Decrease In Ergosterol Content Research Articles

Mechanisms of Litsea cubeba essential oil in the control of Colletotrichum scovillei in pepper (Capsicum annuum L.): Cell membrane/wall perspective

Although Litsea cubeba essential oil (LCEO) is widely used for against fungi, antifungal mechanism of it is not fully understood. In the study, antifungal potential and mechanism of LCEO on Colletotrichum scovillei, the main pathogen fungi caused pepper anthracnose, were studied. Results of gas chromatograph-mass spectrometry (GC-MS) showed that citral (67.37%) and d-Limonene (11.72%) were the major components of LCEO. LCEO was effective in inhibiting germination of C. scovillei spores, and the minimum inhibitory concentration (MIC) was 0.60 mg mL −1. To determine influence of LCEO on cell membrane integrity and permeability of C. scovillei, SEM observation, OD260 nm (nucleic acids), OD280 nm (proteins), extracellular electric conductivity, cell membrane permeability (propidium iodide, PI), cell wall integrity (calcofluor white, CFW), malondialdehyde (MDA) content and ergosterol content were analyzed. Spores exposed to LCEO were partly deformed, collapsed, shrunken and shriveled. The OD260 nm, OD280 nm and the extracellular electric conductivity of LCEO treatment group increased by 2.0, 1.5 and 1.2 times, respectively, at 12 h after treatment of LCEO (0.6 mg mL−1). Besides, high accumulation of MDA content indicated that LCEO significantly enhanced membrane lipid peroxidation. Moreover, LCEO induced a decrease in ergosterol content of C. scovillei. These findings suggest that LCEO exhibited potent inhibitory activities against C. scovillei, and the cell membrane permeability and cell wall integrity of C. scovillei treated by LCEO were significantly destroyed.

Antifungal mode of action of bay, allspice, and ajowan essential oils and their constituents against Colletotrichum gloeosporioides via overproduction of reactive oxygen species and downregulation of ergosterol biosynthetic genes

This study was conducted to investigate the antifungal activity of 135 plant essential oils (EOs) and their constituents against Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. Among the EOs, bay (Pimenta racemosa), allspice (Pimenta dioica), and ajowan (Trachyspermum ammi) showed 100 % inhibition of C. gloeosporioides at 10, 5, and 5 μl/paper disc, respectively. Of their constituents, (-)-α-pinene, (+)-α-pinene, thymol, eugenol, isoeugenol, 4-allylphenol, and carvacrol exhibited 100 % inhibition of growth at 10, 10, 10, 5, 5, 2.5, and 1.25 μl/paper disc, respectively. In an antifungal bioassay of artificial blends, eugenol, 4-allylphenol, and thymol were determined to be the major contributors to the antifungal activity of bay, allspice, and ajowan EOs. Confocal laser scanning microscopy images indicate the overproduction of reactive oxygen species (ROS) and cell membrane disruption in C. gloeosporioides treated with the EOs or their constituents. In addition, a decrease in ergosterol content and downregulation of ergosterol biosynthetic genes were confirmed in treated C. gloeosporioides. Based on these results, we attribute the antifungal actions of bay, allspice, and ajowan EOs and their active constituents to ROS generation and downregulation of ergosterol biosynthetic genes.

Towards understanding the effect of heavy metals on mycobiont physiological condition in a widespread metal-tolerant lichen Cladonia rei

Heavy metals present in the environment can cause a variety of injury symptoms in various organisms including lichens. Most studies examined metal-induced stress under controlled laboratory conditions, and little is known about actual response of lichens in their natural habitat. This study aims to recognize the effect of heavy metal accumulation (total and intracellular) on lichen physiological and biochemical parameters specifically related to the functioning of fungal component. Cladonia rei was used as a model species due to its common occurrence both in unpolluted and extremely polluted sites. We observed a decline in the fungal metabolism which was expressed by a decrease in ergosterol content and an increase in cell membrane damage as a result of increased Zn, Cd, Cu and Ni accumulation. Additionally, the results indicated that increased accumulation of xenobiotics (Pb and As) caused reduction of glutathione (GSH) concentrations and increased membrane lipid peroxidation. Therefore, we conclude that GSH does not provide high oxidative stress protection in C. rei which is somewhat against its insensitivity to pollution. The reduced pool of GSH could be explained by its oxidation to glutathione disulphide induced by heavy metal stress or its use for phytochelatin (PC) synthesis. The content of secondary metabolites was not related to heavy metal accumulation and remained at a relatively stable level. This indicates that the decline in the physiological condition did not weaken the mycobiont of C. rei enough to inhibit the synthesis of secondary metabolites and their precursors were supplied at a sufficient level. Thus, the potential function of main secondary metabolites as extracellular metal immobilizers and antioxidants is still possible even in individuals growing at extremely polluted sites. Despite the evident heavy metal stress, C. rei copes well and spreads easily through extremely polluted environments, which underlines its unique pioneering abilities in highly disturbed sites.

