The influence of ortho- and para-diphenoloxidase substrates on pigment formation in black yeast-like fungi.

Dothideaceous black yeast-like fungi (BYF) are known to synthesise DHN-melanin that is inhibited by the systemic fungicide tricyclazole. The final step of the DHN melanin pathway is the conjoining of 1,8-DHN molecules to form the melanin polymer. There are several candidate enzymes for this step, including phenoloxidases such as tyrosinase and laccases, peroxidases, and perhaps also catalases. We analysed the type polyphenoloxidases that are involved in biosynthesis of BYF melanins. For that purpose we used substrates of o-diphenoloxidases (EC 1.10.3.1.): 4-hydroxyphenyl-pyruvic acid, L-beta-phenyllactic acid, tyrosine, pyrocatechol, 3,4-dihydroxyphenylalanine and homogentisic acid, as well as substrates of p-diphenoloxidases (EC 1.10.3.2.): syringaldazine, resorcinol, p-phenylenediamine, phloroglucinol, guaiacol and pyrogallic acid. Fourteen strains of black yeasts originating from different natural biotopes were investigated. The tested strains could be divided into four groups based on their ability to produce dark pigments when cultivated on aromatic substrates of o- and on p-diphenoloxidases. It was established that syringaldazine, pyrogallic acid and 4-hydrophenyl-pyruvic acid, beta-phenyllactic acid optimally promote melanin biosynthesis. Average intensity of pigmentation of all strains studied was minimal when guaiacol was used as a substrate. The present investigation indicates that the melanisation process may involve more enzymes and more substrates than those commonly recognised. Black yeasts are likely to contain a multipotent polyphenoloxidase.

Effect of melanins from black yeast fungi on proliferation and differentiation of cultivated human keratinocytes and fibroblasts.

The effects of melanin preparations from black yeast fungi (BYF) on the proliferation and differentiation of normal cultivated human skin keratinocytes and embryonic pulmonary fibroblasts have been investigated. Melanin preparations in the range of 5-0.1 microg/ml were optimally active, with a more pronounced effect on keratinocyte than on fibroblast proliferation. Of 17 dihydroxynaphthalene (DHN) natural melanin preparations and two commercial dihydroxyphenylalanine (DOPA) melanin preparations, only one preparation--DOPA melanin (of animal origin) significantly stimulated proliferation of keratinocytes at 5 microg/ml; four preparations (DHN melanin from BYF) significantly inhibited proliferation of these cells at 5 or 1 microg/ml. The remaining preparations had no significant effect. Similarly, of the 17 preparations of DHN melanin from BYF, one preparation significantly stimulated fibroblast proliferation, and four significantly inhibited proliferation at 5 microg/ml, one at all the concentrations, and three from 1 down to 0.1 microg/ml. These melanin preparations were also shown to affect the in vitro differentiation of keratinocytes.

Exopolysaccharides and capsules in human pathogenic Exophiala species.

The black yeasts Exophiala spinifera and E. dermatitidis produce extracellular slimes, which may be either in the form of a well-delimited capsule or of diffusely exuded exopolysaccharides (EPS). The optimal conditions for their production were studied. The presence or absence of polysaccharide material can be used for recognition of the two species. Five-day-old cultures grown on potato glucose agar at 24 degrees C were observed in India ink, and positive identification for E. spinifera was obtained when significant halos were seen around yeast cells. In contrast, E. dermatitidis had irregular EPS with a fibrillar substructure made visible by alcian blue staining. Other Exophiala species produce insignificant amounts of extracellular mucus or none at all. The diagnostic method is particularly useful with yeast-like primary cultures, which often consist entirely of budding cells and lack the characteristic structures of the filamentous Exophiala synanamorph.

Photoprotective activity of melanin preparations in human skin exposed to UV irradiation: dependence on previous photoexposure.

