A monoclonal antibody to glucosylceramide inhibits the growth of Fonsecaea pedrosoi and enhances the antifungal action of mouse macrophages

Fungal glucosylceramides (GlcCer) are conserved lipid components in a large variety of pathogenic and non-pathogenic fungal species, but their biological functions are still unclear. Recent studies demonstrate that GlcCer are immunologically active components inducing the production of antifungal antibodies. In this work, a major GlcCer was purified and characterized from mycelial forms of Fonsecaea pedrosoi, the most frequent causative agent of chromoblastomycosis. As determined by fast atom bombardment mass spectrometry (FAB-MS) analysis, the purified molecule was identified as the conserved structure N-2'-hydroxyhexadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. A monoclonal antibody (Mab) against this structure was used in indirect immunofluorescence with the different morphological stages of F. pedrosoi. Only the surface of young dividing cells was recognized by the antibody. Treatment of F. pedrosoi conidia with the Mab against GlcCer resulted in a clear reduction in fungal growth. In addition, the Mab also enhanced phagocytosis and killing of F. pedrosoi by murine cells. These results suggest that, in F. pedrosoi, GlcCer seem to be cell wall components targeted by antifungal antibodies that directly inhibit fungal development and also enhance macrophage function, supporting the use of monoclonal antibodies to GlcCer as potential tools in antifungal immunotherapy.     

Comparison of Fonsecaea pedrosoi sclerotic cells obtained in vivo and in vitro: ultrastructure and antigenicity

The parasitic form of Fonsecaea pedrosoi from the hyperkeratotic layer of the skin was obtained from four patients with chromoblastomycosis. Primary cultures containing hyphae and conidia were successfully converted into sclerotic cells in the presence of 800 microM propranolol and low pH as described before. The morphology of sclerotic cells of F. pedrosoi obtained in vivo and in vitro was analyzed by light and electron microscopy. Their antigenicity was also compared by immunofluorescence microscopy and ELISA assays, using serum samples from untreated patients infected with F. pedrosoi. Due to the similarity of the sclerotic cells obtained in vivo and in vitro, the latter can be more adequately in studies of host-parasite interactions in chromoblastomycosis.     

Dimorphism-associated variations in the lipid composition of Candida albicans

Yeast and mycelial forms of Candida albicans ATCC 10231, growing together in 12 h and in 96 h cultures, were separated and their lipids were extracted and characterized. The total lipid content of the yeast forms was always lower than that of the mycelial forms. In 12 h cultures the lipids from the two morphological forms consisted mainly of polar compounds, viz, phospholipids and glycolipids. In 96 h cultures both the yeast and mycelial forms accumulated substantial amounts of apolar compounds, mainly steryl esters and triacylglycerols. The mycelial forms were more active than the yeast forms in this respect. Major differences in the lipid composition between the two morphological forms involved the contents of sterols and complex lipids that contain sterols. As a rule, the yeast lipids contained much larger proportions of free sterols than the mycelial lipids. However, the mycelial lipids contained several times more sterols than the yeast forms but bound as steryl glycosides, esterified steryl glycosides and steryl esters. Steryl glycosides and esterified steryl glycosides occurred in yeast lipids only in traces, if at all. The major steryl glycoside in the mycelial forms was unequivocally identified as cholesteryl mannoside. At both phases of growth the apolar and polar lipid fractions from the mycelial forms contained higher levels of polyunsaturated fatty acids (18:2 and 18:3) but lower levels of oleic acid (18:1) than the corresponding fractions from the yeast forms. The lipid content and composition of 12 h and 96 h yeast and mycelial forms of C. albicans KCCC 14172, a clinical isolate, were almost identical with those of C. albicans ATCC 10231.     

