Candida albicans biofilms: comparative analysis of room-temperature and cryofixation for scanning electron microscopy.

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde-based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room-temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room-temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze-substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.

Glycoconjugates and polysaccharides of fungal cell wall and activation of immune system / Glicoconjugados e polissacarídios de parede celular de fungos e ativação do sistema imune

The fate of organochlorine 14C-dicofol in activated sludge process was investigated. Results showed that the major part of radioactivity remained adsorbed on biological sludge. Consequently, its final disposal deserves special attention. The small amounts of dicofol, biotransformed or not, which remained in the treated effluent could contaminate receiving bodies.

Glicoproteínas, glicoesfingolipídios e polissacarídios, expostos nas camadas mais externas da parede celular dos fungos, estão envolvidos em diferentes tipos de interações com o ambiente extracelular. Essas moléculas são componentes essenciais desses organismos, contribuindo para a estrutura, integridade, crescimento celular, diferenciação e sinalização. Alguns são compostos imunologicamente ativos com potencial para regular a patogênese e a resposta imune do hospedeiro, Algumas dessas estruturas podem ser especificamente reconhecidas por anticorpos presentes no soro de pacientes, sugerindo uma possível utilização como ferramenta no diagnóstico das infecções fúngicas.

The opportunistic fungal pathogen Scedosporium prolificans: carbohydrate epitopes of its glycoproteins.

Isolated from the mycelium of Scedosporium prolificans were complex glycoproteins (RMP-Sp), with three structurally related components (HPSEC). RMP-Sp contained 35% protein and 62% carbohydrate with Rha, Ara, Man, Gal, Glc, and GlcNH(2) in a 18:1:24:8:6:5 molar ratio. Methylation analysis showed mainly nonreducing end- of Galp (13%), nonreducing end- (9%), 2-O- (13%), and 3-O-subst. Rhap (7%), nonreducing end- (11%), 2-O- (10%), 3-O- (14%), and 2,6-di-O-subst. Manp units (13%). Mild reductive beta-elimination of RMP-Sp gave alpha-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1-->3)-alpha-d-Manp-(1-->2)-d-Man-ol, with Man-ol substituted at O-6 with beta-d-Galp units, a related pentasaccharide lacking beta-d-Galp units, and beta-d-Galp-(1-->6)-[alpha-d-Manp-(1-->2)]-d-Man-ol in a 16:3:1w/w ratio. Traces of Man-ol and Rha-ol were detected. ESI-MS showed HexHex-ol and Hex(3-6)Hex-ol components. Three rhamnosyl units were peeled off successively from the penta- and hexasaccharide by ESI-MS-MS. The carbohydrate epitopes of RMP-Sp differ from those of the glycoprotein of Pseudallescheria boydii, a related opportunistic pathogen.

Glycoconjugates and polysaccharides of fungal cell wall and activation of immune system.

Glycoproteins, glycosphingolipids and polysaccharides exposed at the most external layers of the wall are involved in several types of interactions of fungal cells with the exocellular environment. These molecules are fundamental building blocks of organisms, contributing to the structure, integrity, cell growth, differentiation and signaling. Several of them are immunologically active compounds with potential as regulators of pathogenesis and the immune response of the host. Some of these structures can be specifically recognized by antibodies from patients' sera, suggesting that they can be also useful in the diagnosis of fungal infections.

Human antibodies against a purified glucosylceramide from Cryptococcus neoformans inhibit cell budding and fungal growth.

A major ceramide monohexoside (CMH) was purified from lipidic extracts of Cryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a beta-glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography. The purified antibodies to CMH (mainly immunoglobulin G1) bound to different strains and serological types of C. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogold-antibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the beta-glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains of C. neoformans inhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal beta-glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro.

Analysis of peptidogalactomannans from the mycelial surface of Aspergillus fumigatus.

