A multifunctional mannosyltransferase family in Candida albicans determines cell wall mannan structure and host-fungus interactions

Abstract

The cell wall proteins of fungi are modified by N- and O-linked mannosylation and phosphomannosylation, resulting in changes to the physical and immunological properties of the cell. Glycosylation of cell wall proteins involves the activities of families of endoplasmic reticulum and Golgi-located glycosyl transferases whose activities are difficult to infer through bioinformatics. The Candida albicans MNT1/KRE2 mannosyl transferase family is represented by five members. We showed previously that Mnt1 and Mnt2 are involved in O-linked mannosylation and are required for virulence. Here, the role of C. albicans MNT3, MNT4, and MNT5 was determined by generating single and multiple MnTDelta null mutants and by functional complementation experiments in Saccharomyces cerevisiae. CaMnt3, CaMnt4, and CaMnt5 did not participate in O-linked mannosylation, but CaMnt3 and CaMnt5 had redundant activities in phosphomannosylation and were responsible for attachment of approximately half of the phosphomannan attached to N-linked mannans. CaMnt4 and CaMnt5 participated in N-mannan branching. Deletion of CaMNT3, CaMNT4, and CaMNT5 affected the growth rate and virulence of C. albicans, affected the recognition of the yeast by human monocytes and cytokine stimulation, and led to increased cell wall chitin content and exposure of beta-glucan at the cell wall surface. Therefore, the MNT1/KRE2 gene family participates in three types of protein mannosylation in C. albicans, and these modifications play vital roles in fungal cell wall structure and cell surface recognition by the innate immune system.

FIGURE 1.

Members of C. albicansMNT1 participate in phosphomannosylation. A, C. albicans MNT1 family members restore the ability of S. cerevisiae ktr6Δ null mutant to bind AB. S. cerevisiae BY4741 (wild type, WT), NGS1 (Scktr6Δ), NGS2 (Scktr6Δ + CaMNT1), NGS3 (Scktr6Δ + CaMNT2), NGS4, (Scktr6Δ + CaMNT3), NGS5 (Scktr6Δ + CaMNT4), and NGS6 (Scktr6Δ + CaMNT5) were grown in either SC medium (open bars) or SC medium added with 2% galactose and 3% raffinose (closed bars), and the ability of the cells to bind the AB was determined. The results obtained with all of the mutant strains are significantly different of those generated with the wild type strain (p < 0.01). B, C. albicans null mutants lacking of both CaMNT3 and CaMNT5 have a reduced ability to bind AB. C. albicans NGY152 (wild type), NGY158 (Camnt1Δ), NGY145 (Camnt2Δ), NGY146 (Camnt3Δ), NGY313 (Camnt4Δ), NGY147 (Camnt5Δ), NGY337 (Camnt1-Camnt2Δ), NGY335 (Camnt1-Camnt2Δ + CaMNT1), NGY336 (Camnt1-Camnt2Δ + CaMNT2), NGY509 (Camnt3-Camnt4Δ), NGY510 (Camnt3-Camnt4Δ + CaMNT3), NGY511 (Camnt3-Camnt4Δ + CaMNT4), NGY1227 (Camnt3-Camnt5Δ), NGY1228 (Camnt3-Camnt5Δ + CaMNT3), NGY1229 (Camnt3-Camnt5Δ + CaMNT5), NGY516 (Camnt4-Camnt5Δ), NGY517 (Camnt4-Camnt5Δ + CaMNT4), NGY518 (Camnt4-Camnt5Δ + CaMNT5), NGY527 (Camnt3-Camnt4-Camnt5Δ), NGY528 (Camnt3-Camnt4-Camnt5Δ + CaMNT3), NGY529 (Camnt3-Camnt4-Camnt5Δ + CaMNT4), NGY530 (Camnt3-Camnt4-Camnt5Δ + CaMNT5) NGY535 (Camnt1-Camnt2-Camnt3-Camnt4-Camnt5Δ), NGY536 (Camnt1-Camnt2-Camnt3-Camnt4-Camnt5Δ + CaMNT1), and NGY537 (Camnt1-Camnt2-Camnt3-Camnt4-Camnt5Δ + CaMNT2) were grown in YPD medium, and the ability to bind AB was measured before (open bars) or after β-elimination of O-linked mannans with 0.1 m NaOH (closed bars). The data represent the means ± S.D. of three independent assays performed in triplicate. *, p < 0.05.

