The Buoyancy of Cryptococcus neoformans Is Affected by Capsule Size

Induction of capsule synthesis decreases C. neoformans cell density. (A) Image representative of three independent repetitions of Percoll density gradients, showing the density of C. neoformans H99 juxtaposed with acapsular mutant cap59, both grown in Sab or MM. (B) Representative data from three independent experiments, depicting a line interpolation of the density factor with the cell densities of the bead standards to calculate the cell densities of the gradients run in parallel. (C) Histogram depicting a decrease in cell density of H99 cells grown in MM compared to Sab, due to capsule induction. cap59 mutant cells are significantly denser than normal H99 cells grown in MM. Each data point on the histogram represents an independent replicate (n = 3); error bars represent SD about the mean. (D) Representative data depict (i) the capsule radii and (ii) the cell body radii of C. neoformans H99 and cap59 grown under different medium conditions (MM or Sab) (n = 3). Strain cap59 cells grown in MM and Sab do not have a capsule; therefore, the capsule radii were not quantified. Error bars represent SD about the mean. One-way ANOVA was used for the comparisons of cell density, capsule density, and cell body radii of C. neoformans cap59 and H99 grown under different conditions. The following symbols were used to annotate the statistical significance of the results: ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Removal of C. neoformans polysaccharide capsule increases cell density. (A) (i) Image representative of five independent repetitions of Percoll density gradients, comparing the cell densities of irradiated (γ) and nonirradiated C. neoformans (H99) grown in MM for 10 days with a standard of colored uniform density beads. (ii) Image representative of four independent Percoll density gradients of encapsulated C. neoformans H99 strains grown in MM for 10 days before and after DMSO extraction. (B) Representative data from independent experiments, depicting a line interpolation of the density factor (df) with the cell densities of the bead standards, to calculate the cell densities of C. neoformans before and after extraction of the capsule, run in parallel. (C) A histogram depicting cell density of C. neoformans before and after capsule extraction by γ rays and DMSO. The experiments were performed 3 to 4 times independently as indicated by the symbols on the bar graph; error bars represent SD about the mean. One-way ANOVA was used to determine differences between the densities of strains H99 and cap59 grown in MM for 10 days with strains treated with DMSO and gamma radiation for capsule removal, respectively. (D) Representative data depict (i) the capsule radii and (ii) the cell body radii of C. neoformans before and after gamma irradiation capsule shedding and DMSO extraction (n = 3). Error bars represent SD about the mean. One-way ANOVA was used for the comparisons of cell densities and capsule densities and cell body radii of C. neoformans strain H99 to those of C. neoformans strain H99 after capsule removal by DMSO and gamma irradiation. The following symbols were used to annotate the statistical significance of the results: ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

The densities of C. neoformans and C. gattii correlate with the capsule radii. (A) Density (in grams per cubic centimeter) significantly correlates with the capsule size (in micrometers). Data points are labeled such that, e.g., “H99 MM 10 days” represents C. neoformans strain H99 grown in MM for 10 days. (B) No linear relationship was found in comparisons of the cell body size data (radii) to the density data. The density values were collated from all the experiments performed under a specific condition (n = 3 to 5). The cell size data were taken from a single experiment corresponding to each condition whose results were found to be representative of the replicates.

Encapsulated C. neoformans settles more slowly in seawater. (A) An image representative of cuvettes (3 ml) containing PBS (left) and seawater (right) imaged at different time points shows that H99 grown in MM settles faster in PBS. The relative displacement of cell sedimentation was calculated as (f − u)/f, where f is the area of the tube and u is the area from the bottom of the tube to the upper menisci of layers of settling cells. At 5 min, the relative displacement of cell sedimentation in seawater was 0, since all the cells were floating. In contrast, by 5 min, a large population of yeast cells suspended in PBS had already sedimented. At 3 h, the relative displacement value for sedimentation in PBS was 1, as all the cells had completely settled. (B) Plot of the normalized displacement of the upper menisci of cells settling in seawater and PBS. A line is drawn through the mean value of relative displacement at a given time intervals and the error bars represent standard deviation about the mean (n = 3 independent experiments). At certain time intervals, the error bar is smaller than the symbol and was not been plotted.