RNA-dependent sterol aspartylation in fungi

Diverting aminoacyl-transfer RNAs (tRNAs) from protein synthesis is a well-known process used by a wide range of bacteria to aminoacylate membrane constituents. By tRNA-dependently adding amino acids to glycerolipids, bacteria change their cell surface properties, which intensifies antimicrobial drug resistance, pathogenicity, and virulence. No equivalent aminoacylated lipids have been uncovered in any eukaryotic species thus far, suggesting that tRNA-dependent lipid remodeling is a process restricted to prokaryotes. We report here the discovery of ergosteryl-3β-O-l-aspartate (Erg-Asp), a conjugated sterol that is produced by the tRNA-dependent addition of aspartate to the 3β-OH group of ergosterol, the major sterol found in fungal membranes. In fact, Erg-Asp exists in the majority of “higher” fungi, including species of biotechnological interest, and, more importantly, in human pathogens likeAspergillus fumigatus. We show that a bifunctional enzyme, ergosteryl-3β-O-l-aspartate synthase (ErdS), is responsible for Erg-Asp synthesis. ErdS corresponds to a unique fusion of an aspartyl-tRNA synthetase—that produces aspartyl-tRNAAsp(Asp-tRNAAsp)—and of aDomain of Unknown Function 2156, which actually transfers aspartate from Asp-tRNAAsponto ergosterol. We also uncovered that removal of the Asp modifier from Erg-Asp is catalyzed by a second enzyme, ErdH, that is a genuine Erg-Asp hydrolase participating in the turnover of the conjugated sterol in vivo. Phylogenomics highlights that the entire Erg-Asp synthesis/degradation pathway is conserved across “higher” fungi. Given the central roles of sterols and conjugated sterols in fungi, we propose that this tRNA-dependent ergosterol modification and homeostasis system might have broader implications in membrane remodeling, trafficking, antimicrobial resistance, or pathogenicity.

Identification of Candidate Ergosterol-Responsive Proteins Associated with the Plasma Membrane of Arabidopsis thaliana

The impact of fungal diseases on crop production negatively reflects on sustainable food production and overall economic health. Ergosterol is the major sterol component in fungal membranes and regarded as a general elicitor or microbe-associated molecular pattern (MAMP) molecule. Although plant responses to ergosterol have been reported, the perception mechanism is still unknown. Here, Arabidopsis thaliana protein fractions were used to identify those differentially regulated following ergosterol treatment; additionally, they were subjected to affinity-based chromatography enrichment strategies to capture and categorize ergosterol-interacting candidate proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Mature plants were treated with 250 nM ergosterol over a 24 h period, and plasma membrane-associated fractions were isolated. In addition, ergosterol was immobilized on two different affinity-based systems to capture interacting proteins/complexes. This resulted in the identification of defense-related proteins such as chitin elicitor receptor kinase (CERK), non-race specific disease resistance/harpin-induced (NDR1/HIN1)-like protein, Ras-related proteins, aquaporins, remorin protein, leucine-rich repeat (LRR)- receptor like kinases (RLKs), G-type lectin S-receptor-like serine/threonine-protein kinase (GsSRK), and glycosylphosphatidylinositol (GPI)-anchored protein. Furthermore, the results elucidated unknown signaling responses to this MAMP, including endocytosis, and other similarities to those previously reported for bacterial flagellin, lipopolysaccharides, and fungal chitin.

Phytosterol Content and Fatty Acid Pattern of Ten Different Nut Types

Ten different nut kinds (almonds, Brazil nuts, cashews, hazelnuts, macadamias, peanuts, pecans, pine nuts, pistachios, and walnuts) were evaluated for their total oil and phytosterol content as well as their fatty acid composition. The total oil content was the predominant component; mean values oscillated between 45.2 % (cashews) and 74.7 % (macadamias). Mean total phytosterol content ranged from 71.7 mg (Brazil nuts) to 271.9 mg (pistachios) per 100 g oil. ß-sitosterol was the major sterol (mean >71.7 mg/100 g oil) followed by minor contents of campesterol, ergosterol, and stigmasterol. Almonds, cashews, hazelnuts, macadamias, and pistachios were high in monounsaturated fatty acids (MUFA; > 55 %). MUFA- and polyunsaturated fatty acid (PUFA)-rich nuts were peanuts and pecans, whereas Brazil nuts, pine nuts, and walnuts had the highest PUFA content (> 50 %); the high unsaturated/saturated fatty acid ratio ranged from 4.5 to 11.8. However, the fatty acid pattern of every nut is unique.

Biochemical Characterization of Coriander Cakes Obtained by Extrusion

This study was designed to examine the effect of operating conditions such as nozzle diameter on fatty acid, sterol, and tocol composition of coriander cakes. Eight fatty acids were identified, with petroselinic acid accounting for 75–77% of the total fatty acids, followed by linoleic, oleic, and palmitic acids, accounting for 12-13%, 5%, and 3%, respectively, of the total fatty acids.β-Sitosterol was the major sterol in all oils with 33–35% of total sterols. The next major sterols in all oils were stigmasterol (24% of total sterols) and Δ7-stigmasterol (15% of total sterols). Coriander cake contained higher amounts of total tocotrienol whereγ-tocotrienol was the main compound.

