Characterization of two novel cryptococcal mannoproteins recognized by immune sera

Abstract

Host defenses against the encapsulated yeast Cryptococcus neoformans involve both humoral and cell-mediated immunity. Mannoproteins (MPs) are a heterogeneous class of immunodominant glycoproteins which have been only incompletely characterized. In this study, we report on the molecular features of two novel MPs that are recognized by serum antibodies during cryptococcosis. After fractionation of extracellular cryptococcal products, MPs reacted more strongly than other components with sera from C. neoformans-infected AIDS patients. Further fractionation and Western blot analysis of MPs evidenced the presence of highly reactive bands with molecular masses of 250, 125, 115, and 84 kDa. The 115- and 84-kDa bands contained significant amounts of N-linked oligosaccharides, as shown by decreased molecular mass after peptide-N-glycosidase F treatment. N-terminal amino acid sequences of the two bands were used to search C. neoformans nucleotide databases. Homologous genomic sequences were used to synthesize DNA probes and isolate cDNA clones containing the full-length genes, which were designated MP84 and MP115. Both genes showed the presence of a serine/threonine-rich region, a potential site for heavy glycosylation. MP84 and MP115 showed homology with, respectively, polysaccharide deacetylases and carboxylesterases from other organisms. Recombinant, deglycosylated proteins expressed in Escherichia coli still reacted with sera from patients, albeit more weakly than natural MPs, indicating that at least some of the reactive epitopes were retained in the recombinant forms. In conclusion, we identified two novel MPs that are important targets of antibody responses during cryptococcosis. These data may be useful to devise alternative immunity-based strategies to control the disease.

FIG. 1.

Reactivity of human sera against C. neoformans CneF determined by ELISA. Sera were obtained from 12 HIV-positive cryptococcosis patients (black triangles). Sera obtained from laboratory workers (gray triangles) and other healthy volunteers (white triangles) were used as controls. All serum samples were used at a dilution of 1:200. One experiment, representative of three, each performed in duplicate, is shown. Bars represent means ± standard deviations. OD, optical density.

FIG. 2.

ELISA inhibition by different fractions obtained from C. neoformans CneF. Plates were coated with CneF, and pooled plasma from cryptococcosis patients was mixed with putative inhibitors before addition to the wells. Whole CneFs from Cap 67 and B. dermatitidis supernatants were used as positive and negative controls, respectively. (A) Inhibitory effects of ConA-binding or nonbinding fractions. (B) Purification of the ConA-binding fraction into three peaks by DEAE anion-exchange chromatography. Bars indicate pooled fractions obtained using a linear gradient of NaCl (right-hand axis). (C) Inhibitory effects of the different DEAE peaks. One experiment, representative of three, each performed in duplicate, is shown.

FIG. 3.

SDS-PAGE and Western blot analysis of DEAE peak 3. Material (2 μg) from peak 3 was run in 12% polyacrylamide gels and stained with Coomassie (A). Proteins resolved by electrophoresis were also transferred to nitrocellulose membranes and incubated with biotin-conjugated ConA (B) with a pool of sera from patients (C) or with a pool of sera from experimentally infected mice (D). BSA was included as a control. Panel E shows the effects of PNGase F on material from peak 3 using immunoblotting with a pool of sera from patients. To obtain a better resolution of the bands of interest, this gel was run for longer than usual (5 versus 2 h), which explains the different band pattern in this panel relative to the other panels. Pooled human and murine sera were used at a dilution of 1:100 in TTBS. Numbers on the left indicate the molecular masses in kilodaltons.

FIG. 4.

Analysis of MP115 and MP84 amino acid (a.a.) sequences. The signal sequence (shaded), catalytic domain (hatched), and Ser/Thr-rich region (cross-hatch) are shown. Sites of N-glycosylation are indicated by a Y. A putative GPI anchor site (ω) in the C-terminal portion of MP84 is also shown.

FIG. 5.

Alignment of similar amino acid sequences from MP84, MP98, and d25. White lettering on a black background indicates identity. Amino acids within the box indicate the polysaccharide deacetylase domain.

FIG. 6.

Analysis of recombinant MP84 and MP115 proteins. SDS-PAGE was followed by Coomassie staining (A), ConA blot (B), Western blot with a pool of sera from patients (C), Western blot with a pool of normal sera from volunteers (D), Western blot with a pool of sera from infected mice (E), or Western blot with a pool of sera preinfection mice (F). All pooled sera were used at a 1:100 dilution. Purified recombinant 84-kDa and 115-kDa proteins were loaded onto lanes 1 and 2, respectively. Numbers on the left indicate the molecular masses in kilodaltons.