Capsule structural heterogeneity and antigenic variation in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a human pathogenic fungus with a capsule composed primarily of glucuronoxylomannan (GXM) that is important for virulence. Current views of GXM structure postulate a polymer composed of repeating mannose trisaccharide motifs bearing a single beta(1,2) glucuronic acid with variable xylose and O-acetyl substitutions to form six triads. GXM from different strains is notoriously variable in triad composition, but it is not known if the polymer consists of one or more motif-repeating units. We investigated the polymeric organization of GXM by using mass spectrometry to determine if its compositional motif arrangement was similar to that of bacterial capsular polysaccharides, namely, a polymer of a single repeating unit. The results were consistent with, and confirmatory for, the current view that the basic unit of GXM is a repeating mannose trisaccharide motif, but we also found evidence for the copolymerization of different GXM repeating units in one polysaccharide molecule. Analysis of GXM from isogenic phenotypic switch variants suggested structural differences caused by glucuronic acid positional effects, which implied flexibility in the synthetic pathway. Our results suggest that cryptococcal capsule synthesis is fundamentally different from that observed in prokaryotes and employs a unique eukaryotic approach, which theoretically could synthesize an infinite number of structural combinations. The biological significance of this capsule construction scheme is that it is likely to confer a powerful avoidance strategy for interactions with the immune system and phagocytic environmental predators. Consistent with this premise, the antigenic variation of a capsular epitope recognized by a nonprotective antibody was observed under different growth conditions.

FIG. 1.

Diagram of identified structural repeating units for GXM (based on data from reference 5). Repeating units designated M1 to M4 are highly abundant in specific serotypes, listed in parentheses. M5 and M6 do not confer serotype specificity. Mannose, open circles; glucuronic acid, half-filled diamond; xylose, open triangles.

FIG. 2.

Fluorophore-assisted carbohydrate electrophoresis of GXM from strain B-3501 under different hydrolysis conditions at 95°C. Lane 1, 0.3 M TFA for 5 h; lane 2, 0.1 M TFA for 1 h; lane 3, 0.5 M TFA for 1 h; lane 4, 0.5 M TFA for 5 h; lane 5, no TFA for 5 h. The dark band at the lower edge of the figure is unreacted fluorophore. *, visible oligosaccharide bands in lane 1, 3, and 4.

FIG. 3.

Representative mass spectra of the GXM hydrolysis products from strains B-3501 (serotype D), 24067 (serotype D), H99 (serotype A), I23 (serotype B), and 106.97 (serotype C). The bottom right panel shows a close-up of the I23 spectrum between a m/z of 700 and 1,400.

FIG. 4.

The MS-MS spectra of m/z of 1,341 and 1,473 from B-3501 GXM and a m/z of 868 from I23 GXM. The fragmentation patterns show the loss of Man, GlcA, and Xyl from the ions, confirming the oligosaccharide composition. (A) A m/z of 1,341, singly charged ion; (B) m/z of 1,473, singly charged ion; (C) m/z of 868, doubly charged ion.

FIG. 5.

Mass spectrum m/z of 1,001 from B-3501 GXM. This ion represents an oligosaccharide of 2,004 g/mol mass, owing to its double ionization. The double ionization is observable by the difference of a m/z of 0.5 between ionized peaks, instead of the anticipated m/z of 1 for singly ionized peaks. The predicted composition is Man₉GlcA₃, equivalent to three M6 repeating units.

FIG. 6.

¹H-NMR and light scattering analyses of GXM from smooth and mucoid phenotypic switch variants, strain RC-2, do not suggest differences in the repeating units or molecular masses. (A) ¹H-NMR of GXM from the smooth phenotypic variant. (B) Zimm plot of light scattering data obtained from the GXM from the smooth phenotypic variant. (C) ¹H-NMR of GXM from the mucoid phenotypic variant. (D) Zimm plot of light scattering data obtained from the GXM from the mucoid phenotypic variant.

FIG. 7.

The viscosity of the smooth GXM solution decreased more rapidly than the mucoid GXM solution in the presence of salt. (A and B) The relative viscosity increment was determined for various concentrations of GXM from the smooth (A) or mucoid (B) variants. GXM was dissolved in NaCl solutions of different ionic strengths. Ionic strength is listed to the right of the linear regression line for each concentration series. (C and D) Comparison of the relative viscosity increment for GXM from the smooth (solid bars) and mucoid (gray hatched bars) variants at densities of 5 × 10⁻⁵ g/cm³ (C) or 2 × 10⁻⁴ g/cm³ (D). The accurate reproducibility of sample flow times results in minimal standard error for η_i, which is not visible when plotted.

FIG. 8.

Expression of epitopes in GXM for MAbs 12A1 and 21D2 under different growth conditions. (A and B) Percentage of cells stained positive for either MAb 12A1 (solid bars) or 21D2 (gray hatched bars) as determined by FACS analysis. Error bars represent the standard errors. (A) Serotype D strain B-3501; (B) serotype C strain 106.97. The growth conditions are listed by the number of days (D) of incubation and the medium used (agar or broth). Data are averages of two to three experiments. (C and D) Histograms from a representative experiment of the FACS data obtained for MAb 21D2 (thick gray line, unfilled) and MAb 12A1 (thin black line, unfilled) after 3 days of growth on agar (C) and 1 day of growth in broth (D) for strain 106.97. The unstained population is represented by the filled area.