RESUMO
The biological chemistry of hydrogen sulfide (H2S) with physiologically important heme proteins is in the focus of redox biology research. In this study, we investigated the interactions of lactoperoxidase (LPO) with H2S in the presence and absence of molecular dioxygen (O2) or hydrogen peroxide (H2O2). Under anaerobic conditions, native LPO forms no heme-H2S complex upon sulfide exposure. However, under aerobic conditions or in the presence of H2O2 the formation of both ferrous and ferric sulfheme (sulfLPO) derivatives was observed based on the appearances of their characteristic optical absorptions at 638 nm and 727 nm, respectively. Interestingly, we demonstrate that LPO can catalytically oxidize H2S by H2O2 via intermediate formation of relatively short-lived ferrous and ferric sulfLPO derivatives. Pilot product analyses suggested that the turnover process generates oxidized sulfide species, which include sulfate S O 4 2 - and inorganic polysulfides ( H S x - ; x = 2-5). These results indicated that H2S can serve as a non-classical LPO substrate by inducing a reversible sulfheme-like modification of the heme porphyrin ring during turnover. Furthermore, electron paramagnetic resonance data suggest that H2S can act as a scavenger of H2O2 in the presence of LPO without detectable formation of any carbon-centered protein radical species, suggesting that H2S might be capable of protecting the enzyme from radical-mediated damage. We propose possible mechanisms, which explain our results as well as contrasting observations with other heme proteins, where either no sulfheme formation was observed or the generation of sulfheme derivatives provided a dead end for enzyme functions.
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Aim: Melanin has been linked to pathogenesis in several fungi. They often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata. Methods & materials:C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment with denaturant and hot acid. Results:C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Biophysical properties demonstrated that the pigment was melanin. Melanized C. glabrata cells were effectively protected from azoles and amphotericin B, incubation at 42°C and macrophage killing. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and enhances host survival.
Aim: Melanin is a pigment that can help fungi to cause disease. Fungi often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata, a yeast species that can cause human disease. Methods & materials:C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment to isolate melanin. Results:C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Several experiments demonstrated that the black pigment was melanin. Melanized C. glabrata cells were effectively protected from antifungal drugs, incubation at 42°C and killing by cells of the immune system. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and has enhanced survival in contact with immunologic defense cells.
Assuntos
Candida glabrata/patogenicidade , Candidíase/microbiologia , Melaninas/metabolismo , Anfotericina B/farmacologia , Animais , Antifúngicos/farmacologia , Azóis/farmacologia , Candida glabrata/efeitos dos fármacos , Candida glabrata/metabolismo , Candidíase/imunologia , Citocinas/metabolismo , Di-Hidroxifenilalanina/metabolismo , Farmacorresistência Fúngica , Macrófagos/imunologia , Camundongos , Viabilidade Microbiana , VirulênciaRESUMO
Melanization of Histoplasma capsulatum remains poorly described, particularly in regards to the forms of melanin produced. In the present study, 30 clinical and environmental H. capsulatum strains were grown in culture media with or without L-tyrosine under conditions that produced either mycelial or yeast forms. Mycelial cultures were not melanized under the studied conditions. However, all strains cultivated under yeast conditions produced a brownish to black soluble pigment compatible with pyomelanin when grew in presence of L-tyrosine. Sulcotrione inhibited pigment production in yeast cultures, strengthening the hyphothesis that H. capsulatum yeast forms produce pyomelanin. Since pyomelanin is produced by the fungal parasitic form, this pigment may be involved in H. capsulatum virulence.
Assuntos
Histoplasma/efeitos dos fármacos , Histoplasma/metabolismo , Tirosina/farmacologia , Animais , Meios de Cultura/química , Cicloexanonas/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Histoplasma/citologia , Humanos , Concentração de Íons de Hidrogênio , Melaninas/genética , Melaninas/metabolismo , Mesilatos/farmacologia , Pigmentos Biológicos/genética , Pigmentos Biológicos/metabolismo , VirulênciaRESUMO
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.