No abstract

Structural studies of cell wall components of the pathogenic yeast Candida albicans have demonstrated the presence of ␤-1,2-linked oligomannosides in phosphopeptidomannan and phospholipomannan. During C. albicans infection, ␤-1,2-oligomannosides play an important role in host/pathogen interactions by acting as adhesins and by interfering with the host immune response. Despite the importance of ␤-1,2-oligomannosides, the genes responsible for their synthesis have not been identified. The main reason is that the reference species Saccharomyces cerevisiae does not synthesize ␤-linked mannoses. On the other hand, the presence of ␤-1,2-oligomannosides has been reported in the cell wall of the more genetically tractable C. albicans relative, P. pastoris. Here we present the identification, cloning, and characterization of a novel family of fungal genes involved in ␤-mannose transfer. Employing in silico analysis, we identified a family of four related new genes in P. pastoris and subsequently nine homologs in C. albicans. Biochemical, immunological, and structural analyses following deletion of four genes in P. pastoris and deletion of four genes acting specifically on C. albicans mannan demonstrated the involvement of these new genes in ␤-1,2-oligomannoside synthesis. Phenotypic characterization of the strains deleted in ␤-mannosyltransferase genes (BMTs) allowed us to describe the stepwise activity of Bmtps and acceptor specificity. For C. albicans, despite structural similarities between mannan and phospholipomannan, phospholipomannan ␤-mannosylation was not affected by any of the CaBMT1-4 deletions. Surprisingly, depletion in mannan major ␤-1,2-oligomannoside epitopes had little impact on cell wall surface ␤-1,2-oligomannoside antigenic expression.

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.