Engineering of cis-Element in Saccharomyces cerevisiae for Efficient Accumulation of Value-Added Compound Squalene via Downregulation of the Downstream Metabolic Flux.

Transcriptional downregulation is widely used for metabolic flux control. Here, marO, a cis-element of Escherichia coli mar operator, was explored to engineer promoters of Saccharomyces cerevisiae for downregulation. First, the ADH1 promoter (PADH1) and its enhanced variant PUADH1 were engineered by insertion of marO into different sites, which resulted in decrease in both gfp5 transcription and GFP fluorescence intensity to various degrees. Then, marO was applied to engineer the native ERG1 and ERG11 promoters due to their importance for accumulation of value-added intermediates squalene and lanosterol. Elevated squalene content (4.9-fold) or lanosterol content (4.8-fold) and 91 or 28% decrease in ergosterol content resulted from the marO-engineered promoter PERG1(M5) or PERG11(M3), respectively, indicating the validity of the marO-engineered promoters in metabolic flux control. Furthermore, squalene production of 3.53 g/L from cane molasses, a cheap and bulk substrate, suggested the cost-effective and promising potential for squalene production.

Evaluation of the antifungal and biochemical activities of mefentrifluconazole against Botrytis cinerea

Mefentrifluconazole is the first product of a new sub-class of triazoles fungicides, i.e., the isopropanol triazoles, with the broad spectrum and high activity. In this study, the potential and biochemical activities of mefentrifluconazole against Botrytis cinerea were investigated. The frequency distribution of all EC50 values of mefentrifluconazole against mycelial growth and germ tube elongation of 106 isolates formed unimodal curve, with the mean EC50 values of 0.124 ± 0.025 and 0.015 ± 0.008 μg/mL, respectively. The effect of mefentrifluconazole against gray mold was determined on detached leaves of cucumber in vivo, the treatment of mefentrifluconazole at 200 μg/mL provided 100% preventative efficacy and 72.7% curative efficacy. No evident correlation was detected between the sensitivity of B. cinerea to mefentrifluconazole and that to tebuconazole, difenoconazole, myclobutanil, hexaconazole, triadimefon, flusilazole and pyrisoxazole (P > 0.05). Mefentrifluconazole treatment resulted in the increase of mycelium branch, the decrease of ergosterol content and the changes of the permeability of cell membrane. These studies evaluated the potential of mefentrifluconazole to control gray mold and helped us to understand the possible biochemical activity of mefentrifluconazole against B.cinerea.

Kalopanaxsaponin A induces reactive oxygen species mediated mitochondrial dysfunction and cell membrane destruction in Candida albicans.

Candidiasis causes high morbidity and mortality among immunocompromised patients. Antifungal drug resistance and cytotoxicity highlight the need of effective antifungal therapeutics. In this study, we found that kalopanaxsaponin A (KPA), a triterpenoid saponin natural product, could inhibit the proliferation of various Candida species, and exerted a fungicidal effect against C. albicans. To further explore its antifungal action mode, spectrofluorophotometer, fluorescence microscopy and transmission electron microscopy were performed, showing that KPA treatment induced the accumulation of intracellular reactive oxygen species (ROS), resulting in mitochondrial dysfunction. Meanwhile, KPA treatment also broke down the membrane barrier of C. albicans causing the leakage of intracellular trehalose, the entrance of extracellular impermeable substance and the decrease of ergosterol content. Both ROS accumulation and membrane destruction contributed to the death of C. albicans cells. Our work preliminarily elucidated the potential mechanisms of KPA against C. albicans on a cellular level, and might provide a potential option for the treatment of clinical candidiasis.

