Fig 1.
Cellular and capsular size of C. neoformans in different media.
Cells from strain H99 were inoculated into Sabouraud (A), capsule inducing medium (10% Sabouraud with MOPS at pH 7.3) (B) and 5% Sabouraud with MOPS + 5% FBS + sodium azide (C). After incubation at 37°C with shaking, total size and capsule size was visualized by suspending the cells in India ink. Total size (D), cell body size (E) and capsule size (F) distribution after incubation as described above. The asterisks indicate significant differences compared to Sabouraud control (p<0.05). Sab, Sabouraud medium.
Fig 2.
Effect of different factors on the cellular growth of C. neoformans.
Cells from H99 strain were incubated on Sabouraud overnight and transferred to different media to evaluate the effect of several factors on the titan-like cell formation. (A) Cells were incubated in Sabouraud or capsule inducing medium (10% Sab, pH 7.3) supplemented with 15 μM sodium azide in the presence or absence of 5% serum (FBS) and cultures were incubated at 37°C overnight. Pictures after suspension of the cells in India Ink were taken, and the total cell size was measured and plotted (B) Effect of sodium azide (black bars) on cell size in Sabouraud or TCM medium. As a control, the same media were inoculated without sodium azide (white bars). Cells were incubated at 37°C overnight. Pictures after suspension of the cells in India Ink were taken, and the total cell size was measured and plotted (C) Effect of shaking effect on titan-like cell formation. The yeasts were incubated at 37°C for 24 h in flasks with Sabouraud or TCM medium in both conditions (black bars, shaking) and (gray bars, no shaking). Pictures after suspension of the cells in India Ink were taken, and the total cell size was measured and plotted (D) Effect of CO2 on cell growth. The cells were incubated in Sabouraud (white bars) and TCM (black bars) with and without 5% of CO2. In both cases, the cells were grown at 37°C without shaking for 24 h in a 96-microdilution plate. Then, the plate was directly observed in the microscope, and the cell body size of was measured and plotted.
Fig 3.
Comparison of titan cells obtained in vivo with enlarged cells obtained in vitro.
Measurements of cell body (A), capsule (B) and total cell sizes (C) of cells isolated from infected mice (in vivo) and cultured in TCM and Saboraud in vitro. D and E show the permeability index of 70 and 40 kDa fluorescent labelled dextrans respectively in the same type of samples (see M&M).
Fig 4.
Effect of serum on cryptococcal cell growth.
H99 cell suspensions were inoculated in Sabouraud (A), Sabouraud supplemented with 5% serum (B) or TCM (C). The cells were incubated overnight at 37°C in 5% CO2. Then, the cells were suspended in India Ink and pictures of the cell body size were taken. The scale in A applies to all the pictures. (D) Effect of purified serum lipids on cell growth. The cells (initial density 104 cells/mL) were grown in TCM medium in which the serum was replaced by different dilutions (40, 100 and 200) of the lipid extract. As controls, the same media was supplemented with PBS. Cultures in TCM are also included for reference. Cell body size was determined after 24 hours at 37° C with CO2 and shaking. The asterisks indicate significant differences compared to Sabouraud control. (E) Effect of phosphatydilcholine on titan-like cell formation. Cells were incubated as in (A), but serum was replaced by 0.1 mM of phosphatidylcholine (PC). A parallel control with the same concentration of ethanol was carried out in parallel. The graph shows the cell body size.
Fig 5.
Influence of cell density on titan-like cell formation in C. neoformans.
(A). Cells from H99 strain inoculated in Sabouraud (white bars) or TCM (black bars) at different concentrations (103, 104, 105 and 106 cells/mL) in 96-well plates. Cell body size was measured after incubation overnight at 37°C with CO2. The asterisks indicate significant differences. B) Effect of conditioned media on cell body size. Cells from H99 strain were incubated in different conditioned TCM medium denominated (see material and methods) as TCS (supernatant of titan-like cells TCM cultures), RCS (supernatant of regular size TCM cultures), TCM + TCS (a 1:1 mixture of fresh TCM with TCS), TCM + RCS (a 1:1 mixture of fresh TCM with RCS). As controls we used a diluted TCM in H2O (1:1) and fresh TCM inoculated with 104 cells/mL (TCM) and with 106 cells/mL. (C) Cells from H99 strain were grown in TCM supplemented with 30 uM and 15 uM of the quorum sensing peptide Qsp1 (NFGAPGGAYPW). As controls, TCM supplemented with 30 uM of an inactive Qsp1 (NFGAPGAAYPW), with 30 uM of a scrambled Qsp1 peptide (AWAGYFPGPNG). TCM without any supplementation was used as control. Cell body size was measured after overnight incubation at 37°C with 5% CO2. D) Titan-like cell formation of qsp1 mutant. Cells from the KN99 (wild type) and qsp1 mutant were inoculated in TCM at 104 and 106 cells/mL, and cell body size was determined after 18 h of incubation at 37°C in the presence of 5% CO2.
Fig 6.