Carbohydrate and lipid components of hyphae and conidia of human pathogenFonsecaea pedrosoi

The carbohydrate and lipid components of mycelium and conidia ofFonsecaea pedrosoi (Brumpt) were analysed by paper, thin-layer and gas-chromatography, mass spectrometry and ultraviolet spectroscopy. Glucose, mannose, galactofuranose, rhamnose and glucosamine were polysaccharide components identified inF. pedrosoi. Significant changes in the carbohydrate pattern occurred during the conversion of mycelium into conidia. Rhamnose was predominant in conidia whereas galactose was prominent in mycelium. Palmitic, stearic, oleic, linoleic, and arachidonic acids were the fatty acids identified in the total lipid fraction. Palmitic and oleic acids were major fatty acids. Marked alterations in the fatty acid constituents were observed between the cell types ofF. pedrosoi. Arachidonic acid was detected only in conidia and linoleic acid was preferentially identified in mycelium. Differences in the sterol composition was also associated with morphogenesis inF. pedrosoi. Two main sterols, ergosterol and another less polar sterol, not fully characterized, were found in mycelium whereas in conidia only the latter sterol was present.     

Phagocytosis of Fonsecaea pedrosoi conidia, but not sclerotic cells caused by Langerhans cells, inhibits CD40 and B7-2 expression

Fonsecaea pedrosoi is the major etiological agent of chromoblastomycosis, a chronic, suppurative, granulomatous mycosis usually confined to skin and subcutaneous tissues, presenting a worldwide distribution. The host defense mechanisms in chromoblastomycosis have not been extensively investigated. Langerhans cells (LC) are bone-marrow-derived, dendritic antigen-presenting cells of the epidermis, which constitutively express major histocompatibility complex (MHC) class II, and comprise 1-3% of total epidermal cells. LC are localized in suprabasal layers of the epidermis and in mucosa, where they play important roles in skin immune responses. The purpose of the present study was to evaluate the interaction of F. pedrosoi conidia or sclerotic cells with LC purified from BALB/c mice skin. We demonstrate here that LC phagocytose F. pedrosoi conidia but not sclerotic cells in the first 3 h of interaction, inhibiting hyphae formation during 12-hour coculture from both forms, internalized or not. Also, LC maturation, analyzed using CD40 and B7-2 expression, was inhibited by conidia, but not by sclerotic cells, indicating an important innate immunity function of LC against F. pedrosoi infection in these mice.     

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.     

Beneficial effects of HIV peptidase inhibitors on Fonsecaea pedrosoi: promising compounds to arrest key fungal biological processes and virulence

BACKGROUND: Fonsecaea pedrosoi is the principal etiologic agent of chromoblastomycosis, a fungal disease whose pathogenic events are poorly understood. Current therapy for chromoblastomycosis is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the fact that endemic countries and regions are economically poor. PURPOSE AND PRINCIPAL FINDINGS: In the present work, we have investigated the effect of human immunodeficiency virus (HIV) peptidase inhibitors (PIs) on the F. pedrosoi conidial secreted peptidase, growth, ultrastructure and interaction with different mammalian cells. All the PIs impaired the acidic conidial-derived peptidase activity in a dose-dependent fashion, in which nelfinavir produced the best inhibitory effect. F. pedrosoi growth was also significantly reduced upon exposure to PIs, especially nelfinavir and saquinavir. PIs treatment caused profound changes in the conidial ultrastructure as shown by transmission electron microscopy, including invaginations in the cytoplasmic membrane, disorder and detachment of the cell wall, enlargement of fungi cytoplasmic vacuoles, and abnormal cell division. The synergistic action on growth ability between nelfinavir and amphotericin B, when both were used at sub-inhibitory concentrations, was also observed. PIs reduced the adhesion and endocytic indexes during the interaction between conidia and epithelial cells (CHO), fibroblasts or macrophages, in a cell type-dependent manner. Moreover, PIs interfered with the conidia into mycelia transformation when in contact with CHO and with the susceptibility killing by macrophage cells. CONCLUSIONS/SIGNIFICANCE: Overall, by providing the first evidence that HIV PIs directly affects F. pedrosoi development and virulence, these data add new insights on the wide-spectrum efficacy of HIV PIs, further arguing for the potential chemotherapeutic targets for aspartyl-type peptidase produced by this human pathogen.     

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.     

