Robert Cherniak

Structure and antigenic activity of the capsular polysaccharide of Cryptococcus neoformans serotype A

Abstract The glucuronoxylomannan (GXM) of C. neoformans serotype A contained molar ratios of xylose: mannose: glucuronic acid: O -acetyl of 2:5:1:2. The xylose and glucuronic acid occurred as single pyranosyl residues and were oxidized by periodate; the Smith-degraded product was an insoluble mannan. Mannose was linked (1→3) with three out of five mannose residues (1→2)-substituted with one glucuronosyl and two single xylosyl end groups. A repeating octasaccharide was proposed as the structure. GXM was separated into species-specific and serotype-specific components by chromatography on a serotype-D-antibody agarose column, but these showed no obvious differences in molar ratios of the monosaccharides. Decreased serological activity was observed when GXM was de- O -acetylated or carboxy-reduced, but not when carboxy-esterified. The Smith-degraded mannan was inactive. Autohydrolysis caused a gradual release of xylose and some mannose, and a decline in serological activity. Xylose and glucuronic acid were implicated in a single antigenic group since selective inactivation of either residue led to loss of more than half of the activity. Extensive crossreactions were observed with the GXMs from C. laurentii and Tremella mesenterica , but not with types II or XIV pneumococcal polysaccharides.

A galactoxylomannan antigen of Cryptococcus neoformans serotype A

Abstract The capsular polysaccharide of Cryptococcus neoformans serotype A was fractionated into two chemically and serologically distinct heteroglycans by differential precipitation with cetyltrimethylammonium bromide (CTAB). The major, viscous, acidic glucuronoxylomannan (GXM, 88% w/w) was precipitated with CTAB, while a previously undetected galactoxylomannan (GalXM, 12% w/w) remained in solution. GalXM is characterized by ( i ) molar ratios of galactose:mannose: xylose:glucuronic acid of 1.9:1.8:1.0.2 and 2% of O -acetyl; ( ii ) a molecular weight of 275,000 ± 25,000, estimated by gel-permeation chromatography; ( iii ) extensive degradation by NaIO 4 ; ( iv ) precipitation in gel by a lectin, concanavalin A, indicating nonreducing mannosyl termini; and ( v ) a distinct, immunoprecipitin arc in counterimmunoelectrophoresis. GalXM was further purified by gel-permeation or ion-exchange chromatography.

Structural characterization of the galactoxylomannan of Cryptococcus neoformans Cap67

The galactoxylomannan (GalXM) obtained from the culture supernatant of an acapsular mutant of Cryptococcus neoformans Cap67 was purified by Concanavalin A affinity, ion-exchange, and gel-filtration chromatographies. The structure of GalXM was determined by methylation analysis and by 1D and 2D NMR spectroscopic studies of the intact polysaccharide and of the oligosaccharide fragments generated by Smith degradation and by acetolysis. GalXM is a complex polysaccharide with an alpha-(1-->6) -galactan backbone. The polysaccharide is branched at c-3 of alternate Gal units of the backbone. C-3 is the point of attachment of the oligosaccharide side chains comprised of alpha-D-Man- (1-->3)-alpha-D-Man-(1-->4)- beta-D-Gal-substituted with zero to three terminal beta-Xyl residues as shown in the following structure: [formula: see text].