Antifungal and aflatoxin B1 inhibitory efficacy of nanoencapsulated Pelargonium graveolens L. essential oil and its mode of action

The study reports the antifungal and aflatoxin B1 (AFB1) inhibitory mode of action of nanoencapsulated Pelargonium graveolens L. essential oil (Ne-PGEO). Nanoencapsulation of PGEO was prepared using chitosan nanoparticles and interactions were confirmed by scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) analysis. Chemical composition of PGEO was explored by gas chromatography-mass spectrometry (GC-MS) analysis where citronellyl formate (19.2%), linalool (15.2%), menthone (16.7%), and isomenthone (12.2%) were identified as the major compounds. Ne-PGEO inhibited the growth and aflatoxin B1 production by Aspergillus flavus at 1.00 μLmL−1, which was lower than the free PGEO at 1.25 μLmL−1. The antifungal and aflatoxin B1 inhibitory mechanism of Ne-BPEO were related to a decrease in ergosterol content, carbon source utilization, enhancement of ions leakage, and impairment in the functioning of the ver-1 gene. In addition, the Ne-PGEO effectively preserves the organoleptic (color, taste, odour, and texture) and nutritional properties of the model food (maize grain). In view of enhanced antifungal, anti-aflatoxin activity, and sensory attributes, the results suggested that Ne-PGEO could be used as a plant-based food preservative to enhance the shelf-life of food commodities.

Alkylimidazolium Ionic Liquids as Antifungal Alternatives: Antibiofilm Activity Against Candida albicans and Underlying Mechanism of Action.

Candida albicans is an opportunistic pathogen causes fungal infections that range from common skin infections to persistent infections through biofilm formation on tissues, implants and life threatening systemic infections. New antifungal agents or therapeutic methods are desired due to high incidence of infections and emergence of drug-resistant strains. The present study aimed to evaluate (i) the antifungal and antibiofilm activity of 1-alklyl-3-methyl imidazolium ionic liquids ([CnMIM]+[X]–, n = 4, 12 and 16) against Candida albicans ATCC 10231 and two clinical C. albicans strains and (ii) the mechanism of action of promising antifungal ionic liquid on C. albicans. Two of the tested compounds were identified as more effective in preventing growth and biofilm formation. These ionic liquid compounds with –dodecyl and –hexadecyl alkyl groups effectively prevented biofilm formation by fluconazole resistant C. albicans 10231 and two other clinical C. albicans strains. Although both the compounds caused viability loss in mature C. albicans biofilms, an ionic liquid with –hexadecyl group ([C16MIM]+[Cl]–) was more effective in dispersing mature biofilms. This promising ionic liquid compound ([C16MIM]+[Cl]–) was chosen for determining the underlying mode of action on C. albicans cells. Light microscopy showed that ionic liquid treatment led to a significant reduction in cell volume and length. Increased cell membrane permeability in the ionic liquid treated C. albicans cells was evident in propidium iodide staining. Leakage of intracellular material was evident in terms of increased absorbance of supernatant and release of potassium and calcium ions into extracellular medium. A decrease in ergosterol content was evident when C. albicans cells were cultured in the presence of antifungal ionic liquid. 2′,7′-Dichlorodihydrofluorescein acetate assay revealed reactive oxygen species generation and accumulation in C. albicans cells upon treatment with antifungal ionic liquid. The effect of antifungal ionic liquid on mitochondria was evident by decreased membrane potential (measured by Rhodamine 123 assay) and loss of metabolic activity (measured by MTT assay). This study demonstrated that imidazolium ionic liquid compound exert antifungal and antibiofilm activity by affecting various cellular processes. Thus, imidazolium ionic liquids represent a promising antifungal treatment strategy in lieu of resistance development to common antifungal drugs.

