Abstract
Sporothrix schenckii is the etiological agent of sporotrichosis, the main subcutaneous mycosis in Latin America. Melanin is an important virulence factor of S. schenckii, which produces dihydroxynaphthalene melanin (DHN-melanin) in conidia and yeast cells. Additionally, l-dihydroxyphenylalanine (l-DOPA) can be used to enhance melanin production on these structures as well as on hyphae. Some fungi are able to synthesize another type of melanoid pigment, called pyomelanin, as a result of tyrosine catabolism. Since there is no information about tyrosine catabolism in Sporothrix spp., we cultured 73 strains, including representatives of newly described Sporothrix species of medical interest, such as S. brasiliensis, S. schenckii, and S. globosa, in minimal medium with tyrosine. All strains but one were able to produce a melanoid pigment with a negative charge in this culture medium after 9 days of incubation. An S. schenckii DHN-melanin mutant strain also produced pigment in the presence of tyrosine. Further analysis showed that pigment production occurs in both the filamentous and yeast phases, and pigment accumulates in supernatants during stationary-phase growth. Notably, sulcotrione inhibits pigment production. Melanin ghosts of wild-type and DHN mutant strains obtained when the fungus was cultured with tyrosine were similar to melanin ghosts yielded in the absence of the precursor, indicating that this melanin does not polymerize on the fungal cell wall. However, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. In conclusion, at least three species of the Sporothrix complex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence.
INTRODUCTION
Melanins are polymers with diverse molecular structures, typically black or dark brown, formed by the oxidative polymerization of phenolic and indolic compounds. They are produced by a broad range of organisms, from bacteria to humans. Several fungi can produce melanins, and the functions of these pigments are related to microbial survival under several unfavorable environmental and host conditions (10, 14). The major melanin type encountered among fungi is 1,8-dihydroxynaphthalene melanin (DHN-melanin), which is synthesized from acetyl coenzyme A via the polyketide pathway. This form of melanin is synthesized by several plant and human fungal pathogens. In addition to DHN-melanin, certain fungi can also produce melanin via dihydroxyphenylalanine (DOPA), in which tyrosinases or laccases hydroxylate tyrosine via DOPA to dopaquinone, which then auto-oxidizes and polymerizes, resulting in a polyphenolic heteropolymer that is black (9). Some fungi produce a soluble melanin from l-tyrosine through p-hydroxyphenylpyruvate and homogentisic acid. This soluble pigment is called pyomelanin, and it is similar to the alkaptomelanin produced by humans. Aspergillus fumigatus, Madurella mycetomatis, and Yarrowia lipolytica are examples of fungi that can produce this type of soluble pigment (4, 18, 20).
Sporothrix schenckii is a dimorphic fungal pathogen that is the primary cause of sporotrichosis, a cosmopolitan subcutaneous mycosis that can affect humans and other animals, such as dogs, cats, horses, and armadillos (2). More recently, additional species of Sporothrix, including Sporothrix brasiliensis and Sporothrix globosa, have been identified as important causes of human or mammalian sporotrichosis (11). When cultured at 25°C to 30°C, the fungus produces smooth and wrinkled mycelial colonies that are initially white to creamy but turn brown to black after a few days of cultivation due to the production of DHN-melanin on conidial cells (17). Although yeast colonies grown at 37°C do not turn black even after long periods of incubation, DHN-melanin is also produced within the cell walls of yeast both in vitro and during infection (12). Moreover, l-DOPA can be used to enhance melanin production on conidial and yeast cells as well as to induce hyphal melanization (1).
Since there is no information about tyrosine metabolism or pyomelanin production in species of the Sporothrix complex, we first decided to evaluate whether this fungus was capable of pyomelanin synthesis and then examined its potential impact on the biology of the fungi.
MATERIALS AND METHODS
Strains.
Seventy-three Sporothrix sp. strains were used to investigate the capacity of the species to produce pyomelanin. All strains were identified by morphological and biochemical characteristics at both 25°C and 36°C, and the identifications were confirmed by molecular criteria (11). The strains were identified as S. schenckii (14 strains), S. brasiliensis (58 strains), and S. globosa (1 strain). Reference strains were included: three S. schenckii reference strains (ATCC 16345, ATCC 32285, and ATCC 32286) and one S. brasiliensis strain (CBS 120339). All strains are maintained in the pathogenic fungal culture collection of the Laboratorio de Micologia/IPEC/Fiocruz (WDCM 951). Strain IPEC 26449, previously characterized by our group as a high-melanin-producing strain (1), was further studied to examine conditions for pigment production and its functions. In addition, a DHN-melanin-deficient mutant strain, Mel-14, which has a mutation in the polyketide synthase gene (17), was also studied to determine if DHN-melanin was involved in pyomelanin formation.
