Biosynthesis and Functions of a Melanoid Pigment Produced by Species of the Sporothrix Complex in the Presence of L-Tyrosine

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Rodrigo Almeida-Paes,a Susana Frases,b,f Glauber de Sousa Araújo,b,f Manoel Marques Evangelista de Oliveira,a Gary J. Gerfen,c Joshua D. Nosanchuk,d,e and Rosely Maria Zancopé-Oliveiraa Laboratório de Micologia, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz, Rio de Janeiro, Brazila; Instituto de Biofísica Prof. Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazilb; Departments of Physiology and Biophysics,c Medicine, Division of Infectious Diseases,d Microbiology and Immunology,e Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA; and Laboratório de Biotecnologia, Instituto Nacional de Metrologia, Normalização e Qualidade Industrial, Rio de Janeiro, Brazilf

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 represen- tatives 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 Sporo- thrix complex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence.

elanins are polymers with diverse molecular structures, typ- initially white to creamy but turn brown to black after a few days of Mically black or dark brown, formed by the oxidative poly- cultivation due to the production of DHN-melanin on conidial merization of phenolic and indolic compounds. They are pro- cells (17). Although yeast colonies grown at 37°C do not turn duced by a broad range of organisms, from bacteria to humans. black even after long periods of incubation, DHN-melanin is also Several fungi can produce melanins, and the functions of these produced within the cell walls of yeast both in vitro and during pigments are related to microbial survival under several unfavor- infection (12). Moreover, L-DOPA can be used to enhance mela- able environmental and host conditions (10, 14). The major mel- nin production on conidial and yeast cells as well as to induce anin type encountered among fungi is 1,8-dihydroxynaphthalene hyphal melanization (1). melanin (DHN-melanin), which is synthesized from acetyl coen- Since there is no information about tyrosine metabolism or zyme A via the polyketide pathway. This form of melanin is syn- pyomelanin production in species of the Sporothrix complex, we thesized by several plant and human fungal pathogens. In addition first decided to evaluate whether this fungus was capable of pyo- to DHN-melanin, certain fungi can also produce melanin via di- melanin synthesis and then examined its potential impact on the hydroxyphenylalanine (DOPA), in which tyrosinases or laccases biology of the fungi. hydroxylate tyrosine via DOPA to dopaquinone, which then MATERIALS AND METHODS auto-oxidizes and polymerizes, resulting in a polyphenolic het- eropolymer that is black (9). Some fungi produce a soluble mela- Strains. Seventy-three Sporothrix sp. strains were used to investigate the capacity of the species to produce pyomelanin. All strains were identified nin from L-tyrosine through p-hydroxyphenylpyruvate and ho- by morphological and biochemical characteristics at both 25°C and 36°C, mogentisic acid. This soluble pigment is called pyomelanin, and it and the identifications were confirmed by molecular criteria (11). The is similar to the alkaptomelanin produced by humans. Aspergillus strains were identified as S. schenckii (14 strains), S. brasiliensis (58 fumigatus, Madurella mycetomatis, and Yarrowia lipolytica are ex- strains), and S. globosa (1 strain). Reference strains were included: three S. amples of fungi that can produce this type of soluble pigment (4, schenckii reference strains (ATCC 16345, ATCC 32285, and ATCC 32286) 18, 20). and one S. brasiliensis strain (CBS 120339). All strains are maintained in 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, Received 1 August 2012 Accepted 26 September 2012 cats, horses, and armadillos (2). More recently, additional species Published ahead of print 5 October 2012 of Sporothrix, including Sporothrix brasiliensis and Sporothrix glo- Address correspondence to Rodrigo Almeida-Paes, rodrigo.paes@ipec.fiocruz.br. bosa, have been identified as important causes of human or mam- Copyright © 2012, American Society for Microbiology. All Rights Reserved. malian sporotrichosis (11). When cultured at 25°C to 30°C, the doi:10.1128/AEM.02414-12 fungus produces smooth and wrinkled mycelial colonies that are