Mode of action and anti-Candida activity of Artemisia annua mediated-synthesized silver nanoparticles

Candida albicans is a polymorphic opportunistic commensal that causes both superficial and systemic fungal infections especially in immunocompromised patients. Biologically synthesized silver nanoparticles (AgNPs) have emerged as potential antifungal agents. The present work evaluates the antifungal activity of Artemisia annua synthesized AgNPs against three Candida species (C. albicans ATCC 90028, C. tropicalis ATCC 750 and C. glabrata ATCC 90030). The in vitro effect of AgNPs was investigated for fungal growth, sterol content, secretion of hydrolytic enzymes and yeast-to-hyphal transition. The green synthesized AgNPs were effective against all the three species with minimum inhibitory concentration (MIC) in the range 80–120μgml−1. Candida glabrata showed greater sensitivity for AgNPs followed by Candida tropicalis and C. albicans. AgNPs at 4MIC were as effective as fluconazole (FLC) and caused only 5% haemolysis while FLC caused 50% haemolysis at the same concentration. The secretion of hydrolytic enzymes was the lowest in case of AgNP exposed C. glabrata. Yeast-to-hyphal transition was significantly reduced in treated C. albicans cells and showed disfigured morphology in SEM images. The decrease in ergosterol content was slightly higher (94%) in both C. glabrata and C. tropicalis in comparison to C. albicans (69%). Green synthesized AgNPs thus have immense potential as an antifungal and can play a crucial role in the management of Candida infections especially those caused by C. glabrata.

Mevalonate Diphosphate Decarboxylase MVD/Erg19 Is Required for Ergosterol Biosynthesis, Growth, Sporulation and Stress Tolerance in Aspergillus oryzae.

Mevalonate diphosphate decarboxylase (MVD; EC 4.1.1.33) is a key enzyme of the mevalonic acid (MVA) pathway. In fungi, the MVA pathway functions as upstream of ergosterol biosynthesis, and MVD is also known as Erg19. Previously, we have identified Aoerg19 in Aspergillus oryzae using bioinformatic analysis. In this study, we showed that AoErg19 function is conserved using phylogenetic analysis and yeast complementation assay. Quantitative reverse transcription–PCR (qRT-PCR) indicated that Aoerg19 expression changed in different growth stages and under different forms of abiotic stress. Subcellular localization analysis showed that AoErg19 was located in the vacuole. Overexpression of Aoerg19 decreased the ergosterol content in A. oryzae, which may due to the feedback-mediated downregulation of Aoerg8. Consistent with the decrease in ergosterol content, both Aoerg19 overexpression and RNAi strains of A. oryzae are sensitive to abiotic stressors, including ergosterol biosynthesis inhibitor, temperature, salt and ethanol. Thus, we have identified the function of AoErg19 in A. oryzae, which may assist in genetic modification of MVA and the ergosterol biosynthesis pathway.

Nanoencapsulated plant-based bioactive formulation against food-borne molds and aflatoxin B1 contamination: Preparation, characterization and stability evaluation in the food system

A novel synergistic formulation (TML) based on the combination of thymol (T), methyl cinnamate (M), and linalool (L) has been prepared using the mixture design assay. Nanoencapsulation of developed formulation TML (Ne-TML) was prepared and characterised by SEM, XRD and FTIR. The Ne-TML was assessed for its antifungal and anti-aflatoxin B1 potential in vitro and in the food systems (Pennisetum glaucum L.), and also examined its effects on organoleptic properties. The Ne-TML cause complete inhibition of growth and AFB1 production at 0.3 μl/ml and 0.2 μl/ml. In-situ results revealed that Ne-TML exhibited maximum protection from fungal (75.40%) and aflatoxin B1 contamination (100%) at 0.3 μl/ml during six months of storage. The speculated antifungal mode of action of Ne-TML was related to the decrease in ergosterol content, membrane ions leakage, impairment in carbon-source utilization, mitochondrial functioning, anti-oxidative defence system (SOD, CAT, and GR) and Ver-1 gene of aflatoxin B1 biosynthesis.

Assessment of chemically characterised Myristica fragrans essential oil against fungi contaminating stored scented rice and its mode of action as novel aflatoxin inhibitor

The study reports chemically characterised Myristica fragrans essential oil (MFEO) as plant based food preservative against fungal and aflatoxin B1 (AFB1) contamination of scented rice varieties. The chemical profile of MFEO revealed elemicin (27.08%), myristicine (21.29%) and thujanol (18.55%) as major components. The minimum inhibitory and minimum aflatoxin inhibitory concentrations of MFEO were 2.75 and 1.5 mg/ml, respectively. The MFEO was efficacious against a broad spectrum of food deteriorating fungi. MFEO caused decrease in ergosterol content of fungal plasma membrane and enhanced leakage of cellular ions, depicting plasma membrane as the site of action. The MFEO caused reduction in cellular methylglyoxal content, the aflatoxin inducer. This is the first report on MFEO as aflatoxin suppressor. The essential oil may be recommended as plant based food preservative after large scale trials and reduction in methylglyoxal suggests its application for development of aflatoxin resistant varieties through green transgenics.

