Abstract
The most distinctive feature of the human pathogenic fungus is a polysaccharide capsule that is essential for virulence and is composed primarily of glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). GXM mediates multiple deleterious effects on host immune function, yet relatively little is known about its physical properties. The average mass of Cryptococcus neoformans GXM from four antigenically different strains ranged from 1.7 to 7 x 10(6) daltons as calculated from Zimm plots of light-scattering data. GalXM was significantly smaller than GXM, with an average mass of 1 x 10(5) daltons. These molecular masses imply that GalXM is the most numerous polysaccharide in the capsule on a molar basis. The radius of gyration of the capsular polysaccharides ranged between 68 and 208 nm. Viscosity measurements suggest that neither polysaccharide altered fluid dynamics during infection since GXM behaved in solution as a polyelectrolyte and GalXM did not increase solution viscosity. Immunoblot analysis indicated heterogeneity within GXM. In agreement with this, scanning transmission electron microscopy of GXM preparations revealed a tangled network of two different types of molecules. Mass per length measurements from light scattering and scanning transmission electron microscopy were consistent and suggested GXM molecules self-associate. A mechanism for capsule growth is proposed based on the extracellular release and entanglement of GXM molecules.
Highlights
The polysaccharide capsule that surrounds the fungus promotes survival within the host [5]
The capsule may contain members of the large family of mannoproteins, which are present in the cell wall and are secreted [6, 7]
Knowledge on the structures and physical properties of GXM, GalXM, and the capsule is essential in understanding their role in cryptococcal pathogenesis and for the development of GXM-based therapeutic approaches
Summary
GXM Isolation—C. neoformans was grown in Sabouraud dextrose broth at 30 °C with shaking at 150 rpm. GalXM-containing fractions, identified by a positive reaction in the phenol-sulfuric acid assay for carbohydrates [29], were combined, ultraconcentrated as before, and dialyzed against water for 7 days. Molecular Mass Determination of GXM and GalXM—The polysaccharides were diluted in sterile-filtered, degassed ultra-pure water to the desired concentrations from a freshly made 5 mg/ml stock solution. Electrophoretic Analysis of GXM—A 0.5% (w/v) agarose solution was made by dissolving the agarose in 1ϫ TAE buffer (40 mM Tris base, 20 mM sodium acetate, and 1 mM EDTA in ultrapure water) using microwave heating. GXM and dextran (high fraction; East Kodak Co.) were dissolved in ultrapure water at a concentration of 10 mg/ml for 3– 4 days at room temperature. GalXM was dissolved in ultrapure water at a concentration of 100 mg/ml for 2 days at room temperature. These terms and definitions are in accordance with the 1989 International Union of Pure and Applied Chemistry guidelines for polymers [34]
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