Media.
Defined chemical medium (minimal medium) for Sporothrix sp. growth consisted of 15 mM glucose, 10 mM MgSO4, 29.4 mM KH2PO4, 13 mM glycine, and 3.0 mM thiamine (pH 5.5). Minimal medium with l-tyrosine was made by adding 10 mM l-tyrosine to minimal medium. Agar plates were made by adding 20 g/liter to the corresponding medium. All chemicals used to prepare culture media were purchased from Sigma-Aldrich (St. Louis, MO).
Screening for pigment production.
The 73 Sporothrix sp. strains were inoculated onto minimal medium agar plates with and without l-tyrosine. Cultures were incubated at both 25°C and 36°C and checked daily for pigment production over a 20-day period. Also, cultures at 36°C were checked for the presence of yeast cells, and they were used only if at least 90% yeast cells were observed.
Influence of sulcotrione and glyphosate.
In order to check if pigment production could be suppressed or abolished by melanin synthesis inhibitors, sulcotrione [2-(2-chloro-4-mesylbenzoyl)cyclohexane-1,3-dione] and glyphosate [N-(phosphonomethyl)glycine] were added to minimal medium with l-tyrosine at concentrations of 16 and 100 mg/liter, respectively. Sulcotrione inhibits 4-hydroxyphenyl-pyruvate-dioxygenase (4-HPPD), impacting pyomelanin formation, whereas glyphosate blocks the shikimate acid pathway for aromatic amino acid synthesis.
Autopolymerization.
Three different flasks with 50 ml minimal medium with l-tyrosine were prepared. A suspension of 1 × 108 conidia from S. brasiliensis strain IPEC 26449 was prepared, and 100 μl was inoculated into one flask. A second suspension of 1 × 108 conidia was boiled for 45 min, and 100 μl was then added to a flask. An aliquot was also plated onto a potato dextrose agar plate to confirm fungal death. The third flask was left uninoculated. All flasks were incubated in the dark for 15 days at 25°C on a rotary incubator at 150 rpm. After incubation, flasks were visually and photometrically (absorbance at 340 nm) examined for the presence of pigment in the supernatants.
Pigment measurement.
An aliquot of 0.5 ml of the supernatant was taken daily from each flask and stored at −20°C. After all samples were collected, they were centrifuged (2,300 × g), and supernatant absorbances at 340 nm were measured with an enzyme-linked immunosorbent assay (ELISA) plate reader (μQuant; Bio-Tek).
Growth curves.
Strains IPEC 26449 and Mel-14 were inoculated into flasks with 50 ml of minimal medium with l-tyrosine, without l-tyrosine, and with both l-tyrosine and sulcotrione at an initial concentration of 1.0 × 104 cells/ml. Flasks were incubated at 25°C and 36°C on a rotary incubator at 150 rpm. Serial dilutions of the different cultures were performed daily during 14 days, 100 μl of each dilution was spread onto plate count agar (Bio-Rad), and cultures were incubated at 25°C. After 7 days of incubation, colonies were counted for CFU determinations. Additionally, daily aliquots of each culture were obtained and assessed for pigmentation, as described above.
Measurements of zeta potential.
In order to calculate the charge of the melanoid pigment, an extraction procedure was performed on 15-day-old cultures incubated with l-tyrosine, according to a protocol described previously for pyomelanin purification from Aspergillus fumigatus cultures (18). In brief, supernatants were filtered through 0.22-μm membranes, acidified to pH 2.0 using 0.5 mol/liter HCl, and left overnight at room temperature (RT). The precipitated pigment was harvested by centrifugation (12,800 × g) and resuspended in sterile distilled water. After purification, pigment was suspended in 10 mM KCl and analyzed by using a zeta-potential analyzer (ZetaPlus; Brookhaven Instruments Corp., Holtsville, NY). At least five different charge measurements were performed for each sample.
EPR analysis.
