Compositional analysis of the major capsular polysaccharides of Cryptococcus neoformans by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

Cryptococcus neoformans is a predominantly saprophytic yeast that can cause serious infections, mostly in individuals with acquired immunodeficiency syndrome (AIDS). A prominent virulence factor of Cryptococcus neoformans is its capsule. This is constituted mainly of glucans; the capsular polysaccharide, glucuronoxylomannan (GXM) (composed of mannose, xylose, glucuronic acid), and at least two minor carbohydrate antigens, galactoxylomannan (GalXM) and mannoproteins (MP). In this paper we present an optimized HPAEC-based method for the rapid and effective determination of the complete carbohydrate constituents of GXM and GalXM. Cryptococcus neoformans strain A 9759 serotype A and the acapsular mutant CAP 67 were grown in a totally dialyzable synthetic medium at 30 degrees C. Culture supernatants were subjected to tangential filtration (10,000-M-r-cut-off) to separate high molecular weight cryptococcal products from medium constituents. The supernatants were dialyzed against phosphate buffer saline (PBS) containing sodium azide (0.02%) and concentrated to a carbohydrate concentration of 1.5 mg/mL. GXM and GalXM were isolated and purified as described in the experimental section. For neutral sugar and uronic acid composition analysis, 2.5 mg polysaccharide fractions were hydrolyzed with 2 M trifluoroacetic acid (TFA, 100 degrees C, 6 h), followed by lyophilization, then the residues were analyzed by HPAEC coupled with pulsed amperometric detection (PAD). Separations were carried out using either a CarboPac PA 100 or a CarboPac PA 10 column and matching guard column (all from Dionex, Sunnyvale, CA, USA). Employing PA 100, as well as PA 10 column, baseline separation of the neutral monosaccharides galactose, glucose, xylose, mannose and glucuronic acid were obtained. Monosaccharide and uronic acid compositions of GXM and GalXM determined by this method were found to be highly accurate.