Evaluation of melanin production by Sporothrix luriei

There is a paucity of studies on the cell biology of Sporothrix luriei, the less common of the pathogenic Sporothrix species worldwide. The production of DHN-melanin, eumelanin, and pyomelanin were evaluated on the mycelial and yeast forms of the S. luriei ATCC 18616 strain. The mycelial form of this species produced only pyomelanin, which protected the fungus against environmental stressors such as ultraviolet light, heat, and cold. The yeast form was unable to produce any of the tested melanin types. The lack of melanin in the parasitic form of S. luriei may be an explanation for its low frequency in human infections.

The first documented S. luriei infection occurred in 1956 (Ajello & Kaplan 1969). Three other human sporotrichosis cases related to S. luriei have been reported (Mercadal-Peyrí et al. 1965, Alberici et al. 1989, Padhye et al. 1992. The main diagnostic feature in these cases was the presence of fungal eyeglasses-like cells (Padhye et al. 1992). A case in a dog, diagnosed through molecular methods, has also been reported (Oliveira et al. 2011).
Different from other Sporothrix species, the absence of sessile dark-pigmented conidia has been described for S. luriei (Marimon et al. 2008). Sporothrix pigmentation is the consequence of melanin deposition in the fungal cell wall (Almeida-Paes et al. 2017). Melanins are present in the three major pathogenic species of the genus: S. brasiliensis, S. schenckii, and S. globosa (Almeida-Paes et al. 2015), and they protect these species against several stress conditions that they can face in the environment and during parasitism. Moreover, genomic data have revealed that melanin biosynthesis in S. schenckii and S. brasiliensis is similar (Almeida-Paes et al. 2017). To the best of our knowledge, there is no information about melanin in the S. mexicana cell wall. Since it was reported that S. mexicana produces dematiaceous conidia, as does S. schenckii and S. brasiliensis (Marimon et al. 2007), the black pigment observed in S. mexicana conidia is also thought to be related to melanin deposited in the cell wall of this species.
The lack of melanin in S. luriei is a possible hypothesis for its low prevalence in human infections. Therefore, this study aimed to determine whether this species can produce the three major types of fungal melanins (DHNmelanin, eumelanin, and pyomelanin) under well-established in vitro conditions used to study melanisation in other Sporothrix species.
The S. luriei strain INCQS 40253 (ATCC 18616 / CBS 937.72) was used in this study. The S. brasiliensis type strain (CBS 120339) was included as a control for melanin production. Strains were maintained in the mycelial form in Sabouraud dextrose agar at 25ºC and in the yeast form in brain heart infusion agar at 35ºC. Production of DHN-melanin was assessed in a minimal medium (29.4 mM KH 2 PO 4 , 10 mM MgSO 4 , 13 mM glycine, 15 mM dextrose, 3 µM thiamine, pH 5.5). Experiments to detect eumelanin and pyomelanin were performed in minimal medium supplemented with 1 mM L-dopa or 10 mM L-tyrosine, respectively. Tricyclazole (16 mg/L), glyphosate (100 mM), and sulcotrione (16 mg/L) were used to supplement the media to evaluate the blockage of the DHN-melanin, eumelanin, and pyomelanin metabolic pathways, respectively (Almeida-Paes et al. 2009, Teixeira et al. 2010. Both the mycelial and yeast forms of S. luriei and the control S. brasiliensis strains were tested for melanin production at an initial inoculum concentration of 1 × 10 6 conidia or yeasts/mL in the above described media. Fungi were incubated in the dark for 15 days at 25ºC (conidia) or 35ºC (yeasts) on a rotary incubator at 150 rpm. To detect DHN-melanin or eumelanin, cells were harvested from the cultures described above and washed three times in phosphate-buffered saline (PBS) and suspended in 1 M sorbitol/0.1 M sodium citrate solution. Protoplasts were generated by incubating cells at 30ºC in a solution containing 10 mg/mL cell wall-lysing enzymes (from Trichoderma harzianum; Sigma Chemi-cal Co.) for 1 h at room temperature. Protoplasts were washed with PBS and incubated in 4.0 M guanidine thiocyanate for 1 h at room temperature with frequent vortexing. The resulting material was washed three times in PBS and boiled in 6.0 M hydrochloric acid for 1 h. Supernatants of cultures supplemented with L-tyrosine were filtered through 0.22-μM membranes, acidified to pH 2.0 using 0.5 M hydrochloric acid, and left for 24 h at room temperature. The precipitated pyomelanin was harvested by centrifugation (12,800 × g) and resuspended in sterile distilled water.