Pigments from strains IPEC 26449 and Mel-14, extracted as described above, were analyzed by electron paramagnetic resonance (EPR), using a Gunn diode as the microwave source. EPR spectra were obtained with a Varian E112 X-Band model spectrometer using a TE102 resonator and a liquid nitrogen finger Dewar vessel. The parameters for EPR were as follows: modulation frequency of 9.07 GHz, modulation amplitude of 1.6 G, center field of 3,250.0 G, sweep width of 100.0 G, microwave frequency of 9.1 GHz, microwave power of 1.0 mW, time constant of 0.5 s, and temperature of 77 K.
Melanin ghosts.
To generate melanin particles, strains IPEC 26449 and Mel-14 were incubated for 14 days in 100 ml of either minimal medium with l-tyrosine, minimal medium without l-tyrosine, or minimal medium with l-tyrosine and sulcotrione at 30°C with shaking (150 rpm). The fungal cells were collected, washed three times in phosphate-buffered saline (PBS) (pH 7.2), and suspended in 1.0 M sorbitol–0.1 M sodium citrate (pH 5.5). Protoplasts were generated by incubating cells at 30°C in 10 mg/ml of cell wall-lysing enzymes (from Trichoderma harzianum; Sigma Chemical Co.) for 1 h at RT. Protoplasts were then collected by centrifugation (2,300 × g) for 10 min, washed with PBS, and incubated in 4.0 M guanidine thiocyanate for 1 h at RT with frequent vortexing. The resulting material was washed again in PBS, collected by centrifugation, and boiled in 6.0 M HCl for 1 h to hydrolyze cellular contaminants associated with melanin. The debris was collected by centrifugation and washed exhaustively with PBS.
Scanning electron microscopy.
Chemically treated melanin ghosts from Sporothrix cultures were fixed overnight in a 4% glutaraldehyde solution in PBS. Particles were then transferred onto polylysine-coated coverslips and submitted to dehydration. Samples were mounted with gold-palladium and viewed with a Quanta 50 scanning electron microscope.
Susceptibility to UV irradiation.
Strains IPEC 26449 and Mel-14 were cultured with or without l-tyrosine at 25°C for 14 days. After incubation, cells were collected, washed three times, counted on a hemocytometer, and diluted in PBS to achieve 5-ml suspensions of 1.0 × 103 conidia/ml. Baseline viability was determined by plating 100 μl of each suspension onto Sabouraud dextrose agar (Difco) plates. Other plates also inoculated with 100 μl of each suspension were exposed to either 15, 30, 45, 60, 75, or 90 s of UV light (290 μW/cm2) irradiation. All plates were incubated at 25°C during 7 days. Survival rates were determined by counting the number of colonies from irradiated cells relative to the number of colonies from untreated controls.
Susceptibility to oxidants.
Fungal cells (1.0 × 107 conidia) of strains IPEC 26449 and Mel-14 were harvested after 14 days of incubation with or without l-tyrosine, washed three times with PBS, and submitted to chemically generated oxidants, as previously described (22). In brief, nitric oxide and reactive nitrogen intermediates were generated in a solution containing 0.5 mM NaNO2 and 25 mM succinic acid (pH 4.0), and oxygen-derived oxidants were generated in a solution containing 0.5 mM ferric ammonium sulfate, 61.8 μM hydrogen peroxide, and 1.0 mM epinephrine bitartrate. All chemicals were purchased from Sigma-Aldrich. After 1, 2, 3, and 4 h of incubation at 36°C with the above-mentioned oxidants, Sporothrix cells were plated onto plate count agar (Bio-Rad) to determine viability, as measured by the number of viable colonies. Aliquots of untreated cells were also plated as controls. Survival rates were calculated by comparing the number of colonies subjected to various time intervals of exposure to these reaction mixtures with the colony number of untreated cells.
Resistance to amphotericin B.
The MIC of amphotericin B for strain IPEC 26449 was determined by broth microdilution in RPMI 1640 medium, as previously described (7). Yeast cells from this strain (2.5 × 105 yeast cells) were harvested after 14 days of incubation in minimal medium with l-tyrosine. Control cells consisted of Sporothrix yeast cells cultured on minimal medium or minimal medium with l-DOPA (21). All cells were washed three times with PBS and incubated with 0.5 or 2 times the MIC found for this strain by the broth microdilution assay. After 2, 6, and 24 h of incubation, serial dilutions were performed and plated onto plate count agar (Bio-Rad). Survival rates were calculated as described above.
