Galactomannan is a characteristic polysaccharide of the human filamentous fungal pathogen Aspergillus fumigatus that can be used to diagnose invasive aspergillosis. In this study, we report the isolation of a galactomannan fraction associated to membrane preparations from A. fumigatus mycelium by a lipid anchor. Specific chemical and enzymatic degradations and mass spectrometry analysis showed that the lipid anchor is a glycosylphosphatidylinositol (GPI). The lipid part is an inositol phosphoceramide containing mainly C 18 -phytosphingosine and monohydroxylated lignoceric acid (2OH-C 24:0 fatty acid). GPI glycan is a tetramannose structure linked to a glucosamine residue: Man␣1-2Man␣1-2Man␣1-6Man␣1-4GlcN. The galactomannan polymer is linked to the GPI structure throught the mannan chain. The GPI structure is a type 1, closely related to the one previously described for the GPI-anchored proteins of A. fumigatus. This is the first time that a fungal polysaccharide is shown to be GPI-anchored.Aspergillus fumigatus has become over the past decade the most prevalent airborne fungal pathogen causing fatal invasive infections in immunocompromised patients (1). One of the characteristic components of this fungus is composed of a linear mannan chain with a tetra-␣-mannoside repeating unit and side chains of ␤1-5 galactofuranoside residues (2). The galactomannan from A. fumigatus has been described as a free polysaccharide found in the culture medium, and it is also covalently associated to the cell wall through the ␤1-3 glucan net and plays a role in the structural organization of the cell wall (2, 3). A monoclonal antibody directed against the galactofuranose side chain has been used to detect the presence of this polymer in the sera of patient with invasive aspergillosis (4, 5). Biosynthesis of the galactomannan is totally unknown in this fungus. During the search of lipid molecules that could be an anchor for the galactomannan, we isolated a membrane bound fraction that contained galactomannan. We investigated the nature of the membrane anchor of the galactomannan by GLC-MS 2 (gas liquid chromatography-mass spectrometry) and ES-MS-MS (electrospraytandem mass spectrometry) and found that the lipid anchor was a glycosylphosphatidylinositol. EXPERIMENTAL PROCEDURES Fungal Culture and Membrane PreparationA. fumigatus, strain CBS 144-89 was grown in a 15-L fermenter in 2% glucose and 1% mycopeptone (Biokar Diagnostics, Pantin, France) for 24 h at 25°C as described previously (6). Total membrane preparation from mycelium was done as described previously (7). Extraction and Purification of the LipogalactomannanLipids and polar glycolipids were extracted from membrane preparation by an organic extraction. A chloroform/methanol 1/1 (v/v) solution was added to the membrane suspension to reach a final concentration of chloroform/methanol/water (10:10:3 v/v/v). The mixture was stirred for 2 h at room temperature to extract lipids, then centrifuged at 10,000 ϫ g for 10 min. The pellet was resuspended in the same solvent, and the e...

Protection against reinfection with noncapsulated Gram-negative bacteria, such as Shigella, an enteroinvasive bacterium responsible for bacillary dysentery, is mainly achieved by Abs specific for the O-Ag, the polysaccharide part of the LPS, the major bacterial surface Ag. The use of chemically defined glycoconjugates encompassing oligosaccharides mimicking the protective determinants carried by the O-Ag, thus expected to induce an efficient anti-LPS Ab response, has been considered an alternative to detoxified LPS-protein conjugate vaccines. The aim of this study was to identify such functional oligosaccharide mimics of the S. flexneri serotype 2a O-Ag. Using protective murine mAbs specific for S. flexneri serotype 2a and synthetic oligosaccharides designed to analyze the contribution of each sugar residue of the branched pentasaccharide repeating unit of the O-Ag, we demonstrated that the O-Ag exhibited an immunodominant serotype-specific determinant. We also showed that elongating the oligosaccharide sequence improved Ab recognition. From these antigenicity data, selected synthetic oligosaccharides were assessed for their potential to mimic the O-Ag by analyzing their immunogenicity in mice when coupled to tetanus toxoid via single point attachment. Our results demonstrated that induction of an efficient serotype 2a-specific anti-O-Ag Ab response was dependent on the length of the oligosaccharide sequence. A pentadecasaccharide representing three biological repeating units was identified as a potential candidate for further development of a chemically defined glycoconjugate vaccine against S. flexneri 2a infection.

The D'A'B'(E')C'DAB(E)C decasaccharide representative of a dimer of a frame-shifted pentasaccharide repeating unit of the O-specific polysaccharide of Shigella flexneri 2a was synthesized as its methyl glycoside by condensing a pentasaccharide donor (D'A'B'(E')C') and a pentasaccharide acceptor (DAB(E)C-OMe). Several convergent routes to these two building blocks, involving either the AB linkage or the BC linkage as the disconnection site, were evaluated in comparison to the linear strategy. The latter was preferred. It is based on the use of the trichloroacetimidate chemistry. The target branched oligosaccharide was designed to probe the recognition at the molecular level of the natural polysaccharide by protective monoclonal antibodies.