Stud Mycol 61(1): 39-49 2008
DOI: 10.3114/sim.2008.61.03
Copyright © 2008 CBS Fungal Biodiversity Centre
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The influence of ortho- and para-diphenoloxidase substrates on pigment formation in black yeast-like fungi
N.A. Yurlova1,*,
G.S. de Hoog2 and
L.G. Fedorova1
1 State Chemical-Pharmaceutical Academy, 14, Prof. Popov St., St.
Petersburg, 197376, Russian Federation
2 CBS Fungal Biodiversity Centre, P.O. Box 85167, NL-3508 AD Utrecht, The
Netherlands
*
Correspondence: N.A. Yurlova,
nadezhda.yurlova{at}mail.ru
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Abstract
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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
[EC]
.): 4-hydroxyphenyl-pyruvic acid,
L-β-phenyllactic acid, tyrosine, pyrocatechol, 3,4-dihydroxyphenylalanine
and homogentisic acid, as well as substrates of p-diphenoloxidases (EC
1.10.3.2
[EC]
.): 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, β-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.
Keywords Black yeast-like fungi / Dothideales / dothideaceous black yeasts / 1,8-dihydroxynaphthalene-melanin / phenoloxidases / o-diphenoloxidases / p-diphenoloxidases
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INTRODUCTION
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Black yeast-like fungi (BYF) are either of basidiomycetous or ascomycetous
relationship. The basidiomycetes are classified in the genera
Moniliella and Trichosporonoides, of which a precise
phylogenetic position has as yet not been established. Most species of these
genera are of industrial significance and are rarely seen in clinical
practice. In the ascomycete order Chaetothyriales, mainly comprising
the family Herpotrichiellaceae, the genus Exophiala is the
preponderant yeast-like anamorph (de Hoog
et al. 2000). The order contains numerous human
pathogens, with a wide spectrum of clinical pictures
(Vitale & de Hoog 2002,
de Hoog et al. 2005).
The majority of these infections are cutaneous or mild pulmonary, but rarely
they may be devastating and fatal. These infections are very difficult to
treat because in vivo the species are frequently more resistant
antimycotics than in vitro
(Vitale & de Hoog 2002,
de Hoog et al. 2005).
The pathology of these black yeasts and their relatives is poorly understood
(de Hoog et al. 2000,
2005).
In contrast, the ascomycete order Dothideales (anamorph genus
Aureobasidium and its relatives) mainly comprises saprobic fungi,
which are only exceptionally involved in human disease. Aureobasidium
pullulans is industrially important because of its production of
extracellular polysaccharides (EPS), which are applied in biotechnology
(Deshpande et al.
1992). The EPS concerned comprise pullulan, a
poly-
-1,6-maltotriose, and aubasidan, a related glucan with
-1,4-D, β-1,6-D and β-1,3-D-glycosidic bonds. A separate
variety, Aureobasidium pullulans var. aubasidani was
described for the strains producing aubasidan-like components
(Yurlova & de Hoog
1997).
Dothidealean black yeast-like fungi were found to be predominant in soils
highly contaminated with radionuclides emitted during the Chernobyl accident
(Zhdanova et al.
1994,
2007). They play an important
role in blackening of rock and architectural surfaces, in the destruction of
marble and limestone (Sterflinger & Krumbein
1995,
1997). The fungi show active
growth in extreme ecological niches, surviving low humidity, high temperature,
high solar irradiation, presence of long lived radionuclides, and absence of
traditional sources of nutrition and energy. The presence of melanin pigments,
which possess a wide protective action, provides the dark-coloured fungi a
competitive advantage under harsh environmental conditions. The pigments
contain stable organic free radicals
(Lyakh 1981). Fungal melanins
may occur as electron-dense granules located in the fungal cell wall, polymers
in the cytoplasm, as extracellular polymers in the medium surrounding the
fungus, or in any combination (Butler &
Day 1998). In the scientific literature there is information on
biological activity of melanins as radioprotectors, antitumor remedies and as
growth stimulators of plant seeds (Lyakh
1981). It had been suggested that these pigments might be useful
as topical sunscreens and sunlight-protective coatings for plastics. DOPA
melanins (of animal and biotechnological origin) have also been recommended
for use in cosmetics (Della-Cioppa et
al. 1990).