Peptidogalactomannans (pGMs) from mycelium of two strains of Aspergillus fumigatus were fractionated by Cetavlon precipitation and size exclusion chromatography and their carbohydrate structures analysed using methylation-fragmentation analysis, partial acetolysis and 13C-nuclear magnetic resonance spectroscopy. The most significant difference between the pGMs of the two strains was the degree of branching and the proportion of non-reducing ends of alpha-D-Manp and beta-D-Galf units. Methylation data showed that the pGM from AF 2109 contained alpha-D-Manp and beta-D-Galf non-reducing end units in a proportion of 3:1 while, in contrast, the proportion of these structures in pGM from AF 2140 was 7:1, resulting in a highly branched structure. The immunoreactivity of the pGM fractions was tested by indirect immunofluorescence. The fractions were also tested in an ELISA system with rabbit antiserum raised to whole cells of A. fumigatus NCPF 2140 and with serum from patients with either proven aspergilloma or ABPA. The carbohydrate moiety of the pGM appears to be responsible for the antigenicity. Periodate treatment, partial acid hydrolysis and beta-elimination removed most of the antibody binding capacity.

Heart muscle cells share common neutral glycosphingolipids with Trypanosoma cruzi.

Neutral glycosphingolipids were isolated from mouse heart muscle cells and their structures were analyzed. The molecular compositions of these glycosphingolipids were examined using column chromatography, HPTLC, GC-MS and fast atom bombardment-mass spectrometry (FAB-MS). Monohexosylceramides are a mixture of glucosyl- and galactosylceramides in a ratio of 1:1, sphingosine as the long chain base and as fatty acyl groups mainly C16, C18 saturated and C22 and C24 hydroxy fatty acids. Dihexosylceramide, identified as lactosylceramide contains C18 sphingosine and C18, C20 and C22 were the major fatty acids. No evidence for the occurrence of hydroxylated fatty acids in this glycolipid could be obtained from the GC-MS data. Our results clearly demonstrated that Trypanosoma cruzi and heart muscle cells have similar glycosphingolipid structures. In addition, heart muscle cells neutral glycosphingolipids have been shown to be immunoreactive. Antibodies reactive with each of the immunogenic glycolipids from heart cells or T. cruzi epimastigotes were present in the sera of human patients with Chagas disease as detected by ELISA. These cross-reactive antigens could be involved in the Chagasic autoimmunity.

Monohexosylceramides of Trypanosoma dionisii.

A detailed knowledge of the primary structure of neutral glycosphingolipids isolated and purified from Trypanosoma dionisii has been elucidated using a combination of techniques--as column chromatography, HPTLC and GC-MS together with fast atom bombardment spectrometry. The ceramide monohexoside fraction (CMH) contained both glucosyl- and galactosylceramides in a ratio of 1:1, sphingosine and as fatty acyl groups mainly C-24 saturated and 2-hydroxy fatty acids. A close similarity between Trypanosoma cruzi and T. dionisii monohexosylceramides was reported.

Amphotericin B-induced carbohydrate changes on the Trypanosoma cruzi surface membrane.

Changes in the cell surface carbohydrates of Trypanosoma cruzi epimastigotes induced by Amphotericin B (AmB) were assessed by chemical methods and by agglutination assay employing a panel of highly purified lectins of various sugar specificities. Escherichia coli K12 with mannose-sensitive fimbriae was also used as an agglutination probe. Amphotericin B caused a decrease in the total carbohydrate content of all glycoconjugate fractions isolated. Exposure to AmB strongly affected the mannose/galactose ratio (1:5) in the CHCl3/methanol/H2O soluble fraction. These sugars in 1.4:1 ratio were the major hexose components of control cells. The decrease in the mannose content (48 to 15%) after AmB treatment agrees with the marked decrease in the T. cruzi cell surface receptors for fimbriated E. coli K12. Also, an increase in the galactose content (74%) as compared with control cells (34%) is in agreement with the peanut agglutinin and Euonymus europaeus lectins agglutination results. Differences in the cell surface carbohydrates induced by AmB could be associated with alterations in the membrane structure and organization.

Structural characterization of neutral glycosphingolipids from Trypanosoma cruzi.

The major neutral glycosphingolipids from Trypanosoma cruzi ceramide mono- and dihexosides (CMH and CDH, respectively) were analysed after chromatographic purification using 1H 500 MHz nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The ceramide monohexoside fraction (CMH) contained both glucosyl- and galactosylceramides. After peracetylation, the CMH fraction was separated into 2 subfractions, CMH-COH and CMH-Cn, containing either hydroxy fatty acids or n-fatty acids. In the CMH-COH fraction glucose and galactose were present in a ratio of 2:1, whereas this ratio was 1:1 in the CMH-Cn fraction. The CDH fraction was identified as lactosylceramide with sphingosine as the long chain base and 16:0, 18:0, and 24:0, 24:4 fatty acids as major components.