Cell-wall glucans of Cryptococcus neoformans CAP 67

Purified cell walls derived from Cryptococcus neofromans Cap 67, an acapsular mutant, consisted of 86% Glc and 7.3% GlcNAc. The integrity of the cell walls was disrupted in three successive extractions with 60% 4-methylmorpholine N-oxide (4-MMNO) at 120 degrees. Four 4-MMNO-soluble D-glucopyranans were isolated. Released within 0.5 h was water-insoluble Gi-1, followed by two water-soluble Gs fractions and water-insoluble Gi-2 over 17.5 h. A 4-MMNO-insoluble residue, containing 27% of GlcNAc, was also isolated. Gi-1 and Gi-2 were isolated as precipitates during dialysis of 4-MMNO extracts and were each reduced with NaBH4 to permit their investigation in alkaline solution. Gs-1 and Gs-2 were separated by ion-exchange chromatography of the water-soluble fractions. The structures of the D-glucopyranans were determined by 13C-n.m.r. spectroscopy and by g.l.c.-mass spectrometry of their per-O-methylated derivatives. Gi-1 was a (1----3)-alpha-D-glucopyranan (97%) with some (1----4)-D-glucosidic linkages (3%) and no chain-branching. Gs-1 and Gs-2 were (1----6)-beta-D-glucopyranans branched at O-3 (10-12%) with beta-D-Glcp-(1----3)-beta-D-Glcp side chains. Gs-2 may have approximately 2% more chain branching than Gs-1. Gi-2 was a D-glucopyranan with 80% of its structure like that of Gi-1, and 20% like that of Gs-1 and -2; the water-insolubility of Gi-2 suggests that these structures were covalently linked. Almost identical D-glucopyranans were obtained from aged cultures that had thickened walls (as observed by electron microscopy).

High-field NMR spectroscopy of cystocarpic and tetrasporic carrageenans from Iridaea undulosa

Abstract High-field 1 H and 13 C NMR spectroscopy has been used to characterize the fine structure of the cystocarpic and tetrasporic carrageenans from Iridaea undulosa . The proportion of different carrageenans from the κ-family in cystocarpic samples were calculated. In the 13 C NMR spectra of the tetrasporic samples, twelve signals of approximately equal intensity were observed, indicating a nearly “perfect” λ-structure.

Role of Mannoprotein in Induction and Regulation of Immunity to Cryptococcus neoformans

ABSTRACT Our previous observations showed that mannoprotein (MP) induces early and massive production of interleukin-12 (IL-12) in vitro. This study was designed to investigate whether this phenomenon could be applied in vivo and to determine the biological significance of MP inCryptococcus neoformans infection. The results reported here show that MP treatment induces IL-12 secretion by splenic macrophages and IL-12 p40 mRNA in the brain. During C. neoformans infection, MP reinforced IL-12 and IFN-γ secretion that coincided with enhanced antifungal activity of natural effector cells, early resolution of the inflammatory process, and clearance of fungal load from the brain. These studies show that MP is a key inflammatory mediator that induces a protective immune response againstC. neoformans infection. This information can be used to facilitate the design of a rational approach to manipulate the immune response to C. neoformans.

Potent Inhibition of Neutrophil Migration by Cryptococcal Mannoprotein-4-Induced Desensitization

Cryptococcal capsular Ags induce the production of proinflammatory cytokines in patients with cryptococcal meningitis. Despite this, their cerebrospinal fluid typically contains few neutrophils. Capsular glucuronoxylomannan is generally considered to mediate the inhibition of neutrophil extravasation. In the current study, culture supernatant harvested from the nonglucuronoxylomannan-producing strain CAP67 was found to be as potent as supernatant from wild-type strains in preventing migration. We identified capsular mannoprotein (MP)-4 as the causative agent. Purified MP-4 inhibited migration of neutrophils toward platelet-activating factor, IL-8, and fMLP, probably via a mechanism involving chemoattractant receptor cross-desensitization, as suggested by its direct chemotactic activity. Supporting this hypothesis, MP-4 elicited Ca2+ transients that were inhibited by preincubation with either fMLP, IL-8, or C5a, but not platelet-activating factor, and vice versa. Moreover, MP-4 strongly decreased the neutrophil surface expression of L-selectin and induced shedding of TNF receptors p55/p75, whereas CD11b/18 increased. Finally, MP-4 was clearly detectable in both serum and cerebrospinal fluid of patients suffering from cryptococcal meningitis. These findings identify MP-4 as a novel capsular Ag prematurely activating neutrophils and desensitizing them toward a chemoattractant challenge.