2,4-dichlorophenoxyacetic acid-induced oxidative stress: Metabolome and membrane modifications in Umbelopsis isabellina, a herbicide degrader.

The study reports the response to herbicide of the 2,4-dichlorophenoxyacetic acid (2,4-D)–degrading fungal strain Umbelopsis isabellina. A comparative analysis covered 41 free amino acids as well as 140 lipid species of fatty acids, phospholipids, acylglycerols, sphingolipids, and sterols. 2,4-D presence led to a decrease in fungal catalase activity, associated with a higher amount of thiobarbituric acid-reactive substances (TBARS). Damage to cells treated with the herbicide resulted in increased membrane permeability and decreased membrane fluidity. Detailed lipidomic profiling showed changes in the fatty acids composition such as an increase in the level of linoleic acid (C18:2). Moreover, an increase in the phosphatidylethanolamine/phosphatidylcholine ratio was observed. Analysis of fungal lipid profiles revealed that the presence of 2,4-D was accompanied by the accumulation of triacylglycerols, a decrease in ergosterol content, and a considerable rise in the level of sphingolipid ceramides. In the exponential phase of growth, increased levels of leucine, glycine, serine, asparagine, and hydroxyproline were found. The results obtained in our study confirmed that in the cultures of U. isabellina oxidative stress was caused by 2,4-D. The herbicide itself forced changes not only to membrane lipids but also to neutral lipids and amino acids, as the difference of tested compounds profiles between 2,4-D—containing and control samples was consequently lower as the pesticide degradation progressed. The presented findings may have a significant impact on the basic understanding of 2,4-D biodegradation and may be applied for process optimization on metabolomic and lipidomic levels.

Nanoencapsulated Illicium verum Hook.f. essential oil as an effective novel plant-based preservative against aflatoxin B1 production and free radical generation

The study reports efficacy of Illicium verum essential oil (IvEO) against food borne moudls and its nanoencapsulation for enhancing antifungal and antiaflatoxigenic potency. Chemical characterization of the IvEO showed anethole (89.12%) as major compound followed by estragole (4.859%). The IvEO showed broad fungitoxic spectrum against common food borne moulds. It's minimum inhibitory concentration (MIC) and minimum aflatoxin B1 inhibitory concentration (MAIC) against aflatoxigenic strain Aspergillus flavus LHP-PV-1 were 0.7, and 0.5 μL/mL respectively. Morphological observations of treatment sets by SEM and TEM along with decrease in ergosterol content and enhanced leakage of Ca2+, K+ and Mg2+ ions denoted fungal cell membrane as site of action. The IvEO showed promising free radical scavenging activity and favourable safety profile with high LD50 value on mice. The IvEO also exhibited considerable protection of Pistacia vera from fungal contamination and complete protection from aflatoxin B1 contamination in storage containers. Nanoencapsulated IvEO in gel form and lyophilized form exhibited enhanced efficacy as fungal inhibitor and aflatoxin suppressor. The chemically characterised IvEO may be recommended as plant based preservative having favourable safety and its nanocapsules may be of industrial significance as shelf life enhancer of food items. This is the first report on in situ antiaflatoxigenic efficacy and nanoencapsulation of IvEO.

