Immunomodulatory Effects of Serotype B Glucuronoxylomannan from Cryptococcus gattii Correlate with Polysaccharide Diameter

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-kappa B 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.