Identification of sialic acids on the cell surface of Candida albicans.

The cell-surface expression of sialic acids in two isolates of Candida albicans was analyzed by thin-layer and gas chromatography, binding of lectins, colorimetry, sialidase treatment and flow cytofluorimetry with fluorescein-labeled lectins. N-acetylneuraminic acid (NANA) was the only derivative found in both strains of C. albicans grown in a chemically defined medium. Its identification was confirmed by mass spectrometry in comparison with an authentic standard. The density of sialic acid residues per cell ranged from 1. 6x10(6) to 2.8x10(6). The surface distribution of sialic acids over the entire C. albicans was inferred from labeling with fluorescein-Limulus polyphemus and Limax flavus agglutinins and directly observed by optical microscopy with (FITC)-Sambucus nigra agglutinin (SNA), abrogated by previous treatment of yeasts with bacterial sialidase. Sialidase-treated yeasts generated beta-galactopyranosyl terminal residues that reacted with peanut agglutinin. In C. albicans N-acetyl-neuraminic acids are alpha2,6- and alpha2,3-linked as indicated by yeast binding to SNA and Maackia amurensis agglutinin. The alpha2,6-linkage clearly predominated in both strains. We also investigated the contribution of sialic acids to the electronegativity of C. albicans, an important factor determining fungal interactions in vivo. Adhesion of yeast cells to a cationic solid phase substrate (poly-L-lysine) was mediated in part by sialic acids, since the number of adherent cells was significantly reduced after treatment with bacterial sialidase. The present evidence adds C. albicans to the list of pathogenic fungi that synthesize sialic acids, which contribute to the negative charge of fungal cells and have a role in their specific interaction with the host tissue.

Cell surface carbohydrates in Crithidia deanei: influence of the endosymbiote.

The cell surface of the symbiote-containing and symbiote-free strains of Crithidia deanei were characterized comparatively by using 22 highly purified lectins with specificities for N-acetyl glucosamine, N-acetyl galactosamine, galactose, mannose-like residues, fucose and sialic acid. The specificity of the cell surface carbohydrate in both strains were analyzed by agglutination and lectin-binding assays. C. deanei with or without endosymbiote was specifically agglutinated by lectins from Triticum vulgaris (WGA) and Aaptos papillata suggesting the presence of D-GlcNAc residues. However, agglutination was stronger with the symbiote-free cells than with symbiote-containing organisms. The D-GalNAc-binding lectin from Wistaria floribunda also reacted most effectively with symbiote-free flagellates. Among D-Gal-binding lectins it was observed that those from Axinella polypoides and Ricinus communis I selectively agglutinated symbiote-free cells. In contrast the lectin from Arachys hypogaeae bound preferentially to the symbiote containing organisms. Both strains of C. deanei agglutinated strongly with the lectins concanavalin A and that from Lens culinaris (lectins for D-mannose-like residues). Conversely no cell agglutination occurred with the L-fucose-binding lectins Lotus tetragonolobus and Ulex europeus. The pattern of agglutination induced by the lectin from Limulus polyphemus of symbiote-free organisms was similar to that of symbiote-containing cells indicating the presence of sialic acid on the cell surface of both strains of C. deanei. These results indicate that the presence of the endosymbiote changes the lectin-binding sites at C. deanei surface membrane.

Identification of sialic acids on the cell surface of hyphae and yeast forms of the human pathogen Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, when grown in a synthetic medium, expresses at the cell surface of both yeast and mycelial forms acidic glycoconjugates containing N-acetylneuraminic acid units. Sialic acids were extracted using mild hydrolytic conditions, and were identified by thin-layer and gas chromatography, standard colorimetry, reaction with periodate-resorcinol and mass spectrometry. Their surface location was inferred from fluorescent-lectin (Limulus polyphemus agglutinin) binding to whole cells abrogated by previous treatment with neuraminidase. Expression of sialic acids on virulent yeast forms of P. brasiliensis (3.7 x 10(6) residues per cell) may inhibit fungal phagocytosis during early infection, when the immunological response is still being built up.

Changes in fatty acid composition associated with differentiation of Trypanosoma cruzi.

The fatty acid composition during the transformation of Trypanosoma cruzi epimastigotes into metacyclic trypomastigotes (metacyclogenesis) was analysed by gas-liquid chromatography and mass spectrometry. Significant qualitative and quantitative changes in the fatty acid composition occurred during incubation of epimastigotes derived from LIT medium in the triatomine artificial urine (TAU). Metacyclogenesis was also followed by alterations in the fatty acid pattern but these were considerably less pronounced when compared to the pattern obtained for TAU-incubated epimastigotes. These results suggest that changes in the lipid composition precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.

