Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, USA, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, USA, Campus Box 8230, St. Louis, USA
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 β1,2-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 (cxt1Δ). 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 β1,2-xylose residues. There was an ~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 β1,2-linked xylose. The GalXM from the mutant strain was also missing a β1,3-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 cxt1Δ is the only strain identified to date with structural alterations of the capsule polysaccharide GalXM.
Fig. 1B, galactoxylomannan, GalXM
13C NMR, 1H NMR, methylation, NMR-2D, GC-MS, acid hydrolysis, biological assays, Southern blotting, extraction, acetylation, reduction, cell growth, precipitation, immunofluorescence
This record contains NMR data of wild-type GalXM (Supplementary Table 2). Supplementary Table 3 also contains NMR data of cxt1Δ GalXM.
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6
6,6,6,6 aDGalp 99.2 69.3 70.5 70.5 69.8 67.6
6,6,6,3,3 bDXylp 105.1 74.0 76.4 70.2 66.1
6,6,6,3,4,2 bDXylp 104.4 73.6 76.6 70.2 66.1
6,6,6,3,4,3,3 bDXylp 101.9 73.9 76.6 70.1 66.1
6,6,6,3,4,3 aDManp 102.6 68.6 79.1 66.0 70.5 61.5
6,6,6,3,4 aDManp 100.6 79.2 76.5 67.2 71.0 61.3
6,6,6,3 aDManp 105.1 71.6 81.5 76.8 74.2 62.0
6,6,6 aDGalp 99.1 68.3 80.9 70.1 69.8 67.6
6,6 aDGalp 99.2 69.3 70.5 70.5 69.8 67.6
6,3,4,3 aDManp 103.1 ? ? ? ? ?
6,3,4 aDManp
6,3 aDManp 105.4 72.3 73.1 78.4 74.2 62.0
6 aDGalp 99.1 68.3 80.9 70.1 69.8 67.6
aDGalp 99.2 69.3 70.5 70.5 69.8 67.6
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6
6,6,6,6 aDGalp 4.98 3.85 3.89 4.04 4.20 3.71-3.93
6,6,6,3,3 bDXylp 4.75 3.39 3.56 3.63 3.29-3.99
6,6,6,3,4,2 bDXylp 4.39 3.31 3.46 3.64 3.34-4.02
6,6,6,3,4,3,3 bDXylp 4.54 3.39 3.49 3.68 3.32-4.02
6,6,6,3,4,3 aDManp 5.22 4.25 4.03 3.81 4.04 3.69-3.74
6,6,6,3,4 aDManp 4.99 4.12 4.07 3.87 4.08 3.84-3.86
6,6,6,3 aDManp 4.69 3.84 3.86 4.30 3.94 3.75-3.80
6,6,6 aDGalp 5.01 4.06 4.01 4.29 4.20 3.71-3.93
6,6 aDGalp 4.98 3.85 3.89 4.04 4.20 3.71-3.93
6,3,4,3 aDManp 5.16 4.07 ? ? ? ?
6,3,4 aDManp
6,3 aDManp 4.66 3.64 3.75 4.09 3.89 3.80-3.89
6 aDGalp 5.01 4.06 4.01 4.29 4.20 3.71-3.93
aDGalp 4.98 3.85 3.89 4.04 4.20 3.71-3.93
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6
6,6,6,6 aDGalp 99.2/4.98 69.3/3.85 70.5/3.89 70.5/4.04 69.8/4.20 67.6/3.71-3.93
6,6,6,3,3 bDXylp 105.1/4.75 74.0/3.39 76.4/3.56 70.2/3.63 66.1/3.29-3.99
6,6,6,3,4,2 bDXylp 104.4/4.39 73.6/3.31 76.6/3.46 70.2/3.64 66.1/3.34-4.02
6,6,6,3,4,3,3 bDXylp 101.9/4.54 73.9/3.39 76.6/3.49 70.1/3.68 66.1/3.32-4.02
6,6,6,3,4,3 aDManp 102.6/5.22 68.6/4.25 79.1/4.03 66.0/3.81 70.5/4.04 61.5/3.69-3.74
6,6,6,3,4 aDManp 100.6/4.99 79.2/4.12 76.5/4.07 67.2/3.87 71.0/4.08 61.3/3.84-3.86
6,6,6,3 aDManp 105.1/4.69 71.6/3.84 81.5/3.86 76.8/4.30 74.2/3.94 62.0/3.75-3.80
6,6,6 aDGalp 99.1/5.01 68.3/4.06 80.9/4.01 70.1/4.29 69.8/4.20 67.6/3.71-3.93
6,6 aDGalp 99.2/4.98 69.3/3.85 70.5/3.89 70.5/4.04 69.8/4.20 67.6/3.71-3.93
6,3,4,3 aDManp 103.1/5.16 ?/4.07 ?/? ?/? ?/? ?/?
6,3,4 aDManp
6,3 aDManp 105.4/4.66 72.3/3.64 73.1/3.75 78.4/4.09 74.2/3.89 62.0/3.80-3.89
6 aDGalp 99.1/5.01 68.3/4.06 80.9/4.01 70.1/4.29 69.8/4.20 67.6/3.71-3.93
aDGalp 99.2/4.98 69.3/3.85 70.5/3.89 70.5/4.04 69.8/4.20 67.6/3.71-3.93
report error
Found 1 record.
Displayed record 1
(later renamed to: Cryptococcus neoformans var. neoformans)
]
wustl.edu