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Selective destruction of microscopic fungi through photo-oxidation of ergosterol

     Instituto de Ciencias Básicas—Laboratorio de Alta Tecnología de Xalapa (LATEX). Médicos, 5, Colonia Unidad del Bosque. CP 91010 Xalapa, Veracruz, México

	ABSTRACT

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Ergosterol is an important component of fungal membranes. This sterol can be easily transformed to peroxide of ergosterol by photo-oxidation with singlet oxygen. Cultures of Papalauspora immersa were grown on Czapeck agar medium, and subjected to the following conditions: 1) irradiation with daylight and quartz light (excluding UV light), 2) addition by diffusion of yellowish eosine (0.1 mg/mL), and 3) the control (no yellowish eosine, under darkness conditions). Fungal growth was completely inhibited after the treatment with quartz light (3 h) and yellow eosine, and no growth was observed in subsequent subcultures. These results suggested that plasma membrane components changed significantly by the transformation of ergosterol to peroxide of ergosterol leading to fungal death. To confirm this, a second experiment on a larger scale was carried out in which the fungus was grown on liquid medium in test tubes, treated, irradiated, and tested for peroxide of ergosterol by ¹HNMR. This peroxide was only found in treated samples. These findings represent a new strategy for developing antifungal agents, based on ergosterol photo-oxidation which might probably be related to the disruption of the plasma membrane, instead of only preventing the ergosterol biosynthesis. The potential application of this strategy for the selective control or prevention of pathogenic fungi is considerable.

Key words: antifungal agents, ergosterol, pathogenic fungi, photo-oxidation

	INTRODUCTION

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Many fungal species cause different diseases affecting humans, plants and animals (Herrera and Ulloa 1990, Agrios 1998). Antifungal agents developed to prevent or to control these diseases have different structure, properties and modes of action. Agents of high biochemical specificity act as inhibitors of mitosis, DNA or RNA synthesis, protein synthesis, respiration, phosphate metabolism, ergosterol biosynthesis, chitin synthesis, and disruption of the plasma membrane function. A large number of antifungal agents involved in the inhibition of ergosterol biosynthesis are related to imidazoles, clotrimazole, and ketoconazole, which can inhibit demethylation of lanosterol (Carlile and Watkinson 1996).

In nature, plasma membranes mainly contain sterols: cholesterol in animals, ß-sitosterol and others in plants, and ergosterol in fungi (Parks and Weete 1991, Deacon 1998, Lehninger et al 1993, Trigos 1998). Ergosterol is a precursor of vitamin D₂ and is known to be a natural antioxidant (Uskokovic et al 1980). This vitamin precursor can easily be transformed into peroxide of ergosterol via photo-oxygenation (Windaus and Brunken 1933, Trigos and Martínez-Carrera 1992). This reaction, which requires visible light, occurs when triplet oxygen is transformed into its reactive singlet state, helped by a photosensitizer. This singlet oxygen interacts with the dienic system from the B ring of ergosterol, allowing a 2 + 4 cycloaddition (Wasserman and Ives 1980).

In this research, we studied the photo-oxidation of ergosterol in three fungal species: Papulaspora immersa Hotson, Emericella rugulosa (Thom et Raper) C. R. Benjamin, and Trichophyton mentagrophytes (Robin) Blanchard. This photo-oxidation led to cellular death and probably affected significantly the plasma membrane structure by the transformation of ergosterol to peroxide of ergosterol. Our findings could represent a new strategy for developing antifungal agents based on ergosterol photo-oxidation. The potential of application of such a strategy for the selective control or prevention of pathogenic fungi is discussed.

	MATERIALS AND METHODS

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Strains – Papulaspora immersa, and Emericella rugulosa were obtained from the strain collection of the Institute of Biology, University of Mexico (UNAM), Mexico, D.F. The strain of Trichophyton mentagrophytes was provided by the Institute of Chemistry, University of Puebla (BUAP). Strains were maintained and subcultured in Czapek's agar (g/L): NaNO₃, 3; K₂HPO₄, 1; MgSO₄.7H₂O, 0.5; KCl, 0.5; FeSO₄.7 H₂O, 0.01; Sucrose, 30; agar, 15. All strains used in this study are also deposited at the LATEX, Xalapa, Veracruz, Mexico.

Culture conditions – A strain of Papulaspora immersa was cultured in humid chambers (14) under aseptic conditions as follows: two glass slides were placed onto wet filter papers within a Petri dish (9 cm), three round agar blocks (replicates) were placed on the glass slides and inoculated with Papulaspora immersa, and then a cover slip was placed over the inoculated agar blocks. Humid chambers were incubated for 5 d at 28 C in the dark. After incubation, experimental treatments were applied to humid chambers, and small pieces of inocula were subcultured on agar plates (3 replicates), incubated for 4 d, and tested for survival.

