Sterols In Fungi Research Articles

Ergosterol distribution controls surface structure formation and fungal pathogenicity.

Ergosterol, the major sterol in fungal membranes, is critical for defining membrane fluidity and regulating cellular processes. Although ergosterol synthesis has been well defined in model yeast, little is known about sterol organization in the context of fungal pathogenesis. We identified a retrograde sterol transporter, Ysp2, in the opportunistic fungal pathogen Cryptococcus neoformans. We found that the lack of Ysp2 under host-mimicking conditions leads to abnormal accumulation of ergosterol at the plasma membrane, invagination of the plasma membrane, and malformation of the cell wall, which can be functionally rescued by inhibiting ergosterol synthesis with the antifungal drug fluconazole. We also observed that cells lacking Ysp2 mislocalize the cell surface protein Pma1 and have abnormally thin and permeable capsules. As a result of perturbed ergosterol distribution and its consequences, ysp2∆ cells cannot survive in physiologically relevant environments such as host phagocytes and are dramatically attenuated in virulence. These findings expand our knowledge of cryptococcal biology and underscore the importance of sterol homeostasis in fungal pathogenesis. IMPORTANCE Cryptococcus neoformans is an opportunistic fungal pathogen that kills over 100,000 people worldwide each year. Only three drugs are available to treat cryptococcosis, and these are variously limited by toxicity, availability, cost, and resistance. Ergosterol is the most abundant sterol in fungi and a key component in modulating membrane behavior. Two of the drugs used for cryptococcal infection, amphotericin B and fluconazole, target this lipid and its synthesis, highlighting its importance as a therapeutic target. We discovered a cryptococcal ergosterol transporter, Ysp2, and demonstrated its key roles in multiple aspects of cryptococcal biology and pathogenesis. These studies demonstrate the role of ergosterol homeostasis in C. neoformans virulence, deepen our understanding of a pathway with proven therapeutic importance, and open a new area of study.

Natamycin sequesters ergosterol and interferes with substrate transport by the lysine transporter Lyp1 from yeast

Natamycin is a polyene macrolide, widely employed to treat fungal keratitis and other yeast infections as well as to protect food products against fungal molds. In contrast to other polyene macrolides, such as nystatin or amphotericin B, natamycin does not form pores in yeast membranes, and its mode of action is not well understood. Here, we have employed a variety of spectroscopic methods, computational modeling, and membrane reconstitution to study the molecular interactions of natamycin underlying its antifungal activity. We find that natamycin forms aggregates in an aqueous solution with strongly altered optical properties compared to monomeric natamycin. Interaction of natamycin with model membranes results in a concentration-dependent fluorescence increase which is more pronounced for ergosterol- compared to cholesterol-containing membranes up to 20 mol% sterol. Evidence for formation of specific ergosterol-natamycin complexes in the bilayer is provided. Using nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy, we find that natamycin sequesters sterols, thereby interfering with their well-known ability to order acyl chains in lipid bilayers. This effect is more pronounced for membranes containing the sterol of fungi, ergosterol, compared to those containing mammalian cholesterol. Natamycin interferes with ergosterol-dependent transport of lysine by the yeast transporter Lyp1, which we propose to be due to the sequestering of ergosterol, a mechanism that also affects other plasma membrane proteins. Our results provide a mechanistic explanation for the selective antifungal activity of natamycin, which can set the stage for rational design of novel polyenes in the future.

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 like Aspergillus 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 a Domain of Unknown Function 2156, which actually transfers aspartate from Asp-tRNAAsp onto 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.

Steroids from Marine-Derived Fungi: Evaluation of Antiproliferative and Antimicrobial Activities of Eburicol.

The most common sterol in fungi is ergosterol, which has frequently been investigated in human pathogenic fungal strains. This sterol, and others isolated from fungal strains, has also demonstrated cytotoxicity against cancer cell lines and antimicrobial activities. Marine fungi can produce high amounts of bioactive compounds. So, a screening was performed to study sterol composition using GC/MS in 19 marine fungal strains and ergosterol was always the major one. One strain, Clonostachys rosea MMS1090, was selected due to its high amount of eburicol and a one strain many compounds approach was performed on seven culture media to optimize its production. After purification and structural identification by NMR, eburicol was assessed against four cancer cell lines, MCF-7, MDA-MB-231, NSCLC-N6-L16 and A549, and seven human pathogenic bacteria Staphylococcus aureus, Bacillus sp., Bacillus cereus, Listeria ivanovii, Escherichia coli, Citrobacter freundii and Salmonella spp. The most significant activity was cytotoxicity against MCF-7 cells (2 µM). This is the first report of such an accumulation of eburicol in the marine fungal strain C. rosea confirming its potential in the production of bioactive lipids.

