Composition and characteristics ofCandida albicans cells differing in resistance to polyene antibiotics

Development of resistance to polyene antibiotics in a highly resistantCandida albicans strain was shown to be accompanied by the complete loss of the ability to synthesize ergosterol and the substitution of other sterol components as well as by higher amounts of free fatty acids. No significant differences in lipid and protein composition have been noted between slightly resistant cultures ofC. albicans and initially susceptible ones. Sterols of resistant cultures (added in the solution and incorporated in the composition of native membranes and liposomes) have the same affinity for polyene antibiotics as do sterols of a sensitive strain. It was found that the resistance of the slightly resistantC. albicans strain did not depend on the cell wall. The ability of some detergents to reduce resistance to polyene antibiotics was shown.     

Effect of polyenic antibiotics on the activity of alkaline phosphatase from Candida albicans]

Polyenic antibiotics (levorin, amphotericin B, nistatin) inhibit in vivo and in vitro the activity of membrane alkaline phosphatase from sensitive Candida albicans strain, and their inhibitory effect is twice lower on the enzyme from the resistant strain. A correlation is observed between the antibiotic concentration and the inhibitory effect on alkaline phosphatase activity. Nistatin is found to be the least efficient inhibitor (among the antibiotics studied) of alkaline phosphatase. The treatment of membranes with polyenic antibiotics does not result in solubilization of membrane proteins nad alkaline phosphatase. The data obtained are considered with respect to the effect of polyenic antibiotics on cell membrane structure.     

Sterol and fatty acid composition of polyene macrolide antibiotic resistant Torulopsis glabrata.

Successive reculturing of Torulopsis glabrata on media containing increasing concentration of the polyene macrolide antibiotics nystalin or lucensomycin resulted in the segregation of cultures resistant to these antibiotics. Isolates resistant to lucensomycin showed good resistance to nystatin, and vice versa. Analysis of the sterols and fatty acids of sensitive and polyene resistant T. glabrata revealed that compositional changes occurred in both classes of lipids upon acquistion of resistance. The sterol composition of nystatin and lucensomycin resistant cultures possessed reduced amounts of, or no ergosterol (the major sterol of the sensitive parent culture), and increased amounts of sterols which were biogenetically more primitive than ergosterol. Resistant cultures in which ergosterol was absent possessed a fatty acid composition that did not differ significantly from the parent sensitive culture grown under identical conditions. Resistant cultures containing significantly reduced amounts of ergosterol were found to possess altered fatty acid compositions. Generally it was observed that these latter cultures possessed fatty acids containing shorter and more saturated chains. These results are considered to indicate that alteration in both lipid and sterol composition is involved in determination of culture resistance to polyene macrolides.     

Ribosome binding to the membranes of Candida albicans]

The in vitro binding of free ribosomes to the membranes of polyenoic antibiotics-sensitive and resistant strains of Candida albicans, considerably differing in the composition of their sterol components, was studied. It was shown that the extent and type of membrane-ribosome interaction is similar in both cases. The dependence of binding on ribosome concentration in the incubation mixture is double-phase, as was shown for different membrane fractions of both sensitive and resistant strains. The role of lipid membrane components, particularly that of sterols in the membrane-ribosome interactions is discussed.     

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.     

Role of sterols in the interaction of polyene antibiotics with lipid membranes

The problem whether the membrane sterols are indirect acceptors of polyenic antibiotics or they play the role of substances providing conditions (at the expense of putting in order the membrane phospholipids) for formation of conductive complexes (ionic canals) from the antibiotic molecules is discussed. The comparative study on the ability of sterols of various structure (ergosterol, 7-dehydrocholesterol, cholesterol, 5 alpha-cholestan-3 beta-ol) to interact with the membrane phospholipids and to increase the sensitivity of such membranes to amphotericin B showed no correlation between the levels of these properties. The value of the changes in the cross elasticity module (E) of artificial bilayer lipid membranes from egg lecithin on introduction of the above sterols into their composition was used as the criterion for the interaction level. The absence of correlation between the above properties of the sterols indicated that the role of the sterols in interaction of polyenic antibiotics with the membranes could not be considered as the only effect of the sterols on putting in order the phospholipids, which confirmed the hypothesis on the acceptor function of the sterols with respect to polyenic antibiotics. The study of the effect of amphotericin B on the elastic properties of the cholesterol-containing bilayer membranes isolated from egg lecithin showed tha the values of the longitudinal and cross elasticity modules of the membranes did not change during introduction into the membranes of the ionic canals.     

