Cryptococcus neoformans CAP59 (or Cap59p) is involved in the extracellular trafficking of capsular glucuronoxylomannan

Abstract

Several genes are essential for Cryptococcus neoformans capsule synthesis, but their functions are unknown. We examined the localization of glucuronoxylomannan (GXM) in strain B-3501 and in cap59 mutants B-4131 and C536. Wild-type strain B-3501 showed a visible capsule by India ink staining and immunofluorescence with anticapsular monoclonal antibodies (MAbs) 12A1 and 18B7. B-4131, a mutant containing a missense mutation in CAP59, showed no capsule by India ink staining but revealed the presence of capsular polysaccharide on the cell surface by immunofluorescence. The cap59 gene deletion mutant (C536), however, did not show a capsule by either India ink staining or immunofluorescence. Analysis of cell lysates for GXM by enzyme-linked immunosorbent assay revealed GXM in C536 samples. Furthermore, the epitopes recognized by MAbs 12A1, 2D10, 13F1, and 18B7 were each detected in the cytoplasm of all strains by immunogold electron microscopy, although there were differences in location consistent with differences in epitope synthesis and/or transport. In addition, the cells of B-3501 and B-4131, but not those of the cap59 deletant, assimilated raffinose or urea. Hence, the missense mutation of CAP59 in B-4131 partially hampered the trafficking of GXM but allowed the secretion of enzymes involved in hydrolysis of raffinose or urea. Furthermore, the cell diameter and volume for strain C536 are higher than those for strain B-3501 or B-4131 and may suggest the accumulation of cellular material in the cytoplasm. Our results suggest that CAP59 is involved in capsule synthesis by participating in the process of GXM (polysaccharide) export.

FIG. 1.

Phenotypic differences in ultrastructures of strains B-3501 (a), B-4131 (b), and C536 (c). Globose electron-dense inclusion were observed in 0 of 6, 0 of 6, and 8 of 11 cells of strains B3501, B4131, and C536, respectively. *, capsule; Cw, cell wall; Cy, cytoplasm. The arrow points to electron-dense inclusions found in vacuoles of C536. Bar, 1 μm.

FIG. 2.

India ink preparation and immunofluorescence after staining with MAbs 12A1 and 18B7 of strains B-3501, B-4131, and C536. Bar, 5 μm.

FIG. 3.

Analysis of the presence of GXM in cell lysates of B-3501, B-4131, and C536 as determined by capture ELISA. Cell lysates from B-3501 and B-4131 samples include capsular material. O.D., optical density.

FIG. 4.

Intracellular localization of GXM epitopes recognized by MAbs 2D10 (a), 12A1 (b), 13F1 (c), 18B7 (d), 5C11 (e), and ricin 45 (f) in strains B-3501, B-4131, and C536 as determined by immunogold labeling. *, capsule; Cw, cell wall; Cy, and cytoplasm. Bar, 1 μm.

FIG. 5.

Localization of the 12A1 epitope (arrows) throughout the cell cytoplasm (a), cell wall (b and c), and capsule (d). Bar, 1 μm.