Biosynthesis and Functions of a Melanoid Pigment Produced by Species of the Sporothrix Complex in the Presence of L-Tyrosine 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 ca- tabolism. 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 me- dium 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 ab- sence 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 December 2012 Volume 78 Number 24 Applied and Environmental Microbiology p. 8623–8630 aem.asm.org 8623 Almeida-Paes et al. 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.