Statistics.
All the experiments described here were repeated at least three times to calculate means and standard deviations. There were no significant variations between iterations of each experimental condition. Data were analyzed with Student's t test by using SPSS 17.0 software.
RESULTS
Production of melanoid pigment from l-tyrosine.
After incubation at 25°C in minimal medium with l-tyrosine, 72 of 73 Sporothrix strains produced a dark brown pigment diffusible into the agar after 9 to 12 days of growth (Table 1). The assessment of the plates at 20 days revealed differences in pigment intensity among strains (Fig. 1), as 8 strains (11.1% of positive strains) produced small amounts of pigment, similar to the plate shown in Fig. 1A; 16 (22.2%) generated a light brown color on the agar surface, similar to the plate shown in Fig. 1B; 29 (40.3%) yielded a dark brown color on agar, as shown in Fig. 1C; and 19 (26.4%) strains were heavily melanized, as represented in Fig. 1D. The 73 strains were able to synthesize pigment on culture media at 37°C, although the level of pigment expression was lower than at 25°C for most strains. At 20 days of cultivation at 37°C, 11 strains (15.1%) produced only small amounts of melanoid pigment, 36 (49.3%) of the cultures were light brown, 24 (32.9%) were dark brown, and only 2 (2.7%) were black. Since most Sporothrix strains grown at 25°C produced more pigment, the mycelial Sporothrix form was used in subsequent experiments.
Table 1.
Profile of dark pigment production of 73 strains of the Sporothrix complex used in this study
| Strain | Species | Pigment productiona |
|
|---|---|---|---|
| 25°C | 37°C | ||
| CBS 120339 | S. brasiliensis | + | ++ |
| IPEC 17307 | S. brasiliensis | +++ | +++ |
| IPEC 17521 | S. brasiliensis | ++++ | +++ |
| IPEC 17585 | S. brasiliensis | ++++ | +++ |
| IPEC 17608 | S. brasiliensis | +++ | +++ |
| IPEC 17692 | S. brasiliensis | +++ | +++ |
| IPEC 17920 | S. brasiliensis | ++++ | ++ |
| IPEC 18202-3 | S. brasiliensis | +++ | + |
| IPEC 18782A | S. brasiliensis | ++++ | ++ |
| IPEC 18782B | S. brasiliensis | ++ | + |
| IPEC 19777 | S. brasiliensis | ++ | + |
| IPEC 22493-1 | S. brasiliensis | ++ | + |
| IPEC 22582 | S. brasiliensis | +++ | ++ |
| IPEC 25303 | S. brasiliensis | ++ | ++ |
| IPEC 25541 | S. brasiliensis | +++ | ++ |
| IPEC 25644 | S. brasiliensis | ++ | +++ |
| IPEC 25712 | S. brasiliensis | +++ | +++ |
| IPEC 25758 | S. brasiliensis | + | + |
| IPEC 25853 | S. brasiliensis | +++ | ++ |
| IPEC 25976 | S. brasiliensis | +++ | + |
| IPEC 26034 | S. brasiliensis | +++ | ++ |
| IPEC 26156 | S. brasiliensis | + | ++ |
| IPEC 26449 | S. brasiliensis | ++++ | +++ |
| IPEC 27588 | S. brasiliensis | ++ | + |
| IPEC 28831 | S. brasiliensis | ++ | ++ |
| IPEC 29039 | S. brasiliensis | +++ | +++ |
| IPEC 29787 | S. brasiliensis | ++ | ++ |
| IPEC 30650 | S. brasiliensis | + | ++ |
| IPEC 30682-1 | S. brasiliensis | ++++ | ++ |
| IPEC 31047-1 | S. brasiliensis | +++ | ++ |
| IPEC 31515 | S. brasiliensis | ++ | +++ |
| IPEC 31676 | S. brasiliensis | ++ | ++ |
| IPEC 32004 | S. brasiliensis | +++ | +++ |
| IPEC 32406 | S. brasiliensis | ++ | + |
| IPEC 33601 | S. brasiliensis | +++ | ++ |
| IPEC 33611 | S. brasiliensis | ++++ | +++ |
| IPEC 33704 | S. brasiliensis | +++ | ++ |
| IPEC 33822 | S. brasiliensis | +++ | ++ |
| IPEC 33946 | S. brasiliensis | ++++ | ++++ |
| IPEC 34079 | S. brasiliensis | +++ | ++ |
| IPEC 34105 | S. brasiliensis | + | +++ |
