Mem Inst Oswaldo Cruz, Rio de Janeiro, 113(1) January 2018
Evaluation of melanin production by Sporothrix luriei
Fundação Oswaldo Cruz-Fiocruz, Instituto Nacional de Infectologia Evandro Chagas, Laboratório de Micologia, Rio de Janeiro, RJ, Brasil
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.
From 1898 to 2006,sporotrichosis was attributed to a single species Sporothrix schenckii (Kauffman 2006 8 ), or its variety, S. schenckii var. luriei (Padhye et al.1992). With the advance of polyphasic fungal taxonomy, the single so-calledS. schenckii species was separated into four species: S. schenckiisensu stricto, Sporothrix brasiliensis, Sporothrix globosa,and Sporothrix mexicana (Marimon et al. 2007 9 ). Moreover, S. schenckiivar. luriei was elevated to the species level, and it is now called Sporothrix luriei (Marimon et al. 2008 10 ).
The first documentedS. luriei infection occurred in 1956 (Ajello & Kaplan 1969). Threeother human sporotrichosis cases related to S. luriei have been reported(Mercadal-Peyrí et al. 1965 11 , Alberici et al. 1989 2 , Padhye et al. 1992 13 ).The main diagnostic feature in these cases was the presence of fungal eyeglasses-likecells (Padhye et al. 1992 13 ). A case in a dog, diagnosed through molecular methods,has also been reported (Oliveira et al. 2011 12 ).
Different fromother Sporothrix species, the absence of sessile dark-pigmented conidiahas been described for S. luriei (Marimon et al. 2008 10 ). Sporothrixpigmentation is the consequence of melanin deposition in the fungal cellwall (Almeida-Paes et al. 2017 3 ). Melanins are present in the three major pathogenicspecies of the genus: S. brasiliensis, S. schenckii, and S.globosa (Almeida-Paes et al. 2015 6 ), and they protect these species againstseveral stress conditions that they can face in the environment and during parasitism.Moreover, genomic data have revealed that melanin biosynthesis in S. schenckiiand S. brasiliensis is similar (Almeida-Paes et al. 2017 3 ). To thebest of our knowledge, there is no information about melanin in the S. mexicanacell wall. Since it was reported that S. mexicana produces dematiaceousconidia, as does S. schenckii and S. brasiliensis (Marimon etal. 2007), the black pigment observed in S. mexicana conidia is alsothought to be related to melanin deposited in the cell wall of this species.
The lack of melaninin S. luriei is a possible hypothesis for its low prevalence in humaninfections. Therefore, this study aimed to determine whether this species canproduce the three major types of fungal melanins (DHN-melanin, eumelanin, andpyomelanin) under well-established in vitro conditions used to studymelanisation in other Sporothrix species.
The S. lurieistrain 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 melaninproduction. Strains were maintained in the mycelial form in Sabouraud dextroseagar 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 KH2PO4,10 mM MgSO4, 13 mM glycine, 15 mM dextrose, 3 µM thiamine,pH 5.5). Experiments to detect eumelanin and pyomelanin were performed in minimalmedium 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 supplementthe media to evaluate the blockage of the DHN-melanin, eumelanin, and pyomelaninmetabolic pathways, respectively (Almeida-Paes et al. 2009 5 , 2012, Teixeira et al. 2010 14 ).
Both the mycelialand yeast forms of S. luriei and the control S. brasiliensis strainswere tested for melanin production at an initial inoculum concentration of 1× 106 conidia or yeasts/mL in the above described media. Fungiwere 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, cellswere harvested from the cultures described above and washed three times in phosphate-bufferedsaline (PBS) and suspended in 1 M sorbitol/0.1 M sodium citrate solution. Protoplastswere generated by incubating cells at 30ºC in a solution containing 10mg/mL cell wall-lysing enzymes (from Trichoderma harzianum; Sigma ChemicalCo.) for 1 h at room temperature. Protoplasts were washed with PBS and incubatedin 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 hydrochloricacid for 1 h. Supernatants of cultures supplemented with L-tyrosine were filteredthrough 0.22-μMmembranes, acidified to pH 2.0 using 0.5 M hydrochloric acid, and left for 24h at room temperature. The precipitated pyomelanin was harvested by centrifugation(12,800 × g) and resuspended in sterile distilled water.