Sporangiospore lipids of the mycelial fungus Mucor ramannianus incapable of dimorphic growth

Members of the species Mucor ramannianus are believed to be monomorphic. They grow only as a mycelium and are not capable of growth as budding cells, i.e., of dimorphic growth. In our study, we investigated the lipid composition of M. ramannianus sporangiospores, which retained the capability of initiating mycelial growth in the course of long-term cultivation of the spore-forming mycelium. It was demonstrated that sporangiospores contained high concentrations of triacylglycerides (TAG) in their reserve lipids and high concentrations of phosphatidylcholine (PC) in their membrane lipids; low concentrations of methylated ergosterol precursors were detected among sterols. On the basis of the data presented, in order to evaluate the potential of mucor fungi for yeastlike growth, it has been suggested to analyze the qualitative and quantitative characteristics of their sporangiospore lipids and to consider the following criteria as the criteria of sporangiospore capacity for giving rise to yeastlike growth upon spore germination: (1) the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio; (2) the level of ergosterol and the ratio between the methylated and demethylated sterols; as well as (3) the ratios between phospholipids and glycolipids (PL/GL) and (4) between etherified and free sterols (ES/FS).     

裴氏着色霉的扫描电镜研究

用扫描电镜研究了引起着色芽生菌病(chromoblastomycosis)的致病菌之一——裴氏着色霉(Fonsecaea pedrosoi)孢子的个体发生,除一级产孢细胞外,还有二级产孢细胞,喙枝孢型的分生孢子梗也可发育成为喙枝孢型的二级产孢细胞。     

Metabolic characteristics and lipid composition of yeastlike cells and mycelium of Mucor circinelloides var. lusitanicus INMI grown at a high glucose content in the medium

It is shown that the fungus Mucor circinelloides var. lusitanicus INMI grown under aerobic conditions in a medium with a high glucose concentration (20%) is capable of both yeastlike and mycelial growth. In the mycelium, the activity of NAD-dependent isocitrate dehydrogenase was more than twice as high as in yeastlike cells, whereas the isocitrate lyase activity was lower. A number of significant differences were found in the lipid composition of the cells of two different morphological variants. Yeastlike cells contained more polar lipids and free fatty acids and less principal reserve lipids (triacylglycerides) than mycelial cells; the content of gamma-linolenic acid and the degree of lipid unsaturation were significantly lower in these cells than in the mycelium. In yeastlike cells, glycolipids composed the bulk of polar lipids; the proportion of phospholipids (primarily phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) was lower. The relationship between cellular metabolism and the lipid composition of fungal cells of different morphotypes grown at high concentrations of glucose, one of the main inducers of dimorphic growth, is discussed.     

Sporangiospore lipids of the mycelial fungus <Emphasis Type="Italic">Mucor ramannianus</Emphasis> incapable of dimorphic growth

Members of the species Mucor ramannianus are believed to be monomorphic. They grow only as a mycelium and are not capable of growth as budding cells, i.e., of dimorphic growth. In our study, we investigated the lipid composition of M. ramannianus sporangiospores, which retained the capability of initiating mycelial growth in the course of long-term cultivation of the spore-forming mycelium. It was demonstrated that sporangiospores contained high concentrations of triacylglycerides (TAG) in their reserve lipids and high concentrations of phosphatidylcholine (PC) in their membrane lipids; low concentrations of methylated ergosterol precursors were detected among sterols. On the basis of the data presented, in order to evaluate the potential of mucor fungi for yeastlike growth, it has been suggested to analyze the qualitative and quantitative characteristics of their sporangiospore lipids and to consider the following criteria as the criteria of sporangiospore capacity for giving rise to yeastlike growth upon spore germination: (1) the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio; (2) the level of ergosterol and the ratio between the methylated and demethylated sterols; as well as (3) the ratios between phospholipids and glycolipids (PL/GL) and (4) between etherified and free sterols (ES/FS).     

Lipid Composition of Citrus Leaves from Plants Resistant and Susceptible to Citrus Bacterial Canker

The contents and composition of lipids in citrus leaves in relation to their general resistance to infection by strains of Xanthomonas campestris pv. citri (Xcc) were determined. The composition and contents of total polar lipids and phospholipids and the degree of fatty acid unsaturation were significantly different between resistant and susceptible species. Leaves from resistant plants had less phospholipids, but more free sterols than those from susceptible plants. The predominant fatty acids in the phospholipids were palmitic (16:0), linoleic (18:2) and α-linolenic acid (18:3). The degree of fatty acid unsaturation was higher in susceptible plants than in resistant plants. Major phospholipids in citrus leaves were phosphatidylchloline (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI). β-Sitosterol, campesterol and lanosterol were major sterols in the leaves of citrus plants with resistant species having a higher ratio of free sterols to total phospholipids than susceptible species. Differences in lipid metabolism may contribute to differences in Xcc-resistance of citrus leaves.     

