A comparison of the sterol content of multiple isolates of the Candida albicans Darlington strain with other clinically azole-sensitive and -resistant strains

H owell , S.A., M allet , A.I. & N oble , W.C. 1990. A comparison of the sterol content of multiple isolates of the Candida albicans Darlington strain with other clinically azole-sensitive and -resistant strains. Journal of Applied Bacteriology 69 , 692–696.
The mechanism of azole resistance of Candida albicans NCPF 3310 (the deposited culture of the Darlington strain) has been investigated but never fully explained. Seven isolates of this strain, from various sources, were examined by gas chromatography-mass spectrometry to detect changes in the sterol composition following passage through many laboratories over several years. Five of the seven, including one recently isolated from the patient, were found to be similar to each other in sterol content, containing large amounts of fecosterol. Of the remaining two, one was thought to be a sensitive variant, both produced only small quantities of fecosterol and resembled the normal clinical strains and other azole-resistant strains in sterol content. The sterol composition of the Darlington strain was unique and apparently stable to prolonged in vitro experimentation and passage through the patient.     

Defective sterol C5-6 desaturation and azole resistance: a new hypothesis for the mode of action of azole antifungals

Two azole resistant isolates of Saccharomyces cerevisiae carried mutations allelic to erg 3 and were blocked to differing degrees at the C5-6 desaturation step of ergosterol biosynthesis. When treated with the sterol 14 alpha-demethylation inhibitor fluconazole the wild-type sensitive strain accumulated lanosterol and 14 alpha-methyl-erogosta-8,24(28)-dien-3 beta, 6 alpha-diol (14-methyl-3,6 diol). The stringent desaturase mutant, A2, accumulated 14 alpha-methyl-8,24(28)-dien-3 beta-ol (14-methyl fecosterol) and lanosterol as the major sterol components when treated with fluconazole. Resistant isolate A3 accumulated 14-methyl-3,6-diol, 14-methyl fecosterol, and lanosterol and was only partially blocked at sterol C5-6 desaturation. We conclude that functional sterol C5-6 desaturase is required for the synthesis of 14-methyl-3,6-diol under conditions of azole inhibition. We present a new hypothesis for the mode of action of azole antifungals based on the inability of 14-methyl-3,6-diol to support growth, and suggest that growth can occur through utilisation of 14-methyl fecosterol, produced by a combination of azole inhibition and defective sterol C5-6 desaturation.     

Characterisation of atovaquone resistance in Leishmania infantum promastigotes

Atovaquone, an antiparasitic agent, could possibly represent an alternative therapy after relapse following classical treatment for visceral leishmaniasis. Atovaquone-resistant strains were selected in vitro by stepwise drug pressure to study the mechanism of resistance in Leishmania. Characteristics of a promastigote strain resistant to 250 microg/ml of atovaquone were compared with those of the wild type (WT) strain. Resistant strains were shown to have a high level of resistance (45 times). They were stable in drug-free medium for 6 months, and showed no cross-resistance with other antileishmanial drugs. Rhodamine uptake and efflux were studied. They were not modified in the resistant strain, indicating the absence of P-glycoprotein overexpession. The effect of atovaquone on membrane lipidic composition was determined in both WT and atovaquone-resistant promastigotes. Analysis of lipid composition of the atovaquone-resistant strain showed that sterol biosynthesis was decreased in atovaquone-resistant parasites. Cholesterol was found to be the major membrane sterol as opposed to the WT strain. Cholesterol, due to its ordering effect, could decrease membrane fluidity and subsequently block the passage of atovaquone through the membrane. Increased membrane cholesterol content and altered drug membrane fluidity resulted from possible decrease of ergosterol biosynthesis by atovaquone, incorporation of cholesterol by promastigotes in the culture medium, solubilisation of atovaquone by cholesterol and co-passage of the two compounds or influence of dimethylsulfoxide. These results indicate that different cellular alterations may participate in the resistant phenotype, by altering drug membrane permeability.     

