Characterization of Fonsecaea pedrosoi melanin.

The constituents of the melanin complex from mycelial forms of Fonsecaea pedrosoi were partially characterized. The pigment was mainly accumulated on large alkali-extractable, electron-dense cytoplasmic bodies (melanosomes) and, apparently, on the outer layer of the cell wall as external deposits within verrucose outgrowths. Using electron microscopy and Thiéry's periodate/thiosemicarbazide/silver proteinate staining method, glycogen-like particles were also detected at the periphery of the cells. Melanin constituents comprised aromatic and aliphatic/glycosidic structures with a predominance of the latter. Infrared spectra showed the presence of hydroxyl, carbonyl and carboxyl groups. The aliphatic/glycosidic moiety consisted of fatty acids and polysaccharides with protein, in a ratio protein/polysaccharide 1:15. Rhamnose, mannose, galactose and glucose (in the ratio 1:2:4:3.5) were the constituents of the polysaccharide. Lipid components included even-numbered, saturated and unsaturated fatty acids (in the ratio 2:1) ranging from C16 to C18. Palmitic and oleic acids were the prominent fatty acids. Aspartic and glutamic acids, leucine, glycine and alanine were the major amino acids. Non-pigmented cells of F. pedrosoi were studied for comparison with the pigmented forms: they did not accumulate acid-insoluble precursors of melanin.     

Isolation of Phialophora verrucosa and Fonsecaea pedrosoi from nature in Japan

Seventeen strains of Phialophora verrucosa and one strain of Fonsecaea pedrosoi were isolated from natural sources such as rotting wood, pine bark, pine logs or soil in Japan. These saprophytic isolates were compared morphologically, biologically and serologically with human isolates and their virulence for rats was studied. There were no distinct differences between the isolates from the two sources.     

Defense mechanisms of mice against Fonsecaea pedrosoi infection

Defense mechanisms of a host against Fonsecaea pedrosoi infection were studied histopathologically using athymic nude (nu/nu) mice of BALB/c background and their heterozygous (nu/+) littermates. Thirty male nu/nu and 30 nu/+ mice, weighing 16–19 g, were employed in this experiment. The nu/nu or nu/+ mice were divided into 3 groups consisting of 10 each. Furthermore, 4 nu/nu mice were supplemented to investigate effects of lymph node cell transfer. Subglobose cells of F. pedrosoi Tsuchiya strain were obtained from a culture in brain heart infusion glucose (1%) broth with reciprocal shaking at 37 °C for 17 days, and then 0.02, 0.1 and 0.5% cells suspensions were prepared. Each cell suspension was allotted to one group of the nu/nu or nu/+ mice. 0.1 ml of the cell suspension was inoculated into a tail vein, then one mouse from each group was sacrificed 1, 2, 4, 6, 8, 10, 14, 18, 21 and 25 days after inoculation. In both the nu/nu and nu/+ mice, the brain, kidneys and heart were affected severely with the strain in that order. Histopathologically, the defense mechanisms of the nu/+ mice against the fungus infection consisted chiefly of 2 steps: first, of non-immune phagocytosis by polymorphonuclear leucocytes (PMNs), and second, of granuloma formation induced by cell-mediated immunity. Those of the nu/nu mice consisted only of one step: phagocytosis by PMNs. A difference in susceptibility to the strain between the nu/nu and nu/+ mice changed according to the amount of the fungal cells inoculated. When inoculated with the 0.02% cell suspension, the resistance of the nu/nu mice was stronger than that of the nu/+ mice. In contrast, when inoculated with the 0.5% cell suspension, the former was affected more severely than the latter. There were little differences in the susceptibility to the strain between the nu/nu and nu/+ mice inoculated with the 0.1% cell suspension. These data seem to indicate that the phagocytic function of PMNs of the nu/nu mice was more active than that of the nu/+ mice, and the nu/nu mice inoculated with the 0.5% cells suspension (beyond the phagocytic capacity) lost resistance against the fungus infection. When the nu/nu mice were transferred with lymph node cells before inoculation of the strain, granulomata were formed to prevent hyphae from growing freely in the tissue.     

Characterization of an ecto-ATPase activity in Fonsecaea pedrosoi

In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5′-triphosphate (ATP) at a rate of 84.6 ± 11.3 nmol Pi h⁻¹ mg⁻¹ mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 ± 27.6 nmol Pi h⁻¹ mg⁻¹) in the presence of 5 mM MgCl₂, with values of V _max and apparent K _m for Mg-ATP²⁻corresponding to 541.9 ± 48.6 nmol Pi h⁻¹ mg⁻¹ cellular dry weight and 1.9 ± 0.2 mM, respectively. The Mg²⁺-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A₁ (V-ATPases)_, and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg²⁺-stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4′-diisothiocyanostylbene-2,2′-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P₂ purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.     

