A preliminary investigation on the chemical composition of the cell surface of five enteropathogenic Escherichia coli serotypes.

The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy. Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins. Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues. The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100 degree C for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon. Phosphate, total sugar and protein contents were determined. Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose. Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction. Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction.

Identification of plasmenylethanolamine as a major component of the phospholipids of strain DM 28c of Trypanosoma cruzi.

A novel phospholipid has been purified from strain Dm 28c of Trypanosoma cruzi, and characterized by fast atom bombardment mass spectrometry and nuclear magnetic resonance spectroscopy as a plasmenylethanolamine with a hexadec-l-enyl group in the sn-1 position and an approximately equimolar mixture of octadecenoate and octadecadienoate esterified to the sn-2 hydroxyl. The purified plasmenylethanolamine reacted positively when probed with sera from patients with chronic Chagas' disease. Since plasmenylethanolamines of similar structure are abundant in mammalian cardiac and neuronal tissues, cross reactions between these epitopes may be a factor in the mechanism of autoimmune pathology in the chronic phase of Chagas' disease.

Structural characterization of neutral glycosphingolipids from Fusarium species.

Glycosphingolipids were extracted from hyphae of Fusarium solani and from an unnamed Fusarium species, and were purified by silica and Iatrobead column chromatography. Their structures were determined by compositional analysis, nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry and by fast atom bombardment mass spectrometry of the native and peracetylated materials, which defined their sugar, long-chain base and fatty acid compositions. The locations of the double bonds in the bases were established by 2D NMR spectroscopy and by novel mass spectrometric approaches, including collisional activation of the protonated and lithium-cationized glycosphingolipids, and of the sphingadienene-derived fragment ion at m/z 276. From these results we propose that the structures of the glycosphingolipids from F. solani and Fusarium sp. are N-2'-hydroxyoctadecanoyl-1-O-beta-D-glucopyranosyl-9-methyl-4, 8-sphingadienine and N-2'-hydroxyoctadecenoyl-1-O-beta-D-glucopyranosyl-9-methyl-4, 8-sphingadienine, respectively.

The structures of polysaccharides and glycolipids of Aspergillus fumigatus grown in the presence of human serum.

A study was made of polysaccharides and glycosphingolipids isolated from Aspergillus fumigatus grown in media supplemented with human serum from healthy donors. Fractionation of Cetavlon-precipitated polysaccharides on Sephacryl S-400 gave rise to an excluded fraction (Fraction I) with molecular weight of > 400 kDa and an included peak (Fraction II) with an average molecular weight of 30-80 kDa. Fraction I comprises about 5% of total polysaccharide and was identified as a glycogen-like molecule. Its structure was deduced from methylation data, treatment with amyloglucosidase, a red-brown coloration produced with an iodine solution and by 1H and 13C-NMR spectroscopy. It was previously suggested that higher amounts of glycogen-like polysaccharide (20%) were present in A. fumigatus grown in serum-free medium. Fraction II was identified as a galactomannan and was the main polysaccharide of A. fumigatus grown in serum-free medium. Fraction II was identified as a galactomannan and was the main 13C-NMR spectroscopy combined with partial acetolysis and methylation analysis. The 13C-NMR spectrum of the galactomannan showed a much greater complexity in the beta-D-gal f and alpha-D-man p C-1 regions, than was evident for galactomannan from serum-free cultures previously described, reflecting differences in the glycosylation pattern, stimulated in serum-supplemented medium. No differences in A. fumigatus glycosphingolipid could be detected between serum-containing and serum-free growth conditions. Our results demonstrate that the change in polysaccharide structure is a more specific response to the altered growth conditions and not merely a symptom of more general changes.

Structural determination of N-2'-hydroxyoctadecenoyl-1-O-beta-D-glucopyranosyl-9-methyl-4, 8-sphingadienine from species of Aspergillus.

Ceramide monohexosides from Aspergillus fumigatus 2140 and 2109 strains and Aspergillus versicolor 550 strain, obtained by silica gel 60, and Iatrobeads chromatography were analysed using high-resolution 1D-, 2D-1H-NMR and 13C-NMR spectroscopy and fast atom bombardment mass spectrometry (FAB-MS). The ceramide monohexoside fraction (CMH) from A. fumigatus 2140 and A. versicolor 550 was identified as glucosylceramide, whereas glucose and galactose were present at a ratio of 1:1 in the CMH of A. fumigatus 2109. The major glycosphingolipid has a particular ceramide composition consisting of 9-methyl-4,8-sphingadienine linked to a 2-hydroxyoctadec-3-enoic acid. Although the structures presently described are similar to those of monohexosylceramides from other fungi, including edible ones, this is the first report on their occurrence in species pathogenic in humans.