Chemically characterized Mentha cardiaca L. essential oil as plant based preservative in view of efficacy against biodeteriorating fungi of dry fruits, aflatoxin secretion, lipid peroxidation and safety profile assessment

The study reports Mentha cardiaca essential oil (EO) as plant based preservative against fungal and aflatoxin contamination of stored dry fruits. Mycoflora analysis of the dry fruits revealed Aspergillus favus LHP-PV-1 as the most aflatoxigenic isolate with highest Aflatoxin B1 content. M. cardiaca EO showed broad fungitoxic spectrum inhibiting the tested moulds contaminating dry fruits. It's minimum inhibitory concentration (MIC), minimum aflatoxin inhibitory concentration (MAIC) and minimum fungicidal concentration (MFC) against A. favus LHP-PV-1 were recorded to be 1.25, 1.0 and 2.25 µL/mL respectively. The EO caused decrease in ergosterol content and enhanced leakage of Ca2+, K+ and Mg2+ ions from treated fungal cells, depicting fungal plasma membrane as the site of antifungal action. The EO showed promising DPPH free radical scavenging activity (IC50 value:15.89 µL/mL) and favourable safety profile with LD50 value (7133.70 mg/kg body wt.) when estimated through acute oral toxicity on mice. Carvone (61.62%) was recorded as the major component of the oil during chemical characterisation through GC-MS. Based on strong antifungal, antiaflatoxigenic and antioxidant potential, the chemically characterised M. cardiaca EO may be recommended as safe plant based preservative and shelf life enhancer of food items. This is the first report on antifungal and antiaflatoxigenic activity of M. cardiaca EO.

The Fungicidal Activity of Thymol against Fusarium graminearum via Inducing Lipid Peroxidation and Disrupting Ergosterol Biosynthesis.

Thymol is a natural plant-derived compound that has been widely used in pharmaceutical and food preservation applications. However, the antifungal mechanism for thymol against phytopathogens remains unclear. In this study, we identified the antifungal action of thymol against Fusarium graminearum, an economically important phytopathogen showing severe resistance to traditional chemical fungicides. The sensitivity of thymol on different F. graminearum isolates was screened. The hyphal growth, as well as conidial production and germination, were quantified under thymol treatment. Histochemical, microscopic, and biochemical approaches were applied to investigate thymol-induced cell membrane damage. The average EC50 value of thymol for 59 F. graminearum isolates was 26.3 μg·mL−1. Thymol strongly inhibited conidial production and hyphal growth. Thymol-induced cell membrane damage was indicated by propidium iodide (PI) staining, morphological observation, relative conductivity, and glycerol measurement. Thymol induced a significant increase in malondialdehyde (MDA) concentration and a remarkable decrease in ergosterol content. Taken together, thymol showed potential antifungal activity against F. graminearum due to the cell membrane damage originating from lipid peroxidation and the disturbance of ergosterol biosynthesis. These results not only shed new light on the antifungal mechanism of thymol, but also imply a promising alternative for the control of Fusarium head blight (FHB) disease caused by F. graminearum.

Impaired ergosterol biosynthesis mediated fungicidal activity of Co(II) complex with ligand derived from cinnamaldehyde

In this study, we have used aldehyde function of cinnamaldehyde to synthesize N, N′-Bis (cinnamaldehyde) ethylenediimine [C20H20N2] and Co(II) complex of the type [Co(C40H40N4)Cl2]. The structures of the synthesized compounds were determined on the basis of physiochemical analysis and spectroscopic data (1H NMR, FTIR, UV–visible and mass spectra) along with molar conductivity measurements. Anticandidal activity of cinnamaldehyde its ligand [L] and Co(II) complex was investigated by determining MIC80, time-kill kinetics, disc diffusion assay and ergosterol extraction and estimation assay. Ligand [L] and Co(II) complex are found to be 4.55 and 21.0 folds more efficient than cinnamaldehyde in a liquid medium. MIC80 of Co(II) complex correlated well with ergosterol inhibition suggesting ergosterol biosynthesis to be the primary site of action. In comparison to fluconazole, the test compounds showed limited toxicity against H9c2 rat cardiac myoblasts. In confocal microscopy propidium iodide (PI) penetrates the yeast cells when treated with MIC of metal complex, indicating a disruption of cell membrane that results in imbibition of dye. TEM analysis of metal complex treated cells exhibited notable alterations or damage to the cell membrane and the cell wall. The structural disorganization within the cell cytoplasm was noted. It was concluded that fungicidal activity of Co(II) complex originated from loss of membrane integrity and a decrease in ergosterol content is only one consequence of this.

Plasma Membrane Proteolipid 3 Protein Modulates Amphotericin B Resistance through Sphingolipid Biosynthetic Pathway.