Concentration of ergosterol in small-grained naturally contaminated and inoculated cereals

Ergosterol (ERG) is a major sterol constituent of most fungi. Its concentration is negligible in higher plants, but can be used as a chemical marker of the presence of fungal contaminations. In this study, ERG concentration was assessed in randomly collected samples of naturally contaminated grain (wheat, barley and oat) and in samples of grain (wheat, barley, triticale and oat) harvested after inoculation of heads with conidia of different Fusarium species. Wheat samples were analysed at three stages of grain development. The lowest ERG concentration was found in non-inoculated samples at the first stage of grain development. This concentration was increasing with grain ripening. In naturally contaminated samples collected after harvest, ERG concentration was lower in wheat than in barley and oat. ERG concentrations in inoculated samples varied significantly, but were always significantly higher than in naturally contaminated samples. In the above cereal samples it was much lower than the levels assayed in laboratory cultures inoculated with fungi from genus Fusarium. The content of ERG was also analyzed in milling products of small-grained cereals and other foodstuffs, where a considerable variation was observed. The lowest ERG amounts were assayed in flours with a high degree of purification, while the highest ones in case of flours and products with a low purification rate. The results indicate the potential application of HPLC combined with microwave-assisted extraction both when assaying samples with low ERG concentrations (naturally contaminated) and those characterized with high contents of fungal biomass (strongly infected, artificially inoculated). It also facilitates analyses of fungal biomass in technological processes, where results may be expected to vary considerably.

Posaconazole Is a Potent Inhibitor of Sterol 14α-Demethylation in Yeasts and Molds

ABSTRACT Posaconazole (POS; SCH 56592) is a novel triazole that is active against a wide variety of fungi, including fluconazole-resistant Candida albicans isolates and fungi that are inherently less susceptible to approved azoles, such as Candida glabrata. In this study, we compared the effects of POS, itraconazole (ITZ), fluconazole (FLZ), and voriconazole (VOR) on sterol biosynthesis in strains of C. albicans (both azole-sensitive and azole-resistant strains), C. glabrata, Aspergillus fumigatus, and Aspergillus flavus. Following exposure to azoles, nonsaponifiable sterols were extracted and resolved by liquid chromatography and sterol identity was confirmed by mass spectroscopy. Ergosterol was the major sterol in all but one of the strains; C. glabrata strain C110 synthesized an unusual sterol in place of ergosterol. Exposure to POS led to a decrease in the total sterol content of all the strains tested. The decrease was accompanied by the accumulation of 14α-methylated sterols, supporting the contention that POS inhibits the cytochrome P450 14α-demethylase enzyme. The degree of sterol inhibition was dependent on both dose and the susceptibility of the strain tested. POS retained activity against C. albicans isolates with mutated forms of the 14α-demethylase that rendered these strains resistant to FLZ, ITZ, and VOR. In addition, POS was a more potent inhibitor of sterol synthesis in A. fumigatus and A. flavus than either ITZ or VOR.

Sterols and linear alkylbenzenes in marine sediments from Admiralty Bay, King George Island, South Shetland Islands

Selected sterols (coprostanol, epicoprostanol, cholesterol, cholestanol), stanone (5ßcoprostanone) and linear alkylbenzenes (LABs) were measured in the surface sediments near Ferraz station sewage outfalls, in Admiralty Bay, King George Island, South Shetland Islands. during the summer of 1997/98 using GC-FID and GC-MS. Total sterol concentrations varied between 0.21 and 10.4 μg g−1 dry sediment. Cholesterol was the major sterol at all sites, except at the sewage outfall, where coprostanol predominated. The concentration of coprostanol varied between 0.03 and 6.14 μg g−1 dry sediment, but the majority of the samples contained levels below 0.13 μg g−1 dry sediment. The parameters coprostanol+epicoprostanol in total sterols, coprostanol/epicoprostanol ratio versus % of cholesterol in total sterols and versus 5ß-coprostanone concentration were used to identify the sewage impacted locations in the study area. Only sites extending to 50m from the sewage outfall exhibited a sterol signal indicating sewage input. Total LABs varied from <0.60 to 11.8 ng.g−1 dry sediment with the maximum level at the sewage outfall. Faeces from different species of seals all contained large amounts of cholesterol and some 5ßcoprostanone. Relatively low levels of coprostanol and high levels of cholesterol observed in distant sites could be attributed to natural sources such as marine mammals.

Sterol composition of itraconazole-resistant and itraconazole-susceptible isolates of Aspergillus fumigatus

Sterol composition of four clinical isolates of Aspergillus fumigatus resistant to itraconazole was determined by gas chromatography – mass spectrometry and compared with that of four susceptible strains. For all strains, the major sterol was ergosterol. Sterol compositions were qualitatively and quantitatively similar for the resistant and susceptible strains. These results suggest that itraconazole resistance is not related, for the strains studied, to alterations in the ergosterol synthesis pathway.Key words: Aspergillus fumigatus, itraconazole resistance, sterol composition.