Melanin Protects Paracoccidioides brasiliensis from the Effects of Antimicrobial Photodynamic Inhibition and Antifungal Drugs

Abstract

c Paracoccidioidomycosis (PCM) is a public health concern in Latin America and South America that when not correctly treated can lead to patient death. In this study, the influence of melanin produced by Paracoccidioides spp. on the effects of treatment with antimicrobial photodynamic inhibition (aPI) and antifungal drugs was evaluated. aPI was performed using toluidine blue (TBO) as a photosensitizer and a 630-nm light-emitting diode (LED) light. The antifungals tested were itraconazole and amphotericin B. We evaluated the effects of each approach, aPI or antifungals, against nonmelanized and melanized yeast cells by performing susceptibility tests and by quantifying oxidative and nitrosative bursts during the experiments. aPI reduced nonmelanized cells by 3.0 log units and melanized cells by 1.3 log units. The results showed that melanization protects the fungal cell, probably by acting as a scavenger of nitric oxide and reactive oxygen species, but not of peroxynitrite. Melanin also increased the MICs of itraconazole and amphotericin B, and the drugs were fungicidal for nonmelanized and fungistatic for melanized yeast cells. Our study shows that melanin production by Paracoccidioides yeast cells serves a protective function during aPI and treatment with itraconazole and amphotericin B. The results suggest that melanin binds to the drugs, changing their antifungal activities, and also acts as a scavenger of reactive oxygen species and nitric oxide, but not of peroxynitrite, indicating that peroxynitrite is the main radical that is responsible for fungal death after aPI. P aracoccidioidomycosis (PCM) is a systemic disease endemic to Latin America and South America that can be fatal if not treated (1). In Brazil, PCM is a serious health problem and is the most important cause of death among the systemic mycoses in immunocompetent patients (1-3). The etiological agents of PCM, dimorphic fungi that present a filamentous form at 25°C and yeast forms at 37°C, belong to the Paracoccidioides species complex, which includes four known phylogenetic lineages, Pb01-like (or Paracoccidioides lutzii), S1, PS2, and PS3 (Paracoccidioides brasiliensis) (4-7). Thermal dimorphism is a survival strategy to resist the host environment (5, 7). It includes secretion of proteases and melanin synthesis as putative virulence factors contributing to the protection of yeast cells from environmental stressors (8).Currently, there has been much discussion about the role of melanin as a putative virulence factor of several fungal species, such as Histoplasma capsulatum, Sporothrix schenckii, and Cryptococcus neoformans (8-11). Melanin is a multifunctional polymer synthesized from 1,8-dihydroxynaphthalene (DHN) by the pentakide pathway or formed when cells grow in the presence of phenolic compounds, such as L-3,4-dihydroxyphenylalanine (L-DOPA) (12). Typically, it is a brown to black pigment formed through the activity of the phenoloxidase enzyme (laccase) on phenolic compounds (8, 13). Its influence on the biology of fungal cells is better ...

In vitro susceptibilities of Neoscytalidium spp. sequence types to antifungal agents and antimicrobial photodynamic treatment with phenothiazinium photosensitizers

“…Arthroconidia of N. dimidiatum isolates were more tolerant to APDT with all the four phenothiazinium PS than the hyaline isolates (see Table 5). The protective effect of melanins and melanin-like pigments against conventional antifungal drugs [61][62][63] and also to APDT [64][65][66][67] was observed previously both in conidia and in vegetative yeast cells. Pigmented conidia of Penicillium and Metarhizium are much more resistant to APDT with different PS than the colorless mutants [65][66].…”

Melanins Protect Sporothrix brasiliensis and Sporothrix schenckii from the Antifungal Effects of Terbinafine

“…One of the described functions for melanins produced by pathogenic fungi is the protection against antifungal drugs. This was observed for eumelanin producing fungi like Cryptococccus neoformans [ 22 , 31 ], Paracoccidioides brasiliensis [ 32 , 33 ], Histoplasma capsulatum [ 22 ], and Penicillium marneffei [ 34 ]. However, DHN-melanin producing strains of Fonsecaea monophora strains expressing melanin without precursors were not found to protect against antifungals [ 35 ] whereas DHN-melanin from Madurella mycetomatis and Aspergillus fumigatus were able to reduce the effects of ketoconazole and itraconazole [ 36 ].…”

ParacoccidioidesSpp.: Virulence Factors and Immune-Evasion Strategies

“…complex on the effects of treatment with antimicrobial photodynamic inhibition (aPI) and antifungal drugs was evaluated [132]. These authors demonstrated that aPI can reduce the viability of Paracoccidioides spp.…”