Cryptococcus neoformans glucuronoxylomannan fractions of different molecular masses are functionally distinct

Abstract

Aims: Glucuronoxylomannan (GXM) is the major polysaccharide component of Cryptococcus neoformans. We evaluated in this study whether GXM fractions of different molecular masses were functionally distinct.

Materials & methods: GXM samples isolated from C. neoformans cultures were fractionated to generate polysaccharide preparations differing in molecular mass. These fractions were used in experiments focused on the association of GXM with cell wall components of C. neoformans, as well as on the interaction of the polysaccharide with host cells.

Results & conclusion: GXM fractions of variable molecular masses bound to the surface of a C. neoformans acapsular mutant in a punctate pattern that is in contrast to the usual annular pattern of surface coating observed when GXM samples containing the full molecular mass range were used. The polysaccharide samples were also significantly different in their ability to stimulate cytokine production by host cells. Our findings indicate that GXM fractions are functionally distinct depending on their mass.

Figure 1. Dynamic light-scattering analysis of glucuronoxylomannan fractions obtained by ultrafiltration

Polysaccharide dimensions were proportional to the molecular mass cut-off used for fractionation, as concluded from the analysis of glucuronoxylomannan samples in the ranges of (A) 1–10, (B) 10–100, (C) 100–300 and (D) >300 kDa.

Figure 2. Serologic properties of glucuronoxylomannan fractions of different molecular masses

(A) Reactivity of polysaccharide fractions distributed in different molecular mass ranges with monoclonal antibody 18B7. The dose-dependent profile of serologic reactivity was similar for all fractions. (B) Incorporation of the different GXM fractions by acapsular Cryptococcus neoformans cells for further serological detection by flow cytometry with monoclonal antibody 18B7. Similar levels of fluorescent intensity were observed for all fractions. The isolated histogram on the left (black line) represents the background fluorescence levels of cap67Δ cells that were not coated with GXM fractions. GXM: Glucuronoxylomannan. For color image please see online at www.futuremedicine.com/doi/full/10.2217/FMB.13.163

Figure 3. Surface distribution of GXM in acapsular (cap67Δ) Cryptococcus neoformans cells after incubation of cells with different fractions of the polysaccharide

GXM detection was based on the serologic reactivity of yeast cells with four different antibodies to the polysaccharide, listed on the left. Molecular mass ranges are indicated on the bottom. Cryptococcus neoformans cells are shown under the differential interferential contrast and red fluorescence modes (gray and black panels, respectively). Scale bar in bottom-right 18B7 panel represents 20 μm. GXM: Glucuronoxylomannan.

Figure 4. Sequential incubations of acapsular Cryptococcus neoformans cells with different polysaccharide fractions

Cryptococcus neoformans cells were sequentially coated with fractions of increasing or decreasing molecular masses. (A–D) represent sequential incubations of cap67Δ cells with fractions of increasing molecular masses, starting with (A) the 1–10 kDa sample alone, followed by (B–D) samples of higher masses and (E & J) the full molecular mass range. (F–I) show sequential incubations of acapsular cells with fractions with decreasing molecular masses, starting with (F) the fraction >300 kDa alone, followed by (G–I) samples of lower masses. Scale bar represents 20 μm. GXM: Glucuronoxylomannan.

Figure 5. Binding of glucuronoxylomannan fractions with variable molecular masses to murine macrophages

The phagocytes were exposed to the different molecular mass samples of GXM and then incubated with monoclonal antibody 18B7 for flow cytometry analysis. The percentage of GXM-containing macrophages as a function of time is shown in (A), while the intensity of fluorescent reactions is shown in (B). Polysaccharide-binding was also tested in macrophages obtained from WT mice and knockout animals lacking the GXM receptors TLR2 and CD14. (C) Analyses of the percentage of GXM-containing macrophages and (D) fluorescence levels of the phagocytes indicate similar profiles of GXM binding to all phagocytes, independently of the molecular mass of the polysaccharide. In all cases, binding efficacy was higher after the 5-min period of incubation. Data are representative of two independent experiments producing similar results. GXM: Glucuronoxylomannan; TLR: Toll-like receptor; WT: Wild-type.

Figure 6. Glucuronoxylomannan fractions of variable molecular masses differentially stimulate cytokine responses in host cells

(A) In vitro cytokine production by mouse peritoneal macrophages stimulated with the different polysaccharide fractions. Control systems were stimulated with PBS. The cytokine response to GXM fractions varied considerably depending on the polysaccharide molecular mass. (B) Analysis of mice lung (n = 5) levels of TNF-α and IL-10 in response to lower- (10–100 kDa) or higher- (>300 kDa) mass GXM fractions, as well as to PBS or the full-mass-range GXM sample, revealed that the polysaccharide preparations of different molecular masses are distinct in their ability to induce cytokines. *Statistical significance in comparison with control (PBS-stimulated) systems; ***Statistical differences between GXM-treated systems; Black asterisks: p < 0.001; Red asterisks: p < 0.01. ND: Not detected; NS: Not significant; PBS: Phosphate-buffered saline. For color image please see online at www.futuremedicine.com/doi/full/10.2217/FMB.13.163