Fluvastatin potentiates the activity of caspofungin against Aspergillus fumigatus in vitro

Statins (anticholesterol drugs) inhibit HMG-CoA reductase, which is a key rate-limiting enzyme in the synthesis of sterols in fungi. We therefore investigated the in vitro inhibitory activity of various statins against Aspergillus fumigatus alone and in combination with antifungal drugs. Fresh conidial suspensions from 10 clinical isolates of A. fumigatus were obtained. The MIC and minimum fungicidal concentration (MFC) were determined by the Clinical and Laboratory Standards Institute M-38A protocol and the fungicidal activity by time–kill study. Fluvastatin (FST) showed good activity (MIC, 2 mg/L; MFC, 4 mg/L) against A. fumigatus; other statins had no activity (MIC ≥256 mg/L). FST enhanced the activity of caspofungin (CFG) against A. fumigatus; subinhibitory concentrations of FST in combination with CFG showed >99.9% killing of A. fumigatus conidia, whereas either drug alone showed poor activity at subinhibitory concentrations. FST potentiated the antifungal activity of CFG but displayed no specific interaction with voriconazole or amphotericin B.

Simultaneous determination of free ergosterol and ergosteryl esters in Cordyceps sinensis by HPLC

The caterpillar-shaped Chinese medicinal mushroom Cordyceps sinensis consists of the fruiting body (CsA) and the host caterpillar (CsB). Ergosterol is a principal sterol in fungi and can indicate the level of mycelia of C. sinensis. Ergosterol is present in two forms, as free ergosterol and esterified ergosterol, which have different physiological functions. The relative abundances of free to esterified ergosterol are different among the various species. In the present study, a gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of free and esterified ergosterol in CsA and CsB of C. sinensis. The results showed that CsA and CsB had similar ergosterol compositions, but the level of ergosteryl esters in CsB was much higher than that in CsA, indicating that CsA and CsB might be in different growth phase or have different physiological functions for the growth and multiplication of C. sinensis.

Production of Polyclonal Antibody against Ergosterol Hemisuccinate Using Freund's and Titermax Adjuvants

Ergosterol is the principal sterol in fungi and an important component of cell membranes. This sterol has been used previously to measure fungal growth in foods. In an attempt to develop antibodies for an immunoassay for ergosterol, we first derivatized the compound to its hemisuccinate (Erg-HS) and conjugated it to bovine serum albumin (Erg-HS-BSA). Six white female New Zealand rabbits were immunized by subcutaneous injections of Erg-HS-BSA conjugate followed by four booster injections. Freund's and Hunter's TiterMax adjuvants were used for stimulating strong and prolonged responses. An indirect enzyme-linked immunosorbent assay (ELISA) was used to assess the titers. Antibody titers to Erg-HS rose much earlier and were markedly higher when Freund's adjuvant was used than when TiterMax was used. Although titers as high as 6,400 were detectable by indirect ELISA, binding of these antibodies to an Erg-HS-BSA solid phase could not be inhibited by free ergosterol in a competitive indirect ELISA. The antibody was also evaluated for applicability in an enzyme-linked immunocytochemical assay for a variety of fungi but failed to detect the presence of ergosterol in the membranes of these organisms. Thus, although antibodies could be generated that showed high specificity for the Erg-HS-BSA conjugate, they could not detect free ergosterol.

Use of gas chromatography/mass spectrometry selected‐ion recording for sterol content pattern recognition in fungal classification

Chemotaxonomic studies are frequently limited by the volume of data obtained and the difficulty of reducing this to statistically interpretable results. This paper describes the use of a straightforward procedure for relating the pattern of occurrence of sterols in fungi, analysed by mass spectrometry, to their classification.