Experimental treatments – These experiments were done with Papulaspora immersa. A 10 mL aqueous yellowish eosine solution (0.1 mg/mL) was prepared and added by diffusion to filter papers. Incubated humid chambers (14) were subjected to the following conditions: 1) irradiation with daylight for 1, 2, and 3 h, with or without yellowish eosine solution (6); 2) irradiation with quartz light as energy source excluding UV light for 1, 2, and 3 h (filtered <400 nm, at a distance of 30 cm, 28 C), with or without yellowish eosine solution as sensitizer (6); and 3) the controls kept in the dark, with (1) or without (1) yellowish eosine solution. After treatments, all samples were incubated at 28 C for 4 d in order to check mycelial growth, and subsequently subcultured to test mycelial recovery. In addition, experiments to confirm data obtained were also carried out for Emericella rugulosa and Trichophyton mentagrophytes, under the following conditions: an irradiation period with quartz light excluding UV light for 4.5 h, with yellowish eosine solution.

Large-scale conversion to peroxide of ergosterol – Ten test tubes (18 x 200 mm) containing 7 mL of liquid Czapek medium (no agar) were inoculated with Papulaspora immersa, and incubated at room temperature in the dark for 8 d. After incubation, 15 mL of aqueous yellowish eosin solution were added to each test tube. All test tubes were irradiated with halogen quartz light excluding UV light for 10 h (filtered <400 nm, at a distance of 20 cm, 28 C). After irradiation, the mycelium was frozen and extracted with ethyl acetate in order to determine peroxide of ergosterol by ¹HNMR.

Photo-oxidation of ergosterol – Two solutions were prepared to compare the oxidation rate of ergosterol and cholesterol, in order to show differences in the reaction time. Solution A contained 15 mg of ergosterol, and 3 mg of yellowish eosine in 30 mL of ethanol. Solution B had 15 mg of cholesterol, and 3 mg of yellowish eosine in 30 mL of ethanol. Both solutions were irradiated with a halogen quartz lamp at a distance of 30 cm, maintained at 28 C, and monitored through thin layer chromatography to assess reactions. The transformation of ergosterol was faster (5 min) than that of cholesterol (4 h). In the case of the ergosterol transformation, the presence of peroxide of ergosterol was determined by ¹HNMR and ¹³CNMR. ¹HNMR (200 MHz, CDCl₃) ppm: 6.54 (1H, d, J = 8.3 Hz, H-6); 6.27 (1H, d, J = 8.3 Hz, H-7); 5.19 (2H, m, J = 6.6 Hz, H-22 y H-23); 3.98 (1H, m, H-3); 2.2 to 1.1 methylene and methyn protons group; 1.0 (3H, d, J = 6.6 Hz, H-21); 0.91 (3H, d, J = 7.3 Hz, H-28); 0.90 (3H, s, H-19); 0.83 (3H, d, J = 6.6 Hz, H-27); 0.82 (3H, s, H-18); 0.81 (3H, d, J = 6.6 Hz, H-26). ¹³CNMR (50 MHz, CDCl₃) ppm: 135.9 (C-6); 135.7 (C-22); 132.8 (C-23); 131.2 (C-7); 83.4 (C-5); 80.2 (C-8); 67.0 (C-3); 56.7 (C-17); 52.2 (C-14); 51.6 (C-9); 45.1 (C-13); 43.3 (C-24); 40.2 (C-20); 39.8 (C-12); 37.4 (C-4); 37.4 (C-10); 35.2 (C-2); 33.6 (C-25); 30.6 (C-1); 29.2 (C-16); 23.9 (C-15); 21.3 (C-21); 21.1 (C-11); 20.4 (C-26); 20.1 (C-27); 18.4 (C-28); 18.1 (C-19); 13.4 (C-18).

	RESULTS

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Effects of all treatments on the mycelial growth are shown in Table I. Irradiation with daylight for 1, 2, and 3 h, with or without yellowish eosine solution, did not inhibit mycelial growth. This also happened with quartz light irradiation for 1, 2, and 3 h, without yellowish eosine solution. In the absence of irradiation (darkness), with or without yellowish eosine solution, mycelial development was observed. However, when samples were irradiated with quartz light, in the presence of yellowish eosine and incubated for 4 d at 28 C, we observed that: 1) There was mycelial growth in samples treated for 1 h; 2) There was a partial inhibition of fungal growth (i.e., 3 out of 9 samples did grow) in samples treated for 2 h; and 3) There was a complete inhibition of fungal growth in samples treated for 3 h. All samples, treated and incubated, were subcultured to be tested for mycelial recovery. The same pattern was observed, as expected. Fungal growth was recorded in all samples, with the exception of those samples containing yellowish eosine and irradiated with quartz light for 3 h (Fig. 1). These data indicated that a combination of quartz light and yellowish eosine inhibited mycelial growth completely when appropriate periods of exposure were used.

