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.

capsular polysaccharides, Xylose, Cryptococcus neoformans, cryptococcosis, gene deletion

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,2	bDGlcpA	103.9	74.6	77.6	73.8	78.5	?
3,3	aDManp	102.3	79.8	79.8	68.6	75.5	63.2
3	aDManp	104.2	72.2	80.9	68.6	75.7	63.2
2	bDXylp	105.5	75.2	77.9	71.6	67.5
	aDManp	103.1	80.4	78.5	69.2	76.0	63.2


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,2	bDGlcpA	4.45	3.34	3.45	3.56	3.66	-
3,3	aDManp	5.19	4.24	4.07	3.79	3.94	3.78-3.85
3	aDManp	5.14	4.18	3.93	3.77	3.88	3.78-3.85
2	bDXylp	4.34	3.27	3.39	3.61	3.23-3.95
	aDManp	5.14	4.18	4.00	3.67	3.81	3.78-3.85


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,2	bDGlcpA	103.9/4.45	74.6/3.34	77.6/3.45	73.8/3.56	78.5/3.66	
3,3	aDManp	102.3/5.19	79.8/4.24	79.8/4.07	68.6/3.79	75.5/3.94	63.2/3.78-3.85
3	aDManp	104.2/5.14	72.2/4.18	80.9/3.93	68.6/3.77	75.7/3.88	63.2/3.78-3.85
2	bDXylp	105.5/4.34	75.2/3.27	77.9/3.39	71.6/3.61	67.5/3.23-3.95
	aDManp	103.1/5.14	80.4/4.18	78.5/4.00	69.2/3.67	76.0/3.81	63.2/3.78-3.85

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O[C@@H]2[C@H](O)[C@@H](O[C@H]3[C@H](O)[C@@H](CO)O[C@H](*)[C@H]3O[C@@H]3OC[C@@H](O)[C@H](O)[C@H]3O)O[C@H](CO)[C@H]2O)[C@H]1O[C@@H]1O[C@H](C(=O)O)[C@@H](O)[C@H](O)[C@H]1O

794.662 g/mol (C₂₉H₄₆R₂O₂₅, R = next and previous repeats, not counted in mol weight)