Granulomatous reactions induced by lipids extracted from Fonsecaea pedrosoi, Fonsecaea compactum, Cladosporium carrionii and Phialophora verrucosum

Granulomatous reactions induced by lipid extracts from the dermatophyte fungi Fonsecaea pedrosoi, Fonsecaea compactum, Cladosporium carrionii and Phialophora verrucosum, the causal organisms of chromoblastomycosis, were studied. Charcoal particles coated with the lipid extracts were prepared and injected intravenously into mice. Inflammation was characterized by an intense mononuclear cell infiltrate that lodged in the lung from 4 to 8 d after inoculation.     

Heat-shock response in Fonsecaea pedrosoi, a pathogenic fungus.

Using two-dimensional electrophoresis we have investigated the heat-shock response in a pathogenic fungus, Fonsecaea pedrosoi. Fungal cultures were transferred from 37 to 45 degrees C for either 30 or 90 min and then returned back to the initial temperature. Analysis of the total proteins resolved on two-dimensional gels indicated important changes in the accumulation of several peptides according to the duration of treatment and the temperature. The 30-min incubation at 45 degrees C resulted in the induction of several new proteins, whereas other proteins were either increased or decreased. These inductions and repressions of proteins (called heat-shock and heat-stroke proteins, respectively) were either specific to this time period or still present after a 90-min incubation. In addition, the 90-min incubation period led to the enhancement of several proteins, which were therefore called late heat-shock proteins to distinguish them from the early ones detected after 30 min. Finally, when cultures were shifted back to 37 degrees C most of the heat-shock proteins decreased or disappeared; in parallel, most of the heat-stroke proteins were reinduced at this time. These results are in good agreement with previous studies on the heat-shock response and provide additional evidence that this phenomenon is highly conserved among species.     

Cytokine Profile of a Self-Healing Fonsecaea pedrosoi Infection in Murine Model

Chromoblastomycosis is a chronic infectious disease of the skin and subcutaneous tissue. However, the host-defence response to this fungal infection has not been investigated thoroughly. This study was carried out to analyse the sequential events and the change of local cytokine release in a murine model infected with Fonsecaea pedrosoi in footpad. The anti-inflammatory Th2 cytokine IL-10 demonstrated an upward trend up to 7 days post infection followed by a steady decline. The titers of TNF-α (a pro-inflammatory Th1 cytokine) increased up to 7 days post infection followed by a relatively steady-state until full recovery. The anti-inflammatory cytokine IL-4 showed a similar pattern as TNF-α. The pro-inflammatory cytokine IFN-γ did not increased until 7 days post infection, while demonstrated an upward trend up to 30 days when the mice reached a full recovery from infection.     

Chromoblastomycosis due to Fonsecaea pedrosoi and F. monophora in Cuba

We report two cases of chromoblastomycosis due to Fonsecaea pedrosoi and F. monophora in otherwise healthy Cuban males. Direct microscopic examination of biopsies revealed muriform cells, the hallmark of chromoblastomycosis. The suspected agents were recovered in culture, identified on the basis of morphological criteria and confirmed by sequencing of the internal transcribed spacer regions of rDNA. Final treatment consisted of surgical excision. The patients were successfully cured since there was no relapse after a follow-up of more than a year. In vitro antifungal susceptibility testing of both isolates showed that itraconazole and posaconazole had potent activity. High MICs of amphotericin B (2 μg/ml), fluconazole (>64 μg/ml), anidulafungin (8 μg/ml) and caspofungin (8 μg/ml) were found.     

裴氏着色霉致着色芽生菌病1例并文献复习

报道1例裴氏着色霉致左手背及腕部着色芽生菌病,通过文献复习统计并分析该病在我国的流行病学及临床特点.患者男,48岁,左手背部红斑、疣状增生伴瘙痒10 a.皮损组织病理检查示真皮中下层有炎细胞及多核巨细胞,PAS染色见硬壳小体.经真菌培养和分子鉴定为裴氏着色霉.经伊曲康唑600 mg每日口服,治疗3个月后好转.     

Biology and pathogenesis of Fonsecaea pedrosoi, the major etiologic agent of chromoblastomycosis

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. The disease is epidemiologically important in several regions, which has stimulated studies focused on the biology and pathogenic potential of its major causative agent. In this review, we summarize the current knowledge on the biological aspects of F. pedrosoi, including cell differentiation and pathogenic mechanisms during the interaction of fungi with different hosts' elements.     

Morphometric and densitometric study of the biogenesis of electron-dense granules in Fonsecaea pedrosoi

Fonsecaea pedrosoi is a polymorphic pathogenic fungus, etiological agent of chromoblastomycosis, that synthesizes a melanin-like pigment. Although this pigment has been described as a component of the outer layers of the cell wall, electron-dense cytoplasmic bodies have also been visualized. In this work, we have correlated the appearance of intracellular electron-dense granules with the melanization process in F. pedrosoi. For this, conidial forms were grown under conditions where melanin was not synthesized. Afterwards, cells were incubated in Hank's medium supplemented with bovine fetal serum, at 37 degrees C, to stimulate the pigment production. The genesis of cytoplasmic bodies, with different stages of electron density, was demonstrated by transmission electron microscopy. The appearance of fungal acidic compartments, visualized by confocal laser scanning microscopy in cells stained with acridine orange, was time coincident with the formation of electron-dense granules observed by transmission electron microscopy. The quantification of granule numbers as well as morphometric and densitometric studies were performed.     

