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Research Article | Host-Microbe Biology

The Buoyancy of Cryptococcus neoformans Is Affected by Capsule Size

Raghav Vij, Radames J. B. Cordero, Arturo Casadevall
J. Andrew Alspaugh, Editor
Raghav Vij
aDepartment of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
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  • ORCID record for Raghav Vij
Radames J. B. Cordero
aDepartment of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
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Arturo Casadevall
aDepartment of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
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J. Andrew Alspaugh
Duke University Medical Center
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DOI: 10.1128/mSphere.00534-18
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  • FIG 1
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    FIG 1

    The cell density of C. neoformans and C. gattii serotypes. (A) Image representative of 3 to 4 independent repetitions of Percoll density gradients, comparing the cell densities of C. neoformans (serotypes A, D, and AD) and C. gattii (serotypes B and C) to that of density bead markers (DBM). (B) Representative data from 4 independent experiments, depicting the line interpolation of the density factor (minimum [min] and maximum [max]) calculated by pixel area per the following formulae: (f − a1/f and f − a2/f). The df (min, max) values of the density marker beads are used to estimate the cell density of the cells run in parallel gradients. (C) Histogram depicting the differences in cell densities of different serotypes of C. neoformans (serotypes A, AD, and D) and C. gattii (serotypes B and C and variants VGI and VGIIa). The experiment was performed 3 to 4 times, as indicated by the symbols on the bar graph. Error bars represent the SD about the mean. (D) Representative data of capsule (i) and cell body (ii) radii of different serotypes and strains (n = 3). Error bars represent the SD about the mean. One-way analysis of variance (ANOVA) was used for the comparisons of the cell densities and capsule and cell body radii of different strains and serotypes of C. neoformans and C. gattii to the respective measurements of strain H99 of C. neoformans. All comparisons were made to strain H99 of C. neoformans. 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.

  • FIG 2
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    FIG 2

    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.

  • FIG 3
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    FIG 3

    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.

  • FIG 4
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    FIG 4

    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.

  • FIG 5
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    FIG 5

    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.

  • FIG 6
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    FIG 6

    Effect of melanization on C. neoformans cell density. (A) Image representative of four independent repetitions of Percoll density gradients, comparing the densities of H99 in MM and of H99 in MM with l-DOPA (mel) and a 1:1 mixture of the cells. The white cells (H99) banded slightly above the melanized black cells (mel), as can be seen by the visualization of the gradient that contained the mixture. (B) Representative data from four 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) (i) A histogram depicting density of melanized and nonmelanized C. neoformans to compare the densities of melanized and nonmelanized cultures started from the same Sab preculture. The experiment was performed using replicates (n = 4), as indicated by the symbols on the bar graph. A paired t test found the pairing results to be significant (**) and found consistent and significant differences (*) between nonmelanized and melanized cells. (ii) A histogram depicting the density of melanized and nonmelanized cells after removal of capsule by gamma radiation. The experiment was performed in pairs, such that the paired cultures were inoculated from the same Sab preculture and were treated with gamma radiation (1,500 Gy) together. A paired t test found the pairing results to be significant (*) and found consistent and significant differences (*) between nonmelanized and melanized cells treated with gamma radiation. (D) Representative data depict (i) the cell body radii and (ii) the capsule radii of melanized and nonmelanized C. neoformans cells (n = 2). Error bars represent SD about the mean. t test was used for the comparisons of capsule and cell body radii of melanized and nonmelanized cells of strain H99 of C. neoformans. 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.

Tables

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  • Supplemental Material
  • TABLE 1

    List of C. neoformans and C. gattii strains used

    SpeciesStrain(s)Reference or source
    C. neoformansH99John Perfect (Durham, NC)
    cap5954
    ATCC 24067ATCC (Manassas, VA)
    C. neoformans hybridsMAS92-20355
    C. gattiiNIH444, ATCC 3260955
    106.9355
    VGI, WM179ATCC (Manassas, VA)
    VGIIa, R265ATCC (Manassas, VA)

Supplemental Material

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  • FIG S1

    Binding of capsular antibodies does not alter C. neoformans cell density. (A) Image representative of independent repetitions of Percoll density gradients, comparing the densities of C. neoformans H99 with and without antibody incubation with (i) 18B7 and (ii) ES1. (B) Representative data from 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) A histogram depicting density of C. neoformans H99 incubated with capsular antibodies. Data represent capsular antibodies (i) 18B7 (n = 4) and (ii) ES1 (n = 2) at different concentrations (0.1, 1, 10, and 20 μg/ml). Download FIG S1, TIF file, 1.7 MB.

    Copyright © 2018 Vij et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S2

    Effect of complement binding, osmotic shock, melanin-binding antibody, and growth in rich lipid media on cell density. Images of Percoll density gradients are presented to compare the densities of C. neoformans H99 upon (A) complement binding, (B) osmotic shock, (C) 6D2 antibody binding, and (D) growth in lipid-rich media. These conditions did not affect the cell density of Cryptococcus neoformans (H99) significantly. Experiments were done once. Download FIG S2, TIF file, 2.0 MB.

    Copyright © 2018 Vij et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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The Buoyancy of Cryptococcus neoformans Is Affected by Capsule Size
Raghav Vij, Radames J. B. Cordero, Arturo Casadevall
mSphere Nov 2018, 3 (6) e00534-18; DOI: 10.1128/mSphere.00534-18

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The Buoyancy of Cryptococcus neoformans Is Affected by Capsule Size
Raghav Vij, Radames J. B. Cordero, Arturo Casadevall
mSphere Nov 2018, 3 (6) e00534-18; DOI: 10.1128/mSphere.00534-18
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    • ABSTRACT
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KEYWORDS

Cryptococcus neoformans
buoyancy
capsular polysaccharide
yeast density

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