Core N-Glycan Structures Are Critical for the Pathogenicity of Cryptococcus neoformans by Modulating Host Cell Death.

Cryptococcus neoformans is a human-pathogenic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised individuals. To investigate the roles of N-glycan core structure in cryptococcal pathogenicity, we constructed mutant strains of C. neoformans with defects in the assembly of lipid-linked N-glycans in the luminal side of the endoplasmic reticulum (ER). Deletion of ALG3 (alg3Δ), which encodes dolichyl-phosphate-mannose (Dol-P-Man)-dependent α-1,3-mannosyltransferase, resulted in the production of truncated neutral N-glycans carrying five mannose residues as a major species. Despite moderate or nondetectable defects in virulence-associated phenotypes in vitro, the alg3Δ mutant was avirulent in a mouse model of systemic cryptococcosis. Notably, the mutant did not show defects in early stages of host cell interaction during infection, including attachment to lung epithelial cells, opsonic/nonopsonic phagocytosis, and manipulation of phagosome acidification. However, the ability to drive macrophage cell death was greatly decreased in this mutant, without loss of cell wall remodeling capacity. Furthermore, deletion of ALG9 and ALG12, encoding Dol-P-Man-dependent α-1,2-mannosyltransferases and α-1,6-mannosyltransferases, generating truncated core N-glycans with six and seven mannose residues, respectively, also displayed remarkably reduced macrophage cell death and in vivo virulence. However, secretion levels of interleukin-1ß (IL-1ß) were not reduced in the bone marrow-derived dendritic cells obtained from Asc- and Gsdmd-deficient mice infected with the alg3Δ mutant strain, excluding the possibility that pyroptosis is a main host cell death pathway dependent on intact core N-glycans. Our results demonstrated N-glycan structures as a critical feature in modulating death of host cells, which is exploited by as a strategy for host cell escape for dissemination of C. neoformansIMPORTANCE We previously reported that the outer mannose chains of N-glycans are dispensable for the virulence of C. neoformans, which is in stark contrast to findings for the other human-pathogenic yeast, Candida albicans Here, we present evidence that an intact core N-glycan structure is required for C. neoformans pathogenicity by systematically analyzing alg3Δ, alg9Δ, and alg12Δ strains that have defects in lipid-linked N-glycan assembly and in in vivo virulence. The alg null mutants producing truncated core N-glycans were defective in inducing host cell death after phagocytosis, which is triggered as a mechanism of pulmonary escape and dissemination of C. neoformans, thus becoming inactive in causing fatal infection. The results clearly demonstrated the critical features of the N-glycan structure in mediating the interaction with host cells during fungal infection. The delineation of the roles of protein glycosylation in fungal pathogenesis not only provides insight into the glycan-based fungal infection mechanism but also will aid in the development of novel antifungal agents.

Use of the cysteine-repressible HpMET3 promoter as a novel tool to regulate gene expression in Hansenula polymorpha.

OBJECTIVES: The promoter of HpMET3, encoding an ATP sulfurylase, was evaluated for its potential as a repressible promoter to downregulate the expression of target genes in the thermotolerant, methylotrophic yeast Hansenula polymorpha. RESULTS: The expression of lacZ under the control of the 0.6 kb HpMET3 promoter was efficiently downregulated by cysteine, but not by methionine or sulfate. The HpMET3 promoter was used to generate a conditional mutant of the HpPMT2 gene encoding an O-mannosyltransferase, which is involved in post-translational protein modification. The addition of 0.5 mM cysteine adversely affected the growth of the conditional HpMET3(p)-Hppmt2 mutant strain by downregulating transcription of HpPMT2 to approx. 40 % of the normal levels, indicating that the HpPMT2 gene is essential for cell viability. However, the HpMET3 promoter was neither induced nor repressed in the heterologous host Saccharomyces cerevisiae. CONCLUSION: Our results reveal that the cysteine-repressible HpMET3 promoter is a useful tool that downregulates the expression of various genes in H. polymorpha.