Fonsecaea pedrosoi: lipid composition and determination of susceptibility to amphotericin B.

Conidia and mycelial cells of Fonsecaea pedrosoi ATCC 46428 were obtained for analyses of lipid composition. Total lipids, phospholipids, sterols, and qualitative sterols and fatty acid composition were determined. A higher lipid content was detected in conidia than in mycelial cells of Fonsecaea pedrosoi, which could not be attributed to total sterols and phospholipids. In both forms of this fungus, ergosterol was the only sterol detected. The minimal inhibitory concentration of amphotericin B was lower for conidia than for mycelium.     

Delayed-type Hypersensitivity Response to Crude and Fractionated Antigens from Fonsecaea pedrosoi CMMI 1 Grown in Different Culture Media

Chromoblastomycosis is a subcutaneous fungal disease caused by dematiaceous fungi, especially by Fonsecaea pedrosoi, regarded as its major causative agent in Brazil. In recent years there has been a decline in the use of skin testing for delayed-type hypersensitivity (DTH) in epidemiological surveys of fungal infections, mainly because of the unpredictability of positive reactions and lack of specificity of the antigens used. The aim of the present study was to assess delayed-type skin tests in guinea pigs experimentally infected with F. pedrosoi using exoantigens prepared from two culture filtrates. Sixteen adult male guinea pigs were inoculated intratesticularly with fungal cells and submitted to sensitivity assays 4 weeks after inoculation. They received an intradermal injection with crude and fractionated antigens from Alviano’s and Smith’s cultures, and were assessed 24 and 48 h thereafter. Except for one animal, all of them had positive indurations after 48 h. There were no statistical differences between the measurements at 24 and 48 h for each exoantigen used, neither among the induration measurements at 48 h when different preparations were compared. Our results suggest that a delayed-type skin test using antigens produced in synthetic media may be useful for the assessment of primary exposure to chromoblastomycosis.     

Study of the conidial development and cleistothecium-like structure of some strains of Fonsecaea pedrosoi

The reproduction of twenty-seven strains of Fonsecaea pedrosoi was studied. The denticulate type (asexual reproduction) showed three morphological variations: medium-size, long and sessile forms. These forms can be used to characterize the different strains isolated especially for epidemiological purposes.Only one strain (MR 1335) isolated from a chromomycosis case in Martinique showed a cleistothecium-like structure. This formation was compared to that observed in Phialophora verrucosa, another agent of chromomycosis, and to the cleistothecium of Dictyotrichiella mansonii, the perfect state of Wangiella mansonii which is a saprophyte fungus.     

Inhibition of Nitric Oxide Production by Macrophages in Chromoblastomycosis: A Role for Fonsecaea pedrosoi Melanin

Chromoblastomycosis is a chronic and progressive deep mycosis that is usually found in tropical and subtropical areas. Fonsecaea pedrosoi is considered its most frequent etiologic agent and causes a typical granulomatous inflammatory response, whose degree reflects the immune status of the host. Since macrophages play a fundamental role in the control of the infection, this study aimed at investigating the production of oxygen reactive specimens, the phagocytic capacity and the production of nitric oxide (NO) by macrophages employing in vitro assays and an in vivo model of chromoblastomycosis. Our results demonstrated that, during the infection, peritoneal macrophages show an increased phagocytic capacity and H₂O₂ production, but also a reduced ability to produce NO. Moreover, F. pedrosoi stimulated H₂O₂ production in vitro but not the synthesis of NO. The incubation of IFNγ and LPS-stimulated macrophages with melanin, obtained from the fungus, inhibited NO production. Examination of the liver and spleen of infected animals, at day 30 or 60 following inoculation, showed a progressive increase in the number and size of granulomas, indicating that macrophages are properly mobilized and activated. Our data suggest that the inability of the host to clear F. pedrosoi, leading to a chronic disease, is due, at least in part, to the inhibition of NO synthesis by macrophages by fungus-produced melanin.     

Scanning electron microscopic observation of the parasitic forms of Fonsecaea pedrosoi in a human skin lesion

The parasitic form of Fonsecaea pedrosoi in the superficial hyperkeratotic layer of the skin, in a patient with chromoblastomycosis, was observed by a scanning electron microscope using some technical improvements. The fungus elements were comprised of branched septate hyphae with spherical cells and sclerotic cells. It was observed that the sclerotic cells, divided into two or three parts, formed a germ tube.     

Induction of yeast-like cells in a strain of Fonsecaea pedrosoi cultured under very acidic conditions

Yeast-like cells with a conidiogenesis of the annellidic type were obtained by culturing a strain of Fonsecaea pedrosoi, the principal agent of chromomycosis, under very acidic conditions (_pH 2.5). These annellides resemble those of such pathogenic black yeasts as Exophiala jeanselmei.