Endoplasmic reticulum alpha-glycosidases of Candida albicans are required for N glycosylation, cell wall integrity, and normal host-fungus interaction

Abstract

The cell surface of Candida albicans is enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc(3)Man(9)GlcNAc(2) N-glycan is processed by alpha-glucosidases I and II and alpha1,2-mannosidase to generate Man(8)GlcNAc(2). This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose. In Saccharomyces cerevisiae, CWH41, ROT2, and MNS1 encode for alpha-glucosidase I, alpha-glucosidase II catalytic subunit, and alpha1,2-mannosidase, respectively. We disrupted the C. albicans CWH41, ROT2, and MNS1 homologs to determine the importance of N-oligosaccharide processing on the N-glycan outer-chain elongation and the host-fungus interaction. Yeast cells of Cacwh41Delta, Carot2Delta, and Camns1Delta null mutants tended to aggregate, displayed reduced growth rates, had a lower content of cell wall phosphomannan and other changes in cell wall composition, underglycosylated beta-N-acetylhexosaminidase, and had a constitutively activated PKC-Mkc1 cell wall integrity pathway. They were also attenuated in virulence in a murine model of systemic infection and stimulated an altered pro- and anti-inflammatory cytokine profile from human monocytes. Therefore, N-oligosaccharide processing by ER glycosidases is required for cell wall integrity and for host-fungus interactions.

FIG. 1.

Cell and colony morphology in the Cacwh41Δ, Carot2Δ, and Camns1Δ null mutants. (A) Cell morphology after growth at 30°C for 16 h in YPD medium, demonstrating clumping of cells in the Cacwh41Δ (HMY19), Carot2Δ (HMY12), and Camns1Δ (HMY5) null mutants. Scale bars, 10 μm. (B and C) Colony morphology after 5 days growth at 30°C on YPD agar plates (B) or solid Spider medium (C). Scale bars, 1 mm.

FIG. 2.

N-glycosylation defects in Cacwh41Δ, Carot2Δ, and Camns1Δ null mutants. The electrophoretic mobility of β-N-acetylhexosaminidase under nondenaturing conditions was examined. The strains tested include NGY152 (wild type), HMY19 (Cacwh41Δ), HMY20 (Cacwh41Δ + CaCWH41), HMY12 (Carot2Δ), HMY13 (Carot2Δ + CaROT2), HMY5 (Camns1Δ), and HMY6 (Camns1Δ + CaMNS1).

FIG. 3.

Sensitivity of Cacwh41Δ, Carot2Δ, and Camns1Δ null mutants to cell-wall-perturbing agents. Wild type (▴), null mutants (○), and reintegrant controls (•) strains were tested for sensitivity to cell-wall-perturbing agents by using the microdilution method. The strains tested were the Cacwh41Δ (HMY19) (A), Carot2Δ (HMY12) (B), and Camns1Δ (HMY5) (C) null mutants. Error bars indicate the means ± the standard deviation (n = 3). The results are pooled data from duplicate experiments.

FIG. 4.

Activation of the cell integrity pathway in glycosidase null mutants assessed by Western analysis. Protein extracts were prepared from cells in mid-exponential phase. As a positive control for activation of the cell integrity pathway, the strains were treated with Calcofluor White (100 μg/ml) as indicated. Extracts are from the following strains: NGY152 (wild type), HMY19 (Cacwh41Δ), HMY20 (Cacwh41Δ + CaCWH41), HMY12 (Carot2Δ), HMY13 (Carot2Δ + CaROT2), HMY5 (Camns1Δ), and HMY6 (Camns1Δ + CaMNS1). Equal loading was confirmed by Ponceau S staining and determining the intensity of nonspecific bands.

FIG. 5.

Cytokine stimulation by Cacwh41Δ, Carot2Δ, and Camns1Δ null mutants. Human PBMC were stimulated 24 h with 10⁶ yeast cells/ml and the TNF (A) and IL-6 and IL-10 (B) concentrations were determined. The experiments for panel A were carried out in the absence (□) or presence (▪) of laminarin, a blocking agent of the β-glucan/dectin-1 recognition pathway. The strains tested are NGY152 (wild type), HMY19 (Cacwh41Δ), HMY20 (Cacwh41Δ + CaCWH41), HMY12 (Carot2Δ), HMY13 (Carot2Δ + CaROT2), HMY5 (Camns1Δ), and HMY6 (Camns1Δ + CaMNS1). The results are pooled data from four volunteers. Error bars indicate the means ± the standard deviation. *, Significant differences in the mutant compared to the wild type (P < 0.05).