EC Accepts, published online ahead of print on 19 June 2009

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Eukaryotic Cell doi:10.1128/EC.00013-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

PRODUCTION OF EXTRACELLULAR POLYSACCHARIDES BY CAP MUTANTS OF CRYPTOCOCCUS NEOFORMANS

Jan Grijpstra, Gerrit J Gerwig, Han Wösten, Johannis P Kamerling, and Hans de Cock^*

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

^* To whom correspondence should be addressed. Email: h.decock{at}uu.nl.

The human pathogen Cryptococcus neoformans causes meningo-encephalitis. 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, 59, 60 and 64 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 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 GLC MS 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. 1D ¹H NMR confirmed that the CAP mutants secrete GalXM and also showed the presence of O-acetylated polymers. Taken together, this is the first time that it is shown that CAP mutants secrete GXM-like polymers in addition to GalXM. The low amount of this GXM-like polymer, 1-5% of the total amount of secreted polysaccharides, may explain the acapsular phenotype.