A fungal protein organizes both glycogen and cell wall glucans

Abstract

Glycogen is a glucose storage molecule composed of branched α-1,4-glucan chains, best known as an energy reserve that can be broken down to fuel central metabolism. Because fungal cells have a specialized need for glucose in building cell wall glucans, we investigated whether glycogen is used for this process. For these studies, we focused on the pathogenic yeast Cryptococcus neoformans, which causes ~150,000 deaths per year worldwide. We identified two proteins that influence formation of both glycogen and the cell wall: glycogenin (Glg1), which initiates glycogen synthesis, and a protein that we call Glucan organizing enzyme 1 (Goe1). We found that cells missing Glg1 lack α-1,4-glucan in their walls, indicating that this material is derived from glycogen. Without Goe1, glycogen rosettes are mislocalized and β-1,3-glucan in the cell wall is reduced. Altogether, our results provide mechanisms for a close association between glycogen and cell wall.

Keywords: Cryptococcus neoformans; cell wall; glycogen; glycogenin; glycosyltransferase.

Fig. 1.

Glycogen synthesis in S. cerevisiae (Sc) and C. neoformans (Cn). (A) Schematic of glycogen synthesis and degradation in Sc and Cn. (B) Serially diluted Cn cells of the indicated strains, grown on SD+Gal for 72 h and exposed to iodine vapor. (C) Sc glg1 glg2 (Sc ΔΔ) or WT cells were transformed as indicated at right with control vector (CV) or plasmids encoding Cn Glg1 or Goe1 (two independent transformants for each). Transformants were grown on SD-Trp+Gal for 72 h and exposed to iodine.

Fig. 2.

Mutation of Glg1 impacts glycogen levels. (A) Partial T-Coffee alignments of glycogenin and Goe1 sequences (64). Red arrow, conserved lysine; blue arrow, acceptor tyrosine. (B) Iodine assay of the indicated strains, grown on SD+Gal for 72 h at 37 °C and exposed to iodine vapor. (C and D) Enzymatic quantitation of glycogen content in cell supernatants. Data are shown as mean + SEM of 3 (C) or 6 (D) biological replicates; ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA. Limits of detection (LOD), 0.043 µg (C) and 0.038 µg (D). (E) Electron micrographs of Thiery-stained cells. CW, cell wall; PM, plasma membrane; arrowheads, example rosettes. Images are to the same scale. (Scale bar, 500 nm.)

Fig. 3.

Goe1 affects glycogen abundance, rosette localization, and cell wall integrity. (A) Iodine assay of the indicated strains grown on SD+Gal ± sorbitol at 37 °C for 96 h. (B) Electron micrographs of Thiery-stained cells. (Scale bar, 500 nm.) (C) Quantitation of rosette density within 500 nm of the cytosol-PM interface (Materials and Methods). Symbols, value for individual images (10 per strain); black bar, mean; ****P < 0.0001 by t test.

Fig. 4.

Goe1 is likely a plasma membrane protein with an extracellular N terminus. (A) Schematic of possible Goe1 topologies. (B) α-Goe1 immunoblot, 25 µg total protein loaded per lane. First three lanes: lysates of the indicated strains. Last three lanes: lysates of WT cells either incubated with biotin (intact + B), lysed prior to incubation with biotin (lysate + B), or never exposed to biotin (no B). Molecular weights of standards (center lane) are indicated at right. (C) α-Goe1 immunoblot of samples labeled as above. Cells were treated with Triton X-100 at the indicated % prior to biotin labeling. P, pellet fractions (25 µg/lane); red triangle, biotinylated species of Goe1 (see text). S, supernatant fractions (16 µL/lane), except final lane (25 µg pellet for size reference).

Fig. 5.

Goe1 affects α-1,4 and β-1,3-glucans in the cell wall. (A) Enzymatic quantitation of α-1,4-glucan in the alkali-insoluble cell wall fraction. Data are shown as mean + SEM of three biological replicates; ns, not significant; ***P < 0.001; ****P < 0.0001 by ANOVA; nd, not detected; LOD = 0.058. (B) NMR spectra of alkali-insoluble cell wall fractions. Peaks assigned as in Lowman et al. Red box and Inset, β-1,3-glucan peak. The signals in the region between 3 and 4 ppm on the right side of the spectra correspond to carbohydrate ring protons and cannot be assigned based on 1D proton NMR alone. The peaks near 5.4 ppm are of unknown origin. (C and D) Enzymatic quantitation of β-1,3-glucan in the alkali-insoluble (AI, Panel C) or alkali-soluble (AS, Panel D) cell wall fractions of the indicated strains. Mean + SEM of three biological replicates shown; ns, not significant; **P < 0.01 by ANOVA; nd, not detected; LOD = 0.046 in (C); LOD = 0.081 in (D).

Fig. 6.

Loss of Glg1 or Goe1 reduces cryptococcal pathogenesis. (A) Intracellular survival in THP-1 cells shown as mean ± SEM of two biological replicates; *P < 0.05 by two-way ANOVA; CFU, colony-forming units. (B and C) Lung burden resulting from 9-d infection with 1.25 × 10⁴ cells of each indicated strain. Horizontal bar, median CFU; **P < 0.01; ****P < 0.0001 by ANOVA. The red dotted line indicates initial inoculum.

Fig. 7.

Schematic of Goe1 and Glg1 products in the cell. The product of Glg1 (α-1,4-glucan, orange hexagon) and the putative glycan product of Goe1 (teal hexagon) are implicated in both glycogen and cell wall synthesis in WT. Goe1’s product affects cell wall architecture either (A) directly, by its incorporation into the wall (WT and glg1Δ) or (B) indirectly; in this example, Goe1 glycosylates an intracellular target (red squiggle), a protein involved in cell wall construction.