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Eukaryotic Cell, August 2009, p. 1165-1173, Vol. 8, No. 8
1535-9778/09/$08.00+0     doi:10.1128/EC.00013-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Production of Extracellular Polysaccharides by CAP Mutants of Cryptococcus neoformans{triangledown}

Jan Grijpstra,1 Gerrit J. Gerwig,2 Han Wösten,1 Johannis P. Kamerling,2 and Hans de Cock1*

Microbiology, Institute of Biomembranes, Department of Biology, Faculty of Science,1 Bio-Organic Chemistry, Bijvoet Center, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands2

Received 8 January 2009/ Accepted 12 June 2009

The human pathogen Cryptococcus neoformans causes meningoencephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). How capsular polysaccharides are synthesized, transported, and assembled is largely unknown. Previously, it was shown that mutations in the CAP10, CAP59, CAP60, and CAP64 genes result in an acapsular phenotype. Here, it is shown that these acapsular mutants do secrete GalXM and GXM-like polymers. GXM and GalXM antibodies specifically reacted with whole cells and the growth medium of the wild type and CAP mutants, indicating that the capsule polysaccharides adhere to the cell wall and are shed into the environment. These polysaccharides were purified from the medium, either with or without anion-exchange chromatography. Monosaccharide analysis of polysaccharide fractions by gas-liquid chromatography/mass spectrometry showed that wild-type cells secrete both GalXM and GXM. The CAP mutants, on the other hand, were shown to secrete GalXM and GXM-like polymers. Notably, the GalXM polymers were shown to contain glucuronic acid. One-dimensional 1H nuclear magnetic resonance confirmed that the CAP mutants secrete GalXM and also showed the presence of O-acetylated polymers. This is the first time it is shown that CAP mutants secrete GXM-like polymers in addition to GalXM. The small amount of this GXM-like polymer, 1 to 5% of the total amount of secreted polysaccharides, may explain the acapsular phenotype.

* Corresponding author. Mailing address: Microbiology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Phone: (31)-30-2532267. Fax: (31)-30-2513655. E-mail: h.decock{at}uu.nl

{triangledown} Published ahead of print on 19 June 2009.

Eukaryotic Cell, August 2009, p. 1165-1173, Vol. 8, No. 8
1535-9778/09/$08.00+0     doi:10.1128/EC.00013-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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