Identification of 24-methylene-24,25-dihydrolanosterol as a precursor of ergosterol in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus

Abstract Study of the plasma membrane sterol composition in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus revealed the presence of ergosterol, lanosterol, dehydroergosterol, fecosterol, episterol and 24-methylene-24,25-dihydrolanosterol (eburicol), a C-31 derivative. The growth of both yeasts in the presence of ketoconazole led to a decrease by 85% of the ergosterol content while the levels of lanosterol and eburicol increased. This suggests that in the biosynthetic pathway of ergosterol in Schizosaccharomyces species, the transmethylation process on the C-24 may occur directly on lanosterol and not only on zymosterol. On the other hand, it cannot be excluded that in the genus Schizosaccharomyces two routes exist from lanosterol to ergosterol: the classical one via a direct C-14, C-4 demethylation of lanosterol and the second one via the formation of a C-31 derivative followed by demethylations.     

Study of the resistance of Candida guilliermondii to polyene antibiotics

250 Candida guilliermondii strains resistant to the polyene antibiotics nystatin, levorin and amphotericin B were obtained using UV irradiation. When the mutant strains became resistant to one of the polyene antibiotics, their resistance to the other ones changed. Phenotypic analysis showed that the resistance of the strains to polyene antibiotics did not make them susceptible to a rise in osmotic pressure and to a change of the temperature of incubation. Some of the polyene-resistant strains were stained in a medium with methylene blue. Analysis of the sterol composition in the mutants by UV spectroscopy showed that the resistance to polyene antibiotics sometimes involved changes in the sterol composition. Two new UV spectrum types were recorded for the sterols of the mutant strains; they differed from the UV spectrum for the sterols of the parent sensitive strain.     

Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism.

Organelles of the yeast Saccharomyces cerevisiae were isolated and analyzed for sterol composition and the activity of three enzymes involved in sterol metabolism. The plasma membrane and secretory vesicles, the fractions with the highest sterol contents, contain ergosterol as the major sterol. In other subcellular membranes, which exhibit lower sterol contents, intermediates of the sterol biosynthetic pathway were found at higher percentages. Lipid particles contain, in addition to ergosterol, large amounts of zymosterol, fecosterol, and episterol. These sterols are present esterified with long-chain fatty acids in this subcellular compartment, which also harbors practically all of the triacylglycerols present in the cell but very little phospholipids and proteins. Sterol delta 24-methyltransferase, an enzyme that catalyzes one of the late steps in sterol biosynthesis, was localized almost exclusively in lipid particles. Steryl ester formation is a microsomal process, whereas steryl ester hydrolysis occurs in the plasma membrane and in secretory vesicles. The fact that synthesis, storage, and hydrolysis of steryl esters occur in different subcellular compartments gives rise to the view that ergosteryl esters of lipid particles might serve as intermediates for the supply of ergosterol from internal membranes to the plasma membrane.     

Lipid composition of cells of heterothallic strains in the developmental cycle of Blakeslea trispora

Lipid compositions in mycelium and spores of Blakeslea trispora heterothallic strains were studied. Distinctions between the strains in the ability to synthesize linolenic acid and in optimal growth temperature were demonstrated. The (-) strain grew at a higher temperature and was unable to synthesize linolenic acid, whereas the (+) strain accumulated this acid up to 20% of total fatty acids. The distinctions between the strains remained at different developmental stages (mycelium and spores). A higher thermophilicity of the (-) strain correlated with a high sterol content, which is typical of thermophilic fungi. The lipid compositions of heterothallic strains studied differed in lipid content, their fractional composition, the degree of unsaturation, and carotenoid composition.     