Reactivity of chagasic sera with crude and highly purified glycosphingolipid fractions from Trypanosoma cruzi epimastigotes.

The reactivities of sera from patients with Chagas disease or from T. cruzi-immunized rabbits with two different lipid preparations of T. cruzi were assessed using epimastigote antigens. Serum reactivities were determined using a quantitative enzyme-linked immunosorbent assay (ELISA). Antigen 1 represents the lower phase obtained from crude lipid extract after Folch partition (LCL). Antigen 2 is a highly purified glycosphingolipid fraction (GSL). The LCL antigen discriminated quite well the reactivities of Chagasic patients' sera and sera from healthy individuals, as well as between the serum from a T. cruzi-immunized rabbit (TIRS) and normal rabbit serum (NRS). A strong reactivity with GSL was obtained with TIRS. Reactivity with GSL was also obtained with human Chagasic sera. Compared to a group of normal individuals, the reactions of antibodies directed against lipid antigens were considerably increased in sera of patients with Chagas disease. Chagasic sera did not differentiate between glycolipids with terminal beta-glucosyl or beta-galactosyl non-reducing units. They discriminated, however, glucosylceramides with differences in the ceramide structure. To determine the specificity of Chagasic sera, antibodies isolated on LCL-immunosorbent (LCL-Ch Abs) as well as on laminin-immunosorbent (Lam-Ch Abs) were tested against laminin and LCL antigens. We found that Lam-Ch Abs reacted with murine laminin, whereas the reaction was negative with LCL. In contrast, the LCL-Ch Abs reacted either with LCL antigens or with laminin. The reactivity with laminin was strong in comparison with LCL.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycolipid and protein profiles in trypanosomatids.

A comparative study of glycolipid and protein composition in Trypanosoma cruzi and non-pathogenic trypanosomatids was carried out using Triton X-114 extraction. Protein profiles were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE), and glycolipids were detected using high-performance thin-layer chromatography (HPTLC). Hydrophilic protein profiles were similar in non-pathogenic protozoa. Endotrypanum schaudinni, Crithidia luciliae and T. mega showed five characteristic protein bands ranging between 30 and 66 kDa. In the hydrophobic phase, a band of 50 kDa was present only in T. mega. Strain-specific protein distribution was detected in T. cruzi clone Dm28c and T. cruzi G and Y strains; clone Dm28c had five typical hydrophilic proteins at between 24 and 45 kDa, the G strain had two bands at 45 kDa in the hydrophilic phase and the Y strain had a major protein band at 24 kDa in both phases. T. dionisii and T. cruzi clone Dm28c showed a characteristic distribution of three hydrophilic proteins of approx. 45 kDa. Qualitative analysis of glycolipid composition showed that the T. cruzi strains and Dm28c clone and T. dionisii had four orcinol-positive spots, whereas in the other non-pathogenic trypanosomatids only three glycolipids were detected.

Glycolipid and protein profiles of normal and Trypanosoma cruzi infected heart muscle cells.

Using Triton X-114, glycolipids and proteins were extracted from heart muscle cells (HMC) infected with Trypanosoma cruzi clone Dm28c and from uninfected HMC, and analysed by SDS-PAGE and high-performance thin-layer chromatography (HPTLC). Two major differences were observed: (a) two proteins with a molecular mass of 92 kDa and 69 kDa were present in the uninfected cells but absent from the infected cells and (b) a 70-90 kDa protein band was detected only in parasitized cells. These differences would seem to constitute alterations taking place during the process of cell recognition and/or parasite interiorization. No differences were observed in the respective glycolipid compositions, of control and infected cells analysed by HPTLC. A glycolipid with the same mobility as the neutral glycolipid isolated from epimastigotes of T. cruzi was detected in the uninfected cells. This finding may lend support to the previously described hypothesis that molecular mimicry is implicated in the cardioneuropathology of Chagas' disease.