Melanins are produced by a variety of higher organisms including humans,
but microbes are the melanin producers of choice in biotechnology. Melanin
harvest from mammalian tissues sometimes may reach up to 8–10 mg/kg of
raw material, while that of fungi can be 100–1000 times higher
(Lyakh 1981). Differences were
established between the absorption spectra of black yeast melanins and
commercial Sepia melanin, which have been kept in the dark and photomodified
by daylight irradiation. These data indicate perspectives of some black yeast
melanins as photoprotectors and stimulators of skin regeneration
(Blinova et al. 2003,
Turkovskij & Yurlova 2002). Some black yeast melanins have higher
UV-defensive activity than commercial melanins and they stimulate human skin
regeneration significantly (Yurlova
2001, Turkovskij & Yurlova 2002). The photochemical properties
of these melanins were found to be dependent on both the producing strain and
the condition of its cultivation.
Melanins are high-molecular weight pigments formed by the oxidative
polymerisation of phenolic compounds. The phenolic compounds from which the
fungal melanin polymers are derived include tyrosine via
3,4-dihydroxyphenylalanine (DOPA) in various fungi and other microorganisms;
-glutaminyl-3,4-dihydroxybenzene (GDHB) or catechol in Basidiomycetes,
and 1,8-dihydroxynaphthalene (DHN) in Ascomycetes
(Bell & Wheeler 1986).
Dothideaceous species that have been found to synthesise DHN-melanin include
Aureobasidium pullulans (Siehr
1981), Cladosporium cladosporioides
(Latgé et al.
1988), Hortaea werneckii, Phaeotheca triangularis, and
Trimmatostroma salinum (Kogej
et al. 2004). Chaetothyrialean species include
Cladophialophora carrionii, C. bantiana, Exophiala jeanselmei and
E. mansonii (Taylor et
al. 1987). The authors mentioned above used the inhibitor
tricyclazole to test the fungi for the presence of 1,8-dihydroxynaphthalene
(DHN)-melanin biosynthesis.
The final step in the DHN melanin pathway is the conjoining of 1,8-DHN
molecules to form the melanin polymer. There are a number of candidate enzymes
for this step, including phenoloxidases such as tyrosinase and laccases,
peroxidases, and perhaps also catalases
(Butler & Day 1998). DHN
appears to be polymerised to melanin via a laccase but not much is know about
this enzyme and its function in the melanin pathway
(Bell & Wheeler 1986). The
aim of the present study was to analyze the influence of ortho- and
para-diphenoloxidase substrates on pigment formation in black yeasts and to
determine the type polyphenoloxidases that are involved in biosynthesis of
black yeast melanins.
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MATERIALS AND METHODS
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Diphenoloxidase substrates
Stock cultures (Table 1)
were maintained on 2 % malt extract agar (MEA) slants. The low molecular
weight aromatic compounds tested are listed in
Table 2. The formation of
melanin from low molecular weight aromatic compounds was determined by a
modified auxanographic technique in which plates of Czapek agar (CzA) (in 90
x 15 mm Petri dishes) were divided in half diametrically
(Fig. 1). One side of the plate
was spread with a suspension of seven-day-old culture cultivated on 2 % MEA at
24 °C. Simultaneously three substrate assay cups were placed on each side.
Each cup on each side received 0.1 mL of a solution of aromatic substrate
(Table 2) in 0.1 M phosphate
buffer (pH 7.0 or 7.2). The other half of the plate served as control for
spontaneous oxidation of aromatic compounds. Plates were incubated at 24
°C and observed at intervals for 1 to 7 ds for development of a
black-brown colour. The intensity of growth and pigmentation was estimated
visually, and the intensity of growth and pigmentation of strain
Aureobasidium pullulans
CBS 105.22 = VKM
F-179 (T) cultivated on 4 % MEA was listed as 100 %. The intensity of the
pigmentation was represented according to five-grade scale: 100 % (black), 75
% (dark-brown, dark olive-green or dark grey), 50 % (brown or grey), 25 %
(light brown or green-brown), and 0 % (yellow or white or pinkish).