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TABLE 1 Profile of dark pigment production of 73 strains of the TABLE 1 (Continued) Sporothrix complex used in this study Pigment productiona Pigment productiona Strain Species 25°C 37°C Strain Species 25°C 37°C ATCC 32285 S. schenckii ϩϩϩϩ ϩϩϩ CBS 120339 S. brasiliensis ϩϩϩATCC 32286 S. schenckii ϩϩϩ ϩϩϩ IPEC 17307 S. brasiliensis ϩϩϩ ϩϩϩ IOC 1113 S. schenckii ϩϩϩ ϩϩ IPEC 17521 S. brasiliensis ϩϩϩϩ ϩϩϩ IPEC 23249 S. schenckii ϩϩ ϩϩ IPEC 17585 S. brasiliensis ϩϩϩϩ ϩϩϩ IPEC 23250 S. schenckii Ϫ ϩϩϩ IPEC 17608 S. brasiliensis ϩϩϩ ϩϩϩ IPEC 23251 S. schenckii ϩϩ IPEC 17692 S. brasiliensis ϩϩϩ ϩϩϩ IPEC 23252 S. schenckii ϩϩϩϩ ϩ IPEC 17920 S. brasiliensis ϩϩϩϩ ϩϩ IPEC 23253 S. schenckii ϩϩϩϩ ϩϩϩ IPEC 18202-3 S. brasiliensis ϩϩϩ ϩ IPEC 24372-1 S. schenckii ϩϩ ϩϩϩ IPEC 18782A S. brasiliensis ϩϩϩϩ ϩϩ IPEC 25374 S. schenckii ϩϩϩ ϩϩ IPEC 18782B S. brasiliensis ϩϩ ϩ IPEC 25457 S. schenckii ϩϩϩϩ ϩϩϩ IPEC 19777 S. brasiliensis ϩϩ ϩ IPEC 27722 S. schenckii ϩϩϩϩ ϩϩϩ IPEC 22493-1 S. brasiliensis ϩϩ ϩ Mel-14 S. schenckii ϩϩϩϩ ϩϩϩ ϩϩϩ ϩϩ IPEC 22582 S. brasiliensis a Ϫ, no pigment production; ϩ, low-level pigment production; ϩϩ, light brown IPEC 25303 S. brasiliensis ϩϩ ϩϩ pigment production; ϩϩϩ, dark brown pigment production; ϩϩϩϩ, black pigment IPEC 25541 S. brasiliensis ϩϩϩ ϩϩ production. IPEC 25644 S. brasiliensis ϩϩ ϩϩϩ IPEC 25712 S. brasiliensis ϩϩϩ ϩϩϩ IPEC 25758 S. brasiliensis ϩϩ IPEC 25853 S. brasiliensis ϩϩϩ ϩϩ the pathogenic fungal culture collection of the Laboratorio de Micologia/ IPEC 25976 S. brasiliensis ϩϩϩ ϩ IPEC/Fiocruz (WDCM 951). Strain IPEC 26449, previously characterized IPEC 26034 S. brasiliensis ϩϩϩ ϩϩ by our group as a high-melanin-producing strain (1), was further studied IPEC 26156 S. brasiliensis ϩϩϩto examine conditions for pigment production and its functions. In addi- IPEC 26449 S. brasiliensis ϩϩϩϩ ϩϩϩ tion, a DHN-melanin-deficient mutant strain, Mel-14, which has a mu- IPEC 27588 S. brasiliensis ϩϩ ϩ tation in the polyketide synthase gene (17), was also studied to determine IPEC 28831 S. brasiliensis ϩϩ ϩϩ if DHN-melanin was involved in pyomelanin formation. IPEC 29039 S. brasiliensis ϩϩϩ ϩϩϩ Media. Defined chemical medium (minimal medium) for Sporothrix IPEC 29787 S. brasiliensis ϩϩ ϩϩ sp. growth consisted of 15 mM glucose, 10 mM MgSO4, 29.4 mM IPEC 30650 S. brasiliensis ϩϩϩKH2PO4, 13 mM glycine, and 3.0 mM thiamine (pH 5.5). Minimal me- IPEC 30682-1 S. brasiliensis ϩϩϩϩ ϩϩ dium with L-tyrosine was made by adding 10 mM L-tyrosine to minimal IPEC 31047-1 S. brasiliensis ϩϩϩ ϩϩ medium. Agar plates were made by adding 20 g/liter to the corresponding IPEC 31515 S. brasiliensis ϩϩ ϩϩϩ medium. All chemicals used to prepare culture media were purchased IPEC 31676 S. brasiliensis ϩϩ ϩϩ from Sigma-Aldrich (St. Louis, MO). IPEC 32004 S. brasiliensis