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Eukaryotic Cell doi:10.1128/EC.00398-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Parallel -helix proteins required for accurate capsule polysaccharide synthesis and virulence in the yeast Cryptococcus neoformans

Dept. of Biochemistry and Biophysics; Dept. of Pharmaceutical Chemistry; University of California; 600 16^th St.; San Francisco, CA 94158-2200

^* To whom correspondence should be addressed. Email: hiten{at}biochem.ucsf.edu.

	Abstract

The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an -1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel -helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional NMR spectroscopy of purified GXM from the mutants indicated that both the wildtype GXM structure as well as novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wildtype capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. Knockout mutants of PBX1 and PBX2 were severely attenuated for virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered.