Abstract
The opportunistic yeast Cryptococcus neoformans causes serious disease in humans and expresses a prominent polysaccharide capsule that is required for its virulence. Little is known about how this capsule is synthesized. We previously identified a beta1,2-xylosyltransferase (Cxt1p) with in vitro enzymatic activity appropriate for involvement in capsule synthesis. Here, we investigate C. neoformans strains in which the corresponding gene has been deleted (cxt1Delta). Loss of CXT1 does not affect in vitro growth of the mutant cells or the general morphology of their capsules. However, NMR structural analysis of the two main capsule polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), showed that both were missing beta1,2-xylose residues. There was an approximately 30% reduction in the abundance of this residue in GXM in mutant compared with wild-type strains, and mutant GalXM was almost completely devoid of beta1,2-linked xylose. The GalXM from the mutant strain was also missing a beta1,3-linked xylose residue. Furthermore, deletion of CXT1 led to attenuation of cryptococcal growth in a mouse model of infection, suggesting that the affected xylose residues are important for normal host-pathogen interactions. Cxt1p is the first glycosyltransferase with a defined role in C. neoformans capsule biosynthesis, and cxt1Delta is the only strain identified to date with structural alterations of the capsule polysaccharide GalXM.
Highlights
Do not eradicate the organism from neural tissues [1]
Cxt1p plays a primary role in GalXM synthesis and is involved in GXM production; its deletion leads to attenuation of growth in a mouse model of infection
The pathways involved in synthesis of C. neoformans capsule polysaccharides have proven difficult to elucidate, and to date no glycosyltransferases have been clearly implicated in this process [26]
Summary
Mans CAP67 strain as previously described [14]. A separate replacement cassette was generated in the same manner, except. Strain Backcrosses—To obtain a cxt1⌬ strain in an encapsulated strain background, the CAP67 cxt1⌬ strain was crossed to JEC20 on V8 agar as described in Ref. 17, and the resulting spores were plated on solid medium containing nourseothricin. The precipitate was recovered by centrifugation and the pellet was dissolved overnight in 150 ml of 2 M NaCl. The resulting solution was dialyzed (3,500 MWCO) against 4 liters of diH2O for 5 days with daily water changes, frozen, and lyophilized to dryness. An aliquot of each sample was suspended in 200 l of dimethyl sulfoxide and stirred for 2 days before permethylation by the method of Ciucanu and Kerek [23] Following permethylation, this material was hydrolyzed with 2 M trifluoroacetic acid (2 h in a sealed tube at 121 °C), reduced with NaBD4, and acetylated using acetic anhydride/trifluoroacetic acid. The lungs were harvested and serial dilutions were plated on YPD agar to quantitate colony forming units
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