The pathogenic yeast Candida albicans has the ability to synthesize unique sequences of ␤-1,2-oligomannosides that act as adhesins, induce cytokine production, and generate protective antibodies. Depending on the growth conditions, ␤-1,2-oligomannosides are associated with different carrier molecules in the cell wall. Structural evidence has been obtained for the presence of these residues in the polysaccharide moiety of the glycolipid, phospholipomannan (PLM). In this study, the refinement of purification techniques led to large quantities of PLM being extracted from Candida albicans cells. A combination of methanolysis, gas chromatography, mass spectrometry, and nuclear magnetic resonance analyses allowed the complete structure of PLM to be deduced. The lipid moiety was shown to consist of a phytoceramide associating a C 18 /C 20 phytosphingosine and C 25 , C 26 , or mainly C 24 hydroxy fatty acids. The spacer linking the glycan part was identified as a unique structure: -Man-P-Man-Ins-P-. Therefore, in contrast to the major class of membranous glycosphingolipids represented by mannose diinositol phosphoceramide, which is derived from mannose inositol phosphoceramide by the addition of inositol phosphate, PLM seems to be derived from mannose inositol phosphoceramide by the addition of mannose phosphate. In relation to a previous study of the glycan part of the molecule, the assignment of the second phosphorus position leads to the definition of PLM ␤-1,2-oligomannosides as unbranched linear structures that may reach up to 19 residues in length. Therefore, PLM appears to be a new type of glycosphingolipid, which is glycosylated extensively through a unique spacer. The conferred hydrophilic properties allow PLM to diffuse into the cell wall in which together with mannan it presents C. albicans ␤-1,2-oligomannosides to host cells.Sphingolipids are ubiquitous and essential components of living cells found mainly on the outer leaflet of plasma membranes (1-3). Along with their role in membrane permeability and fluidity, they have also been shown to act as second messengers produced in response to various stress situations, which regulate basic processes such as cell cycle control, apoptosis (4), cell-cell interactions (1), and immune response (5).Sphingolipids are ceramide structures composed of a long chain base whose amino group is amide-linked to various fatty acids. This basic structure as well as those derived by the addition of polar groups such as phosphocholine or carbohydrates to the ceramide may vary according to the species (6 -8).It has been established that sphingolipids from fungal and mammalian cells display important differences in their fine structure and biosynthetic pathways (9). Fungal species incorporate phytosphingosine (PHS) 1 and presumably dihydrosphingosine in the ceramide moieties instead of the sphingosine used mainly by mammals and also have longer fatty acids (C 24 -C 26 instead of C 16 -C 18 ) (9). Fungi then preferentially add inositol to the ceramide group, leading to the family of ...

Studies on Candida albicans phospholipomannan have suggested a novel biosynthetic pathway for yeast glycosphingolipids. This pathway is thought to diverge from the usual pathway at the mannose-inositol-phospho-ceramide (MIPC) step. To confirm this hypothesis, a C. albicans gene homologue for the Saccharomyces cerevisiae SUR1 gene was identified and named MIT1 as it coded for GDP-mannose:inositol-phospho-ceramide mannose transferase. Two copies of this gene were disrupted. Western blots of cell extracts revealed that strain mit1⌬ contained no PLM. Thin layer chromatography and mass spectrometry confirmed that mit1⌬ did not synthesize MIPC, demonstrating a role of MIT1 in the mannosylation of C. albicans IPCs. As MIT1 disruption prevented downstream ␤-1,2 mannosylation, mit1⌬ represents a new C. albicans mutant affected in the expression of these specific virulence attributes, which act as adhesins/immunomodulators. mit1⌬ was less virulent during both the acute and chronic phases of systemic infection in mice (75 and 50% reduction in mortality, respectively). In vitro, mit1⌬ was not able to escape macrophage lysis through down-regulation of the ERK1/2 phosphorylation pathway previously shown to be triggered by PLM. Phenotypic analysis also revealed pleiotropic effects of MIT1 disruption. The most striking observation was a reduced ␤-mannosylation of phosphopeptidomannan. Increased ␤-mannosylation of mannoproteins was observed under growth conditions that prevented the association of ␤-oligomannosides with phosphopeptidomannan, but not with PLM. This suggests that C. albicans has strong regulatory mechanisms associating ␤-oligomannoses with different cell wall carrier molecules. These mechanisms and the impact of the different presentations of ␤-oligomannoses on the host response need to be defined.

In a series of studies, we have shown that Candida albicans synthesizes a glycolipid, phospholipomannan (PLM), which reacted with antibodies specific for ␤-1,2-oligomannosides and was biosynthetically labeled by [ 3 H]mannose, [3 H]palmitic acid, and [ 32 P]phosphorus. PLM has also been shown to be released from the C. albicans cell wall and to bind to and stimulate macrophage cells. In this study, we show by thin layer chromatography scanning of metabolically radiolabeled extracts that the C. albicans PLM corresponds to a family of mannose and inositol co-labeled glycolipids. We describe the purification process of the molecule and the release of its glycan fraction through alkaline hydrolysis. Analysis of this glycan fraction by radiolabeling and methylation-methanolysis confirmed the presence of inositol and of 1,2-linked mannose units. NMR studies evidenced linear chains of ␤-1,2-oligomannose as the major PLM components. Mass spectrometry analysis revealed that these chains were present in phosphoinositolmannosides with degrees of polymerization varying from 8 to 18 sugar residues. The PLM appears as a new type of eukaryotic inositol-tagged glycolipid in relationship to both the absence of glucosamine and the organization of its glycan chains. This first structural evidence for the presence of ␤-1,2-oligomannosides in a glycoconjugate other than the C. albicans phosphopeptidomannan may have some pathophysiological relevance to the adhesive, protective epitope, and signaling properties thus far established for these residues.