As expected, the control S. brasiliensis strain produced the three melanin types in both morphologies, as described previously (Supplementary data, Figure). In contrast, the chemical treatment with enzymes, denaturant, and hot acid dissolved S. luriei mycelial and yeast cells without generating dark particles retaining the shape and size of the conidia, hyphae, or yeast cells (Fig. 1A). However, small dystrophic particles, similar to those observed when the DHN-melanin synthesis was blocked by tricyclazole in S. brasiliensis or S. schenckii, were observed in both fungal morphologies, even in the absence of this inhibitor (Fig. 1B). The S. luriei yeast form was also unable to produce pyomelanin under the in vitro conditions employed herein. However, supernatants of S. luriei mycelial cultures supplemented with L-tyrosine turned black after 10 days of growth at 25ºC (Fig. 1C). This pigment was acid resistant, and its synthesis was specifically blocked by sulcotrione, thereby confirming this pigment to be pyomelanin.
Since the S. luriei mycelial form produced pyomelanin, we hypothesised that this pigment would be involved in protection against harsh environmental conditions. To check this hypothesis, S. luriei conidia were harvested from cultures with and without L-tyrosine, adjusted to 1 × 10 8 conidia/mL, and submitted to either 15, 30, 45, 60, or 75 seconds of ultraviolet (UV) light (290 µW/cm 2 ). In addition, conidia were incubated for 24 h at 38ºC and stored without cryoprotectants at 4ºC for six months to evaluate heat and cold protection, respectively. Six measurements were taken in each of these experiments. The results were analysed with the Mann-Whitney test using GraphPad 5 software. As depicted in Fig. 2A, melanised conidia submitted to UV light had more colony forming units than non-melanised conidia (p < 0.05). Moreover, only melanised conidia survived UV exposures longer than 60 s. Melanised S. luriei conidia were also more resistant to heat and cold (p < 0.05 for both experiments) than non-melanised cells (Fig. 2B).
The presence of pyomelanin in the mycelial form of S. luriei may be a result of the better adaptation of this species to environmental conditions, which agrees with the protection that this pigment confers to the fungus  against abiotic stress factors. The degree of protection against UV radiation observed in this study was similar to that observed with S. brasiliensis pyomelanin and other fungal melanin types (Almeida-Paes et al. 2012).
Melanins were not found in the S. luriei yeast cell wall. Its low incidence as an agent of sporotrichosis (Zhang et al. 2015) and the requirement of a high S. luriei inoculum to achieve virulence in an experimental infection model using the same strain as in the present study (Fernández-Silva et al. 2012) may result from the lack of melanin in the parasitic form of this species. Under the same conditions that other Sporothrix species are able to produce DHN-and eumelanin (Almeida-Paes et al. 2009), only small acid-resistant particles that did not have the shape and size of S. luriei cells were observed. Besides the three melanin types studied in this work, some fungi produce other pigments, such as γ-glutaminyl-3,4-dihydroxy-benzene-melanin, catechol melanin, p-aminophenol melanin, deoxybostrycoidinmelanin, and asp-melanin. The observed particles are not likely to be related to these uncommon types of fungal melanins, since they are expressed in sexual reproduction structures and/or require exogenous compounds for production (Toledo et al. 2017). The black acid-resistant structures of S. luriei are similar to those produced by S. schenckii and S. brasiliensis when the DHN-pathway is inhibited with tricyclazole (Almeida-Paes et al. 2009), suggesting that melanin synthesis in S. luriei is blocked by an unknown mechanism. These dysmorphic particles resemble the melanosome-like structures observed in S. schenckii (Almeida-Paes et al. 2017). One hypothesis is that they are polymerisation products of accumulated intermediary metabolites of a hindered melanin synthesis pathway. Since information on the whole genome of S. luriei is unavailable, a search for mutations or missing genes related to melanin synthesis was not possible.
Due to the paucity of available S. luriei strains (Marimon et al. 2008), we were able to study melanisation in only one strain. This was also a limitation in other important studies on S. luriei taxonomy and virulence (Marimon et al. 2008, Oliveira et al. 2011, Fernández-Silva et al. 2012. Future studies with more strains are necessary to gain a better understanding of S. luriei cell biology and pathogenesis.