The Glycosylphosphatidylinositol-Anchored DFG Family Is Essential for the Insertion of Galactomannan into the β-(1,3)-Glucan-Chitin Core of the Cell Wall of Aspergillus fumigatus

Abstract

The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. At the cell wall level, enzyme activities are involved in postsynthesis polysaccharide modifications such as cleavage, elongation, branching, and cross-linking. Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to participate in cell wall biosynthesis and specifically in polysaccharide remodeling. Among these proteins, the DFG family plays an essential role in controlling polar growth in yeast. In the filamentous fungus and opportunistic human pathogen Aspergillus fumigatus, the DFG gene family contains seven orthologous DFG genes among which only six are expressed under in vitro growth conditions. Deletions of single DFG genes revealed that DFG3 plays the most important morphogenetic role in this gene family. A sextuple-deletion mutant resulting from the deletion of all in vitro expressed DFG genes did not contain galactomannan in the cell wall and has severe growth defects. This study has shown that DFG members are absolutely necessary for the insertion of galactomannan into the cell wall of A. fumigatus and that the proper cell wall localization of the galactomannan is essential for correct fungal morphogenesis in A. fumigatusIMPORTANCE The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It is composed mainly of polysaccharides that are synthetized by protein complexes. Enzymes involved in postsynthesis polysaccharide modifications, such as cleavage, elongation, branching, and cross-linking, are essential for fungal life. Here, we investigated in Aspergillus fumigatus the role of the members of the Dfg family, one of the 4 GPI-anchored protein families common to yeast and molds involved in cell wall remodeling. Molecular and biochemical approaches showed that DFG members are required for filamentous growth, conidiation, and cell wall organization and are essential for the life of this fungal pathogen.

Keywords: Aspergillus fumigatus; cell wall; glycobiology.

FIG 1

Expression profiles of seven DFG genes in different stages of A. fumigatus development assayed by qRT-PCR. RNA was extracted from freshly harvested resting (0 h), swollen (4 h), or germinated (6 to 8 h) conidia and mycelium grown in Sabouraud liquid medium and sporulating mycelium grown on Sabouraud solid medium. The relative expression levels of individual DFG genes were analyzed with the 2^ΔΔCT method with the EF1α gene used as an internal control for normalization. Microscopy analysis of developmental stages was followed by staining with calcofluor white. (Values represent means and standard deviations of results from three different experiments. Bars: time zero h [0h], 4h, 6h, and 8h, 1 μm; 16h, 10 μm; 24h, 20 μm.)

FIG 2

Growth of DFG deletion mutant strains on solid medium. (A) Radial growth of the parental and DFG deletion mutant strains on malt agar medium (48 h at 37°C). (B) Mycelial morphology of the parental strain and Δdfg mutants grown on malt agar medium. Hyphae were stained with calcofluor white (bar, 10 μm). (C) Mycelial dry weight of the Δdfg mutants obtained after 48 h of growth on malt agar medium at 37°C. (Values represent means and standard deviations of results from three different experiments; statistically significant differences [P < 0.001] are indicated by an asterisk.)

FIG 3

Morphology of the Δdfg mutants in liquid medium. (A) Visual aspect of Δdfg cultures in Sabouraud medium (10⁶ conidia/50 ml medium) after 24 h at 37°C (bar, 1 cm). (B) Mycelial dry weight of the Δdfg mutants quantified after 24 h of growth in Sabouraud medium at 37°C (10⁷ conidia/50 ml medium). (Values represent means and standard deviations of results from four different experiments; statistically significant differences [P < 0.001] are indicated by an asterisk. ns, not significant.)

FIG 4

Conidial germination of the Δdfg strains. (A) Germination kinetics of the parental and Δdfg mutants. (B) Calcofluor white staining of swollen conidia of the parental strain and Δdfg mutants (incubated in liquid Sabouraud medium for 4 h at 37°C). The estimated proportion (50%) of intracellularly fluorescent swollen conidia was determined by counting 200 conidia for the Δdfg5/2/1/3/4/7 mutant. (Bar, 2 μm.)

FIG 5

Sensitivity of the parental and Δdfg strains to drugs. The levels of sensitivity of the Δdfg mutants to calcofluor white (CFW [50 μg/ml]) and azole compounds (voriconazole [100 ng/ml] and itraconazole [50 ng/ml]) were determined after 72 to 96 h of growth at 37°C on RPMI agar medium.

FIG 6

Labeling of the galactomannan present at the cell surface of the Δdfg mutant complemented strain and the parental strain by an anti-Galf antibody. Mycelia grown for 16 h at 37°C in Sabouraud medium were fixed with p-formaldehyde and subjected to immunolabeling with anti-Galf antibody and anti-rat FITC secondary antibody. (Bar, 100 μm.) Both fluorescence and bright-field images are shown for the Δdfg3 mutant.

FIG 7

Impact of DFG deletion on galactomannan localization. (A) Galactomannan content in the alkali-insoluble fraction of the cell wall (estimated as the amount of mannose in the alkali-insoluble fraction). (B) Lipogalactomannan content extracted from membrane preparation. (C) Galactomannan levels estimated in the culture supernatant (quantified by sandwich enzyme-linked immunosorbent assay [ELISA] using a Platelia kit). Values represent means and standard deviations of data from three different experiments. Statistically significant differences (P < 0.05) are indicated by an asterisk.

FIG 8

Functional complementation of yeast dcw1^ts/Δdfg5 mutant by AfDFG3. (A) Growth on YNB plate medium after 3 days at 30°C and 37°C. (B) Calcofluor white staining of the cells after 2 days of culture in YNB liquid medium at 30°C and 37°C. An average of 15% of enlarged yeast cell (>15 μm) were estimated in the dcw1ts/∆dfg5 mutant by counting 400 cells (bar, 6 μm).