Effect of terrilytin on antifungal antibiotic activity]

The antifungal activity of terrilitine, an enzymatic preparation of microbial origin and its effect on the activity of antifungal polyenic antibiotics and griseofulvine were studied in vitro. It was found with the method of serial dilutions in Sabourand's liquid medium that terrilitine was active against C. albicans and certain dermatophytes. In combination with amphotericin B, amphoglucamine, mycoheptine, levorin, nystatin or griseofulvin it increased the activity of these antibiotics 2-16 times.     

Influence of lipid composition on the sensitivity of Candida albicans to antifungal agents

Cerulenin, a specific inhibitor of fatty acid and sterol biosyntheses, inhibited growth and lipid synthesis in C. albicans, which on supplementing the growth medium with optimum concentrations of fatty acids was reversed. Significant changes in the levels of phospholipids and sterols were observed in fatty acid-supplemented cells. Altered phospholipids and their fatty acid profile rendered cells more resistant to miconazole and thereby more permeable to [3H]proline. Thus it appears that fatty acid composition plays an important role in determining the permeability susceptibility of C. albicans to drugs.     

Sensitivity of Candida strains to polyenic antibiotics in the treatment of oral candidiasis

Sensitivity of 146 clinical strains of Candida albicans to nystatin, levorin and amphoglucamine was studied on solid media with the replica method. The strains were isolated from 79 patients with candidiasis of the oral mucosa. It was found that sensitivity of the fungi to the polyenic antibiotics was different which should be considered in treatment of candidiasis. On the basis of the mean MICs for the clinical strains and their distribution by the MICs it was shown that the activity level of levorin and amphoglucamine was higher than that of nystatin. During the treatment resistance of the Candida strains to the polyenic antibiotics increased and cross resistance developed which required application of other treatment means.     

Effect of amigluracil and levorin on membrane permeability and protein synthesis in Candida albicans protoplasts]

It was shown that amigluracyl, a water soluble derivative of methacyl which decreased the nephrotoxic effect of polyens activated the membrane permeability in Candida albicans for a mixture of 14C-amino acids but had no significant effect on protein synthesis in this microorganism. The level of inhibition of the membrane permeability in C. albicans for the amino acids and protein synthesis in the fungus by levorin did not practically depend on the presence of amigluracyl in the incubation medium. The minimum levorin concentration inhibiting the growth of Candida albicans in the presence or absence of levorin was 0.039 gamma/ml. Therefore, amigluracyl may be used in combination with polyenic antibiotics for the treatment of mycoses.     

The effects of temperature acclimation on membrane sterols and phospholipids of Neurospora crassa

Experiments were conducted to examine the effects of temperature acclimation on sterol and phospholipid biosynthesis in Neurospora crassa. Cultures grown at high (37 degrees C) and low (15 degrees C) temperatures show significant differences in free and total sterol content, sterol/phospholipid ratios and distribution of major phospholipid species in total lipids and two functionally distinct membrane fractions. The ratio of free sterols to phospholipids in total cellular lipids from 15 degrees C cultures was found to be about one-half that found at 37 degrees C, whereas sterol/phospholipid ratios of mitochondrial and microsomal membranes were found to be higher at the low growth temperature. Total sterol and phospholipid biosynthetic rates showed parallel reductions in cultures acclimating to a shift from 37 to 15 degrees C growth conditions. Distribution of [14C]acetate label into free sterols was significantly lower under these conditions, however; indicating an increase in the conversion rate of sterols to sterol esters at the lower temperature. Mitochondrial and microsomal membrane fractions showed distinct phospholipid distributions which also differed from total lipid distributions at the two growth temperatures. In each case there was a consistent decrease in phosphatidylcholine and a corresponding increase in phosphatidylethanolamine as growth temperatures were lowered.     

Action of polyene antibiotics on actinomycetes with a varying lipid makeup]

The effect of polyenic antibiotics, such as nystatin, levorin, amphotericin B and mycoheptin on 93 representatives of various species of the ray fungi was studied. It was shown that resistance of the actinomycetes to the polyens was connected with the absence or insufficient content of sterols (0.001--0.008 per cent in the dry mycelium). On addition of cholesterol to the nutrient media (100 microgram/ml) it was included into the membranes of some cultures and their sensitivity increased 2--60 times. Resistance of Actinomyces sp. LIA 0775 grown on the media with fats differing in their composition decreased 2--4 times. In these cases the culture lipids were characterized by lower content of phospholipids (35--45 per cent from the total lipids as compared to 70--80 per cent when grown on the control medium without fats) and significantly increased content of unsaturated fatty acids (3--4 times).     

