earticle

영어

The human fungal pathogen Cryptococcus neoformans assembles O-linked mannosyl glycans on its proteins in two types; Ktr3p-meidated major-O-glycans without xylose and Cap6-mediated minor-O-glycans without xylose, respectively1. The C. neoformans ktr3Δcap6Δ double mutant strain was constructed to block completely O-mannosylation extension in the Golgi. The ktr3Δcap6Δ strain accumulated the O-glycans carrying only a single mannose and displayed defective growth under several stress conditions, accompanied with completely attenuated virulence in a mouse model of cryptococcosis. By RNA-Seq-based transcriptome analysis, we investigated the effect of defective O-mannosylation on the stress-responsive signaling pathways associated with virulence. The comparative transcriptome analysis of the wild-type (WT) and ktr3Δcap6Δ cells under normal growth and tunicamycin (TM) treatment conditions, respectively, revealed the decreased induction of Mpk1p-and calcineurin-Crz1-mediated cell wall integrity (CWI) signaling pathways. Furthermore, we examined the possible change of physiochemical properties of cell surface in the ktr3Δcap6Δ cells, by profile analysis of N-linked glycans assembled on cell wall proteins, flow cytometry analysis of cell wall components, and filipin-staining analysis of erogosterol. Although no apparent difference in the total amount and overall length of N-glycans was observed, the N-glycan profile of ktr3Δ cap6Δ showed a marginal change with the decreased neutral glycan peaks and increased acidic glycan peaks compared to that of the WT strain. We also observed the slightly increased amount of chitin in ktr3Δ cap6Δ, which might reflect the compensation mechanism of defective CWI. Notably, the ktr3Δ cap6Δ cells showed defective trafficking of ergosterol, an immunoactive fungal molecule, from the ER to the plasma membrane. In conclusion, our results demonstrate the importance of extended O-glycan structure for cell wall integrity signaling and physiochemical properties of cell surface, which critically affect the survival in the host cell environments and the interaction with host cells of C. neoformans during infection process.