| IPEC 34196 | S. brasiliensis | +++ | +++ |
| IPEC 34249 | S. brasiliensis | +++ | ++ |
| IPEC 34255 | S. brasiliensis | ++ | ++ |
| IPEC 34316 | S. brasiliensis | +++ | ++ |
| IPEC 34328 | S. brasiliensis | ++++ | ++ |
| IPEC 34498 | S. brasiliensis | +++ | ++ |
| IPEC 34566 | S. brasiliensis | +++ | ++ |
| IPEC 34567 | S. brasiliensis | + | ++ |
| IPEC 34641 | S. brasiliensis | +++ | +++ |
| IPEC 34798 | S. brasiliensis | ++++ | ++ |
| IPEC 34851 | S. brasiliensis | ++ | ++ |
| IPEC 34910 | S. brasiliensis | + | ++ |
| IPEC 34968 | S. brasiliensis | +++ | ++ |
| IPEC 36062 | S. brasiliensis | ++ | + |
| IPEC 41908-1 | S. brasiliensis | +++ | ++ |
| IPEC 74H | S. brasiliensis | ++++ | ++ |
| IPEC 645H | S. brasiliensis | +++ | ++ |
| IPEC 27135 | S. globosa | ++++ | ++++ |
| ATCC 16345 | S. schenckii | ++++ | +++ |
| ATCC 32285 | S. schenckii | ++++ | +++ |
| ATCC 32286 | S. schenckii | +++ | +++ |
| IOC 1113 | S. schenckii | +++ | ++ |
| IPEC 23249 | S. schenckii | ++ | ++ |
| IPEC 23250 | S. schenckii | − | +++ |
| IPEC 23251 | S. schenckii | + | + |
| IPEC 23252 | S. schenckii | ++++ | + |
| IPEC 23253 | S. schenckii | ++++ | +++ |
| IPEC 24372-1 | S. schenckii | ++ | +++ |
| IPEC 25374 | S. schenckii | +++ | ++ |
| IPEC 25457 | S. schenckii | ++++ | +++ |
| IPEC 27722 | S. schenckii | ++++ | +++ |
| Mel-14 | S. schenckii | ++++ | +++ |
−, no pigment production; +, low-level pigment production; ++, light brown pigment production; +++, dark brown pigment production; ++++, black pigment production.
Fig 1.
Production of a diffusible melanoid pigment by Sporothrix mycelial forms. We noted four intensity levels of agar pigmentation after 20 days of incubation at 25°C. The melanization ranged from minimal pigmentation to light to dark brown or to black, as represented by IPEC 34910 (A), IPEC 31676 (B), IPEC 33611 (C), and IPEC 26449 (D).
Conditions for pigment production.
The pigmentation of culture supernatants occurred only if viable fungal cells were inoculated into minimal medium containing tyrosine (Fig. 2A). No autopolymerization of l-tyrosine occurred in uninoculated flasks. The formation of pigment was inhibited with 16 mg/liter of sulcotrione, while glyphosate was unable to completely inhibit pigmentation, although the pigment isolated was brown rather than black (Fig. 2B). Also, fungal cells grown in the presence of l-tyrosine accumulated this pigment such that it was visible in cell pellets (Fig. 2C).
Fig 2.
Pigment production by strain IPEC 26449 in the presence of l-tyrosine. (A) Heat-killed Sporothrix cells are unable to produce pigment after 20 days of incubation, whereas viable fungal cells develop black pigmentation. (B) Glyphosate (100 mg/liter) was unable to completely inhibit melanin production, whereas sulcotrione (16 mg/liter) abolished pigment formation after 12 days of incubation. (C) Sporothrix cell mass harvested after 20 days of incubation with or without l-tyrosine.
Growth curves performed by using minimal medium with or without l-tyrosine as well as minimal medium with l-tyrosine and sulcotrione indicated that both the pigment precursor and inhibitor had no effect on Sporothrix growth (Fig. 3A). Furthermore, we observed by determinations of the optical density of supernatants at 340 nm that pigment is produced during the stationary phase of fungal growth in the cultures with minimal medium with tyrosine and without sulcotrione (Fig. 3B).
Fig 3.