Induction of differentiation in Herpetomonas samuelpessoai by dimethylsulfoxide.

Dimethylsulfoxide (DMSO) induces in Herpetomonas samuelpessoai, grown in a chemically defined medium at 28 degrees C the transformation of promastigotes into paramastigotes and opisthomastigotes. This effect was dependent on the period of cultivation and the concentration of DMSO. Initially, DMSO induced the appearance of paramastigotes and later, of opisthomastigotes. Approximately 43% opisthomastigotes were obtained after growth of the protozoan for 96 h at 28 degrees C in a medium containing 3% DMSO. No ultrastructural changes were observed on DMSO-treated cells. Based on these results and others previously described, the process of differentiation in Herpetomonas is compared and analyzed with that occurring in Trypanosoma.

Fatty acid composition of amastigote and trypomastigote forms of Trypanosoma cruzi.

The fatty acid composition of total lipids from trypomastigote and amastigote forms of Trypanosoma cruzi and of Vero cells before and after parasite infection were analyzed by gas-liquid chromatography and mass spectrometry. Even-numbered, saturated, monoenoic and polyenoic acids ranging from C-12 to C-18 were characterized in both T. cruzi development stages. Significant changes in the fatty acid composition occurred during the T. cruzi life cycle. Oleic and linoleic acids were prominent in trypomastigote forms, whereas palmitic acid was the major fatty acid of amastigotes. Other differences include higher stearic acid and lower palmitoleic and linolenic acid levels as well as the absence of lauric acid in amastigotes as compared with trypomastigote forms. The fatty acid pattern of Vero cells before T. cruzi infection as compared with that after infection showed mostly qualitative differences. Linoleic and linolenic acids were observed only in T. cruzi infected cells.

Effect of ultraviolet and gamma-radiation on Herpetomonas samuelpessoai.

A study was made about the influence of ultraviolet (UV) and gamma-radiations on Herpetomonas samuelpessoai grown either in a chemically defined or in a complex medium. Cells cultivated in defined medium were more sensitive to UV than those from complex medium, as estimated by inhibition of cellular growth. The effect of gamma-radiation, however, was independent of the media in which the cells were grown. Both radiations interfere with the plasma membrane as analysed by parameters such as excretion of cellular material and concanavalin-A-induced agglutination. Doses of UV which inhibit the cellular growth do not interfere with the plasma membrane. With gamma-radiation, however, doses which inhibit cellular growth also interfere with the plasma membrane. These results suggest that for certain applications UV radiation may be an advantage in vaccine production.

Phagocytosis of the three developmental forms of Trypanosoma cruzi: effect of specific sera.

Studies were carried out aiming at comparing the uptake of the three evolutionary stages of Trypanosoma cruzi by mouse peritoneal macrophages, influenced by specific immunosera. Incorporation of T. cruzi by macrophages was time dependent. In absence of antibody, trypomastigotes are forms more effectively incorporated by macrophages. Pre-incubation of macrophages with specific sera against each of the T. cruzi morphological stages was followed by an increase in the uptake of amastigotes and trypomastigotes but not of epimastigotes. Our results show that amastigotes, in comparison with the other T. cruzi forms are more actively phagocytized in presence of specific serum.

Chemical composition of lipopolysaccharide from Moraxella bovis.

Lipopolysaccharide from Moraxella bovis was isolated and its components characterized by paper and gas-liquid chromatography and mass spectrometry. The endotoxin showed a small amount of protein and contained 2-keto-3-deoxyoctonic acid glucose, mannose, glucosamine, heptose and phosphate. Six fatty acids (palmitic, palmitoleic, margaric, stearic, oleic and arachidic acids) were also identified. Margaric acid was the most prevalent fatty acid present in the lipid fraction.

Cell surface alterations induced by methylene blue and direct electric current in Escherichia coli.

Cell surface properties, including hydrophobicity, zeta potential, carbohydrate and fatty acid components, were altered on treatment of E. coli K12 with methylene blue (MB) and direct electric current (DC). The treatment of fimbriated E. coli cells with MB greatly increased the agglutination of yeast cells when compared to untreated bacteria. However, this increased agglutination was markedly reduced when the bacteria were treated with MB plus DC. These results suggest that MB modifies cell surface components in the absence of light and these alterations are more pronounced when cells are treated simultaneously with MB and DC.

Demonstration of concanavalin A receptors on Leptomonas pessoai cell memebrane.