ϩϩϩ ϩϩϩ Screening for pigment production. The 73 Sporothrix sp. strains were IPEC 32406 S. brasiliensis ϩϩ ϩ inoculated onto minimal medium agar plates with and without L-ty- IPEC 33601 S. brasiliensis ϩϩϩ ϩϩ rosine. Cultures were incubated at both 25°C and 36°C and checked daily IPEC 33611 S. brasiliensis ϩϩϩϩ ϩϩϩ for pigment production over a 20-day period. Also, cultures at 36°C were IPEC 33704 S. brasiliensis ϩϩϩ ϩϩ checked for the presence of yeast cells, and they were used only if at least IPEC 33822 S. brasiliensis ϩϩϩ ϩϩ 90% yeast cells were observed. IPEC 33946 S. brasiliensis ϩϩϩϩ ϩϩϩϩ Influence of sulcotrione and glyphosate. In order to check if pigment IPEC 34079 S. brasiliensis ϩϩϩ ϩϩ production could be suppressed or abolished by melanin synthesis inhib- IPEC 34105 S. brasiliensis ϩ ϩϩϩ itors, sulcotrione [2-(2-chloro-4-mesylbenzoyl)cyclohexane-1,3-dione] IPEC 34196 S. brasiliensis ϩϩϩ ϩϩϩ and glyphosate [N-(phosphonomethyl)glycine] were added to minimal IPEC 34249 S. brasiliensis ϩϩϩ ϩϩ medium with L-tyrosine at concentrations of 16 and 100 mg/liter, respec- IPEC 34255 S. brasiliensis ϩϩ ϩϩ tively. Sulcotrione inhibits 4-hydroxyphenyl-pyruvate-dioxygenase (4- IPEC 34316 S. brasiliensis ϩϩϩ ϩϩ HPPD), impacting pyomelanin formation, whereas glyphosate blocks the IPEC 34328 S. brasiliensis ϩϩϩϩ ϩϩ shikimate acid pathway for aromatic amino acid synthesis. IPEC 34498 S. brasiliensis ϩϩϩ ϩϩ Autopolymerization. Three different flasks with 50 ml minimal me- ϫ 8 IPEC 34566 S. brasiliensis ϩϩϩ ϩϩ dium with L-tyrosine were prepared. A suspension of 1 10 conidia ␮ IPEC 34567 S. brasiliensis ϩϩϩfrom S. brasiliensis strain IPEC 26449 was prepared, and 100 l was inoc- ϫ 8 IPEC 34641 S. brasiliensis ϩϩϩ ϩϩϩ ulated into one flask. A second suspension of 1 10 conidia was boiled ␮ IPEC 34798 S. brasiliensis ϩϩϩϩ ϩϩ for 45 min, and 100 l was then added to a flask. An aliquot was also plated IPEC 34851 S. brasiliensis ϩϩ ϩϩ onto a potato dextrose agar plate to confirm fungal death. The third flask IPEC 34910 S. brasiliensis ϩϩϩwas left uninoculated. All flasks were incubated in the dark for 15 days at IPEC 34968 S. brasiliensis ϩϩϩ ϩϩ 25°C on a rotary incubator at 150 rpm. After incubation, flasks were IPEC 36062 S. brasiliensis ϩϩ ϩ visually and photometrically (absorbance at 340 nm) examined for the IPEC 41908-1 S. brasiliensis ϩϩϩ ϩϩ presence of pigment in the supernatants. IPEC 74H S. brasiliensis ϩϩϩϩ ϩϩ Pigment measurement. An aliquot of 0.5 ml of the supernatant was Ϫ IPEC 645H S. brasiliensis ϩϩϩ ϩϩ taken daily from each flask and stored at 20°C. After all samples were ϫ IPEC 27135 S. globosa ϩϩϩϩ ϩϩϩϩ collected, they were centrifuged (2,300 g), and supernatant absorbances ATCC 16345 S. schenckii ϩϩϩϩ ϩϩϩ at 340 nm were measured with an enzyme-linked immunosorbent assay (ELISA) plate reader (␮Quant; Bio-Tek).