Kinetics of pigment production. (A) Fungal growth is similar on minimal medium (MM), minimal medium with l-tyrosine (MMTyr), and minimal medium with l-tyrosine and sulcotrione (MMTyrSulc). (B) The quantification of pigment by optical density (OD) measurements at 340 nm shows that melanin is formed in the stationary phase of cultures grown on l-tyrosine, but no pigmentation occurs in the absence of l-tyrosine or in the presence of sulcotrione.
EPR analysis.
Both pigment samples extracted from wild-type strain IPEC 26449 and DHN-melanin mutant strain Mel-14 had EPR signals consistent with melanin (Fig. 4). The X-band EPR spectrum of melanin is relatively featureless, but some variation in line shape and/or line width can occur between samples, depending on the structure of the melanin polymer. The two samples were very similar in both spectral features, roughly indicative of the molecular structure, and intensity, indicative of the radical concentration.
Fig 4.
Continuous-wave (Cw) EPR spectrum from extracellular pigment extracted from S. schenckii Mel-14 (A) and S. brasiliensis IPEC 26449 (B).
Zeta potential.
Since melanins are pigments with negative charges, we used a zeta-potential analyzer to analyze pigments produced by both strains IPEC 26449 and Mel-14. We observed a charge of −25.54 ± 5.93 mV on pigment derived from strain IPEC 26449 and a charge of −17.56 ± 4.36 mV on the Mel-14 pigment.
Melanin ghost analysis.
Melanin particles were obtained from S. schenckii strains IPEC 26449 and Mel-14 grown in the presence (Fig. 5A) or absence (data not shown) of l-tyrosine, which were subsequently treated with enzymes and with hot acid. Hyphae from both strains were completely solubilized under all tested culture conditions. Melanin ghosts from strain IPEC 26449 included particles with the same shape and size of fungal conidia. We observed mean diameters of 2.07 ± 0.30 μm in the absence of l-tyrosine and 2.43 ± 0.42 μm in the presence of l-tyrosine. Additionally, we also observed the presence of small dysmorphic black particles on all tested cultures. These particles were cylindrical or spherical, with a mean diameter of 0.634 ± 0.138 μm. Also, they were the main constituent of Mel-14 particles in the presence (Fig. 5B) or absence of l-tyrosine.
Fig 5.
Scanning electron microscopy of melanin ghosts from strains IPEC 26449 (A) and Mel-14 (B) after 15 days of incubation with l-tyrosine. Similar particles were generated on minimal medium without l-tyrosine (not shown).
Protection against UV light.
Since strain Mel-14 is unable to produce DHN-melanin, its susceptibility to UV irradiation was higher than that observed for strain IPEC 26449. Although the addition of tyrosine did not protect Mel-14 from UV damage after 30 min, Mel-14 cells grown with tyrosine were significantly more capable of surviving UV irradiation than Mel-14 cells cultivated without tyrosine (Fig. 6). In contrast, strain IPEC 26449 cells grown in the presence of tyrosine survived after exposure to up to 75 s of irradiation, although only 3% of cells survived this maximal dose. IPEC 26449 cells grown without tyrosine were all eradicated after more than 30 s of UV exposure. Although IPEC 26449 cells grown with or without tyrosine were similarly able to resist UV irradiation for 15 s, cells incubated with tyrosine were significantly more resistant to 30 s of UV exposure than cells grown without tyrosine.
Fig 6.
Survival rates of Sporothrix cells grown with and without l-tyrosine after UV irradiation. Each value represents the mean and standard deviation from at least four different experiments. *, P < 0.05.
Protection against nitrogen-derived oxidants.
Table 2 shows the survival percentages of strain IPEC 26449 cells grown in the presence or absence of l-tyrosine after various time intervals of exposure to nitrogen-derived oxidants. Cells that produced the melanoid pigment in culture medium exhibited significantly higher survival rates than did cells grown on minimal medium without pyomelanin precursors. At all time points, cells grown in the presence of l-tyrosine were more resistant to nitrogen-derived free radicals than cells that were not. It is interesting that the addition of sulcotrione to culture medium with l-tyrosine led to the production of cells with susceptibility to nitrogen-derived oxidants similar to that of cells grown on minimal medium, demonstrating the impact of the inhibition of the pyomelanin synthesis pathway.
Table 2.