The genetic basis of fluconazole resistance development in Candida albicans

Infections by the opportunistic fungal pathogen Candida albicans are widely treated with the antifungal agent fluconazole that inhibits the biosynthesis of ergosterol, the major sterol in the fungal plasma membrane. The emergence of fluconazole-resistant C. albicans strains is a significant problem after long-term treatment of recurrent oropharyngeal candidiasis (OPC) in acquired immunodeficiency syndrome (AIDS) patients. Resistance can be caused by alterations in sterol biosynthesis, by mutations in the drug target enzyme, sterol 14alpha-demethylase (14DM), which lower its affinity for fluconazole, by increased expression of the ERG11 gene encoding 14DM, or by overexpression of genes coding for membrane transport proteins of the ABC transporter (CDR1/CDR2) or the major facilitator (MDR1) superfamilies. Different mechanisms are frequently combined to result in a stepwise development of fluconazole resistance over time. The MDR1 gene is not or barely transcribed during growth in vitro in fluconazole-susceptible C. albicans strains, but overexpressed in many fluconazole-resistant clinical isolates, resulting in reduced intracellular fluconazole accumulation. The activation of the gene in resistant isolates is caused by mutations in as yet unknown trans-regulatory factors, and the resulting constitutive high level of MDR1 expression causes resistance to other toxic compounds in addition to fluconazole. Disruption of both alleles of the MDR1 gene in resistant C. albicans isolates abolishes their resistance to these drugs, providing genetic evidence that MDR1 mediates multidrug resistance in C. albicans.     

Screening of natural immunosuppressors by their ability to modify steroid synthesis in hepatocytes]

In the programme for screening sterol synthesis inhibitors with the use of actinomycetes and fungi 702 strains were tested. The effect of alcohol extracts of the mycelium of fungi and actinomycetes at a dilution of 1/10(3) on sterol synthesis by the Hep G2 hepatome cells was determined by incorporation of 3H acetate into sterols and proteins. Lovastatin (200 pg/ml) was used as the control: the sterol synthesis was decreased by 49 +/- 4% without inhibiting the protein synthesis. A number of the cultures produced compounds inhibiting under the experimental conditions the synthesis of sterols by 70 to 80% with simultaneous inhibition of the protein synthesis at least by 60 to 70%. Three compounds from that group produced by streptomycetes were subjected to a more detailed investigation. The compounds were demonstrated to be active antifungal antibiotics (MIC 0.1-1 mcg/ml). In a dose of 0.1-1 mcg/ml they showed high immunosuppressive activity in models of lymphocyte transformation in mice, whereas cyclosporin was active in a dose of 1 mcg/ml. Therefore, the model for screening hypolipidemic compounds could be considered useful for screening promising natural immunosuppressors.     

Increased in vitro sensitivity of Candida albicans to amphotericin B when grown in mixed culture with Escherichia coli.

The growth of Candida albicans was inhibited by some Escherichia coli strains both in conventional batch cultures and also in a chemostat under conditions of constant addition of fresh medium. Concentrations of 0.2 microgram amphotericin B per millilitre and of 2 microgram nystatin per millilitre, which caused a slight inhibition of C. albicans in pure culture, exerted a strong fungicidal effect when the yeast was placed in mixed cultures with certain strains of E. coli. Candida albicans cells, inhibited by either E. coli or in mixed culture with polyene antibiotics, appeared larger and less uniformly stained by acridine orange than control cells from pure cultures. Addition of chloramphenicol to the mixed cultures, in quantities sufficient to kill the E. coli cells, abolished the increased sensitivity of C. albicans to amphotericin B or nystatin. In preliminary in vivo tests, E. coli did not sensitize C. albicans to the polyene antibiotics.     

Increase in the in vitro susceptibility of Staphylococcus aureus to antimicrobial agents in the presence of Candida albicans.