8624 aem.asm.org Applied and Environmental Microbiology Pyomelanin Production by Sporothrix spp.

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).

Growth curves. Strains IPEC 26449 and Mel-14 were inoculated into Melanin ghosts. To generate melanin particles, strains IPEC 26449 flasks with 50 ml of minimal medium with L-tyrosine, without L-tyrosine, and Mel-14 were incubated for 14 days in 100 ml of either minimal me- and with both L-tyrosine and sulcotrione at an initial concentration of dium with L-tyrosine, minimal medium without L-tyrosine, or minimal 4 1.0 ϫ 10 cells/ml. Flasks were incubated at 25°C and 36°C on a rotary medium with L-tyrosine and sulcotrione at 30°C with shaking (150 rpm). incubator at 150 rpm. Serial dilutions of the different cultures were per- The fungal cells were collected, washed three times in phosphate-buffered formed daily during 14 days, 100 ␮l of each dilution was spread onto plate saline (PBS) (pH 7.2), and suspended in 1.0 M sorbitol–0.1 M sodium count agar (Bio-Rad), and cultures were incubated at 25°C. After 7 days of citrate (pH 5.5). Protoplasts were generated by incubating cells at 30°C in incubation, colonies were counted for CFU determinations. Additionally, 10 mg/ml of cell wall-lysing enzymes (from Trichoderma harzianum; daily aliquots of each culture were obtained and assessed for pigmenta- Sigma Chemical Co.) for1hatRT.Protoplasts were then collected by tion, as described above. centrifugation (2,300 ϫ g) for 10 min, washed with PBS, and incubated in Measurements of zeta potential. In order to calculate the charge of 4.0 M guanidine thiocyanate for1hatRTwith frequent vortexing. The the melanoid pigment, an extraction procedure was performed on 15- resulting material was washed again in PBS, collected by centrifugation, day-old cultures incubated with L-tyrosine, according to a protocol de- and boiled in 6.0 M HCl for1htohydrolyze cellular contaminants asso- scribed previously for pyomelanin purification from Aspergillus fumigatus ciated with melanin. The debris was collected by centrifugation and cultures (18). In brief, supernatants were filtered through 0.22-␮m mem- washed exhaustively with PBS. branes, acidified to pH 2.0 using 0.5 mol/liter HCl, and left overnight at Scanning electron microscopy. Chemically treated melanin ghosts room temperature (RT). The precipitated pigment was harvested by cen- from Sporothrix cultures were fixed overnight in a 4% glutaraldehyde trifugation (12,800 ϫ g) and resuspended in sterile distilled water. After purification, pigment was suspended in 10 mM KCl and analyzed by using solution in PBS. Particles were then transferred onto polylysine-coated a zeta-potential analyzer (ZetaPlus; Brookhaven Instruments Corp., coverslips and submitted to dehydration. Samples were mounted with Holtsville, NY). At least five different charge measurements were per- gold-palladium and viewed with a Quanta 50 scanning electron micro- formed for each sample. scope. EPR analysis. Pigments from strains IPEC 26449 and Mel-14, ex- Susceptibility to UV irradiation. Strains IPEC 26449 and Mel-14 tracted as described above, were analyzed by electron paramagnetic reso- were cultured with or without L-tyrosine at 25°C for 14 days. After incu- nance (EPR), using a Gunn diode as the microwave source. EPR spectra bation, cells were collected, washed three times, counted on a hemocy- 3 were obtained with a Varian E112 X-Band model spectrometer using a tometer, and diluted in PBS to achieve 5-ml suspensions of 1.0 ϫ 10 ␮ TE102 resonator and a liquid nitrogen finger Dewar vessel. The parameters conidia/ml. Baseline viability was determined by plating 100 l of each for EPR were as follows: modulation frequency of 9.07 GHz, modulation suspension onto Sabouraud dextrose agar (Difco) plates. Other plates also amplitude of 1.6 G, center field of 3,250.0 G, sweep width of 100.0 G, inoculated with 100 ␮l of each suspension were exposed to either 15, 30, microwave frequency of 9.1 GHz, microwave power of 1.0 mW, time 45, 60, 75, or 90 s of UV light (290 ␮W/cm2) irradiation. All plates were constant of 0.5 s, and temperature of 77 K. incubated at 25°C during 7 days. Survival rates were determined by count-