Invasive opportunistic fungal infections of humans are common among those suffering from impaired immunity, and are difficult to treat resulting in high mortality. Amphotericin B (AmB) is one of the few antifungals available to treat such infections. The AmB resistance mechanisms reported so far mainly involve decrease in ergosterol content or alterations in cell wall. In contrast, depletion of sphingolipids sensitizes cells to AmB. Recently, overexpression of PMP3 gene, encoding plasma membrane proteolipid 3 protein, was shown to increase and its deletion to decrease, AmB resistance. Here we have explored the mechanistic basis of PMP3 effect on AmB resistance. It was found that ergosterol content and cell wall integrity are not related to modulation of AmB resistance by PMP3. A few prominent phenotypes of PMP3 delete strain, namely, defective actin polarity, impaired salt tolerance, and reduced rate of endocytosis are also not related to its AmB-sensitivity. However, PMP3 overexpression mediated increase in AmB resistance requires a functional sphingolipid pathway. Moreover, AmB sensitivity of strains deleted in PMP3 can be suppressed by the addition of phytosphingosine, a sphingolipid pathway intermediate, confirming the importance of this pathway in modulation of AmB resistance by PMP3.

UPC2A is required for high-level azole antifungal resistance in Candida glabrata.

Candida glabrata, the second most common cause of Candida infections, is associated with high rates of mortality and often exhibits resistance to the azole class of antifungal agents. Upc2 and Ecm22 in Saccharomyces cerevisiae and Upc2 in Candida albicans are the transcriptional regulators of ERG11, the gene encoding the target of azoles in the ergosterol biosynthesis pathway. Recently two homologs for these transcription factors, UPC2A and UPC2B, were identified in C. glabrata. One of these, UPC2A, was shown to influence azole susceptibility. We hypothesized that due to the global role for Upc2 in sterol biosynthesis in S. cerevisiae and C. albicans, disruption of UPC2A would enhance the activity of fluconazole in both azole-susceptible dose-dependent (SDD) and -resistant C. glabrata clinical isolates. To test this hypothesis, we constructed mutants with disruptions in UPC2A and UPC2B alone and in combination in a matched pair of clinical azole-SDD and -resistant isolates. Disruption of UPC2A in both the SDD and resistant isolates resulted in increased susceptibility to sterol biosynthesis inhibitors, including a reduction in fluconazole MIC and minimum fungicidal concentration, enhanced azole activity by time-kill analysis, a decrease in ergosterol content, and downregulation of baseline and inducible expression of several sterol biosynthesis genes. Our results indicate that Upc2A is a key regulator of ergosterol biosynthesis and is essential for resistance to sterol biosynthesis inhibitors in C. glabrata. Therefore, the UPC2A pathway may represent a potential cotherapeutic target for enhancing azole activity against this organism.

Efficacy of Angelica archangelica essential oil, phenyl ethyl alcohol and α- terpineol against isolated molds from walnut and their antiaflatoxigenic and antioxidant activity.

Mold association, aflatoxin B1 contamination as well as oxidative deterioration of agri-food items during storage and processing are some global task for food industries. In view of the adverse effects of some synthetic preservatives on treated food items and subsequently on consumers health, recently plant based chemicals are encouraged by food industries as better alternatives of synthetics. The present study recommends the combination (1:1:1) of Angelica archangelica essential oil: Phenyl ethyl alcohol (PEA): α- terpineol as botanical preservative against molds, aflatoxin contamination and oxidative deterioration of walnut samples. Eight mold species were procured from stored walnut samples, including some aflatoxigenic Aspergillus flavus strains. The combination inhibited growth of aflatoxigenic strain Aspergillus flavus NKDW-7 and aflatoxin B1 production at 2.25 and 2.0μLmL(-1) respectively. The IC50 value of the combination was recorded as 3.89μLmL(-1), showing strong antioxidant potential. The antifungal action of the combination showed > 90% decrease in ergosterol content in plasma membrane of A. flavus at 2.0μLmL(-1). The LD50 of the combination, through oral administration on mice, was 9562.9μLkg(-1) body weight, indication favourable safety profile as a plant based preservative. The combination may be recommended as safe preservative against molds, aflatoxin contamination and oxidative deterioration of walnut samples.