View larger version (90K): [in this window] [in a new window]    FIG. 1. Culture propagation growth after four days. Image letters correspond to the sample letters of Table I. (A–F) treated with natural light; (G–L) with quartz lamp and (M–N) dark. Figs. (D, E, F, J, K and L) with yellowish eosin; (A, B, C, G, H, I and M) controls. (K) Growth was observed in 3 out of 9 inocula. (L) No fungal growth was observed

A large experiment was carried out with Papulaspora immersa using test tubes, yellowish eosine, and irradiation with quartz light (10 h). After treatment, the mycelium was separated from the medium and extracted using ethyl acetate. The solvent was evaporated at reduced pressure and percolated through a 0.04–0.063 particle-size silica gel using an 80:20 mixture of hexane-acetate as eluent. The substance resulting from the reaction mixture was analyzed by ¹HNMR. This analysis provided evidence of the A-B system of the H-6 and H-7 vinyl protons, which showed the presence of peroxide of ergosterol (Fig. 2).

View larger version (25K): [in this window] [in a new window]    FIG. 2. a. A-B system of the H-6 and H-7 vinyl protons, observed in 1H NMR (200 MHz) amplified spectrum of ergosterol peroxide in the reaction mixture from photooxygenation of P. immersa mycelium. b) and c) A-B system of the H-6 and H-7 vinyl protons from authentic samples of ergosterol and ergosterol peroxide (Trigos and Martínez-Carrera 1992, Trigos 1999)

	DISCUSSION

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Singlet oxygen, (¹O₂) constitutes the first excited electronic state of molecular oxygen located at 22.4 kcal/mol above the triplet ground state. The reaction between triplet oxygen and organic molecules is called photo-oxidation. There is a fast and efficient technique used to obtain these reactions, which consists of the dye-sensitized photochemical excitation of triplet oxygen. This dye agent becomes excited after a rapid intersystemic crossover that acts as a sensitizer, transmitting the energy to the triplet oxygen with which it forms ¹O₂, while regenerating the dye agent to its ground state (Margaretha 1982).

As a general rule, singlet oxygen behaves like an ethylene. Three types of reactions of ¹O₂, which have been used in organic synthesis, are usually observed: (a) Diels-Alder as cycloaddition to dyenes; (b) the ene reaction with olefins, and (c) cycloaddition to activated double bonds (Wasserman and Ives 1980).

Cycloaddition reactions occur when particular dienes and polyenes are irradiated in an environment containing oxygen and a triplet sensitizer, such as methylene blue coloring and yellowish eosine (Pine 1987). This is because molecular oxygen, usually in a triplet ground state, is promoted to an excited state known as singlet oxygen, in which all electrons are paired. Singlet oxygen behaves like a dienophile and will bind to the diene to form an endoperoxide through a Diels-Alder-type reaction (Cowan and Drisko 1976, March 1985, Margaretha 1982). Ergosterol can be transformed through photochemical oxygenation into its transanular peroxide by using yellowish eosine as sensitizer, in an ethanol medium (Windaus 1933).

The fungal plasma membrane shows a three-phase composition, consisting of a double layer of phospholipids. As with other eukaryotes, this membrane also contains large amounts of proteins and sterols. Sterols are contained in the double layer of phospholipids, usually in a proportion of 1:5 or 1:10. They play a role in the stabilization of the phospholipids, and also they probably help the integration of membranes. The main sterol present in fungal membranes is ergosterol (Deacon 1998, Lehninger et al 1993, Parks and Weete 1991).

Our results have suggested that there is a correlation between photooxidation of ergosterol and cell death in the fungal species studied: Papulaspora immersa, Emericella rugulosa, and Trychophyton mentagrophytes. This death may be due to a significant change of the plasma membrane structure by the transformation of ergosterol to peroxide of ergosterol, leading to fungal death. However, further studies are needed to show that the fungal death is actually due to the oxidation of ergosterol.

In vitro and in vivo methods used to determine antimycotic activity are similar to those used for testing antibacteriological compounds. It is relatively easy to discover numerous synthetic and natural compounds which, at a low dose, inhibit fungal growth in culture. However, many of these substances are not very effective when tested in vivo. Some of them show a toxicity that is not selective enough to be safe; others prove unable to reach the infected area, or are metabolized too rapidly to be useful. Furthermore, pathogenic fungi show different growth patterns in vitro and in vivo (Foye 1989).

The main target of some antimycotic drugs is the fungal membrane. With the exception of griseofulvin and flucitosine, antimycotic drugs operate either by fixing ergosterol or by inhibiting its biosynthesis. As human beings do not synthesize ergosterol, these drugs do not affect human cell membranes (Jawetz et al 1996).

These findings could represent a new strategy for developing antifungal agents based on ergosterol photo-oxidation, which produces a structural change of the plasma membrane instead of only preventing the ergosterol biosynthesis. The potential application of this strategy for the selective control or prevention of pathogenic fungi is considerable.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Daniel Martínez-Carrera (Colegio de Posgraduados, campus Puebla, México) for interesting discussions and comments, and Dr. Miguel Ulloa (Instituto de Biología de la UNAM., Mexico), for the donation of the strains studied.

	FOOTNOTES

 
¹ Corresponding author, Email: atrigos{at}uv.mx

Accepted for publication January 16, 2002.

	LITERATURE CITED

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