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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 quanti- ﬁcation 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, FIG 2 Pigment production by strain IPEC 26449 in the presence of L-tyrosine. but no pigmentation occurs in the absence of L-tyrosine or in the presence of (A) Heat-killed Sporothrix cells are unable to produce pigment after 20 days of sulcotrione. 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 incuba- with L-tyrosine. Control cells consisted of Sporothrix yeast cells cultured tion with or without L-tyrosine. 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 ing the number of colonies from irradiated cells relative to the number of count agar (Bio-Rad). Survival rates were calculated as described above. colonies from untreated controls. Statistics. All the experiments described here were repeated at least Susceptibility to oxidants. Fungal cells (1.0 ϫ 107 conidia) of strains three times to calculate means and standard deviations. There were no IPEC 26449 and Mel-14 were harvested after 14 days of incubation with or signiﬁcant variations between iterations of each experimental condition. without L-tyrosine, washed three times with PBS, and submitted to chem- Data were analyzed with Student’s t test by using SPSS 17.0 software. ically generated oxidants, as previously described (22). In brief, nitric oxide and reactive nitrogen intermediates were generated in a solution RESULTS 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 Production of melanoid pigment from L-tyrosine. After incuba- ferric ammonium sulfate, 61.8 ␮M hydrogen peroxide, and 1.0 mM epi- tion at 25°C in minimal medium with L-tyrosine, 72 of 73 Sporo- nephrine bitartrate. All chemicals were purchased from Sigma-Aldrich. thrix strains produced a dark brown pigment diffusible into the After 1, 2, 3, and4hofincubation at 36°C with the above-mentioned agar after 9 to 12 days of growth (Table 1). The assessment of the oxidants, Sporothrix cells were plated onto plate count agar (Bio-Rad) to plates at 20 days revealed differences in pigment intensity among determine viability, as measured by the number of viable colonies. Ali- strains (Fig. 1), as 8 strains (11.1% of positive strains) produced quots of untreated cells were also plated as controls. Survival rates were small amounts of pigment, similar to the plate shown in Fig. 1A; calculated by comparing the number of colonies subjected to various time 16 (22.2%) generated a light brown color on the agar surface, intervals of exposure to these reaction mixtures with the colony number of untreated cells. similar to the plate shown in Fig. 1B; 29 (40.3%) yielded a dark Resistance to amphotericin B. The MIC of amphotericin B for strain brown color on agar, as shown in Fig. 1C; and 19 (26.4%) strains IPEC 26449 was determined by broth microdilution in RPMI 1640 me- were heavily melanized, as represented in Fig. 1D. The 73 strains dium, as previously described (7). Yeast cells from this strain (2.5 ϫ 105 were able to synthesize pigment on culture media at 37°C, al- yeast cells) were harvested after 14 days of incubation in minimal medium though the level of pigment expression was lower than at 25°C for

8626 aem.asm.org Applied and Environmental Microbiology Pyomelanin Production by Sporothrix spp.

