Candida albicans is a common, harmless yeast in the human digestive tract that also causes severe systemic fungal infection in hospitalized patients. Its cell-wall surface displays a unique glycolipid called phospholipomannan (PLM). The ability of PLM to stimulate tumor necrosis factor (TNF)-alpha production by J774 mouse cells correlates with the activation of nuclear factor (NF)-kappaB. We examined the involvement of Toll-like receptors (TLRs) in PLM-dependent stimulation. Compared with wild-type cells, which produced large amounts of TNF-alpha after incubation with PLM, the deletion of the TLR4 and TLR6 genes led to a limited alteration of the PLM-induced response. Deletion of the TLR2 gene completely abolished the cell response. Surface expression of PLM is a phylogenic trait of C. albicans, and the recognition of PLM by TLRs, together with the unique pathogenic potential of C. albicans, suggests that this molecule may be a member of the pathogen-associated molecular pattern family.

Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages that perturbation of the GPI biosynthesis results in hypersensitivity to host defences. IntroductionCandida albicans is a ubiquitous commensal and a major opportunistic human pathogen that causes superficial infections such as oropharyngeal candidiasis, which is a frequent complication in patients with human immunodeficiency virus (HIV) infections. In other clinical conditions, such as neutropenia, C. albicans can invade host tissues and cause severe, often fatal, disseminated infections (Odds, 1988). Owing to the associated morbidity and mortality, prophylactic or prolonged antifungal treatments have been used in patients at risk of developing candidaemia, such as patients undergoing chemotherapy or patients with recurrent superficial infections such as HIV. This has led to the emergence of isolates with decreased susceptibility to the commercially available antifungal agents, especially azoles (Fridkin and Jarvis, 1996).In C. albicans, the dimorphic conversion between the yeast and hyphal forms is thought to be critical for pathogenesis, as mutations that block the transition to either form attenuate virulence (Braun and Johnson, 1997;Lo et al., 1997). Several studies have revealed some of the genetic components involved in the control of the morphogenetic switch (Kohler and Fink, 1996;Sharkey et al., 1999;Young et al., 2000). Unlike most laboratories that work on pathways initially described in Saccharomyces cerevisiae, we used Yarrowia lipolytica as a model for morphogenesis. This non-conventional yeast also undergoes complete yeast-to-hyphae transition like C. albicans and can be genetically manipulated (Barth and Gaillardin, 1997;Dominguez et al., 2000). We screened a series of recessive Fil -mutants that were unable to display a morphogenetic switch (Richard et al., 2001) and found a GPI7 homologue, a member of the glycosylphosphatidylinositol (GPI) gene family, which is involved in the biosynthesis of GPI anchors (Benghezal et al., 1995).GPI anchoring is a eukaryotic mechanism for attaching proteins to the cell surface. Proteins destined to be GPI anchored have conserved features, an N-terminal signal sequence for localization to the endoplasmic reticulum (ER) and a C-terminal signal sequence for attachment of the GPI anchor. Shortly after protein synthesis in the ER, SummaryGlycosylphosphatidylinositol (GPI)-anchored proteins are involved in cell wall integrity and cell-cell interactions. We disrupted the Candida albicans homologue of the Saccharomyces cerevisiae GPI7/LAS21 gene, which encodes a GPI anchormodifying activity. In the mutant and on solid media, the yeast-to-hyphae transition was blocked, whereas chlamydospore formation was enhanced. However, the morphogenetic switch was normal in liquid medium. Abnormal budding patterns, cytokinesis and cell shape were observed in both liquid and solid media. The cell wall structure was also modified in the mutants...

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.