this is the first report demonstrating that α-bisabolol inhibits A. fumigatus growth specifically via suppressing fungal 24-SMT. Since this enzyme is a specific fungal enzyme not reported to exist in mammalian cells, α-bisabolol may serve as a lead compound in the development of new antifungal drugs. Fungi were cultured in presence of serial concentrations of α-bisabolol (0.281-9mM) for 3days at 35 °C. Mycelia dry weight was determined as an index of fungal growth and ergosterol content was assessed. Microsomal 24-SMT activity was assayed in presence of α-bisabolol as an inhibitor, lanosterol as a substrate and [methyl-H3] AdoMet (S-Adenosyl methionin). In addition, the expression of erg 6 gene (24-SMT encoding gene) was determined after treatments with various concentrations of α-bisabolol. Our results demonstrated that α-bisabolol strongly inhibited A. fumigatus growth (35.53-77.17%) and ergosterol synthesis (26.31-73.77%) dose-dependently and suppressed the expression of erg 6 gene by 76.14% at the highest concentration of 9mM. α-bisabolol inhibited the activity of 24-SMT by 99% at the concentration of 5mM. Taken together, these results provides an evidence for the first time that α-bisabolol inhibits A. fumigatus Af239 growth via affecting microsomal ∆24-sterol methyltransferase as a crucial enzyme in ergosterol biosynthetic pathway.

Inhibitory effects of cold atmospheric plasma on the growth, ergosterol biosynthesis, and keratinase activity in Trichophyton rubrum

BackgroundDermatophytosis is the most important superficial fungal infection which affects nearly 20% of human population worldwide. Recurrence of disease and emerging resistance of Trichophyton rubrum to synthetic antifungals are the main problems in control of dermatophytosis. The purpose of this study was to evaluate the effect of cold atmospheric plasma (CAP) on T. rubrum growth, ergosterol biosynthesis and keratinase activity. MethodsA CAP system, comprised of helium 98% – oxygen 2% (He/O2), was used. Trichophyton rubrum conidia suspensions were treated with CAP in time periods of 90, 120, 150 and 180 s in 96-well microplates. Fungal growth was evaluated by counting the colony forming unit (CFU). Fungal dry weight, ergosterol biosynthesis and keratinase activity were evaluated in CAP-treated T. rubrum and untreated controls. ResultsT. rubrum growth was significantly inhibited by 62%–91%. CAP strongly suppressed fungal ergosterol biosynthesis by 27%–54%. The keratinase activity was increased by 7.30%–21.88% up to 120 s CAP exposure. ConclusionOur results demonstrated for the first time that CAP inhibits T. rubrum growth, suppresses ergosterol biosynthesis and increases moderately keratinase activity in a dose-dependent manner. Overall, CAP exposure could be a potentially useful method for treatment of clinical cases of human and animal dermatophytoses.

Fungal sterol C22-desaturase is not an antimycotic target as shown by selective inhibitors and testing on clinical isolates

Inhibition of concise enzymes in ergosterol biosynthesis is one of the most prominent strategies for antifungal chemotherapy. Nevertheless, the enzymes sterol C5-desaturase and sterol C22-desaturase, which introduce double bonds into the sterol core and side chain, have not been fully investigated yet for their potential as antifungal drug targets. Lathosterol side chain amides bearing N-alkyl groups of proper length are known as potent inhibitors of the enzymes sterol C5-desaturase and sterol Δ24-reductase in mammalian cholesterol biosynthesis. Here we present the results of our evaluation of these amides for their ability to inhibit enzymes in fungal ergosterol biosynthesis. In the presence of inhibitor(s) an accumulation of sterols lacking a double bond at C22/23 (mainly ergosta-5,7-dien-3β-ol) was observed in Candida glabrata, Saccharomyces cerevisiae, and Yarrowia lipolytica. Hence, the lathosterol side chain amides were identified as selective inhibitors of the fungal sterol C22-desaturase, which was discussed as a specific target for novel antifungals. One representative inhibitor, (3S,20S)-20-N-butylcarbamoylpregn-7-en-3β-ol was subjected to antifungal susceptibility testing on patient isolates according to modified EUCAST guidelines. But, the test organisms showed no significant reduction of cell growth and/or viability up to an inhibitor concentration of 100μg/mL. This leads to the conclusion that sterol C22-desaturase is not an attractive target for the development of antifungals.