Cryptococcus neoformans responds to mannitol by increasing capsule size in vitro and in vivo

Abstract

The polysaccharide capsule of the fungus Cryptococcus neoformans is its main virulence factor. In this study, we determined the effects of mannitol and glucose on the capsule and exopolysaccharide production. Growth in mannitol significantly increased capsular volume compared with cultivation in glucose. However, cells grown in glucose concentrations higher than 62.5 mM produced more exopolysaccharide than cells grown in mannitol. The fibre lengths and glycosyl composition of capsular polysaccharide from yeast grown in mannitol was structurally different from that of yeast grown in glucose. Furthermore, mannitol treatment of mice infected intratracheally with C. neoformans resulted in fungal cells with significantly larger capsules and the mice had reduced fungal dissemination to the brain. Our results demonstrate the capacity of carbohydrate source and concentration to modify the expression of a major virulence factor of C. neoformans. These findings may impact the clinical management of cryptococcosis.

Figure 1

Effect of mannitol and glucose on C. neoformans capsule and polysaccharide production. C. neoformans yeasts were grown in different concentrations of mannitol and glucose and their effects on the capsule were evaluated. (A) India ink staining of C. neoformans yeasts. Scale bar = 10 µm. (B) Capsular volumes were determined by measuring total cell volume (cell body and capsule) and subtracting the cell body volume. White bars show the capsular volumes for cells grown in mannitol, Black solid bars show capsular volumes for cells grown in glucose. Grey bars show the cell body volume for each condition evaluated. Graphs show the average of at least 100 Cn cells evaluated. (C) The quantity of capsular polysaccharide epitopes was determined using a capture ELISA. Graphs show the average of 3 different experiments. (D) The concentration of exopolysaccharide epitopes in supernatants was determined by ELISA showing a different dynamic than the capsular polysaccharide epitope production. Graphs show the average of 3 different experiments.

Figure 2

Distribution of epitopes on the capsule of C. neoformans cells grown in different concentrations of mannitol and glucose. (A) Direct immunofluorescence at similar exposure times show differences in binding and brightness intensity, suggesting a different density of epitopes in each growth condition considered. Scale bar = 10 µm (*p<0.05). (B) Epitope concentration in the capsule obtained by capture ELISA correlated positively with capsular volume from mannitol calculated by india ink staining; however, in concentrations lower than 62.5 mM, epitope concentration was higher. (C) A similar effect was observed for cells grown on glucose. (D) FACS analysis showing the fluorescence intensity of cells after binding of FITC-labeled 18B7 mAb for cells grown in mannitol and (E) glucose. (F) Fluorescence intensity (FL1-H) normalized by cell diameter (FSC-H) values show a higher density of epitopes for both carbon sources when cells were grown in concentrations lower that 62.5 mM.

Figure 3

Determinations of cell charge and glycosyl composition of capsular polysaccharide show different capsular contents. (A) Zeta-potential of cells grown in different concentrations of mannitol and glucose show a similar cell charge for cells grown in both sugars. (B) Glycosyl composition of the capsular polysaccharide of cells grown in different concentrations of mannitol and glucose indicate a different stoichiometry in calpsular composition. Xyl- Xylose; GlcA- Glucuronic acid, Man- Mannose, Gal- Galactose, Glc- Glucose.

Figure 4

Dynamic light scattering (DLS) analysis and multimodal size distribution of capsular-PS from strain H99 grown in different concentrations of (A) mannitol [M] and (B) glucose [G]. The x axis represents the distribution of fibers diameter measured in nanometers; y axis corresponds to the values of percentage intensity weighted sizes obtained from the NNLS algorithm. (C) Correlation between capsule size measured by India ink negative stain and average of capsular PS and multimodal size distribution analysis of capsular PS by DLS.

Figure 5

In vivo role of mannitol and glucose infusions on the outcome of cryptococcosis after intratracheal C. neoformans infection. (A) Lung CFU determinations after 5 days of infection show similar values for mannitol and glucose treated and control mice . (B) Brain CFUs were lower in mannitol treated mice (*p<0.05). The results shown represent one of three independent experiments, each of which produced consistent data. (C, D and E) Mucicarmine staining of lungs from (C) control, (D) mannitol and (E) glucose show yeast capsular polysaccharide stained in pink. Insets on each respective figure display tissue inflammation by H&E. Scale bar = 50 µm.

Figure 6

Direct immunofluorescence with C. neoformans cells isolated from lung and brains of mice 5 days after infection. (A) Yeasts isolated from brain showed a much smaller capsule when compared to cells isolated from lung (B) of control mice. (C) Cells isolated from lungs of mice treated with mannitol had thicker capsules compared to controls. (D) Cells isolated from lungs of animals treated with glucose did not show any difference compared to controls. Scale bar = 10 µm. (E) Ratios of capsule/cell body volumes suggest a much more effective mechanism of mannitol cells in vivo regarding capsular production (*p<0.001).

Figure 7

Inhibition ELISA quantification of GXM levels in the lungs (A) and brains (B) of mice treated with or without glucose or mannitol. The levels correlated with tissue CFU determinations (*p<0.05). Mean values and standard deviations from five different mice are plotted.