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Fig. 1. The effect of the aromatic substrates on pigment production by strains
studied. The left half only of the plate was inoculated (O), the right half
serving as a control (C) for spontaneous oxidation of aromatic substrates.
Cups 1, 2, 3 contained 0.1 mL of a solution of aromatic substrates.
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Tricyclazole inhibition
Each fungus listed in Table
1 was grown in 90 x 15 mm Petri dishes containing 4 % MEA
with tricyclazole, CzA with tricyclazole, and on 4 % MEA (control), and CzA
(control). Tricyclazole was first dissolved in 100 % ethanol and then added to
cooled medium prior to solidification to produce a concentration of
10–50 µg/mL. The final concentration of ethanol was 1.0 %. Control
cultures were established on 4 % MEA and CzA which received only 1.0 %
ethanol. All media were adjusted to pH 7.5 prior to dispensing. Point
inoculation of each fungus was made centrally on the plate in Petri dishes
(inoculation was made by a suspension of seven-day-old culture cultivated on 2
% MEA at 24 °C). The cultures were grown in the dark at 24 °C for 21
d. The intensity of growth and pigmentation was estimated visually, and the
intensity of growth and pigmentation of strain Aureobasidium
pullulans CBS
105.22 = VKM F-179 cultivated on 4 % MEA was accepted as 100 %.
All tests were performed three times in duplicate.
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Fig. 2. The average intensity of pigmentation of some strains by presence of
different phenolic substracts. Substrates of o-diphenoloxidases (EC
1.10.3.1
[EC]
.): 4-hydroxyphenyl-pyruvic acid, L-β-phenyllactic acid,
tyrosine, pyrocatechol, 3,4-dihydroxyphenylalanine and homogentisic acid are
indicated in red colour.
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Thin-layer chromatography (TLC)
Fourteen-day-old Petri dish cultures of A. pullulans VKM F-179 =
CBS 105.22, A.
pullulans VKM F-370, A. pullulans VKPM F-371, A.
pullulans var. aubasidani VKPM F-448, grown on CzA with
(10–50 µg/mL) or without tricyclazole, were cut into small fragments
(about 1 cm3) and extracted in 150 mL acetone for 8 h. The extracts
were subsequently filtered, evaporated under reduced pressure and the
remaining aqueous solutions extracted twice with equal volumes of ethyl
acetate. The ethyl acetate fractions were collected, combined, and residual
water was removed over NaSO4. After the ethyl acetate was
evaporated under reduced pressure, each sample was reconstituted with 1 mL of
ethyl acetate to provide concentrated solutions for chromatographic evaluation
(Taylor et al. 1987,
Kogej et al. 2004).
The concentrated extracts and the standards of flaviolin, 2-hydroxyjuglone
(2-HJ), scytalone, 1,3,8-trihydroxynaphthalene (1,3,8-THN),
1,8-dihydroxynaphthalene were spotted on silica gel-coated TLC plates with
fluorescent indicator (Merck) and developed with ether-hexane-formic acid
(60:39:1). Once separated, metabolites from the extracts were observed in
daylight and under ultraviolet (UV) light at 254 and 365 nm for characteristic
colours and Rf values. The plates were then sprayed with an aqueous
solution of 1 % FeCl3. Once they were dried, they were again
evaluated for colours that appeared in daylight
(Taylor et al. 1987,
Kogej et al.
2004).
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RESULTS
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Fourteen strains of BYF originating from different natural biotopes were
investigated (Table 1). It was
established that syringaldazine, pyrogallic acid (substrates of
p-diphenoloxidases) and 4-hydrophenyl-pyruvic acid, L-β-phenyllactic acid
(substrates of o-diphenoloxidases) optimally promoted melanin biosynthesis
when compared to other groups of substrates investigated. Average intensities
of pigmentation of all strains studied were the lowest when guaiacol
(substrate of p-diphenoloxidases) was used as a substrate
(Fig. 2).