FIG 4 Continuous-wave (Cw) EPR spectrum from extracellular pigment extracted from S. schenckii Mel-14 (A) and S. brasiliensis IPEC 26449 (B). most strains. At 20 days of cultivation at 37°C, 11 strains (15.1%) at 340 nm that pigment is produced during the stationary phase of produced only small amounts of melanoid pigment, 36 (49.3%) of fungal growth in the cultures with minimal medium with tyrosine the cultures were light brown, 24 (32.9%) were dark brown, and and without sulcotrione (Fig. 3B). only 2 (2.7%) were black. Since most Sporothrix strains grown at EPR analysis. Both pigment samples extracted from wild-type 25°C produced more pigment, the mycelial Sporothrix form was strain IPEC 26449 and DHN-melanin mutant strain Mel-14 had used in subsequent experiments. EPR signals consistent with melanin (Fig. 4). The X-band EPR Conditions for pigment production. The pigmentation of spectrum of melanin is relatively featureless, but some variation in culture supernatants occurred only if viable fungal cells were in- line shape and/or line width can occur between samples, depend- oculated into minimal medium containing tyrosine (Fig. 2A). No ing on the structure of the melanin polymer. The two samples autopolymerization of L-tyrosine occurred in uninoculated ﬂasks. were very similar in both spectral features, roughly indicative of The formation of pigment was inhibited with 16 mg/liter of sul- the molecular structure, and intensity, indicative of the radical cotrione, while glyphosate was unable to completely inhibit pig- concentration. mentation, although the pigment isolated was brown rather than Zeta potential. Since melanins are pigments with negative black (Fig. 2B). Also, fungal cells grown in the presence of L-ty- charges, we used a zeta-potential analyzer to analyze pigments rosine accumulated this pigment such that it was visible in cell produced by both strains IPEC 26449 and Mel-14. We observed a pellets (Fig. 2C). charge of Ϫ25.54 Ϯ 5.93 mV on pigment derived from strain IPEC Growth curves performed by using minimal medium with or 26449 and a charge of Ϫ17.56 Ϯ 4.36 mV on the Mel-14 pigment. without L-tyrosine as well as minimal medium with L-tyrosine and Melanin ghost analysis. Melanin particles were obtained from sulcotrione indicated that both the pigment precursor and inhib- S. schenckii strains IPEC 26449 and Mel-14 grown in the presence itor had no effect on Sporothrix growth (Fig. 3A). Furthermore, we (Fig. 5A) or absence (data not shown) of L-tyrosine, which were observed by determinations of the optical density of supernatants subsequently treated with enzymes and with hot acid. Hyphae

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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). from both strains were completely solubilized under all tested addition of tyrosine did not protect Mel-14 from UV damage after culture conditions. Melanin ghosts from strain IPEC 26449 in- 30 min, Mel-14 cells grown with tyrosine were signiﬁcantly more cluded particles with the same shape and size of fungal conidia. capable of surviving UV irradiation than Mel-14 cells cultivated We observed mean diameters of 2.07 Ϯ 0.30 ␮m in the absence of without tyrosine (Fig. 6). In contrast, strain IPEC 26449 cells L-tyrosine and 2.43 Ϯ 0.42 ␮m in the presence of L-tyrosine. Ad- grown in the presence of tyrosine survived after exposure to up to ditionally, we also observed the presence of small dysmorphic 75 s of irradiation, although only 3% of cells survived this maximal black particles on all tested cultures. These particles were cylindri- dose. IPEC 26449 cells grown without tyrosine were all eradicated cal or spherical, with a mean diameter of 0.634 Ϯ 0.138 ␮m. Also, after more than 30 s of UV exposure. Although IPEC 26449 cells they were the main constituent of Mel-14 particles in the presence grown with or without tyrosine were similarly able to resist UV (Fig. 5B) or absence of L-tyrosine. irradiation for 15 s, cells incubated with tyrosine were signiﬁcantly Protection against UV light. Since strain Mel-14 is unable to more resistant to 30 s of UV exposure than cells grown without produce DHN-melanin, its susceptibility to UV irradiation was tyrosine. higher than that observed for strain IPEC 26449. Although the Protection against nitrogen-derived oxidants. Table 2 shows

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 Ͻ 0.05.