As expected, thecontrol S. brasiliensis strain produced the three melanin types in bothmorphologies, 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 retainingthe shape and size of the conidia, hyphae, or yeast cells (Fig.1A). However, small dystrophic particles, similar to those observed whenthe DHN-melanin synthesis was blocked by tricyclazole in S. brasiliensisor S. schenckii, were observed in both fungal morphologies, evenin the absence of this inhibitor (Fig. 1B). The S. lurieiyeast form was also unable to produce pyomelanin under the in vitro conditionsemployed herein. However, supernatants of S. luriei mycelial culturessupplemented with L-tyrosine turned black after 10 days of growth at 25ºC(Fig. 1C). This pigment was acid resistant, and its synthesiswas specifically blocked by sulcotrione, thereby confirming this pigment tobe pyomelanin.
Since the S.luriei mycelial form produced pyomelanin, we hypothesised that this pigmentwould be involved in protection against harsh environmental conditions. To checkthis hypothesis, S. luriei conidia were harvested from cultures withand without L-tyrosine, adjusted to 1 × 108 conidia/mL, andsubmitted to either 15, 30, 45, 60, or 75 seconds of ultraviolet (UV) light(290 µW/cm2). In addition, conidia were incubated for 24 hat 38ºC and stored without cryoprotectants at 4ºC for six months toevaluate heat and cold protection, respectively. Six measurements were takenin each of these experiments. The results were analysed with the Mann-Whitneytest using GraphPad 5 software. As depicted in Fig. 2A, melanisedconidia submitted to UV light had more colony forming units than non-melanisedconidia (p < 0.05). Moreover, only melanised conidia survived UV exposureslonger than 60 s. Melanised S. luriei conidia were also more resistantto heat and cold (p < 0.05 for both experiments) than non-melanised cells(Fig. 2B).
The presence ofpyomelanin in the mycelial form of S. luriei may be a result of the betteradaptation of this species to environmental conditions, which agrees with theprotection that this pigment confers to the fungus against abiotic stress factors.The degree of protection against UV radiation observed in this study was similarto that observed with S. brasiliensis pyomelanin and other fungal melanintypes (Almeida-Paes et al. 2012 4 ).
Melanins were notfound in the S. luriei yeast cell wall. Its low incidence as an agentof sporotrichosis (Zhang et al. 2015) and the requirement of a high S. lurieiinoculum to achieve virulence in an experimental infection model using thesame strain as in the present study (Fernández-Silva et al. 2012 7 ) mayresult from the lack of melanin in the parasitic form of this species. Underthe same conditions that other Sporothrix species are able to produceDHN- and eumelanin (Almeida-Paes et al. 2009 5 ), only small acid-resistant particlesthat 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 otherpigments, such as γ-glutaminyl-3,4-dihydroxy-benzene-melanin,catechol melanin, p-aminophenol melanin, deoxybostrycoidin-melanin, andasp-melanin. The observed particles are not likely to be related to these uncommontypes of fungal melanins, since they are expressed in sexual reproduction structuresand/or require exogenous compounds for production (Toledo et al. 2017 15 ). Theblack acid-resistant structures of S. luriei are similar to those producedby S. schenckii and S. brasiliensis when the DHN-pathway is inhibitedwith tricyclazole (Almeida-Paes et al. 2009 5 ), suggesting that melanin synthesisin S. luriei is blocked by an unknown mechanism. These dysmorphic particlesresemble the melanosome-like structures observed in S. schenckii (Almeida-Paes et al. 2017 3 ). One hypothesis is that they are polymerisation products of accumulatedintermediary metabolites of a hindered melanin synthesis pathway. Since informationon the whole genome of S. luriei is unavailable, a search for mutationsor missing genes related to melanin synthesis was not possible.
Due to the paucityof available S. luriei strains (Marimon et al. 2008 10 ), we were able tostudy melanisation in only one strain. This was also a limitation in other importantstudies on S. luriei taxonomy and virulence (Marimon et al. 2008 10 , Oliveira et al. 2011 12 , Fernández-Silva et al. 2012 7 ). Future studies with more strainsare necessary to gain a better understanding of S. luriei cell biologyand pathogenesis.
To MaríliaMartins Nishikawa for providing the Sporothrix luriei strain used inthis work.
RA-P and RMZ-Oconceived the study and wrote the manuscript; ILRC and MHGF-C performed theexperiments; RA-P analysed the data. All authors read and approved the finalmanuscript.