Developmentally regulated protein expression mediated by dimethylsulfoxide in Herpetomonas samuelpessoai.

We have analyzed the total cell extract, cell surface, and secretory protein profiles related to cellular differentiation triggered by dimethylsulfoxide in the insect trypanosomatid Herpetomonas samuelpessoai. The flagellates were cultivated in chemically defined conditions in the absence or in the presence of 4% DMSO, and the resolved protein bands were detected by SDS-PAGE gels and avidin-Western blotting. The cell-associated proteins showed a complex pattern of around 40 silver-staining bands ranging from 15 to 150 kDa. There were generally minor quantitative differences in the protein profile between the non-treated and the DMSO-treated cells. The cell-surface protein profile revealed by the incubation of live parasites with biotin showed a decrease in the expression of the 120 kDa biotinylated polypeptide observed in the DMSO-treated cells when compared with untreated ones. However, control samples of both systems showed an endogenous biotinylated polypeptide of 63 kDa which also presented gelatinolytic activity. The trypanosomatids released at least 10 polypeptides to the culture medium. A low molecular mass exopolypeptide (35 kDa) was found exclusively in untreated cells, whereas a high-molecular-mass exopolypetide (250 kDa) was preferentially found in DMSO-treated cells.

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.

Purification and immunocytochemical localization of neuraminidase from Tritrichomonas foetus.

Lysis of Tritrichomonas foetus with a solution of the non-ionic detergent Triton X-114 at 0 degree C, followed by low-speed centrifugation, resulted in a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and an aqueous phase. Neuraminidase activity was mostly located in the detergent-insoluble pellet. When the parasites were incubated with bacterial phosphatidylinositol phospholipase C (PI-PLC) prior to detergent solubilization and phase separation neuraminidase activity was predominantly recovered in aqueous phase, rather than in the pellet and detergent phase. The molecular mass determined by gel permeation in high performance liquid chromatography (HPLC) and SDS-PAGE was 80,000 Da. Indirect immunofluorescence microscopy using polyclonal antibodies raised in rabbits against the purified neuraminidase, indicated that the enzyme is exposed on the cell surface. Previous treatment of the cells with PI-PLC significantly reduced antibody binding. Incubation of cryo-sections with the antibodies followed by detection using gold-labelled anti-rabbit IgG confirmed the presence of neuraminidase in the plasma membrane enclosing the cell body and flagella and in the membrane of vesicles preferentially located at the peripheral region of the protozoan.

Cell-surface carbohydrates of Entamoeba invadens.

Cell-surface carbohydrates of Entamoeba invadens trophozoites were analyzed using (a) a panel of highly purified lectins specific for molecules containing N-acetylglucosamine or sialic acid, N-acetylgalactosamine, galactose, mannose-like residues, and fucose; (b) Escherichia coli K-12 with mannose-sensitive fimbria; (c) enzymatic digestion; and (d) scanning electron microscopy. The presence of galactose (D-Gal) and N-acetylgalactosamine (D-GalNAc) was detected in the amoeba. Previous trypsinization induced the appearance of Glycine max (SBA, specific for D-GalNAc residues)-binding sites, whereas such treatment completely abolished the ability of Ricinus communis (RCAI) and Axinalla polypoides (APP, specific for D-Gal) lectins and partially abolished that of Euonymus europaeus (EEL, specific for D-Gal) lectins to agglutinate the trophozoites. The agglutinating activity of E. coli K-12 adheans with the amoeba was markedly increased after trypsin digestion, indicating that mannose units become exposed after enzyme treatment. These findings were essentially confirmed by scanning electron microscopy. After neuraminidase treatment the parasites became strongly agglutinated with SBA and Arachis hypogaea (PNA, specific for D-Gal) and the cell interaction with Wisteria floribunda (WFH, specific for D-GalNAc) was markedly increased. These results suggest that in E. invadens trophozoites, sialic acid residues are linked to D-Gal and D-GalNAc.

Isolation of ergosterol peroxide and its reversion to ergosterol in the pathogenic fungus Sporothrix schenckii.

Ergosterol peroxide, a presumed product of the H2O2-dependent enzymatic oxidation of ergosterol, has been isolated from yeast forms of the pathogenic fungus Sporothrix schenckii. The substance, which may have a role in fungal virulence, has been characterized mainly using spectroscopic methods (1H and 13C nuclear magnetic resonance and high resolution mass spectra). The purified compound showed a molecular formula of C28H44O3, displaying characteristic features of epidioxy sterols and was reverted to ergosterol when submitted to S. schenckii enzymatic extract.

