Extension of O -Linked Mannosylation in the Golgi Apparatus Is Critical for Cell Wall Integrity Signaling and Interaction with Host Cells in Cryptococcus neoformans Pathogenesis.

The human-pathogenic yeast Cryptococcus neoformans assembles two types of O -linked glycans on its proteins. In this study, we identified and functionally characterized the C. neoformans CAP6 gene, encoding an α1,3-mannosyltransferase responsible for the second mannose addition to minor O -glycans containing xylose in the Golgi apparatus. Two cell surface sensor proteins, Wml1 ( W SC/ M id2- l ike) and Wml2, were found to be independent substrates of Cap6-mediated minor or Ktr3-mediated major O -mannosylation, respectively. The double deletion of KTR3 and CAP6 ( ktr3 Δ cap6 Δ) completely blocked the mannose addition at the second position of O -glycans, resulting in the accumulation of proteins with O -glycans carrying only a single mannose. Tunicamycin (TM)-induced phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) was greatly decreased in both ktr3 Δ cap6 Δ and wml1 Δ wml2 Δ strains. Transcriptome profiling of the ktr3 Δ cap6 Δ strain upon TM treatment revealed decreased expression of genes involved in the Mpk1-dependent cell wall integrity (CWI) pathway. Consistent with its defective growth under several stress conditions, the ktr3 Δ cap6 Δ strain was avirulent in a mouse model of cryptococcosis. Associated with this virulence defect, the ktr3 Δ cap6 Δ strain showed decreased adhesion to lung epithelial cells, decreased proliferation within macrophages, and reduced transcytosis of the blood-brain barrier (BBB). Notably, the ktr3 Δ cap6 Δ strain showed reduced induction of the host immune response and defective trafficking of ergosterol, an immunoreactive fungal molecule. In conclusion, O -glycan extension in the Golgi apparatus plays critical roles in various pathobiological processes, such as CWI signaling and stress resistance and interaction with host cells in C. neoformans. IMPORTANCE Cryptococcus neoformans assembles two types of O -linked glycans on its surface proteins, the more abundant major O -glycans that do not contain xylose residues and minor O -glycans containing xylose. Here, we demonstrate the role of the Cap6 α1,3-mannosyltransferase in the synthesis of minor O -glycans. Previously proposed to be involved in capsule biosynthesis, Cap6 works with the related Ktr3 α1,2-mannosyltransferase to synthesize O -glycans on their target proteins. We also identified two novel C. neoformans stress sensors that require Ktr3- and Cap6-mediated posttranslational modification for full function. Accordingly, the ktr3 Δ cap6 Δ double O- glycan mutant strain displays defects in stress signaling pathways, CWI, and ergosterol trafficking. Furthermore, the ktr3 Δ cap6 Δ strain is completely avirulent in a mouse infection model. Together, these results demonstrate critical roles for O -glycosylation in fungal pathogenesis. As there are no human homologs for Cap6 or Ktr3, these fungus-specific mannosyltransferases are novel targets for antifungal therapy.