Induction of DNA polymerase α in senescent cultures of normal and Werner's syndrome cultured skin fibroblasts

DNA polymerase α activity was determined following serum stimulation of early and late passages of human diploid fibroblast-like (HDFL) cultures derived from apparently normal donors (two strains) and from a patient with Werner's syndrome (one strain). Induction of this enzyme was observed in both low passage, actively proliferating cultures and in postmitotic “senescent” cultures from all three strains. The maximal polymerase activity of early and late passage cells of each strain were nearly identical when normalized to the number of cells present. However, the activity of the enzyme was observed to be significantly lower in late passage cultures when normalized to total protein content apparently because of enlargement of the senescent cells. The behavior of Werner derived cells was similar to that of the normal cells. The induction of DNA polymerase α in senescent cultures indicates that they retain the capacity to carry out some complex metabolic responses to mitogen stimulation. In addition, these results suggest the possibility that dilution of DNA polymerase α and/or other DNA replication factors may play a role in the onset or maintenance of the postmitotic state in the enlarged senescent HDFL cells.     

Ergosterol biosynthesis pathway in Aspergillus fumigatus

The sterol composition of Aspergillus fumigatus for the biosynthesis of ergosterol is of interest since this pathway is the target for many antifungal drugs in clinical use. The sterol composition of this fungal species was analyzed by gas chromatography-mass spectrometry in different strains (susceptible and resistant to azole drugs). Also, sterols were analyzed in several A. fumigatus mutant strains deficient in enzymatic steps of the ergosterol biosynthesis pathway such as 14-alpha sterol demethylases (Cyp51A and Cyp51B) and C-5 sterol desaturases (Erg3A, Erg3B and Erg3C). All sterols identified from azole-resistant A. fumigatus strains were qualitatively and quantitatively similar to the susceptible strain (CM-237). However, sterol composition of mutants strains were different depending on the lacking enzyme. The analysis of the sterol composition in these mutant strains led to a better understanding of the ergosterol biosynthesis pathway in this important fungus.     

Resistance to amphotericin B associated with defective sterol Δ8→7 isomerase in a Cryptococcus neoformans strain from an AIDS patient

Abstract Two Cryptococcus neoformans strains isolated from an AIDS patient were investigated, a pretreatment isolate (CN1) and a second isolate (CN3) following failure of fluconazole and amphotericin B treatment. No difference in fluconazole sensitivity, but relative resistance to amphotericin B was observed for CN3. The sterol composition of CN3 indicated a defect in sterol Δ ^8→7 isomerase in this strain and depletion of ergosterol, the major sterol of the CN1.     

Acholeplasma multilocale sp. nov., isolated from a horse and a rabbit.

Acholeplasma strains were isolated from the nasopharynx of a horse (strain PN525T [T = type strain]) and the feces of a rabbit (strain B1). One clone of strain PN525T and one clone of strain B1 were examined in detail. These clones were indistinguishable from each other and were serologically distinct from the previously described Acholeplasma and Mycoplasma spp. The strains had the following properties: guanine-plus-cytosine content of 31 mol%; sterol was not required for growth, which occurred under both aerobic and anaerobic conditions; glucose was metabolized; and arginine was hydrolyzed. Strain PN525 (= NCTC 11723) is the type strain of a new species, Acholeplasma multilocale.     

Lipid Composition of Cells of Heterothallic Strains in the Developmental Cycle of <Emphasis Type="Italic">Blakeslea trispora</Emphasis>

Lipid compositions in mycelium and spores of Blakeslea trispora heterothallic strains were studied. Distinctions between the strains in the ability to synthesize linolenic acid and in optimal growth temperature were demonstrated. The (−) strain grew at a higher temperature and was unable to synthesize linolenic acid, whereas the (+) strain accumulated this acid up to 20% of total fatty acids. The distinctions between the strains remained at different developmental stages (mycelium and spores). A higher thermotolerance of the (−) strain correlated with a high sterol content, which is typical of thermophilic fungi. The lipid compositions of heterothallic strains studied differed in lipid content, their fractional composition, the degree of unsaturation, and carotenoid composition.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 449–453.Original Russian Text Copyright © 2005 by Tereshina, Memorskaya, Feofilova.     