Diphenoloxidase substrates
Strains investigated were divided into four groups based on their ability
to produce dark pigments when they were cultivated on aromatic o- and on
p-diphenoloxidase substrates (Fig.
3).
- Group 1. Exophiala nigra VKM F-2137 and E.
prototropha CBS
534.94 produced black pigments when they were cultivated on all 12
aromatic substrates used, including o- and on p-diphenoloxidase substrates.
The intensity of pigmentation of strains Exophiala nigra F-2137 =
CBS 535.94 and
E. prototropha CBS
534.94, cultivated on each of the 12 aromatic substrates, was
listed as 100 %.
- Group 2. Strains utilising 8–10 aromatic substrates and
synthesising dark pigments (Fig.
3A). This group includes two strains. A. pullulans
SPChPhA 129(11), growing and synthesizing black or dark brown or brown
pigments when five substrates of o-diphenoloxidases (4-hydroxyphenyl-pyruvic
acid, L-β-phenyllactic acid, tyrosine, 3,4-dihydroxyphenylalanine,
homogentisic acid) and five substrates of p-diphenoloxidases (syringaldazine,
resorcinol, p-phenylenediamine, phloroglucinol, pyrogallic acid) were used for
cultivation. Kabatiella lini
CBS 125.21 produced
black or dark-brown or brown pigment when five substrates of
o-diphenoloxidases (4-hydroxyphenyl-pyruvic acid, L-β-phenyllactic acid,
tyrosine, pyrocatechol, homogentisic acid) and three substrates of
p-diphenoloxidases (syringaldazine, resorcinol, phloroglucinol) were used for
cultivation.
- Group 3. Strains utilising 5–7 aromatic substrates and
synthesizing dark pigments when cultivated on CzA with aromatic substrates
(Fig. 3B). This group includes
mostly strains of Aureobasidium pullulans: VKPM F-371, VKM F-179, VKM
F-1125 produced black or dark brown or dark olive-green or dark-grey, brown or
grey, light brown or green-brown pigments on two substrates of
o-diphenoloxidases (L-β-phenyllactic acid, 4-hydroxyphenyl-pyruvic acid)
and on four substrates of p-diphenoloxidases (p-phenylenediamine,
syringaldazine, pyrogallic acid, guaiacol). Strain A. pullulans
var. aubasidani VKPM F-448 produced brown or light brown or green
brown pigment on three substrates of o-diphenoloxidases
(4-hydroxyphenyl-pyruvic acid, pyrocatechol, homogentisic acid) and four
substrates of p-diphenoloxidases (syringaldazine, pyrogallic acid,
phloroglucinol, guaiacol). Strain A. pullulans SPChPhA 2320 formed
pigment on four substrates of o-diphenoloxidases (tyrosine,
L-β-phenyllactic acid, 4-hydroxyphenyl-pyruvic acid, homogentisic acid)
and two substrates of p-diphenoloxidases (syringaldazine, pyrogallic
acid).
- Group 4. Strains synthesizing dark pigments only on 1–3
aromatic substrates when cultivated on CzA with aromatic substrates
(Fig. 3C). Strains included
A. pullulans VKPM F-370 (light brown pigmentation) and H.
dematioides VKM F-2836 (dark olive-green pigmentation), producing pigment
only when substrates of p-diphenoloxidases (pyrogallic acid, resorcinol) were
used. Aureobasidium pullulans VKPM F-2204 produced pigment of equal
intensity (brown or light brown) on p-diphenoloxidases (pyrogallic acid) and
on o-diphenoloxidases (pyrocatechol) substrates. Strain VKPM F-2205 gave light
brown pigmentation on two substrates of p-diphenoloxidases
(p-phenylenediamine, pyrogallic acid) and on pyrocatechol (substrate of
o-diphenoloxidases).