Lipid composition and effect of amphotericin B on yeast cells of Paracoccidioides brasiliensis

Yeast cells of Paracoccidioides brasiliensis strain SN, were obtained for analysis of lipid composition. Total lipids, phospholipids, sterols, and qualitative sterols and fatty acid composition were determined. Such analysis were made on cells cultured in the presence or absence of amphotericin B and on non proliferating cell suspensions exposed to the antibiotic. Marked alterations in lipid contents were observed in this different conditions. The major alterations were the reduction of total lipids, sterols, and palmitoleic acid in both, proliferating and non proliferating antibiotic exposed cells. The effect of amphotericin B was evaluated also in terms of viability and release of intracellular substances, at different times of exposure. The minimal inhibitory concentration (MIC) determined for that strain of this fungus was 0.2 g/mL.     

Lipid composition of the entomopathogenic fungus Nomuraea rileyi

The total lipid content, neutral and polar lipid composition, and fatty acid profiles were determined for the different developmental stages of the entomopathogenic fungus, Nomuraea rileyi. In general, the lipid composition of N. rileyi was found to be very similar to that of other Fungi Imperfecti. Triacylglycerides and sterols were the major neutral lipids and phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were the major polar lipids. Phosphatidylcholine, resolved in conidial and mycelial samples, was not detected in hyphal body samples. The major fatty acids were palmitic, oleic, and linoleic. Stearic acid was detected as a minor component of hyphal body samples. During conidiogenesis the overall lipid composition was altered in (1) a reduction in total lipid content, (2) an increase in the polar/neutral lipid ratio, and (3) an increase in the degree of fatty acid unsaturation.     

Lipid Composition of Plasma Membranes and Tonoplasts Isolated from Etiolated Seedlings of Mung Bean (Vigna radiata L.)

The lipid composition of plasma membranes and tonoplasts from etiolated mung bean hypocotyls was examined in detail. Phospholipids, sterols, and ceramide monohexoside(s) were the major lipid classes in both membranes. The content of phospholipids on a protein basis was higher in the tonoplast, but the content of total sterols was similar in both membranes. Accordingly, the sterol to phospholipid molar ratio in the plasma membrane was higher than that of the tonoplast. Phosphatidylethanolamine and phosphatidylcholine comprised the major phospholipids in both membranes. Phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol were identified as minor phospholipid components. The content of phosphatidylinositol and phosphatidylglycerol was relatively high in the tonoplast, comprising 11 and 5% of the total phospholipids, respectively. Although special care was taken against the degradative action of phospholipase D and phosphatidic acid phosphatase during the isolation of these membranes, by adding EDTA, EGTA, KF, choline, and ethanolamine to the homogenizing medium, significant amounts of phosphatidic acid, about 15% of the total phospholipids, were detected in the plasma membrane. On the other hand, the content of phosphatidic acid in tonoplasts and other membrane fractions was very low. This fact may indicate that high levels of phosphatidic acid occur naturally in plasma membranes. Phosphatidylglycerol in both membranes and phosphatidylinositol in the tonoplast contained high levels of palmitic acid, which comprised more than 50% of the total fatty acids. Significant differences were observed in the sterol compositions of plasma membranes and tonoplasts. More than 90% of the sterols in the plasma membrane were unesterified, while the tonoplast was enriched in glycosylated sterols, especially acylated sterylglycosides. Ceramide monohexoside was found to be specifically located in these membranes, in particular, in the tonoplast, in which it comprised nearly 17% of the total lipids.     

