Unraveling the Novel Structure and Biosynthetic Pathway of O-Linked Glycans in the Golgi Apparatus of the Human Pathogenic Yeast Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an encapsulated basidiomycete causing cryptococcosis in immunocompromised humans. The cell surface mannoproteins of C. neoformans were reported to stimulate the host T-cell response and to be involved in fungal pathogenicity; however, their O-glycan structure is uncharacterized. In this study, we performed a detailed structural analysis of the O-glycans attached to cryptococcal mannoproteins using HPLC combined with exoglycosidase treatment and showed that the major C. neoformans O-glycans were short manno-oligosaccharides that were connected mostly by alpha 1,2-linkages but connected by an alpha 1,6-linkage at the third mannose residue. Comparison of the O-glycan profiles from wild-type and uxs1 Delta mutant strains strongly supports the presence of minor O-glycans carrying a xylose residue. Further analyses of C. neoformans mutant strains identified three mannosyltransferase genes involved in O-glycan extensions in the Golgi. C. neoformans KTR3, the only homolog of the Saccharomyces cerevisiae KRE2/MNT1 family genes, was shown to encode an alpha 1,2-mannosyltransferase responsible for the addition of the second mannose residue via an alpha 1,2-linkage to the major O-glycans. C. neoformans HOC1 and HOC3, homologs of the Saccharomyces cerevisiae OCH1 family genes, were shown to encode alpha 1,6-mannosyltransferases that can transfer the third mannose residue, via an alpha 1,6-linkage, to minor O-glycans containing xylose and to major O-glycans without xylose, respectively. Moreover, the C. neoformans ktr3 Delta mutant strain, which displayed increased sensitivity to SDS, high salt, and high temperature, showed attenuated virulence in a mouse model of cryptococcosis, suggesting that the extended structure of O-glycans is required for cell integrity and full pathogenicity of C. neoformans.