In experiments with mixed cultures of Staphylococcus aureus and Candida albicans both in the absence and in the presence of 5-fluorocytosine (5-FC), we have observed that (1) there is an inhibition of S. aureus growth in mixed cultures with C. albicans in media supplemented with 1 microgram/mL of 5-fc and that 5-FC has no effect on staphylococci in pure cultures; (2) this inhibition occurred with clinically isolated and laboratory strains and could be reversed by specific metabolites; (3) Staphylococcus aureus was inhibited by filtrates of C. albicans cultures treated with 5-FC and this seemed to be favored by some C. albicans filterable product which can affect the cell wall and the permeability of the staphylococcal cells since they become sensitive to 5-FC; (4) nine other commonly used antimicrobials showed an increased inhibitory activity against S. aureus in mixed cultures with C. albicans; and (5) there is a decrease in the number of precipitating antigens of S. aureus and of the activity of alpha toxin when this species was grown with both C. albicans and 5-FC. Our results indicate that the susceptibility of some species to antimicrobials could be significantly modified in the presence of other species. One cannot exclude that a similar phenomenon could happen in hosts under treatment with antibiotics against infection.     

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.     

Studies on the anticandidal mode of action of Allium sativum (garlic)

The mode of action of aqueous garlic extract (AGE) was studied in Candida albicans. The minimum inhibitory concentration (MIC) of AGE against six clinical yeast isolates ranged between 0.8 and 1.6 mg ml-1. Scanning electron microscopy and cell leakage studies showed that garlic treatment affected the structure and integrity of the outer surface of the yeast cells. Growth of C. albicans in the presence of AGE affected the yeast lipid in a number of ways: the total lipid content was decreased; garlic-grown yeasts had a higher level of phosphatidylserines and a lower level of phosphatidylcholines; in addition to free sterols and sterol esters, C. albicans accumulated esterified steryl glycosides; the concentration of palmitic acid (16:0) and oleic acid (18:1) increased and that of linoleic acid (18:2) and linolenic acid (18:3) decreased. Oxygen consumption of AGE-treated C. albicans was also reduced. The anticandidal activity of AGE was antagonized by thiols such as L-cysteine, glutathione and 2-mercaptoethanol. Interaction studies between AGE and thiols included growth antagonism, enzymic inhibition and interference of two linear zones of inhibition. All three approaches suggest that AGE exerts its effect by the oxidation of thiol groups present in the essential proteins, causing inactivation of enzymes and subsequent microbial growth inhibition.     

Sterol and Squalene Content of a Docosahexaenoic-Acid-Producing Thraustochytrid: Influence of Culture Age, Temperature, and Dissolved Oxygen

Thraustochytrid strain ACEM 6063, rich in omega-3 polyunsaturated fatty acids, was cultured at 15°C and 20°C in high (>40%) and low (<5%) dissolved oxygen (DO), and at 25°C in low-DO media. Samples were taken 4, 2, and 0 days before each culture reached peak biomass (T₋₄, T₋₂, and T_p, respectively). Twenty sterols, 13 of which were identified, were detected. Predominant were cholest-5-en-3β-ol, 24-ethylcholesta-5,22E-dien-3β-ol, 24-methylcholesta-5,22E-dien-3β-ol, and 2 coeluting sterols, one of which was 24-ethylcholesta-5,7,22-trien-3β-ol. These 4 sterols comprised 50% to 90% of total sterols. Cultures grown at high DO had simpler sterol profiles than those grown at low DO. Only the 4 sterols mentioned above were present at more than 3% of total sterols in high-DO cultures. In low-DO cultures, up to 6 additional sterols were present at more than 3% of total sterols. Culture age, temperature, and DO influenced squalene and sterol content. Total sterols (as a proportion of total lipids) decreased with increasing culture age. If organisms such as ACEM 6063 are to be used for commercial production of lipid products for human consumption, both their sterol content and factors influencing sterol production need to be characterized thoroughly. Received January 8, 2001; accepted March 6, 2001.     

Reversal of cerulenin-induced inhibition of phospholipids and sterol synthesis by exogenous fatty acids/sterols in Epidermophyton floccosum

Cerulenin, a specific inhibitor of fatty acids and sterol biosynthesis inhibited the growth of Epidermophyton floccosum, which was reversed when growth medium was supplemented with palmitic acid and sterols. Unsaturated fatty acids partially restored the growth. Cerulenin inhibited both phospholipid and sterol biosynthesis (60-70%) at the minimum inhibitory concentration (0.5 microgram/ml) as demonstrated by [32P]orthophosphoric acid and [14C]acetate incorporation into the respective lipids. Cerulenin-induced inhibition of phospholipid and sterol synthesis was dose dependent up to 0.5 microgram/ml. Exogenously supplied fatty acids and sterols restored the biosynthesis of phospholipids in cerulenin-treated cultures, while that of sterols was enhanced. The biosynthesis of both saturated and unsaturated fatty acids was inhibited by cerulenin.