Binding of the Wheat Germ Lectin to Cryptococcus neoformans Suggests an Association of Chitinlike Structures with Yeast Budding and Capsular Glucuronoxylomannan

Abstract

The capsule of Cryptococcus neoformans is a complex structure whose assembly requires intermolecular interactions to connect its components into an organized structure. In this study, we demonstrated that the wheat germ agglutinin (WGA), which binds to sialic acids and ␤-1,4-N-acetylglucosamine (GlcNAc) oligomers, can also bind to cryptococcal capsular structures. Confocal microscopy demonstrated that these structures form round or hooklike projections linking the capsule to the cell wall, as well as capsule-associated structures during yeast budding. Chemical analysis of capsular extracts by gas chromatography coupled to mass spectrometry and high-pH anion-exchange chromatography suggested that the molecules recognized by WGA were firmly associated with the cell wall. Enzymatic treatment, competition assays, and staining with chemically modified WGA revealed that GlcNAc oligomers, but not sialic acids, were the molecules recognized by the lectin. Accordingly, treatment of C. neoformans cells with chitinase released glucuronoxylomannan (GXM) from the cell surface and reduced the capsule size. Chitinase-treated acapsular cells bound soluble GXM in a modified pattern. These results indicate an association of chitin-derived structures with GXM and budding in C. neoformans, which may represent a new mechanism by which the capsular polysaccharide interacts with the cell wall and is rearranged during replication.

Role for Chitin and Chitooligomers in the Capsular Architecture of Cryptococcus neoformans

“…Chitooligomers were recently described by our group as candidate molecules that connect the cell wall to capsular GXM (32). The ability of GXM to bind to ␤-1,4-linked GlcNAc-containing structures was then explored by incubation of the cryptococcal polysaccharide with chitin and elution of bound fractions with increasing concentrations of NaCl.…”

“…These cells were also defective in the synthesis of chitosan, which has also been demonstrated to regulate the retention of cell wall melanin (1). Treatment of C. neoformans acapsular mutants with chitinase affected the incorporation of capsular components into the cell wall (32). Considering that melanin and capsular components are crucial for virulence, these results strongly suggest that GlcNAc-derived molecules are key components of the C. neoformans cell surface.…”

“…To study capsule assembly in C. neoformans, two different reagents related to the metabolism of GlcNAc-containing molecules were used. Chitin and chitooligomers were recently described as cryptococcal components linking the cell wall to the capsule (32). Chitin is not water soluble, but chitooligomers of two to six ␤-1,4-linked GlcNAc units are soluble in aqueous solvents.…”

“…Seemingly, the assembly of the surface envelope of C. neoformans requires the interaction of cell wall components with capsular elements. Some of the cryptococcal cell wall-capsule connectors have been identified, including the structural polysaccharide ␣-1,3-glucan and chitooligomers (29,30,32).…”

“…In a previous study, we demonstrated that ␤-1,4-linked GlcNAc oligomers, which are specifically recognized by the wheat germ agglutinin (WGA), form bridge-like connections between the cell wall and the capsule of C. neoformans (32). In fact, other reports indicate that molecules composed of GlcNAc or its deacetylated derivative play key roles in C. neoformans structural biology.…”