Immunomodulatory effects of serotype B glucuronoxylomannan from Cryptococcus gattii correlate with polysaccharide diameter

Abstract

Glucuronoxylomannan (GXM), the major capsular component in the Cryptococcus complex, interacts with the immune system in multiple ways, which include the activation of Toll-like receptors (TLRs) and the modulation of nitric oxide (NO) production by phagocytes. In this study, we analyzed several structural parameters of GXM samples from Cryptococcus neoformans (serotypes A and D) and Cryptococcus gattii (serotypes B and C) and correlated them with the production of NO by phagocytes and the activation of TLRs. GXM fractions were differentially recognized by TLR2/TLR1 (TLR2/1) and TLR2/6 heterodimers expressed on TLR-transfected HEK293A cells. Higher NF-kappaB luciferase reporter activity induced by GXM was observed in cells expressing TLR2/1 than in cells transfected with TLR2/6 constructs. A serotype B GXM from C. gattii was the most effective polysaccharide fraction activating the TLR-mediated response. This serotype B polysaccharide, which was also highly efficient at eliciting the production of NO by macrophages, was similar to the other GXM samples in monosaccharide composition, zeta potential, and electrophoretic mobility. However, immunofluorescence with four different monoclonal antibodies and dynamic light-scattering analysis revealed that the serotype B GXM showed particularities in serological reactivity and had the smallest effective diameter among the GXM samples analyzed in this study. Fractionation of additional serotype B GXMs, followed by exposure of these fractions to macrophages, revealed a correlation between NO production and reduced effective diameters. Our results demonstrate a great functional diversity in GXM samples from different isolates and establish their abilities to differentially activate cellular responses. We propose that serological properties as well as physical chemical parameters, such as the diameter of polysaccharide molecules, may potentially influence the inflammatory response against Cryptococcus spp. and may contribute to the differences in granulomatous inflammation between cryptococcal species.

FIG. 1.

Quantitative and structural analyses of GXMs from five Cryptococcus isolates. GXM was isolated by formation of polysaccharide gels after concentration of culture supernatants of five different isolates of C. neoformans and C. gattii. (A and B) The volume of gel formation in normalized cultures (A) apparently correlates with the ability of each strain to produce and secrete GXM to the extracellular medium (B). The results are expressed as means ± standard deviations for three different experiments. (C) Correlation properties. (D) Monosaccharide composition of polysaccharides obtained from the five different isolates of C. neoformans and C. gattii. Monosaccharides were identified by GC-MS; the relative amount of each sugar residue in the polysaccharides is shown as a molar percentage. The serotype of each strain is given in parentheses above the bars.

FIG. 2.

NF-κB activation in cells expressing TLRs by GXM. (A) Control systems. Pam₃CSK₄ (P₃C) and FSL-1, but not LPS, activated NF-κB nuclear translocation in cells expressing either TLR2/1 or TLR2/6, as expected. Transfection of HEK293A cells with a plasmid containing no TLR-coding sequences (vector) resulted in unresponsiveness. (B to F) Stimulation of HEK293A cells expressing TLR2/1 (inverted triangles) or TLR2/6 (triangles) by GXM samples resulted in dose-dependent NF-κB activation. Cryptococcus strains (serotypes) from which each GXM sample was isolated are given at the top of each panel.

FIG. 3.

Comparative analysis of the efficacies of GXM samples in the activation of TLR-mediated NF-κB nuclear translocation. Treatment of HEK293A cells expressing either TLR2/1 (A) or TLR2/6 (B) with GXM revealed that the polysaccharide fractions from strain CN23/10993 were significantly more efficient than all others (P < 0.0001) at 1 and 10 μg/ml (asterisks). No significant differences were observed at a higher concentration (100 μg/ml). Strains T₁444, HEC3393, HEC40143, and ATCC 28938 manifested similar efficacies in activating NF-κB nuclear translocation. Strain serotypes are given in the key.

FIG. 4.

Comparative analysis of the efficacies of GXM samples in the induction of NO production by macrophages. Polysaccharide fractions from strain CN23/10993 were significantly more efficient at inducing NO production than all others (P < 0.0001) at 1, 10, and 100 μg/ml. LPS was used as a positive control for NO production by macrophage-like cells; incubation of the phagocytes in the medium alone (no stimulation) was the negative control. Serotypes are given in parentheses for each strain.

FIG. 5.

Reactivities of C. neoformans and C. gattii isolates with four monoclonal antibodies to GXM. Fungal strains and serotypes are given at the top; antibodies are given on the left. Differential interferential contrast (gray) and fluorescence (red) images are shown. The overreaction of CN23/10993 cells with antibody 12A1 (single asterisk) and their lack of reactivity with antibody 13F1 (double asterisks) are highlighted.

FIG. 6.

Diameters of GXM fractions of different isolates of C. neoformans and C. gattii. (A) Distribution of effective diameters of GXM; (B) average diameters.

FIG. 7.

NO induction by GXM fractions of different molecular masses and effective diameters. (A) Stimulation of macrophage-like cells with the GXM fractions results in differential production of NO. Asterisks indicate significant differences after stimulation of phagocytes with GXM fractions (P < 0.0001). (B) Determination of effective diameters of fractions obtained by sequential ultrafiltration through 100-kDa- and 10-kDa-cutoff filtration discs. Asterisks indicate that the differences in effective diameter are statistically significant (P < 0.0001). (C) Correlation analysis of effective diameters of serotype B GXM samples in the 10- to 100-kDa range and their abilities to induce NO.