8628 aem.asm.org Applied and Environmental Microbiology Pyomelanin Production by Sporothrix spp.

TABLE 2 Percent survival rates of S. brasiliensis strain IPEC 26449 cells DISCUSSION cultured under several conditions submitted to nitrogen-derived Sporotrichosis is a mycotic infection with a broad range of clinical oxidants for different timesa manifestations, from localized cutaneous infection to severe dis- Mean % survival Ϯ SD seminated and often fatal disease (2). One of the factors that might Time of Minimal medium with play a role in the development of sporotrichosis is the differential incubation Minimal Minimal medium with 10 mM L-tyrosine and expression of virulence characteristics of the infective Sporothrix (h) medium 10 mM L-tyrosine 16 mg/liter sulcotrione strain (3). Melanins represent important virulence factors of this 1 22.90 Ϯ 6.39 64.56 Ϯ 36.30 23.65 Ϯ 10.80 fungus, offering protection against phagocytosis and killing by 2 5.55 Ϯ 1.47 21.41 Ϯ 7.54 7.01 Ϯ 1.27 human monocytes and murine macrophages (17). S. schenckii and 3 2.51 Ϯ 0.93 7.32 Ϯ 2.42 1.34 Ϯ 0.41 related species produce DHN- and L-DOPA-derived melanin (1, 4 0.51 Ϯ 0.12 2.78 Ϯ 1.95 0.50 Ϯ 0.39 12, 17). Here, we show, for the first time, that at least three species a Results are the means Ϯ standard deviations from at least three different observations. 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 the survival percentages of strain IPEC 26449 cells grown in the presence of L-tyrosine during the stationary phase of fungal presence or absence of L-tyrosine after various time intervals of growth, presents a negative charge, and yields a signal indicative of exposure to nitrogen-derived oxidants. Cells that produced the a stable free radical population. Moreover, its synthesis requires melanoid pigment in culture medium exhibited significantly metabolically active fungal cells, and production is blocked by higher survival rates than did cells grown on minimal medium sulcotrione. This compound is an inhibitor of the enzyme 4-hy- without pyomelanin precursors. At all time points, cells grown in droxyphenylpyruvate dioxygenase (19), which is involved in the the presence of L-tyrosine were more resistant to nitrogen-derived L-tyrosine degradation pathway (18). An S. schenckii strain with a free radicals than cells that were not. It is interesting that the ad- defective polyketide synthase gene and, therefore, which was un- dition of sulcotrione to culture medium with L-tyrosine led to the able to produce DHN-melanin (17) was also able to synthesize production of cells with susceptibility to nitrogen-derived oxi- pyomelanin. This result also supports that the formation of this dants similar to that of cells grown on minimal medium, demon- melanoid pigment is independent of the DHN-melanin metabolic strating the impact of the inhibition of the pyomelanin synthesis pathway. pathway. By the results of our experiments, it was not possible to deter- Protection against oxygen-derived oxidants. When S. brasil- mine if pyomelanin was produced by conidia or hyphae of Sporo- iensis strain IPEC 26449 was exposed to chemically generated ox- thrix spp. This pigment was produced after 9 days of incubation, ygen-derived oxidants for 1 h, there was a statistical difference and it is not possible to maintain a culture containing only conidia between the survival rates of cells cultured with and cells cultured during this time. We believe that both structures produce this without tyrosine (86.8% Ϯ 10.59% and 24.97% Ϯ 10.65%, re- pigment. Up to now, the exact fungal structures containing pyo- spectively; P ϭ 0.04). At 2, 3, or4hofincubation, no statistical melanin have not been reported, since this pigment was described differences in cell survival rates were observed. previously for only two other filamentous fungi, and it was not Resistance to amphotericin B. The MIC of amphotericin B for determined in which structure it was produced (18, 20). strain IPEC 26449 was 1.0 ␮g/ml. Therefore, cells were incubated Some fungi, such as Aspergillus oryzae, are able to produce with 0.5 and 2.0 ␮g/ml amphotericin B, and survival rates were eumelanin through the two-step oxidation of tyrosine to dopaqui- determined after 2, 6, and 24 h. Yeast cells grown with tyrosine none via L-DOPA (9). We demonstrated previously that S. were more resistant to amphotericin B killing than cells grown on schenckii can use L-DOPA for melanogenesis (1), but it is unlikely minimal medium alone (Table 3). After 2 and6hofincubation that the process of melanin synthesis described in the current with a concentration of 0.5 ␮g/ml amphotericin B and after2hof work is based on L-tyrosine conversion to L-DOPA. As we previ- incubation with 2.0 ␮g/ml, a statistical difference was observed for ously demonstrated, L-DOPA-derived melanin accumulates on survival rates between melanized and nonmelanized cells (P val- the fungal cell wall of conidia, yeast cells, and even S. schenckii ues of 0.0393, 0.038, and 0.0059, respectively). The resistance to hyphae. Scanning electron microscopy showed that hyphae cul- 2.0 ␮g/ml amphotericin B conferred by pyomelanin was even tured in the presence of L-tyrosine were completely dissolved after greater than resistance conferred by the L-DOPA-derived melanin denaturant and hot-acid treatments. Moreover, melanin ghosts of after2hofincubation (P ϭ 0.0113). Sporothrix cultures with L-tyrosine were similar to ghosts gener-