Percent survival rates of S. brasiliensis strain IPEC 26449 cells cultured under several conditions submitted to nitrogen-derived oxidants for different timesa
| Time of incubation (h) | Mean % survival ± SD |
||
|---|---|---|---|
| Minimal medium | Minimal medium with 10 mM l-tyrosine | Minimal medium with 10 mM l-tyrosine and 16 mg/liter sulcotrione | |
| 1 | 22.90 ± 6.39 | 64.56 ± 36.30 | 23.65 ± 10.80 |
| 2 | 5.55 ± 1.47 | 21.41 ± 7.54 | 7.01 ± 1.27 |
| 3 | 2.51 ± 0.93 | 7.32 ± 2.42 | 1.34 ± 0.41 |
| 4 | 0.51 ± 0.12 | 2.78 ± 1.95 | 0.50 ± 0.39 |
Results are the means ± standard deviations from at least three different observations.
Protection against oxygen-derived oxidants.
When S. brasiliensis strain IPEC 26449 was exposed to chemically generated oxygen-derived oxidants for 1 h, there was a statistical difference between the survival rates of cells cultured with and cells cultured without tyrosine (86.8% ± 10.59% and 24.97% ± 10.65%, respectively; P = 0.04). At 2, 3, or 4 h of incubation, no statistical differences in cell survival rates were observed.
Resistance to amphotericin B.
The MIC of amphotericin B for strain IPEC 26449 was 1.0 μg/ml. Therefore, cells were incubated with 0.5 and 2.0 μg/ml amphotericin B, and survival rates were determined after 2, 6, and 24 h. Yeast cells grown with tyrosine were more resistant to amphotericin B killing than cells grown on minimal medium alone (Table 3). After 2 and 6 h of incubation with a concentration of 0.5 μg/ml amphotericin B and after 2 h of incubation with 2.0 μg/ml, a statistical difference was observed for survival rates between melanized and nonmelanized cells (P values of 0.0393, 0.038, and 0.0059, respectively). The resistance to 2.0 μg/ml amphotericin B conferred by pyomelanin was even greater than resistance conferred by the l-DOPA-derived melanin after 2 h of incubation (P = 0.0113).
Table 3.
Amphotericin B killing assay with S. brasiliensis strain IPEC 26449 cultured on media without or with different melanin synthesis inducersa
| Time (h) | Mean % survival ± SD |
|||||
|---|---|---|---|---|---|---|
| Minimal medium |
Minimal medium with l-DOPA |
Minimal medium with l-tyrosine |
||||
| 0.5 μg/ml AMB | 2.0 μg/ml AMB | 0.5 μg/ml AMB | 2.0 μg/ml AMB | 0.5 μg/ml AMB | 2.0 μg/ml AMB | |
| 2 | 7.0 ± 0.3 | 1.1 ± 0.5 | 12.5 ± 5.6 | 4.9 ± 1.6 | 70.0 ± 36.2 | 25.8 ± 8.0 |
| 6 | 0.55 ± 0.1 | 0 | 1.8 ± 1.3 | 0 | 8.5 ± 4.4 | 0 |
| 24 | 0.14 ± 0.1 | 0 | 2.0 ± 0.4 | 0 | 4.7 ± 3.8 | 0 |
Results are the means ± standard deviations of percent survival rates from at least three different observations. AMB, amphotericin B.
DISCUSSION
Sporotrichosis is a mycotic infection with a broad range of clinical manifestations, from localized cutaneous infection to severe disseminated and often fatal disease (2). One of the factors that might play a role in the development of sporotrichosis is the differential expression of virulence characteristics of the infective Sporothrix strain (3). Melanins represent important virulence factors of this fungus, offering protection against phagocytosis and killing by human monocytes and murine macrophages (17). S. schenckii and related species produce DHN- and l-DOPA-derived melanin (1, 12, 17). Here, we show, for the first time, that at least three species of the Sporothrix complex (S. schenckii, S. brasiliensis, and S. globosa) are able to produce a third type of melanin, which has been characterized as pyomelanin. This pigment is synthesized in the presence of l-tyrosine during the stationary phase of fungal growth, presents a negative charge, and yields a signal indicative of a stable free radical population. Moreover, its synthesis requires metabolically active fungal cells, and production is blocked by sulcotrione. This compound is an inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase (19), which is involved in the l-tyrosine degradation pathway (18). An S. schenckii strain with a defective polyketide synthase gene and, therefore, which was unable to produce DHN-melanin (17) was also able to synthesize pyomelanin. This result also supports that the formation of this melanoid pigment is independent of the DHN-melanin metabolic pathway.