FIGURE 2.

Electrophoretic mobility shift assays of HexNAcase. The HexNAcase activity was induced as described under “Experimental Procedures,” and protein samples before (A) or after (B) deglycosylation with endoglycosidase H were separated by nondenaturing electrophoresis, and enzyme activity was revealed with naphthyl-GlcNAc and Fast Blue. The strains used were C. albicans NGY152 (wild type, WT), NGY516 (Camnt4-Camnt5Δ), NGY517 (Camnt4-Camnt5Δ + CaMNT4), NGY518 (Camnt4-Camnt5Δ + CaMNT5), NGY527 (Camnt3-Camnt4-Camnt5Δ), NGY528 (Camnt3-Camnt4-Camnt5Δ + CaMNT3), NGY529 (Camnt3-Camnt4-Camnt5Δ + CaMNT4), and NGY530 (Camnt3-Camnt4-Camnt5Δ + CaMNT5). The continuous and dashed lines indicate the electrophoretic mobility of normal and underglycosylated HexNAcase, respectively.

FIGURE 3.

Calcofluor White binding assays. In A, C. albicans cells were stained with Calcofluor White (CFW) and examined under differential interference contrast (DIC) or fluorescence microscopy. Scale bar, 10 μm. In B, the fluorescence associated with cells was quantified using the Openlab system. The data represent the means of three independent assays performed by duplicate, analyzing 50 cells/strain/experiment. *, p < 0.01. The strains were: column 1, NGY152 (wild type, WT); column 2, NGY1227 (Camnt3-Camnt5Δ); column 3, NGY1228 (Camnt3-Camnt5Δ + CaMNT3); column 4, NGY1229 (Camnt3-Camnt5Δ + CaMNT5); column 5, NGY527 (Camnt3-Camnt4-Camnt5Δ); column 6, NGY528 (Camnt3-Camnt4-Camnt5Δ + CaMNT3); column 7, NGY529 (Camnt3-Camnt4-Camnt5Δ + CaMNT4); and column 8, NGY530 (Camnt3-Camnt4-Camnt5Δ + CaMNT5).

FIGURE 4.

Cytokine stimulation by C. albicansMNT1 null mutants. PBMCs were coincubated with either heat-killed (A) or live (B) yeast cells in a ratio monocyte: yeast 5: 1 for 24 h, and the cytokine concentrations were determined. The strains were: NGY152 (wild type, WT), NGY1227 (Camnt3-Camnt5Δ), NGY1228 (Camnt3-Camnt5Δ + CaMNT3), NGY1229 (Camnt3-Camnt5Δ + CaMNT5), NGY516 (Camnt4-Camnt5Δ), NGY517 (Camnt4-Camnt5Δ + CaMNT4), NGY518 (Camnt4-Camnt5Δ + CaMNT5), NGY527 (Camnt3-Camnt4-Camnt5Δ), NGY528 (Camnt3-Camnt4-Camnt5Δ + CaMNT3), NGY529 (Camnt3-Camnt4-Camnt5Δ + CaMNT4), and NGY530 (Camnt3-Camnt4-Camnt5Δ + CaMNT5). The data represent the means ± S.D. *, p < 0.05.

FIGURE 5.

Cytokine stimulation upon blocking of dectin-1 with glucan-P. The strains and conditions are as in Fig. 4, but PBMCs were incubated with 10 μg/ml glucan-P for 60 min at 37 °C prior to stimulation with C. albicans cells. The data represent the means ± S.D. *, p < 0.05. WT, wild type.