TABLE 3 Amphotericin B killing assay with S. brasiliensis strain IPEC 26449 cultured on media without or with different melanin synthesis inducersa Mean % survival Ϯ SD

Minimal medium Minimal medium with L-DOPA Minimal medium with L-tyrosine Time (h) 0.5 ␮g/ml AMB 2.0 ␮g/ml AMB 0.5 ␮g/ml AMB 2.0 ␮g/ml AMB 0.5 ␮g/ml AMB 2.0 ␮g/ml AMB 2 7.0 Ϯ 0.3 1.1 Ϯ 0.5 12.5 Ϯ 5.6 4.9 Ϯ 1.6 70.0 Ϯ 36.2 25.8 Ϯ 8.0 6 0.55 Ϯ 0.1 0 1.8 Ϯ 1.3 0 8.5 Ϯ 4.4 0 24 0.14 Ϯ 0.1 0 2.0 Ϯ 0.4 0 4.7 Ϯ 3.8 0 a Results are the means Ϯ standard deviations of percent survival rates from at least three different observations. AMB, amphotericin B.

December 2012 Volume 78 Number 24 aem.asm.org 8629 Almeida-Paes et al. ated on minimal medium. In addition, pigment formation was FAPERJ grant E-26/103.157/2011. J.D.N. was supported in part by NIH dependent on the viability of Sporothrix cells. In contrast, the pig- grant AI52733. ment formed in L-DOPA supernatant cultures is, at least in part, a REFERENCES result of L-DOPA autopolymerization and does not require met- 1. Almeida-Paes R, et al. 2009. Growth conditions influence melanization abolically active cells for its appearance (15). Thus, L-DOPA-de- of Brazilian clinical Sporothrix schenckii isolates. Microbes Infect. 11:554– rived pigment formation occurs outside fungal cells and deposits 562. along the surface of fungal cells, promoting their darkening. 2. Barros MB, Almeida-Paes R, Schubach AO. 2011. Sporothrix schenckii L-Tyrosine did not interfere with the growth of species of the and sporotrichosis. Clin. Microbiol. Rev. 24:633–654. 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