Leptomonas pessoai promastigotes cultivated in a synthetic medium were agglutinated with concanavalin A (ConA). Agglutination was predominantly of the flagellar-flagellar type, and was inhibited with sucrose or alpha-methyl-D-mannoside. Cell surface polysaccharides and/or glycoproteins were demonstrated by several cytochemical methods at the fine-structural level. A Con A-horseradish peroxidase-diaminobenzidine (DAB) technic was used to detect Con A receptors in the pellicular membrane of the flagellar pocket region and in the flagellar membrane proper. Somewhat less of the Con A-DAB reaction product was observed in the pellicular membrane enclosing the rest of the cell.

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.

Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions.

Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (L-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the D-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or D-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (D-Gal-specific), although the reactivity of other D-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.

Polysaccharide and glycolipid composition in Tritrichomonas foetus.

1. The polysaccharide and glycolipid composition in Tritrichomonas foetus was studied by paper, thin-layer and gas-liquid chromatographic analysis. 2. The carbohydrate components of the polysaccharide were glucose (47%), galactose (34%) and mannose (19%). N-acetylneuraminic acid was the sialic acid derivative characterized in the flagellate whole cells. 3. The sialic acid density was estimated as 2.7 x 10(7) residues/cell. 4. The long-chain base dihydrosphingosine, the carbohydrates galactose (67%), glucose (21%) and mannose (12%) as well as the fatty acids myristic (48%) and palmitic (52%) acids were characterized as components of the total glycolipids of T. foetus. 5. Total glycolipids were fractionated: a galactocerebroside and a ganglioside were identified.

Effect of environmental factors on Fonsecaea pedrosoi morphogenesis with emphasis on sclerotic cells induced by propranolol.

The influence of growth conditions, as well as of propranolol on Fonsecaea pedrosoi morphogenesis was established using the chemically defined media of Czapeck-Dox (CD) and Butterfield (BF). Mycelial growth of F. pedrosoi in both media was obtained at room temperature (25 degrees C) for 14 days, without shaking, whereas conidia formed at 37 degrees C, for 4 days, in shaken cultures and could be isolated free from the mycelium by filtration in gauze. At low pH (2.5-3.0), there appeared sclerotic cells attached to normal hyphae. When propranolol ws added to the CD medium moniliform hyphae were observed, whereas this drug in the BF medium induced formation of sclerotic cells. Ultrastructural examination revealed that the propranolol-induced sclerotic cells were very similar to those observed in infected tissues.

Detection of cellular immunity with the soluble antigen of the fungus Sporothrix schenckii in the systemic form of the disease.

Sporothrix schenckii is the etiologic agent of sporotrichosis, a mycosis of world-wide distribution more commonly occurring in tropical regions. The immunological mechanisms involved in the prevention and control of sporotrichosis are not fully understood but apparently include both the humoral and cellular responses. In the present investigation, cellular immunity was evaluated by in vivo and in vitro tests in mice infected with yeast-like forms of S. schenckii. The disease developed systemically and cellular immunity was evaluated for a period of 10 weeks. The soluble antigen utilized in the tests was prepared from yeast form of the fungus through the sonication (20 min: 10 sonications at 50 W at 2-min intervals). Delayed hypersensitivity and lymphocyte transformation tests showed that the cellular immune response was depressed between the 4th and 6th week of infection when the animals were challenged with the soluble fungal antigen. This depression frequently indicates worsening of the disease, with greater involvement of the host. This is a promising field of research for a better understanding of the pathogeny of this mycosis.

Carbohydrate and lipid components of hyphae and conidia of human pathogen Fonsecaea pedrosoi.

The carbohydrate and lipid components of mycelium and conidia of Fonsecaea 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 in F. 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 of F. 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 in F. 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.

The effect of propranolol on metabolism and on membrane-associated polysaccharide components of Herpetomonas muscarum muscarum.

The effects of propranolol on the metabolism (cell growth, cell differentiation and cell respiration) as well as on membrane-associated polysaccharide components of Herpetomonas muscarum muscarum were investigated. At the dose of 10(-2) and 10(-3) mM the drug increased significantly the number of the more differentiated opisthomastigote form of the protozoan, whereas at higher concentrations (10(-1) mM) total inhibition of growth occurred with gross cell lysis. Propranolol (10(-3) mM) enhanced the oxidation of glycerol and more markedly that of glucose, glutamic acid and proline. The pattern of the composition of membrane-associated polysaccharides was also substantially changed: the drug (10(-3) mM) induced a preferential synthesis of galactose and a marked decrease of xylose and mannose, whereas glucose content remained practically unchanged.