Glycosylphosphatidylinositol-anchored Fungal Polysaccharide in Aspergillus fumigatus

Abstract

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...

Biosynthesis of the Fungal Cell Wall Polysaccharide Galactomannan Requires Intraluminal GDP-mannose

“…Purification and Analysis of Lipogalactomannan-Purification of lipogalactomannan and fractionation on octyl-Sepharose was essentially as described (8). Details are provided in the supplemental material.…”

“…In the latter fungus, galactomannan, a linear ␣-1,2/␣-1,6-mannan with short side chains of ␤-1,5-galactofuran, also substitutes the cell wall ␤1,3/1,6-glucan (6,7). This polysaccharide can additionally be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) 4 (8) or be part of the alkalisoluble fraction of the cell wall, containing the non-covalently associated polysaccharides ␣-1,3-glucan and galactosaminogalactan as well as secreted glycoproteins (3). Finally, circulating galactomannan is also present in patient blood, where it can be detected by the monoclonal antibody EB-A2 for clinical diagnostic of invasive aspergillosis (9,10).…”

Sphingolipids from the human fungal pathogen Aspergillus fumigatus

“…In the GPI anchor, the glucosamine residue is linked to the 4-O position of the inositol ring whereas it is linked in 2-O position in the zwitterionic GPIC. This structural difference explains the sensitivity of LGM to PI-PLC digestion and shown that the Gnt-A transferase is not involved in the GM biosynthesis [54,67]. Despite the presence of GM in membrane and cell wall of both morphotypes of A. fumigatus, conidia and mycelium, its biosynthetic pathway remains poorly understood.…”

“…At the plasma membrane level, SPLs are involved in fungal pathogenesis by regulating plasma membrane fluidity and permeability [66,67,68]. Lipid microdomains are also essential to the trafficking and to the localization of transporter such as efflux pumps that are required for multi-drug resistance [75] or in the modulation of drug-target interaction [70,71].…”

“…This polymer is composed of a main chain of α-mannoside and short side-chains of galactofuranose residues. The GM remains an unique polymer in fungal kingdom since it occurs in three forms: free and released in the culture medium [64], covalently linked to cell wall β-1,3-glucans [66] and membrane bound through a GPI anchor, also called lipogalactomannan (LGM) [67]. The LGM contains the same IPC moiety as GIPC on which the mannan chain is linked to the glucosamine residue (Table I).…”