Photoprotective effects of three melanin preparations (from black yeast fungi and Sepia sp.) were studied. These preparations in aqueous solutions (5 g/ml, dark exposure for 7 days) demonstrated high photomodification capacity upon exposure to visible light in doses of up to 1.8 kJ/m2. Preliminary exposure of these solutions to visible light in a dose of 360 kJ/m2 notably decreased the photoprotective effect of melanins during UV exposure of the skin treated with these solutions (at UV dose of 3.4 kJ/m2). This necessitates empirical selection of the dose and storage condition of melanin preparations for attaining the optimal photoprotective effect.

A new variety of Aureobasidium pullulans characterized by exopolysaccharide structure, nutritional physiology and molecular features.

The black yeast Aureobasidium pullulans (de Bary) Arnaud is known to synthesize the exopolysaccharide pullulan, a poly-alpha-1,6-maltotriose. Nine strains were found to produce additional aubasidan-like EPS, i.e. glucans with alpha-1,4-D-, beta-1,6-D- and beta-1,3-D-glycosidic bonds. These strains had previously been found to deviate in genotypic characters. Additional physiological differences were found: the optimal nitrogen source for exopolysaccharide production in liquid medium was NaNO3 for aubasidan-producing strains, and (NH4)2SO4 for the remaining strains. A new variety, A. pullulans var. aubasidani Yurlova, is described for the strains producing aubasidan-like components. The new variety can be distinguished from A. pullulans var. pullulans by the absence of assimilation of methyl-alpha-D-glucoside and lactose.

Distinction of species in Aureobasidium and related genera by PCR-ribotyping.

Taxonomic markers for differentiation of the anamorph genera Aureobasidium, Hormonema and Kabatiella were developed using PCR-ribotyping with the primers 5.8S-R and LR7 for amplification and the restriction enzymes Alul, Ddel, Hhal, Mspl and Rsal for digestion. Aureobasidium and Hormonema are optimally differentiated with Mspl; Ddel is particularly useful to distinguish Aureobasidium, Kabatiella and Selenophoma. Relationships of the anamorph genera Aureobasidium, Hormonema and Kabatiella with the teleomorph genera Pringsheimia and Dothiora are discussed.

Intraspecific variability and exopolysaccharide production in Aureobasidium pullulans.

Forty seven strains of the black yeasts, Aureobasidium pullulans and Hormonema dematioides, and the type strain of Hormonema macrosporum were examined using PCR-ribotyping and universally primed PCR with subsequent hybridization. Four groups (populations) were distinguished within A. pullulans with PCR-ribotyping, which largely coincided with UP-PCR/hybridization groups. The UP-PCR technique revealed a greater degree of heterogeneity between the groups studied. Five strains identified as Hormonena dematioides on the basis of physiological and morphological data formed a group recognizable with PCR-ribotyping and UP-PCR/hybridization, which also included H. macrosporum. Aureobasidium pullulans is characterized by the absence of RsaI restriction sites in rDNA amplified with primers 5.8S-R and LR7, while Hormonema species possessed several bands after RsaI digestion. For analysis of distance between populations, PCR-ribotyping with AluI and MspI is sufficient. Strains of A. pullulans produce exopolysaccharides in liquid media with different nitrogen sources, while the strains of Hormonema synthesize minor amounts of polysaccharides in media with peptone. Populations of A. pullulans differ slightly from each other in their optimal, medium-dependent production of polysaccharides.

Conidiogenesis, nutritional physiology and taxonomy of Aureobasidium and Hormonema.

Diagnostic criteria for the distinction of the slightly osmophilic species, Aureobasidium pullulans, and the opportunistic pathogen on conifers, Hormonema dematiodies, are provided. Reliable identification requires a combination of characters of conidiogenesis, expansion growth and assimilative abilities. Relationships with species of Kabatiella, and with the teleomorph genera Pringsheimia, Dothidea and Dothiora, all having Dothiora, all having Hormonemia-like cultural states, are discussed. An identification key is provided.