The intracellular location and physiological effects of abnormal sterols in fungi grown in the presence of morpholine and functionally related fungicides

Saccharomyces cerevisiae, when grown in the presence of nonlethal concentrations of fenpropimorph, accumulated 24-methylene ignosterol as its major sterol instead of ergosterol. This abnormal sterol was shown to be present in the microsomal membranes. Membranes containing this sterol were shown to have abnormal physiological properties: the S. cerevisiae plasma membrane was found to have a greater fluidity than normal and 24-methylene ignosterol-containing liposome membranes were found to be more permeable to glucose than those prepared with normal sterols. The phytopathogenic fungi, Botrytis cinerea, Pseudocercosporella herpotrichoides, and Pyrenophora teres, when grown similarly, accumulated a mixture of Δ 8- and Δ 8,14-sterols in place of ergosterol. Liposomes prepared with the total sterol mixture isolated from these fungi and S. cerevisiae after growth in the presence of fenpropimorph were also found to be more permeable to glucose than those prepared with the total sterol mixture isolated from control cultures. The possibility that the ultimate fungitoxicity of morpholine and related fungicides is due to alterations in membrane behavior brought about by the presence of abnormal sterols in the lipid bilayer is discussed.

Fungistatic activity of lucerne saponins and digitonin as related to sterols

The relationship between the growth inhibiting activity of saponins and the presence of sterols in fungi was studied. Spraying of mycelia with lucerne saponins or digitonin resulted in swift lysis of the hyphal tips of those fungi, which contain cholesterol in their cell membrane. The fungistatic activity of the saponins was found to be closely related to the relative amount of cholesterol in the found. Incorporation of exogenous cholesterol did not change the sensitivity of Pythium sp. PRL 2142 to lucerne saponins or digitonin. Similar experiments with M. laidlawii changed this organism from a tolerant to a highly sensitive one to digitonin but not to lucerne saponins, indicating that the growth inhibiting activity of saponins depends not only on the presence but also on the orientation of cholesterol in the cell membrane.

Sterols in Growth and Reproduction of Fungi

Fungi are cukaryotic organisms, which are characterized by cellular or­ ganelles such as nuclei and mitochondria. Eukaryotic organisms are gener­ ally considered to synthesize and cont::tin sterols, whereas prokaryotic orga­ nisms �;t]ch as hacteria (see 20) and hlm'-green algae (74, 103) are gener­ ally considered not to contain or need sterols. Although sterols and related compounds (tetracyclic triterpenes) occur in tremendous variety in the various taxonomic groups of plants and ani­ mals (17, 54, 55, 127), is usually considered to be the principal sterol in fungi. This conception probably arose because of the commercial interest in as provitamin D and as a precursor for the synthesis of clinically important steroids. Yeasts and certain other fungi frequently used in industrial fermentations are readily available sources of ergosterol. Yeasts and a few other fungi are also useful in studies of the biosynthesis of sterols (20, 43-45). Deeper penetrations into these areas demonstrate anew the folly of gener­ alizations based on few data. At least some bacteria (132, 133) and blue­ green algae (34, 124) in the prokaryotic group apparently do synthesize sterols. Another prokaryotic group, the pathogenic Mycoplasma, does not synthesize sterols but has a nutritional requirement for �hem (119). Eukar­ yotic organisms which cannot synthesize sterols hut require them include probably all insects (28), at least some nematodes (29, 71), and some proto­ zoa (20, 75, 78). Adequate charactcrizations of the sterols extracted from fungi are few. Frequently ergosterol contents of fungi are given with no attempt to prove the structure. The use of in physiological experiments with aquatic Phycomycetes on the assumption that is the fungal sterol is unfortunate, because is much more susceptible to autoxidation than sterols lacking the extra double bonds. Furthermore, the elegant study by McCorkindale and co-workers (108) shows that species of the Sapro­ legniales· and Leptomitales may be expected to contain cholesterol, desmos­ terol, 24-methylenecholesterol, or fucosterol rather than compounds such as which have a pair of conjugated double bonds in ring B. Other

NEW FORMS AND SOURCES OF VITAMIN D

Certain facts about vitamin D are widely known. It occurs only rarely in foodstuffs. It is formed in the skin by exposure to ultraviolet rays from the sun or from artificial sources. It is developed in some foodstuffs by their being briefly irradiated. It is produced by irradiating ergosterol, the sterol of fungi. From irradiated ergosterol it can be isolated and crystallized. The fact that vitamin D is not a single chemical substance has only recently been recognized. The erroneous view still commonly held is that ergosterol is the parent substance, or provitamin, from which all vitamin D arises. Ergosterol exhibits four spectral absorbtion bands in the ultraviolet region. These bands were observed in the unsaponifiable fraction of the various materials that become antiricketic on irradiation. Since it is rare that even one band, not to mention such a series of bands, is exactly duplicated by different chemical substances, the