Article Figures & Data
Figures
- Fig 1
Interaction of cryptococcal GXM with chitin as assayed by SEM. (A) SEM analysis of aggregates formed after incubation of yeast cells with chitin in minimal medium. Yeast capsular fibers (spherical forms) are apparently in close contact with the surface of a chitin particle (larger and amorphous particle). At least 10 microscopic fields were examined in each system. Scale bar, 10 μm. (B) Treatment of chitin particles with soluble GXM before incubation with C. neoformans resulted in decreased number of yeast cells per chitin particle. Scale bar, 2 μm. (C) Quantification of the results of the systems illustrated in panels A (Cn + Chitin [where Cn is C. neoformans]) and B (Cn + GXM-Chitin) and also the effects of pretreatments of yeast cells with a monoclonal antibody to GXM (18B7-Cn + Chitin) and of chitin particles with a chitin-binding lectin (Cn + WGA-Chitin). Results are expressed as means ± standard deviations.
- Fig 2
Chitin-GXM interactions involve noncovalent bonds and large GXM fibers. (A) Incubation of C. neoformans cells with chitin followed by exposure to chaotropic molecules (NaCl and urea) or a chelating agent (EDTA) inhibits aggregate formation in comparison with systems that were exposed to PBS. (B) Formation of the mixed aggregates is impaired in the presence EDTA. (C) The use of GXM fractions of different molecular masses to treat chitin before exposure to C. neoformans cells reveals that samples of higher molecular masses are more effective in inhibiting the formation of cell-polysaccharide aggregates. Results are expressed as means ± standard deviations.
- Fig 3
GXM O-acetylation and glucuronic acid-associated negative charges are not involved in interaction with chitin. (A) Incubation of standard (Cn) or de-O-acetylated C. neoformans cells (de-O-Cn) with chitin results in similar levels of association. (B) The use of native, de-O-acetylated (de-O-GXM) or carboxyl-reduced (de-COOH) GXM fractions to treat chitin before exposure to C. neoformans cells reveals that all samples had the same efficacy as inhibitors of formation of cell-polysaccharide aggregates. Results are expressed as means ± standard deviations.
- Fig 4
Requirement of amino groups and N-acetylation for the interaction of chitin with GXM. (A) Replacement of chitin by cellulose results in decreased levels of attachment of C. neoformans (Cn) cells to chitin and reduced sensitivity to inhibition by previous exposure to GXM. (B) A chitooligomer [(GlcNAc)3] and GlcNAc are more efficient than chitosan as inhibitors of the interaction of C. neoformans with chitin, as determined by comparison of systems in which fungal cells were treated with PBS or the amino sugars before incubation with chitin with systems in which cryptococci were treated with minimal medium alone. Results are expressed as means ± standard deviations.
- Fig 5
Formation of glycan complexes composed of GXM and chitin-derived structures during incubation of C. neoformans in culture medium. (A) Flow cytometry-based experimental model designed for detection of glycan complexes. (B) Reactivity of 18B7-coated spheres with TRITC-WGA after incubation with culture supernatants. (C) Analysis of the reactivity of antibody-coated spheres with TRITC-WGA after incubation with sterile medium or after a 30-min incubation of encapsulated or acapsular cells in the medium. (D) Effect of a chitinase inhibitor (3-methylxanthine) on the formation of glycan complexes in cultures of C. neoformans incubated for 30 min at room temperature. Images are representative of three different experiments.
- Fig 6
Formation of glycan complexes composed of GXM and chitin-derived structures during infection of macrophages by C. neoformans. (A) Reactivity of 18B7-coated spheres with TRITC-WGA after incubation with macrophage supernatants, incubated with C. neoformans for the periods indicated at the top of each panel. (B) Kinetics of formation of the hybrid complexes based on the values obtained in panel A. Images are representative of three different experiments.
- Fig 7
Cytokine determination in the lungs of mice exposed to PBS (control), chitooligomer [(GlcNAc)3; GlcNAc Oligo], GXM, or glycan complex structures. Analysis of the production of IL-10 (A), IL-17 (B), and TNF-α (C) revealed that the glycan complex structures are much more efficient than chitin-like structures or GXM alone in their ability to induce pulmonary cytokine responses.
Tables
- Table 1
Analytical models for analysis of the interaction of GXM with chitin
Structural aspect assessed Approach used Molecular specificity of C. neoformans-chitin interaction Pretreatment of the chitin particles with WGA or with soluble GXM; pretreatment of yeasts with anti-GXM Involvement of noncovalent bonds Treatment of chitin-C. neoformans complexes with urea, NaCl, and EDTA Requirement of O-acetyl groups of GXM Pretreatment of the chitin particles with de-O-acetylated GXM and comparison with results obtained with samples of native GXM; treatment of yeast cells with de-O-acetylating agent, followed by incubation with chitin particles Requirement of carboxyl groups of GXM Pretreatment of the chitin particles with carboxyl-reduced GXM, followed by comparison with results obtained with samples of native GXM Influence of GXM molecular mass Treatment of chitin-C. neoformans complexes with EDTA; pretreatment of chitin particles with fractions containing GXM with different molecular masses Requirement of chitin amino groups Replacement of chitin by cellulose in SEM tests Requirement of N-acetyl groups from chitin Pretreatment of yeast cells with chitosan