Tricyclazole inhibition
Following the same subdivision:
- Group 1. Tricyclazole had no apparent effect on growth of the black
yeast strains belonging to the strains of this group, as was observed both on
4 % MEA and on CzA. The strains concerned were blackish or dark brown in
colour, when grown on 4 % MEA containing 10–20 µL/mL tricyclazole,
and on CzA containing 10–20 µL/mL tricyclazole. We observed reddish
pigment only in Group 1 strains (Exophiala nigra VKM F-2137 and
E. prototropha CBS
534.94) when we used higher (40–50 µg/mL) concentrations
of tricyclazole. Other groups (Groups 2–4) of strains studied did not
form reddish or red-brown pigments even they were cultivated in media with
high (30–50 µg/mL) concentrations of tricyclazole.
- Group 2. Tricyclazole had no apparent effect on growth and
pigmentation of A. pullulans SPChPhA 129(11), when grown on 4 % MEA.
It slightly inhibited the growth of this strain on CzA and had no effect on
pigmentation (Fig. 4). The
plant pathogen K. lini
CBS 125.21 was
inhibited by tricyclazole on 4 % MEA and CzA. The intensity of pigmentation
was decreased almost in two times, when K. lini
CBS 125.21 was
grown on CzA with tricyclazole (Fig.
4).
- Group 3. The growth of the strains belonging to this group was
slightly inhibited by tricyclazole both on 4 % MEA and on CzA. Tricyclazole
affected intensity of pigmentation of A. pullulans VKM F-179, VKM
F-1125, SPChPhA 2320, A. pullulans var. aubasidani VKPM
F-448 on CzA. On 4 % MEA an effect was found on pigmentation of only A.
pullulans var. aubasidani VKPM F-448
(Fig. 5).
- Group 4. Tricyclazole had no apparent effect on growth of the
strains A. pullulans VKM F-2204, VKM F-370 of this group, both on 4 %
MEA and on CzA (Fig. 6). The
intensity of pigmentation of the strains VKM F-2204, VKM F-370 grown on 4 %
MEA with tricyclazole was almost the same as on 4 % MEA without tricyclazole.
The strains A. pullulans VKM F-2204, VKM F-2205, VKM F-370 and
Hormonema macrospora VKM F-2452 were yellow or light yellow or
pinkish, when grown on CzA with or without tricyclazole. Hormonema
dematioides VKM F-2836 did not grow at all on CzA
(Fig. 6). Microscopic
comparisons indicated that all strains studied had their normal morphologies
in the presence of tricyclazole.
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Fig. 4. The influence of tricyclazole on growth and pigmentation of strains
belonging to the second group. Y-axis: intensity of growth, intensity of
pigmentation, %. X-axis: 4 % MEA = 4 % malt extract agar; 4 % MEA +TR = 4 %
malt extract agar with 20 µL/mL tricyclazole; CzA = Czapek agar; CzA + TR =
Czapek agar with 20 µL/mL tricyclazole.
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Fig. 5. The influence of tricyclazole on growth and pigmentation of strains
belonging to the third group. Y-axis: intensity of growth, intensity of
pigmentation, %. X-axis: 4 % MEA = 4 % malt extract agar; 4 % MEA +TR = 4 %
malt extract agar with 20 µL/mL tricyclazole; CzA = Czapek agar; CzA + TR =
Czapek agar with 20 µL/mLtricyclazole.
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Fig. 6. The influence of tricyclazole on growth and pigmentation of strains
belonging to the fourth group. Y-axis: intensity of growth, intensity of
pigmentation, %. X-axis: 4 % MEA = 4 % malt extract agar; 4 % MEA + TR = 4 %
malt extract agar with 20 µL/mL tricyclazole; CzA = Czapek agar; CzA + TR =
Czapek agar with 20 µL/mL tricyclazole.