By the results of our experiments, it was not possible to determine if pyomelanin was produced by conidia or hyphae of Sporothrix spp. This pigment was produced after 9 days of incubation, and it is not possible to maintain a culture containing only conidia during this time. We believe that both structures produce this pigment. Up to now, the exact fungal structures containing pyomelanin have not been reported, since this pigment was described previously for only two other filamentous fungi, and it was not determined in which structure it was produced (18, 20).
Some fungi, such as Aspergillus oryzae, are able to produce eumelanin through the two-step oxidation of tyrosine to dopaquinone via l-DOPA (9). We demonstrated previously that S. schenckii can use l-DOPA for melanogenesis (1), but it is unlikely that the process of melanin synthesis described in the current work is based on l-tyrosine conversion to l-DOPA. As we previously demonstrated, l-DOPA-derived melanin accumulates on the fungal cell wall of conidia, yeast cells, and even S. schenckii hyphae. Scanning electron microscopy showed that hyphae cultured in the presence of l-tyrosine were completely dissolved after denaturant and hot-acid treatments. Moreover, melanin ghosts of Sporothrix cultures with l-tyrosine were similar to ghosts generated on minimal medium. In addition, pigment formation was dependent on the viability of Sporothrix cells. In contrast, the pigment formed in l-DOPA supernatant cultures is, at least in part, a result of l-DOPA autopolymerization and does not require metabolically active cells for its appearance (15). Thus, l-DOPA-derived pigment formation occurs outside fungal cells and deposits along the surface of fungal cells, promoting their darkening.
l-Tyrosine did not interfere with the growth of species of the Sporothrix complex on minimal medium with glucose as a carbon source but clearly conferred protection to this fungus in the setting of several lethal conditions. l-Tyrosine is presumably available to the fungus in ulcerative cutaneous lesions of patients, since the amino acid is used to form skin and hair melanin (13), allowing pyomelanin synthesis during infection. It is also likely that tyrosine is available during fungal growth in the environment, as it has important functions in chloroplasts and photosynthesis (6). Given the known functions of melanin, the pigment deposited onto the Sporothrix cell wall is presumably able to absorb UV light, acting like a shield from high-energy photons, preventing DNA damage and cellular death. The polymer is also likely to protect Sporothrix cells against the fungicidal effects of oxygen, as occurred with Aspergillus fumigatus (18), and especially nitrogen-derived oxidants. Protection against nitrogen radicals is important during infection, since amelanotic S. schenckii is susceptible to the fungicidal effects of nitric oxide produced by macrophages (5).
When the susceptibilities of Sporothrix spp. melanized in the presence of l-DOPA or l-tyrosine were analyzed, we observed that the fungus became more resistant than when growth occurred on minimal medium. Importantly, l-tyrosine-derived pyomelanin offered more resistance to antifungal stress than l-DOPA-derived melanin. In Cryptococcus neoformans and Histoplasma capsulatum, l-DOPA-derived melanin binds amphotericin B, reducing its antifungal properties (21). Although the results from this work do not permit a complete conclusion about this observation with Sporothrix cells, our group is currently working on a comparison of the binding affinities of amphotericin B for the different Sporothrix melanin types to better understand this process.
In summary, our results suggest a role for pyomelanin in the virulence of the Sporothrix complex. Interestingly, the beneficial role of tyrosine in Sporothrix is partially supported by findings for other fungal species. The inhibition of tyrosine degradation in Paracoccidioides brasiliensis impairs fungal growth and differentiation for the parasitic yeast phase of the fungus (16). A. fumigatus is able to produce pyomelanin (18), but despite the protection against reactive oxygen intermediates conferred by this pigment, it is not necessary for fungal virulence (8). Our explorations suggest several pathways for protective responses in Sporothrix in the presence of tyrosine, and our findings open up interesting avenues for further exploration into the survival of Sporothrix spp. in the environment and during interactions with diverse hosts.
ACKNOWLEDGMENTS
Financial support was provided by FAPERJ (grant E-26/110.619/2012). R.M.Z.-O. is supported in part by CNPq grant 350338/2000-0 and FAPERJ grant E-26/103.157/2011. J.D.N. was supported in part by NIH grant AI52733.
Footnotes
Published ahead of print 5 October 2012
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