The primary conidia function as sympodial conidiogenous cells to form the secondary conidia. Fonsecaea is a pigmented dematiaceous , filamentous fungus found in rotten wood and soil. It has no known teleomorphic phase. As well as being a saprophyte in nature, it causes infections in humans. Cold-blooded animals living in swamps may also be infected.
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Metrics details. The pathogenic fungus Fonsecaea pedrosoi constitutively produces the pigment melanin, an important virulence factor in fungi. Melanin is incorporated in the cell wall structure and provides chemical and physical protection for the fungus. We evaluated the production of nitric oxide NO in macrophages, the oxidative burst and the inducible nitric oxide synthase i-NOS activity in interactions between activated murine macrophages and F.
Experiments were carried out with or without tricyclazole TC treatment, a selective inhibitor of the dihydroxynaphthalene DHN -melanin biosynthesis pathway in F. The paramagnetisms of melanin and the TC-melanin were analysed by electron spin resonance. Melanised F. Nitrite was not detected in the supernatant of macrophages incubated with melanised fungal cells. However, i-NOS expression was unaffected by the presence of either untreated control F.
In addition, the inhibition of the DHN-melanin pathway by TC improved the oxidative burst capability of the macrophages. The NO-trapping ability of F. Fonsecaea pedrosoi is a soil-borne dimorphic fungus and the major etiological agent of chromoblastomycosis, a chronic disease that can affect immunocompetent hosts. Infection usually occurs after exposure to the fungus via contaminated soil, splinters or sharp farm equipment, and results in long-term inflammation, suppurative granulomatous dermatitis and fibrosis [ 1 , 2 ].
The affected patients are typically low-income workers that engage in agricultural or manual labour in tropical and subtropical countries. Rarely, F. The management of diseases caused by F. Treatment depends on an early diagnosis and the use of systemic antifungal agents and local therapies, including the surgical removal of lesions. Even with treatment, relapses are common [ 4 , 5 ]. Melanins are present in both prokaryotic and eukaryotic organisms.
These ubiquitous dark compounds are formed by the oxidative polymerisation of phenolic or indolic compounds. Melanins have been extensively studied and characterised as negatively charged amorphous compounds with quinone groups, hydrophobic and insoluble in organic solvents [ 7 , 8 ]. Efforts to elucidate the structure of melanins are not yet conclusive due to limitations of the biochemical and biophysical analytical methods available.
Electron spin resonance ESR can characterise pigments, including melanin, and reveals that a typical melanin spectrum falls between and gauss [ 7 — 9 ]. Franzen et al. Inside melanosomes, melanin plays a role in the intracellular storage and regulation of calcium and iron ions [ 11 ]. The anti-phagocytic properties of F. In addition, conidia from F. Macrophages are found in granulomas of chromoblastomycosis lesions and may participate in the antigen presentation and innate immune response against F.
To contain the growth of pathogens, activated macrophages release oxygen and nitrogen reactive intermediates. NO released by the activated macrophages are fungicidal against Histoplasma capsulatum [ 15 ], Cryptococcus neoformans and Sporothrix schenkii [ 16 , 17 ].
The anti-oxidative properties of fungal melanins [ 18 , 19 ], their paramagnetism as revealed by ESR, and the melanin-iron a known magnetic or paramagnetic metal depending on its oxidation state association in F. The aims of the present work were the following: I to characterise the melanin of F. The ESR spectra of the control-melanin and TC-melanin present strikingly similar signals with a peak of gauss with respect to line width, line shape, and g value of 2.
Progressive microwave power saturation shows that the paramagnetic centres in these melanins do not saturate under the experimental conditions. In addition, these experiments reveal that the control-melanin has a higher spin relaxation rate than the TC-melanin Fig.
These observations suggest that the control-melanin is a more compact polymer than TC-melanin. Electron spin resonance of melanins of F. In B , progressive microwave power saturation experiments show that the paramagnetic centres of these melanins do not saturate under the experimental conditions, and also that the control-melanin sample black line, circles has a higher spin relaxation rate than the TC-melanin sample gray line, squares.
The macrophage oxidative burst was analysed by the NBT assay. The activity of oxidative compounds released by activated macrophages was visualised through the precipitation of NBT-formazan dark dye around the fungus in all melanin-deficient systems.
This precipitation occurs in response to superoxide molecules near the fungal cell wall Fig. Formazan precipitation was observed near S. However, activity of the oxidative compounds was not detected in control F. Light microscopy of the fungal interaction with activated murine macrophages. Light micrographs of activated murine macrophages after interaction in a ratio with: A TC-treated F. Fungal cells are marked with arrows.
Immunocytochemistry studies with anti-i-NOS enzymes revealed that these enzymes were active in all models tested: macrophages alone Fig. Such data indicate that i-NOS expression was not inhibited in any tested condition. Phase contrast microscopy A, C and E and confocal immunocytochemistry B, D and F images of activated murine macrophages alone A-B , activated murine macrophages with untreated F.
After 24 h of interaction in cultures with F. A similar reduction was observed when melanin extracted from control F. The growth of TC-treated F. Differences were more prominent at concentrations of 0. Fungal growth after exposure to H 2 O 2 and NO. Graphic representation of the growth of F. Fungal melanins are a hot topic among mycologists and have been extensively characterised as virulence factors.
Melanin pigments can protect pathogenic fungi from the mammalian host innate immune responses providing resistance: I to phagocytosis in C. ESR characterizations of melanins correspond to a peak signal on the spectra near gauss. These data are coherent among several fungi regardless of the specific melanin biosynthetic pathway or even if the fungus is pathogenic, including C.
The ESR characterisation of the samples revealed the presence of paramagnetic centres in both the control-melanin and TC-melanin; however, the control-melanin sample was of a higher intensity indicating that the number of unpaired electrons free radicals was higher.
Thus, these results indicate that the control-melanin is a polymer with more paramagnetic centres than the TC-melanin. In our experiments Fe III was used as a nutrient since we used ferric ammonium citrate as the medium substrate. Fungal melanins are able to reduce Fe III to Fe II , and this oxidative change prevents the formation of oxidative radicals when iron reacts with hydrogen peroxide, thus protecting the fungus from oxidative stress [ 28 ].
Cunha et al. At the time, the stronger binding was attributed to more anionic groups on the surface of the control and melanin's affinity to iron.
Experiments with melanin from C. Thus, it is possible that melanin maximises its antioxidant potential by reducing Fe III to Fe II , ensuring the balance of its redox chemical microenvironment and minimising the effect of oxidation of fundamental structures on fungal growth. The novel findings of this work led us to propose that the melanin of F.
This explains the higher growth rate of the control F. The progressive microwave power saturation ESR experiments, which varied the power of the microwaves on the magnetised sample, showed approximately a two times higher intensity in the control-melanin samples compared to the TC-melanin samples. According to our hypothesis, this suggests that control-melanin has more self-interaction sites as well as interaction sites for associated structures and therefore is more compact.
As indicated by Herbst et al. Hence, the measure of the progressive microwave power saturation curves for similar paramagnetic centres may provide an indirect indication of molecular arrangements. In this study, the profiles observed for control-melanin Fig. Such data are in agreement with the thinner cell wall of untreated F. Our data from interaction assays between fungi and activated murine macrophages suggest that melanin is involved in the protection of the fungus against NO.
Precipitation of formazan in the NBT assay Fig. These experiments also showed that the presence of control-melanin either free in the media or adhered to the fungal cell decreased NO levels as revealed by its direct correlation to the detected nitrite levels.
Further, TC-treatment of F. These data indicate that the inhibition of the melanin pathway, and consequently, the absence of melanin exposed on the cell wall of the fungus, could stimulate the production of NO by activated macrophages. Fungal glucans, the major component of the fungal cell wall, were previously described to activate macrophages which express glucan receptors and promote the synthesis and release of NO [ 31 ]. Nimrichter et al. We conclude that the increase of the macrophages' oxidative response after interaction with TC-treated F.
The inhibition of i-NOS expression by pathogens has been reported in other microorganisms, e. Bocca el al. However, our experiments suggest that the reduction of nitrite levels after the interaction of macrophages and control conidia was not due the inhibition of i-NOS expression, since its expression was detected in all tested conditions in immunofluorescence experiments.
We propose that F. Melanin participates in the storage of calcium and iron in F. In these experiments, our only variables were the F. Consequently, in these systems, no other mechanism can occur to inhibit i-NOS production. Our data suggest a protective role for F. This mechanism impairs the immune system and makes difficult the fungal clearance by macrophages and other phagocytes, which might be related to the recalcitrant nature of chromoblastomycosis lesions and the chronic nature of this disease.
Further investigation is necessary to define the structure of fungal melanins and describe putative chemical reactions that could occur in the infection environment, the products of such reactions and possible target sites for the development of new drugs.
A human isolate of F. All other reagents were acquired from Sigma-Aldrich Brazil , unless otherwise specified. After isolation, melanins 10 mg from F. The trituration was a necessary step in order to diminish the grain size, which otherwise could lead to preferential orientations and to the observation of artifacts in the ESR spectra. The pigments were analysed by ESR spectroscopy coupled to a spin-trapping analysis. The amplitude modulation was kept constant at 3.
Fonsecaea pedrosoi is the main etiologic agent of chromoblastomycosis CBM , one of the most prevalent subcutaneous mycosis in tropical and subtropical countries. CBM is a poorly characterized chronic infection that commonly starts after transcutaneous inoculation of conidia and saprophytic hyphae of F. Recently, we have shown that unlike conidia, hyphae and muriform cells the parasitic morphotype of F. We show here that F. Furthermore, F. Finally, we showed using a murine CBM model that F.
Fonsecaea pedrosoi is a fungal species in the family Herpotrichiellaceae , and the major causative agent of chromoblastomycosis. Fonsecaea is a genus of ascomycetous fungi affiliated with the family Herpotrichiellaceae. Fonsecaea pedrosoi occurs in soil and on plants and trees where it grows as a saprotroph. The closely related species F. Fonsecaea pedrosoi is one of several main causative agents of human chromoblastomycosis , a chronic fungal infection localized to skin and subcutaneous tissue.
Santos, Vanila F. Palmeira, Sonia Rozental, Lucimar F. Kneipp, Leonardo Nimrichter, Daniela S. Alviano, Marcio L. Rodrigues, Celuta S. Fonsecaea pedrosoi is the principal etiologic agent of chromoblastomycosis, a fungal disease whose pathogenic events are poorly understood. Treatment of the disease presents poor effectiveness and serious side effects.
Metrics details. The pathogenic fungus Fonsecaea pedrosoi constitutively produces the pigment melanin, an important virulence factor in fungi. Melanin is incorporated in the cell wall structure and provides chemical and physical protection for the fungus. We evaluated the production of nitric oxide NO in macrophages, the oxidative burst and the inducible nitric oxide synthase i-NOS activity in interactions between activated murine macrophages and F. Experiments were carried out with or without tricyclazole TC treatment, a selective inhibitor of the dihydroxynaphthalene DHN -melanin biosynthesis pathway in F. The paramagnetisms of melanin and the TC-melanin were analysed by electron spin resonance. Melanised F.