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Cryptococcus neoformans is an opportunistic fungal pathogen that causes a life-threatening disease, cryptococcosis, in immunocompromised individuals (7, 9, 10). Disseminated cryptococcosis is a leading cause of death among patients with AIDS (9, 11). Glucuronoxylomannan (GXM) consists of a linear (13)--D-mannopyranan bearing -D-xylopyranosyl (Xyl), -D-glucopyranosyluronic acid (GlcA), and 6-O-acetyl substituents (2, 6, 12-15). Mannitol is a major metabolite of the yeast, and it and GXM are considered major virulence factors (3, 8). C. neoformans can use Xyl, mannose (Man), and mannitol as a sole carbon source, but the biochemical pathways for their incorporation into GXM produced in a chemically defined medium have not been determined. The assimilation of [1-¹³C]Xyl, [1-¹³C]Man, and [1-¹³C]mannitol was studied by determining the distribution of ¹³C in the saccharide residues of purified GXM by one-dimensional ¹³C nuclear magnetic resonance spectroscopy (NMR).

A stock culture (10 ml) of C. neoformans 409, serotype B (1), was prepared by using a chemically defined standard liquid medium containing natural-abundance glucose (5, 12). This culture (0.2 ml) was used to inoculate a second aliquot (10 ml) of defined medium. After 48 h, the culture was centrifuged (5,000 × g for 5 min); the cells were resuspended in prechilled, glucose-free medium, and the centrifugation was repeated. This procedure was repeated three times to remove as much of the natural-abundance glucose as feasible. The washed cells were suspended in fresh medium (25 ml) containing 500 mg of [1-¹³C]Man (Cambridge Isotope Laboratories, Inc.). After 5 days, the culture was autoclaved for 25 min at 121°C, the cells were removed by centrifugation at 18,000 × g for 1 h, and the supernatant fluid was reserved. The pellet was washed with distilled water (3 ml), and the centrifugation was repeated. The combined culture supernatant fluid and wash (~25 ml) was adjusted to 0.2 M NaCl, and 0.75 g of hexadecyltrimethylammonium bromide was added to the stirred solution at 23°C. A 0.05% solution of hexadecyltrimethylammonium bromide (50 ml) was added slowly with stirring, and the precipitate was recovered by centrifugation at 5,000 × g for 15 min at 23°C. The pellet was triturated with 10% ethanol, and the suspension was centrifuged as described above. The pellet was dissolved in 1 M NaCl (12 ml) by stirring overnight or until the precipitate was completely dissolved. GXM was precipitated by slowly adding 3 volumes of 95% ethanol, and the flask was placed at 4°C. The GXM was recovered by centrifugation at 5,000 × g for 15 min at 4°C. The GXM was dissolved in 1 M NaCl (~12 ml) with stirring until it was completely dissolved. The solution was treated by ultrasonic irradiation (Branson 450 Sonifier) at 80% power and a 40% pulse under nitrogen for 2 h below 20°C, dialyzed, and lyophilized (69 mg; 13.8% yield, based on the amount of glucose added). A portion of the GXM was O-deacetylated at pH 11.5 (NH₄OH) for 24 h at 23°C, dialyzed, and lyophilized (4, 5). A similar procedure was used to prepare GXM from [1-¹³C]mannitol (9% yield) or [1-¹³C]Xyl (3.8% yield; one-half scale and 15-day culture) as the sole carbon source.

GXM or O-deacetylated GXM (9 to 20 mg) was exchanged twice in 99.96% D₂O (Isotec Inc.) with intermediate lyophilization. The sample was dissolved in 0.70 ml of 99.96% D₂O and transferred into a 5-mm NMR tube (Wilmad 535-PP). All NMR experiments were performed at 60°C on a Varian 400 VXR spectrometer equipped with a 5-mm switchable probe operated at 100.58 MHz for ¹³C. The spectral width was 25 kHz, and 1,000 data points were collected at 10-s intervals. The data were processed off line by using the FELIX 95.0 software package (Biosym/Molecular Simulations, San Diego, Calif.) on a Silicon Graphics Indy workstation. ¹³C chemical shifts were measured relative to that of sodium 4,4-dimethyl-4-silapentane-1-sulfonate, which was taken as 0.00 ppm.

The NMR spectrum of the O-deacetylated GXM obtained from the experiment using D-[1-¹³C]Man as the sole carbon source showed that the carbon chain of Man was not altered by the yeast during its conversion to the intermediates required for the synthesis of GXM (UDP-GlcA, UDP-Xyl, and GDP-Man) (16, 17). Figure 1a shows that only the anomeric carbon atoms of the three saccharide constituents of GXM are labeled (13). This is consistent with the usual pathways for the assimilation of Man. The NMR spectrum of the O-deacetylated GXM obtained from the experiment using D-[1-¹³C]mannitol as the sole carbon source showed that the carbon chain of mannitol was not altered by the yeast. Figure 1b shows that only the anomeric center (carbon 1) and carbon 6 of the three saccharide constituents of GXM were labeled (13). The carbonyl carbon of GlcA is not shown in Fig. 1b, but it is clearly visible at 178.8 ppm in Fig. 2b (13). Mannitol has an axis of symmetry that makes carbons 1 and 6 indistinguishable during its assimilation. This is consistent with the usual pathways for the assimilation of mannitol. O-deacetylated GXM obtained from the experiment using D-[1-¹³C]Xyl as the sole carbon source showed that the carbon chain was not incorporated intact by the yeast. Figure 1c shows that only the anomeric carbon and carbon 3 of the three saccharide constituents of GXM are labeled (13). This is consistent with the assimilation of Xyl through the pentophosphate pathway (hexose monophosphate shunt). NMR analysis of the three native GXMs showed that the methyl of the O-acetyl substituent was labeled (Fig. 2a, b, and c, 23.1 ppm). The carboxyl carbonyl of GlcA was labeled when [1-¹³C]mannitol was the sole carbon source (Fig. 2b, 177.8 ppm). These results are consistent with the data discussed above.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Proton-decoupled 13C NMR spectra of O-deacetylated GXM. a, [1-13C]Man; b, [1-13C]mannitol; c, [1-13C]Xyl. The chemical shifts of the carbon atoms detected are as follows: C-1, ~102 to 106 ppm; C-3, 77 to 80 ppm; C-6, 63 ppm.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Proton-decoupled 13C NMR spectra of native GXM. a, [1-13C]Man; b, [1-13C]mannitol; c, [1-13C]Xyl. The chemical shifts of the carbon atoms detected are as follows: carbonyl (carboxyl of GlcA and O-acetyl), 180 ppm; C-1, ~102 to 106 ppm; C-3, 77 to 80 ppm; C-6, 63 ppm; methyl (O-acetyl), 21 ppm.

In summary, the carbon chain of Man was incorporated intact into GXM since ¹³C was observed only in carbon 1 of Man, GlcA, and Xyl. The carbon chain of mannitol was incorporated intact into GXM since ¹³C was observed only in carbons 1 and 6 of the GXM sugar residues. This was expected since mannitol has an axis of symmetry. The carbon chain of Xyl was identified only in carbons 1 and 3 of Man, GlcA, and Xyl. This pattern of labeling is consistent with the assimilation of Xyl through the pentophosphate pathway (hexose monophosphate shunt). This study illustrates the use of ¹³C-labeled monosaccharides for the tracing of their assimilation and incorporation into capsular polysaccharides. The preparation of GXMs specifically enriched with ¹³C (13) will be useful for defining conformation models of these important antigenic polysaccharides (18, 19).