Article Figures & Data
Figures
- FIG. 1.
Association of GXM and chitin-like molecules. (A) Binding of GXM to chitin followed by elution with a step gradient of NaCl resulted in different GXM peaks. (B) Experimental model used to detect β-1,4-linked GlcNAc oligomers in complex with GXM in culture supernatants. WGA was used to capture chitooligomers. Lectin-coated wells of ELISA microplates were incubated with culture supernatants for further detection of carbohydrate complexes using a MAb raised to GXM. (C) Chitotriose was added to C. neoformans cultures at different concentrations, and the chitooligomer-GXM complexes were captured by WGA on ELISA microplates and detected by anti-GXM monoclonal antibody. (D) Two peaks corresponding to chitotriose-GXM complexes in C. neoformans culture supernatant could be separated by affinity binding to an agarose-WGA column. AP, alkaline phosphatase.
- FIG. 2.
Morphological and antibody-reacting features of cryptococcal capsules in yeast cells grown in the presence of (GlcNAc)3. India ink counterstaining was used to analyze capsule morphology under control conditions (A) or after growth in the presence of the chitooligomer (6.5 μg/ml) (B). Scale bar, 10 μm. Yeast cells with defective capsules that became permeable to India ink (C; arrows) were not recognized by MAb 18B7 (D). Scale bar, 10 μm. Growth of C. neoformans in the presence of a higher (GlcNAc)3 concentration (25 μg/ml) results in the formation of capsules with aberrant morphology that are not permeable to India ink penetration (E). These structures are still recognized by MAb 18B7 (F). Scale bar, 10 μm. Cellular structures stained in blue and green show, respectively, recognition sites of calcofluor and MAb 18B7.
- FIG. 3.
Analysis of the C. neoformans capsule after growth in minimal medium (A and B) or in the same medium supplemented with 1 M NaCl (C and D). (A and B) India ink counterstaining and fluorescence analysis showing the regular profiles of capsule expression and staining of GXM and chitooligomers by MAb 18B7 (green fluorescence) and WGA (red fluorescence), respectively. (C) India ink counterstaining showing that the vast majority of the yeast cells have no visible capsule. (D) Only the yeast cells with normal capsules are recognized by the antibody to GXM. Most cells show the typical profile of staining of β-1,4-linked GlcNAc oligomers by WGA. Scale bar, 10 μm.
- FIG. 4.
Interaction of GXM with chitooligomers of differential lengths affects the effective diameter of the fungal polysaccharide. A shift of the distribution curves to areas of higher diameter was observed after incubation of GXM in the presence of all β-1,4-linked GlcNAc oligosaccharides in comparison to control systems in the absence of chitooligomers.
- FIG. 5.
Inhibition of glucosamine 6-phosphate synthesis by Nva-FMDP affects the morphology and cell wall-capsule connections in C. neoformans. (A) Major morphological features of control cells (regular morphology) or of yeast cells cultivated in the presence of Nva-FMDP. (B) Distribution of the morphological alterations shown in panel A after growth of C. neoformans with Nva-FMDP. (C) Off-center cell bodies suggesting a possible loose connection between the cell wall and the capsule as induced by the inhibitor. (D) Growth in the presence of increasing concentrations of Nva-FMDP rendered yeast cells more susceptible to capsule release by sonication. Asterisks denote inhibitor concentrations at which capsule release by sonication was significantly more efficient than under control conditions (no inhibitor).
- FIG. 6.
Nva-FMDP interferes with chitin synthesis in C. neoformans cells. In this assay, calcofluor staining (blue) shows a regular chitin expression; lack of calcofluor reactivity indicates defective chitin biosynthesis. Staining in red and green indicates, respectively, detection of chitooligomers and GXM. Panels A to C show the morphological features of India ink-counterstained C. neoformans after growth with Nva-FMDP. Panels D to F show the corresponding fluorescence images. Regions of the cell surface with defective chitin biosynthesis were associated with reduced capsule size (arrows). Panels A and D, B and E, and C and F represent three different experiments prepared under the same conditions and producing similar results. Scale bar, 1 μm.
- FIG. 7.
Capsule fibrils are affected by growth of C. neoformans in the presence of Nva-FMDP. SEM of control cells at low (A) and high (B) magnifications show the regular aspects of capsule fibers. These structures were extracted from yeast cells with DMSO, and their effective diameters were determined by light scattering (C). SEM of fungal cells grown with Nva-FMDP (D and E) showed a reduction in the size of capsular fibers, which was confirmed by light-scattering analysis of DMSO extracts of capsule material (F). Scale bars, 10 μm (A and D) and 1 μm (B and E).
- FIG. 8.
Monosaccharide composition of capsular extracts of C. neoformans after growth under control conditions or in the presence of Nva-FMDP. Capsular extracts from C. neoformans cultivated in the presence of the inhibitor showed eightfold less glucuronic acid and a 10-fold increase in the content of glucose.
- FIG. 9.
WGA reactivity of C. neoformans cells in infected lungs and brains. (A) Tissue sections were stained with fluorescent WGA and analyzed microscopically. Results obtained in vivo were compared with the profiles of lectin reactivity observed in vitro. A limited staining of specific surface sites (localized pattern; single asterisks) was observed in untreated control cells compared to the diffuse pattern (double asterisks) of lectin reactivity in chitinase-treated cells. (B) Quantitative analysis revealed that the same fluorescence pattern observed for chitinase-treated cells in vitro is abundant in infected lungs, whereas the lectin binding profile of untreated control cells is more often detected in infected brains (P < 0.0001).