Amphotericin B-induced damage of Trypanosoma cruzi epimastigotes.

Amphotericin B (AmB) autoxidation resulted in oxygen consumption, superoxide anion formation and production of thiobarbituric acid (TBA)-reactive material (malondialdehyde). Malondialdehyde formation increased after incubation of the drug with ascorbate-ADP-FeCl3. Growth of Trypanosoma cruzi epimastigotes in the presence of AmB induced a decrease in the free fatty acid content of the cells (57% in control cells vs. 7% in AmB-treated cells), and in the proportion of unsaturated fatty acids as well as cell killing. No changes were detected on sterol content. No evidence was found for lipid peroxidation as a mechanism of cell injury by this antibiotic.

Further characterization of carbohydrate-containing fractions from Trypanosoma mega.

Epimastigotes of Trypanosoma mega were submitted to phenol extraction after lipid extraction, providing an extract whose carbohydrate portion (30%) contained fucose, ribose, xylose, mannose, galactose, and glucose. The purified fraction recovered in the void volume of Bio Gel P-150 gave on SDS-PAGE a band of Mr approximately equal to 55,000 positive for protein and carbohydrate and a diffuse band strongly positive for carbohydrate and lipids (Mr approximately equal to 22,000). The structural analysis of the carbohydrate moiety of this fraction by GLC-MS indicated the presence of nonreducing end groups of fucopyranose, mannopyranose, and galactopyranose, 3-O- and 4-O-substituted and 2,3- and 2,4-di-O-substituted galactopyranosyl units. Extraction of this fraction with chloroform/methanol/water provided a soluble fraction that on SDS-PAGE gave rise to a carbohydrate and lipid-positive band (Mr approximately equal to 22,000). This fraction contained fucose, mannose, and galactose (1:1:1). As main branch points, 2,3-di-O-substituted galactopyranosyl units were present according to methylation data. Similar proportions of fucopyranosyl, mannopyranosyl, galactopyranosyl end units were present. The presence of lipids in this fraction was confirmed by methanolysis following isolation and characterization of the corresponding fatty acid methyl esters. Palmitic acid (16:0) and an 18:1 fatty acid were the predominant fatty acids.

Lipid alterations induced by nifurtimox in Trypanosoma cruzi.

The influence of growth in the presence of nifurtimox on total lipids of Trypanosoma cruzi epimastigotes was determined. Nifurtimox-treated organisms showed greater amounts of unsaturated fatty acids as compared with control cells. 18:2 was the major component in the total esterified lipid fraction. Similar results were observed on the free fatty acid composition as detected by trimethylsilyl derivatization of the total lipids.

Isolation and characterization of a neutral glycosphingolipid from the epimastigote form of Trypanosoma mega.

A glycosphingolipid fraction from Trypanosoma mega was isolated after acetylation and was further purified on a silicic acid column. Final purification was by preparative thin-layer chromatography. The carbohydrate components of the glycolipid were fucose and galactose in approximately equimolar amounts. The neutral glycolipid of T. mega has a sphingosine base composition that consists of sphingosine and traces of dihydrosphingosine. Fatty acids forming amide groups with the sphingosine bases were analyzed by gas-liquid chromatography-mass spectrometry and are a mixture of normal and alpha-hydroxy fatty acids. Normal C16:0, C18:0, and 2-hydroxy C18:0 are the predominant fatty acids.

Glycolipid components of epimastigote forms of Trypanosoma cruzi.

Glycosphingolipids were isolated from a lipid extract of epimastigote forms of Trypanosoma cruzi via Florisil and silicic acid column chromatography. The carbohydrate components of neutral glycolipid consisted of mannose and galactose in a ratio of 1:2. The fatty acids of the glycolipid were analyzed by gas liquid chromatography-mass spectrometry (g.l.c.-m.s.). Normal and 2-hydroxy fatty acids were found. The sphingosine bases were C18 dihydrosphingosine and 17-methyl sphingosine.