Identification of a New Family of Genes Involved in β-1,2-Mannosylation of Glycans in Pichia pastoris and Candida albicans

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.

Genome sequence of the recombinant protein production host Pichia pastoris

“…Indeed, we did not find an ortholog of the S. cerevisiae gene MNN1 (encoding the alpha-1,3-mannosyltransferase) in the Pichia genome. However, Pichia glycoproteins can in some cases be modified with b-1,2-mannose residues 30 , reminiscent of antigenic epitopes on the Candida albicans cell wall 31 . We find the patented P. pastoris AMR2 b-mannosyltransferase in the genome, and three homologs, thus providing the basis for reducing the levels of this undesired glycan modification.…”

Conformational Analysis of .BETA.-1,2-Linked Mannobiose to Mannoheptaose, Specific Antigen of Pathogenic Yeast Candida albicans

“…These results suggest that the oligosaccharide is one of the virulence factors in Leishmania species as well as Candida species, and that enzymes involved in the synthesis are potential targets for new therapies for these infections. Recently, Mille et al 25) identified the b-1,2-mannosyltransferase gene family in C. albicans. They showed that none of these sequences contains the conserved sugar-binding region containing the DXD (D, aspartic acid; X, any amino acid) motif (pfam Gly_transf_sug; PF04488) found in many families of glycosyltransferases.…”

Glycannes pariétaux de levures et anticorps spécifiques

“…majorité des mannoprotéines de paroi. Les raisons pour lesquelles ce système est aussi complexe restent inconnues, mais son existence suggère une certaine importance biologique pour C. albicans [17], d'autant que, selon des mécanismes qui restent obscurs, les anticorps anti-β-mannosides protègent des candidoses expérimentales vaginales et systémiques.…”