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Identification of DHN-melanin intermediates
Metabolites from ethyl acetate extracts of A. pullulans VKM F-179
= CBS 105.22, VKM
F-370, VKPM F-371 and A. pullulans var. aubasidani VKPM
F-448 were analysed by TLC to determine if DHN-melanin precursors or related
metabolites were present. Flaviolin/biflaviolin and 2-HJ were detected in the
extracts of 14 ds old cultures of A. pullulans VKM F-370 and A.
pullulans VKPM F-371 when they were grown with tricyclazole; however,
they were not found in culture without tricyclazole
(Table 3). The TLC results
indicated that tricyclazole had blocked the DHN-melanin pathway, causing the
accumulation of 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN) and 1,3,8-THN,
which were autoxidised to flavolin or 3,3-biflaviolin and 2-HJ, respectively
(Table 3). Strains A.
pullulans VKM F-179 and A. pullulans var. aubasidani
VKPM F-448 did not secrete 2-HJ and flaviolin both in the presence of
tricyclazole and without tricyclazole
(Table 3).
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Table 3. Melanin metabolites analysed by TLC in control cultures and in
tricyclazole-inhibited cultures of A. pullulans VKM F-179, VKM F-370,
VKPM F-371 and A. pullulans var. aubasidani VKPM F-448.
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DISCUSSION
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Three out of four black yeast genera analysed (Aureobasidium,
Hormonema and Kabatiella)
(Table 1) belong to the
ascomycetous order Dothideales, while Exophiala is an
anamorph of Chaetothyriales (de
Hoog et al. 1999). Many authors
(Siehr 1981,
Taylor et al. 1987,
Butler & Day 1998,
Butler et al. 2004,
Kogej et al. 2004)
indicated that both types of fungi synthesise a DHN-type melanin. Details of
the DHN-type melanin pathway have been elucidated using a number of different
fungi. Much of what is known about the pathway and its enzymes has come from
the use of melanin-deficient strains and compounds, such as tricyclazole,
which inhibit specific enzymes in the pathway
(Bell & Wheeler 1986,
Butler & Day 1998).
The systemic fungicide tricyclazole
[5-methyl-1,2,4-thiazolo(3,4,b)-benzothiazile] (TR) is an inhibitor of
biosynthesis of melanins, which form via the pentaketide pathway
(Bell & Wheeler 1986). For
example, it is known to strongly inhibit the enzymatic reduction (reductase
enzymes) of 1,3,8-trihydroxynaphthalene (1,3,8-THN) to vermelone. Tricyclazole
has also been shown to weakly inhibit the reduction of
1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN) to scytalone
(Wheeler & Greenblatt
1988).
Flaviolin and 2-hydroxyjuglone (2-HJ) are known as autoxidative products of
1,3,6,8-THN and 1,3,8-THN, respectively
(Fig. 7). The presence of
flaviolin and 2-HJ in fungal cultures, treated with tricyclazole, is usually
accepted as proof that 1,3,6,8-THN and 1,3,8-THN were involved in the
synthesis of DHN-melanin (Butler & Day
1998). Once produced, 1,3,8-DHN is reduced to vermelone, which in
turn is dehydrated to 1,8-dihydroxynaphthalene (DHN)
(Bell & Wheeler 1986,
Taylor et al. 1987)
(Fig. 7). In most cases, these
two reactions are carried out by the same reductase and dehydratase enzymes
that produce 1,3,8-THN from 1,3,6,8-THN. DHN appears to be polymerised to
melanin via a laccase (Butler & Day
1998).
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Fig. 7. Biosynthetic pathway of DHN-melanin and related pentaketide metabolites,
from the scheme shown by Bell & Wheeler
(1986). The first known product
of the pathway is I.3.6.8-THN. This metabolite is reduced lo scytalone, which
is then dehydrated to 1,3.8-THN. Next, 1.3.8-THN is reduced to vermelone,
which is then dehydrated to DHN. The enzyme(s) that catalyze the final
polymerization reaction, oxidation of DHN to melanin, have not yet been
adequately studied but it appears to be a laccase. Tricyclazole (Tr) inhibits
the reduction of 1.3.6,8-THN and 1.3.8-THN to scytalone and vermelone,
respectively. Its strongest inhibitory effect is on the reduction of
1.3.8-THN. This results in the accumulation of flaviolin, 2-HJ, and their
related shunt products, 1.2.4.5
[EC]
.7-pentahydroxynaphthalene (1.2.4.5
[EC]
.7-PHN),
1.2.4.5
[EC]
-tetrahydroxynaphthalene (1.2.4.5
[EC]
-THN), and 1.4.5-trihydroxynaphthalene
(1.4.5-THN) are extremely unstable and have not been isolated from fungi.