Occurrence of N-acetyl- and N-O-diacetyl-neuraminic acid derivatives in wild and mutant Crithidia fasciculata.

The cell-surface expression of sialic acids in wild-type Crithidia fasciculata and three drug-resistant mutants (FU(R)11, TR3, and TFRR1) was analyzed using fluorescein-labeled Limulus polyphemus agglutinin (LPA) binding, glycosidase of known sugar specificity, and thin-layer chromatography (TLC). Gas-liquid chromatography-mass spectrometry (GC-MS) analysis using both electron-impact (EI-MS) and chemical ionization (CI-MS) by isobutane with selected ion monitoring (SIM) was also used. The surface location of sialic acid was inferred from LPA binding to whole cells abrogated by previous treatment with neuraminidase. An exception occurred with the TFRR1 strain, which after incubation with neuraminidase showed increased reactivity with the fluorescent lectin. Both N-acetyl- and N-O-diacetyl-neuraminic acids were identified in the flagellates by TLC, with a clear predominance being noted for the former derivative. However, the content of N-O-diacetyl-neuraminic acid was preferentially found in the TFRR1 strain. The GC-MS analysis of the acidic component of the TFRR1 mutant strain confirmed the occurrence of N-acetyl-neuraminic acid (Neu5Ac) by the presence of the diagnostic ions (m/z values: 684 and 594 for CI-MS and 478, 298, and 317 for EI-MS) and also by comparison with the standard Neu5Ac retention time. GC-MS analysis also showed fragments (m/z values: 654 and 564 for CI-MS and 594, 478, 298, and 317 for EI-MS) expected for the 7-O- and 9-O-acetyl-N-acetyl-neuraminic acids (Neu5,7Ac2 and Neu 5,9Ac2, respectively).

Disturbances in the production of interleukin-1 and tumor necrosis factor in disseminated murine sporotrichosis.

Production of Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) by adherent peritoneal cells from BALB/c mice was measured at week 2, 4, 6, 8 and 10 after intravenous inoculation with 10(6) Sporothrix schenckii yeasts. As compared with age-matched controls, IL-1 and TNF production by adherent peritoneal cells from S. schenckii-infected mice was reduced severely at week 4 and 6 of infection and greater than normal at week 8 and 10. Moreover, between week 4 and 6 of infection there was a depression of delayed type hypersensitivity response to a specific whole soluble antigen, and an increase in fungal multiplication in the livers and spleens of infected mice. Thus, the deficits of cell-mediated immunity in mice with systemic S. schenckii infection may derive, in part, from impaired amplification of the immune response consequent to abnormal generation of IL-1 and TNF.

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.

The cell surface of Phytomonas davidi.

Carbohydrates were located on the surface of Phytomonas davidi using ultrastructural cytochemistry, and agglutination induced by lectins which bind to residues of mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid. The surface charge of the cells was analysed by the binding of cationic particles (colloidal iron and cationized ferritin) to the cell surface and by cell electrophoretic mobility (EPM). Based on observations of binding of cationic particles to the cell surface; a decrease in the binding of these particles to the cell surface; a decrease in the mean EPM of the cells after their incubation in the presence of neuraminidase; and detection of N-acetylneuraminic acid by paper and gas-liquid chromatography, it was concluded that sialic acid residues are exposed on the surface of P. davidi. These residues may be glycolipids or are masked on the cell surface since only after brief trypsinization were the cells agglutinated by the lectin from Limulus polyphemus.

Phagocytosis of Herpetomonas samuelpessoai by mouse peritoneal macrophages: effect of lidocaine, concanavalin A and carbohydrates.

Normal mouse peritoneal macrophages, as well as macrophages treated with Concanavalin A (Con A) and lidocaine, were incubated in the presence of normal, Con A-treated or glutaraldehyde-fixed Herpetomonas samuelpessoai. The percentage of infected macrophages, the mean number of protozoa per macrophage and the phagocytic index was determined after 1 and 2 h of protozoa-macrophage interaction. The uptake of H. samuelpessoai by the macrophages was increased when either the protozoa or the macrophages or both were treated with Con A. The effect of Con A was dependent on its concentration and was inhibited by alpha-methyl-D-mannoside. The uptake of H. samuelpessoai by macrophages was significantly inhibited by xylose, mannose and a polysaccharide isolated from the portozoan. Lidocaine-treated macrophages incorporated less protozoa than normal macrophages. Both Con A and lidocaine induced the formation of a large number of cytoplasmic vacuoles in the macrophages. Glutaraldehyde-fixed protozoa were more easily incorporated by the macrophages than living cell. However, their uptake was not stimulated by treatment of the macrophages with Con A.

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.

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.

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.