Article Figures & Data
Figures
- FIG 1
Methods for estimation of C. neoformans CSH. (A) CSH estimated by MATH assay that quantifies the interaction of C. neoformans cells in a suspension with the hydrocarbon solvent n-hexadecane. CSH% was calculated as the percent change in OD of a C. neoformans cell suspension after vortexing the mixture of cells with n-hexadecane. (B) In addition, we estimated CSH by visualizing the interaction between C. neoformans cells and hydrophobic beads (0.8 μm) in a hemocytometer and counting cells that had >3 beads/100 cells to calculate CSH%. Image created with BioRender.
- FIG 2
CSH of C. neoformans differs by strain. Graphical representation of CSH of C. neoformans and C. gattii strains. (A) Graphical representation of CSH estimated by MATH assay. (B) Graphical representation of CSH estimated by hydrophobic microsphere assay (left). Experiments have been performed 2 to 6 times independently, as indicated by individual data points. Circles indicate data points of CSH of an encapsulated strain of C. neoformans and C. gattii. Error bars represent the standard deviation of the mean. (C) Representative image of a mixture of hydrophobic beads with C. neoformans strain H99 (top) and relatively hydrophobic C. neoformans strain B3501 (bottom) used for the assay. Hydrophobic beads (small spheres, approximately 0.8 μm in diameter) adhere to the cell surface due to the high hydrophobicity of the B3501 cell, covering it almost completely. The hydrophobic beads are all but absent from the surface of H99 cells. Ordinary one-way analysis of variance was used to compare the CSH of C. neoformans strain H99 with the CSH of C. neoformans and C. gattii strains. The following symbols were used to annotate the statistical significance of the results: ***, P ≤ 0.001; ****, P ≤ 0.0001.
- FIG 3
Capsular motifs, and not the cell wall composition, may contribute to the variation in hydrophobicity of C. neoformans. (A) Comparison of the CSH of C. neoformans H99 grown in Sabouraud broth with those of C. neoformans H99 grown in MM and acapsular cap59 strain and comparison of the CSH of strain B3501 with that of acapsular cap67 strain. One-way analysis of variance and t test were used, respectively, to compare the means. Data have been compiled from separate experiments to draw comparisons and study the influence of presence or induction of capsule on the CSH of C. neoformans. Each data point represents a biological replicate, and the error bar represents the SD of the mean. (B) CSH% measured by a hydrophobic bead assay of C. neoformans strain H99 and acapsular strain cap59 grown in capsule induction medium. The experiment was performed in two independent replicates, as represented by data points about the median. (C) Lipophilicity, log P, of dominant carbohydrate motifs in the carbohydrate was predicted by an equation proposed by Mannhold et al. (28). M4 was found to be the most hydrophobic motif and M1 the least. The number of hydroxyl groups on each polysaccharide motif was calculated (below). Glycan notification followed the Symbol Nomenclature for Glycans (SNFG) (71). (D) No significant differences were found when the CSH% of C. neoformans strain K99 was compared to those of chitin deacetylase 1 mutant (cda1Δ) and chitin deacetylase triple-knockout mutant (cda123Δ) strains as measured by hydrophobic microsphere assay. The following symbols were used to annotate the statistical significance of the results: *, P ≤ 0.05.
- FIG 4
CSH of C. neoformans correlates with phagocytosis of C. neoformans by natural predator A. castellanii. Significant positive linear correlation (R2 = 0.5722) between CSH of C. neoformans strains and phagocytosis index by A. castellanii. Phagocytosis index is estimated by fluorescence microscopy as the number of C. neoformans cells labeled by Uvitex internalized per 100 A. castellanii cells. Error bars represent the standard deviation of the mean.
- FIG 5
Binding of protective capsule antibodies influences CSH. Incubation of C. neoformans strain H99 grown in the capsule induction medium (MM) with protective capsular antibody 18B7 significantly increased CSH in a concentration-dependent manner, while 12A1 decreased CSH and 13F1 had no significant effect on CSH. CSH was determined by MATH assay in 2 to 3 biological replicates, as indicated by data points. Error bars represent the standard deviation of the mean. Ordinary one-way analysis of variance was used to compare the CSH of untreated C. neoformans strain H99 with the CSH of H99 cells treated with different antibodies. The following symbols were used to annotate the statistical significance of the results: ns, P > 0.05; *, P ≤ 0.05; ***, P ≤ 0.001.