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In the present investigation we demonstrated, that the DHN-melanin
inhibitor, tricyclazole, inhibited melanin biosynthesis only in some black
yeast strains. Four groups were distinguished, differing by their ability to
produce pigment with o- and p-diphenoloxidase substrates and to be inhibited
by tricyclazole (Table 4).
The effect of tricyclazole on pigment production proved to be more
pronounced when strains were grown on CzA. On this medium 53.3 % of the
strains were inhibited by tricyclazole, whereas only 26.6 % of the strains
decreased their intensity of pigmentation when they were cultivated on 4 % MEA
with tricyclazole (Table 4).
Metabolites from ethyl acetate extracts of biomass from A. pullulans
VKM F-179 = CBS
105.22, VKM F-370, VKPM F-371 and A. pullulans var.
aubasidani VKPM F-448 were analysed by thin-layer chromatography to
determine if DHN-melanin precursors or related metabolites were present.
Flaviolin and 2-HJ were detected only in the extracts of 14-d-old cultures of
A. pullulans VKM F-370, A. pullulans VKPM F-371, when they
were grown on CzA with 10 and 20 µg/mL tricyclazole. However, flaviolin and
2-HJ were not found in acetone extracts of biomasses of these strains, when
they were grown in CzA without tricyclazole
(Table 3). Strains A.
pullulans VKM F-179, A. pullulans var. aubasidani VKPM
F-448 did not secrete 2-HJ and flaviolin, neither in the presence of nor
without tricyclazole (TR) (Table
3) and even when they were cultivated in media with high
(30–50 µg/mL) concentrations of tricyclazole. The halophilic
ascomycetous black yeasts Hortaea werneckii, Phaeotheca triangularis
and Trimmatostroma salinum accumulated 4,8-dihydroxytetralone
(4,8-DHT) in cultures non-inhibited by TR
(Kogej et al. 2004)
(Fig. 7). Small amounts of
4-hydroxyscytalone (4-HS) (Fig.
7) have been reported in wild-type cultures of Curvularia
lunata non-inhibited by TR
(Ri
ner & Wheeler
2003), as well as of scytalone in Thielaviopsis basicola
(Wheeler & Stipanovic
1979) and Sporothrix schenckii
(Romero-Martinez et al.
2000). This means that products which are typical for cultures of
black yeasts inhibited by tricyclazole (TR) were also found in non-inhibited
cultures.
In our earlier investigations (Yurlova
& Sindeeva 1996) we proved the presence of intracellular and
extracellular laccase activity of 14 above mentioned strains of black yeasts.
Tricyclazole decreased laccase activity (Yurlova & Sindeeva 1995).
Tyrosinase, which oxidises tyrosine, was not found in any of the strains
investigated (Table 1) (Yurlova
& Sindeeva 1995). On the basis of the present data we hypothesise that
black yeasts contain a multipotent polyphenoloxidase able to oxidise
substrates characteristic for o-diphenoloxidases and p-diphenoloxidases. Such
kind of multipotent polyphenoloxidase has previously been observed in the
marine bacterium Marinomonas mediterranea
(Fernandez et al.
1999). The melanisation process might involve other enzymes and
more substrates than those commonly recognised. The mechanism of biosynthesis
of black yeast melanins remains to be further elucidated.
|
Acknowledgments
|
|---|
We are grateful to Drs N.N. Stepanichenko and L.N. Ten, Tashkent State
University, Uzbekistan, for assistance in obtaining TLC data.
|
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TOP
Abstract
INTRODUCTION