Metabolic characteristics and lipid composition of yeastlike cells and mycelium of <Emphasis Type="Italic">Mucor circinelloides</Emphasis> var. <Emphasis Type="Italic">lusitanicus</Emphasis> INMI grown at a high glucose content in the medium

It is shown that the fungus Mucor circinelloides var. lusitanicus INMI grown under aerobic conditions in a medium with a high glucose concentration (20%) is capable of both yeastlike and mycelial growth. In the mycelium, the activity of NAD-dependent isocitrate dehydrogenase was more than twice as high as in yeastlike cells, whereas the isocitrate lyase activity was lower. A number of significant differences were found in the lipid composition of the cells of two different morphological variants. Yeastlike cells contained more polar lipids and free fatty acids and less principal reserve lipids (triacylglycerides) than mycelial cells; the content of γ-linolenic acid and the degree of lipid unsaturation were significantly lower in these cells than in the mycelium. In yeastlike cells, glycolipids composed the bulk of polar lipids; the proportion of phospholipids (primarily phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) was lower. The relationship between cellular metabolism and the lipid composition of fungal cells of different morphotypes grown at high concentrations of glucose, one of the main inducers of dimorphic growth, is discussed.     

The major chromoblastomycosis fungal pathogen, Fonsecaea pedrosoi, extracellularly releases proteolytic enzymes whose expression is modulated by culture medium composition: implications on the fungal development and cleavage of key's host structures

We investigated the possible secretion of peptidases by F. pedrosoi, when conidial cells were cultured in two distinct media. Aspartyl proteolytic activity was detected on the Czapeck-Dox-derived supernatant, which was blocked by pepstatin, and only active in extremely acidic conditions. The supernatant obtained after conidia growth in Kauffman medium presented metallopeptidase activity, which was active over a broad pH range and sensitive to 1,10-phenanthroline and EGTA. Additionally, both culture supernatants were able to cleave a wide range of proteinaceous substrates, including important human serum proteins (e.g. albumin and immunoglobulin G) and extracellular matrix components (e.g. fibronectin and laminin). As peptidases participate in different cellular metabolic pathways, we also tested the influence of proteolytic inhibitors on the F. pedrosoi conidia development in vitro. The metallopeptidase inhibitors, 1,10-phenanthroline, EGTA and EDTA, strongly abrogated the growth of conidial forms by approximately 95%, 85% and 60%, respectively. Moreover, 1,10-phenanthroline blocked the differentiation process from conidia to mycelia, an essential step during the F. pedrosoi life cycle. Phenylmethanesulfonyl fluoride, a serine peptidase inhibitor, slightly reduced the conidial growth, whereas proteolytic inhibitors of cysteine (E-64) and aspartic (pepstatin) type peptidases did not alter conidial developmental behavior. In summary, our results showed for the first time the expression of extracellular proteolytic activity by F. pedrosoi conidial cells.     

The role of sterols in morphogenetic processes and dimorphism in fungi

The review considers the fundamental biological problem of fungal dimorphism as an adaptive reaction to adverse impacts. Primary attention is paid to sterols, phospholipids, storage lipids, and fatty acids. The structural and biological functions of sterols are considered, as well as their role in membrane stabilization under stress and their relation to morphogenetic processes in mycelial fungi, of which many are pathogenic. Data on the biosynthesis of the main fungal sterol, ergosterol, are presented, as well as data on the inhibitors of this process and on the mutants deficient in its particular stages. Ergosterol biosynthesis is also considered in terms of its relation to the composition of the fungal cell wall, which is the cell shape-determining structure, and to the intensity of chitin synthesis, a process in which azole derivatives play a role. Data obtained by the authors are presented that show the role of changes in the composition of sterols, phospholipids, storage lipids, and unsaturated fatty acids of resting cells in the induction of yeastlike growth in mucoraceous fungi.     

The role of sterols in morphogenetic processes and dimorphism in fungi

The review considers the fundamental biological problem of fungal dimorphism as an adaptive reaction to adverse impacts. Primary attention is paid to sterols, phospholipids, storage lipids, and fatty acids. The structural and biological functions of sterols are considered, as well as their role in membrane stabilization under stress and their relation to morphogenetic processes in mycelial fungi, of which many are pathogenic. Data on the biosynthesis of the main fungal sterol, ergosterol, are presented, as well as data on the inhibitors of this process and on the mutants deficient in its particular stages. Ergosterol biosynthesis is also considered in terms of its relation to the composition of the fungal cell wall, which is the cell shape-determining structure, and to the intensity of chitin synthesis, a process in which azole derivatives play a role. Data obtained by the authors are presented that show the role of changes in the composition of sterols, phospholipids, storage lipids, and unsaturated fatty acids of resting cells in the induction of yeastlike growth in mucoraceous fungi.