Early Induction of Interleukin-12 by Human Monocytes Exposed to Cryptococcus neoformans Mannoproteins

ABSTRACT Interleukin-12 (IL-12) production by human monocytes stimulated with mannoproteins (MPs) of Cryptococcus neoformans was investigated. The results reported show that secreted or cell-associated MPs induce an early and significant production of IL-12. MPs show different capabilities to quantitatively affect IL-12 production; MP2, an 8.2-kDa MP purified from the culture supernatant ofC. neoformans, appears to be the most potent stimulator. Cytochalasin B inhibits both internalization and IL-12 induction by MP. In addition, a drastic reduction of IL-12 was observed when monocytes were cultured in the absence of normal human serum or treated with soluble mannan. Early production of IL-12 promotes early secretion of gamma interferon by T cells but does not influence the magnitude of the MP-induced lymphoproliferative response. Overall our results identify cryptococcal antigens responsible for rapid and potent induction of IL-12 in monocytes. MPs appear to regulate IL-12 secretion by internalization via the endocytic pathway and by interaction with monocyte receptors or serum factors.

Glucuronoxylomannan of Cryptococcus neoformans serotype B: structural analysis by gas-liquid chromatography-mass spectrometry and 13C-nuclear magnetic resonance spectroscopy.

The major extracellular polysaccharide (glucuronoxylomannan, GXM) from six strains of Cryptococcus neoformans serotype B was characterized by gas-liquid chromatography (g.l.c.), g.l.c.-mass spectrometry (g.l.c.-m.s.), and nuclear magnetic resonance (n.m.r.) spectroscopy. Ultrasonic irradiation (u.i.) was used to reduce the mol.wt. of native GXM from 9.75 x 10(5) to 1.15 x 10(5) without apparent change in its composition (GXM-S). The Xylp:Manp:GlcpA molar ratio of the GXM and GXM-S from the six strains of C. neoformans serotype B is approximately 3.5:3.0:0.6. GXM-S was O-deacetylated (GXM-D) by treatment with NH4OH. The 13C-n.m.r. analysis of GXM-D gave spectra that served as characteristic fingerprints of the structure and also facilitated the assignment of the anomeric carbon resonances to specific structural moieties present in GXM-D. The GXM-D from each serotype B strain was found to be similar by 13C-n.m.r. spectroscopy. The structure contains a linear (1----3)-alpha-D-Manp backbone substituted with 2-O-beta-GlcpA and 2-O-beta-Xylp. beta-Xylp is also O-4 linked to the Manp substituted with GlcpA. In addition, a model for the disposition of the Xylp and GlcpA side chain substituents along the mannopyranan backbone is proposed, based upon results from the combination of g.l.c.-m.s. and 13C-n.m.r. spectroscopy.

Mannoprotein from Cryptococcus neoformans Promotes T-Helper Type 1 Anticandidal Responses in Mice

ABSTRACT We previously demonstrated that mannoprotein (MP) from Cryptococcus neoformans (CnMP) stimulates interleukin-12 production by human monocytes, thus fostering a T-helper type 1 (Th1) protective anticryptococcal response. In this paper we show that CnMP was also able to induce a Candida albicans-directed protective Th1 response. This was demonstrated for mice immunized with CnMP by induction of a delayed-type hypersensitivity (DTH) reaction to C. albicans MP (CaMP) as well as induction of gamma interferon production by CD4+ and CD8+ splenic T cells stimulated in vitro with CaMP. CnMP-immunized mice were also partially protected from lethal systemic challenge with C. albicans, as shown by prolonged median survival times and decreased fungal burden in the kidney. Much evidence supports the validity of these cross-reactive and functional Th1 responses: (i) a non-cross-reactive C. albicans antigen, such as enolase, did not produce a DTH response to CaMP; (ii) passive adoptive transfer of T cells primed with CnMP induced a DTH reaction; (iii) C. neoformans extract elicited a DTH response to CaMP; and (iv) a monoclonal antibody (7H6) directed against a major and immunodominant T-cell-stimulatory 65-kDa MP (MP65) of C. albicans also recognized discrete 100-kDa constituents of C. neoformans extracts, as well as secretory constituents of the fungus. These results suggest the presence of common Th1 antigenic determinants in the mannoproteic material of C. neoformans and C. albicans epitopes, which should be considered in devising common strategies for immunoprophylactic or immunotherapeutic control of the fungi.