Variation of the sterols from Kluyveromyces lactis and from a resistant mutant by culture in the presence of sublethal doses of amphotericin B

A mutant strain of Kluyveromyces lactis resistant to amphotericin B and weakly to nystatin has been isolated from subcultures of the wild strain grown in the presence of sublethal doses of amphotericin B. The mutant and the wild strain were equally sensitive to pimaricin, filipin, and candicidin. The efficacy of fungizone was very low. In comparison with the wild strain the level of sterols was two times lower in the resistant strain but the composition of these sterols was about the same in the two strains. The action of sublethal doses of amphotericin B on the composition of the sterols was the same in these two yeasts and brought a 40% decrease of the total sterol level and a modification in their distribution. This variation cannot fully explain the resistance of the yeast but it may be associated to other changes of the membranes.     

Influence of methylfenpropidine on growth, sterol content and fatty acid composition ofCandida albicans

The effect of methylfenpropidine on growth, lipid contents, sterol and fatty acid composition was investigated in 5 strains ofCandida albicans. The sensitivity of the strains decreased in the order: wild strains >erg ⁺ ade nys ^R >ade nys ^R erg (defective Δ⁸⁻⁷-isomerase) >ade nys ^R erg (defective Δ⁵-desaturase). The presence of the inhibitor influenced fecosterol isomerization, episterol dehydrogenation, zymosterol transmethylation, ignosterol reduction and squalene epoxidation. Methylfenpropidine also induced changes in fatty acid composition, causing a reduction of the palmitic and oleic acid content with a concomitant elevation of stearic, linoleic and linolenic acid levels. The lipid unsaturation index slightly increased. Morphological changes of wild strains were observed after the fungicide treatment.     

Studies on delta8-delta7 isomerization and methyl transfer of sterols in ergosterol biosynthesis of yeast.

The formation of cholesta-7,24-dien-3 beta-ol and its activity as a substrate for the sterol side-chain methyltransferase in yeast have not previously been studied. Experiments with acetone-powder extracts of yeast showed that the sterol is formed from zymosterol by delta8-delta7 isomerization. However, direct conversion of cholesta-7,24-dien-3 beta-ol into zymosterol could not be demonstrated. The reversibility of the reaction was proved by the detection of 3H-incorporation into cholesta-8-en-3 beta-ol (with lathosterol as a carrier) from [3H]H2O in the medium. Incubation of cholesta-7,24-dien-3 beta-ol and S-adenosyl-L-[methyl-14C]methionine with the acetone-powder extract resulted in methylation of the sterol to form episterol. Similar incubation of zymosterol gave fecosterol and episterol, suggesting that fecosterol initially formed by the methylation was isomerized to episterol. In intact cells, however, an alternative pathway (zymosterol yields cholesta-7,24-dien-3 beta-ol yields episterol) may also operate. The relative importance of the two pathways is not known.     

A comparison of the yield of Vi and O antigens during the adaptation of Salmonella typhi strains to cultivation under starvation conditions

S. typhi strains Ty(2)4446 and Vi-1S underwent multiple passages in f synthetic liquid starvation culture medium consisting of water with salts and glucose added. In the process of the adaptation of the cultures to these stress conditions (starvation stress) the increasing yield of biomass from passage to passage was observed. Differences in the accumulation of Vi- and O-antigens were noted in two strains under study. In the cultures of strain Ty(2)4446 an insignificant increase in the antigen content from passage to passage was observed, while in the cultures of strain Vi-1S an increase in the content of Vi- and O-antigens was 4- to 5-fold. With the adaptation of the culture the Vi-antigen to O-antigen ratio changed from 1:57 to 1:20 for strain Ty(2)4446 and from 1:2.7 tp 1:2.2 for strain Vi-1S. Strain Ty(2)4446 had an advantage over strain Vi-1S with respect to the synthesis of Vi-antigen. These data are indicative of the expediency of using not only strain Ty(2)4446, but also strain Vi-1S for the preparation of typhoid vaccine, especially the one based on Vi-antigen.     

Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains

The phospholipid and sterol composition of the plasma membranes of five fluconazole-resistant clinical Candida albicans isolates was compared to that of three fluconazole-sensitive ones. The three azole-sensitive strains tested and four of the five resistant strains did not exhibit any major difference in their phospholipid and sterol composition. The remaining strain (R5) showed a decreased amount of ergosterol and a lower phosphatidylcholine:phosphatidylethanolamine ratio in the plasma membrane. These changes in the plasma membrane lipid and sterol composition may be responsible for an altered uptake of drugs and thus for a reduced intracellular accumulation of fluconazole thereby providing a mechanism for azole resistance.     

Analysis of lipids by gas-liquid chromatography and complementary methods in four strains of Aedes aegypti mosquitoes

Fatty acid composition of total lipids, neutral lipids and phospholipids of strains of Aedes aegypti were determined. The fatty acid composition of the strains differed quantitatively with regard to the relative percentage of commonly occurring fatty acids. Gas-liquid chromatography of fatty acid methyl esters showed 18:1 (oleic or elaidic) to be the predominant fatty acid. The fatty acid was identified as oleic by argentation thin-layer chromatography. A modified colorimetric method was used to determine tissue-free fatty acids. The lipids were predominantly triacylglycerol with lesser amounts of free fatty acids and decreasing amount of sterol ester, sterol, monoacylglycerol, diacylglycerol and hydrocarbons. The data show considerable lipid differences between the Caribbean strains (Les Cayes, Haiti, and San Juan, Puerto Rico) and the Jakarta (Indonesia) strain. The Shimba Hills (Kenya) strain was more similar to Jakarta than to the Caribbean strains. The results obtained with the different strains are discussed in relation to the established oral susceptibility to Dengue 1 and Dengue 2, yellow fever, and genetic analysis by isoenzyme studies.     

Phospholipid and fatty acid compositions of Alteromonas putrefaciens and A. haloplanktis

The phospholipid and fatty acid composition of Alteromonas putrefaciens S29 (non-halophilic type) and A. haloplanktis S5B (halophilic type) was determined. Major phospholipids of both strains were the same when they were grown in media containing optimum salt concentrations. However, the fatty acid composition of phos-pholipids in strain S29 was remarkably different from that of strain S5B. Strain S29 contained iso-C15: 0 and eicosapentaenoic acid (20: 5) as constituent fatty acids of phospholipids and also contained sterol ester and wax as neutral lipids. In contrast, strain S5B did not contain branched and polyunsaturated fatty acids, and neither sterol ester nor wax were detected.     

Changes in the levels and composition of sterols in different tissues of Lolium temulentum plants during floral development

Free, esterified and glycosylated sterols were analysed separately from the shoot apices, leaves, leaf sheaths and stems of Lolium temulentum L. (strain Ceres) plants during floral development. Short-day grown plants (50 days old) were induced to flower by exposure to a single long day. The four major sterols found by GC-MS analysis were sitosterol, cholesterol, campesterol and stigmasterol. The sterol levels in the shoot apex were much higher than those in the leaf, leaf sheath and stem. A much greater proportion of cholesterol was found in the shoot apex than in other tissues and this may reflect a specific association of cholesterol with meristematic and/or reproductive tissues.
During the inductive treatment, the sterol levels decreased in all four tissues. The major effect during early differentiation was the occurrence of transient increases in the free and esterified sterol levels in the leaf and the stem tissues. The steryl ester content peaked 24 h before the appearance of double ridges, followed by a peak in free sterol content at the double ridge stage. Similar changes could not be detected in the shoot apices. This is the first report of the sterol composition of developing shoot apices, and the results emphasize the dynamic nature of sterol metabolism during reproductive growth of L. temulentum.