Nuclear staining of titan-like cells.
Cells of regular size and titan-like cells were obtained by inoculating H99 strain in TCM at 106 and 104 cells/mL respectively. After incubating overnight at 37°C with 5% CO2, the cells were fixed and stained with DAPI as described in material and methods. A and B show the microscopic appearance of the nucleus of cells of regular size (A), and titan-like cells (B). C-E, Analysis of nuclear staining by flow cytometry. C) FFS/SSC scatter plot of cells of regular size, D) same graph of titan-like cells. In C and E, we defined a gate to clearly separate cells of regular size (cells in blue) and titan-like cells (cells in light red). The fluorescence intensity (histograms) of the cells from these two gates is represented in panel E. Blue histogram, fluorescence from the cells shown in the gate in panel C, and light red histogram, fluorescence from the cells shown in the gate in panel D.
Fig 7.
Influence of PKC pathway on titan-like cell formation.
The influence of the PKC pathway was evaluated by its inhibition with three different agents: Bisindolylmaleimide I (A), Calphostin C (B), Staurosporine (C). Genestein, a tyrosine kinase inhibitor, was used as control (D). The experiments in A, B and D were performed the same days, so they share the same control. However, for clarity, the graphs corresponding to each inhibitor have been separated. The experiments were repeated on three days, and the data from the three experiments is plotted. Cells of the H99 strain were incubated with these different agents overnight in TCM at 37°C with 5% CO2 without shaking and cell body size was measured. The asterisks indicate statistical significant differences.
Fig 8.
Titan-like cell induction in different strains.
Four strains of each species (serotypes) were inoculated into Sabouraud (black symbols) or TCM (grey symbols) at a density of 104 cells/mL and grown overnight at 37°C with CO2. (A) C. neoformans var. grubii (serotype A); (B) C. neoformans var neoformans (serotype D); (C) C. neoformans A/D; (D) C. gattii. (E) MATa and MATα strains: NE822/NE824 (serotype D), 3259/3260 (serotype A) and JEC20/JEC21 (serotype D). Cell body size was measured in all cases. The red lines represent the mean and standard error.
Fig 9.
Titan-like cell formation in different mutants.
(A) Cells from the capsule deficient mutants gat201, cap59, cap60 and ada2 (H99 background) were inoculated in Sabouraud and TCM medium at a density of 104 cells/mL in 96-well plates. Cell body size was determined after overnight incubation at 37°C with CO2. (B) Role of adenylate cyclase on cell growth of C. neoformans. Cells from the wild-type strain H99, the adenylate cyclase mutant (cac1) and the reconstituted strain (cac1/CAC1) were incubated as described above. (C) Role of carbonic anhydrases on cell growth. Cells from the wild type strain H99 and KN99 (3260) and the can1 and can2 mutants were used for this purpose. Cell body size was determined after overnight incubation at 37°C with CO2 in all cases. The asterisks indicate significant differences (see M&M). The red lines represent the mean and standard error.
Fig 10.
Interaction of titan-like cells with murine-like macrophages.
A) Cells from H99-GFP strain were incubated in TCM inoculated at 3x104 cells/mL (titan-like cells), 106 cells/mL (regular cells in TCM), and Sabouraud. Then, phagocytosis experiments with RAW264.7 cells were performed and quantified by flow cytometry as described in material and methods. Statistical differences are highlighted. B) Effect of preincubation of macrophages with titan-like cells on the phagocytosis of regular cells. Titan-like cells were obtained by incubation of C. neoformans in TCM as described in material and methods, and were exposed to RAW264.7 macrophages at 1:1 ratio. The incubation was performed in media containing the opsonizing mAb 18B7 or in its absence. As control, macrophages were preincubated with growth medium with or without the mAb. The plates were incubated for 1h at 37°C in the presence of 5% CO2, and then the cells were washed to remove titan-like cells. Next, C. neoformans cells (H99-GFP) of regular size grown in Sabouraud were added to the macrophages at 1:1 ratio in the presence of mAb 18B7, and phagocytosis was performed for 1 h at 37°C in CO2. Phagocytosis percentage was quantified by flow cytometry as described in M&M. ns: no statistical difference. The experiment was performed in triplicates in three different days.
Fig 11.
Gene expression changes by RNAseq in titan-like cells obtained in vitro.
Transcriptomic profiling of the transition to titan-like cells. A) Number of genes induced or repressed after 7 h (blue) or 18 h (red). The number of genes whose expression is modified at both times is denoted at the intersection of the circles. Scale is only approximated. B) Gene Ontology analysis of genes induced or repressed at 7 h (upper panel) or 18 h (lower panel). All classifications correspond to “biological function” terms, except the central graph in the lower panel, which correspond to “cellular components”. Numbers in the histograms denote the p-value for each assignment. See Materials and Methods for details.
Table 1.
Genes that were significantly overexpressed (fold>2 with P<0.05) at 7 or 18 h in titan-like cells were subjected to Gene Ontology analysis.
For unassigned genes, additional Blastp comparisons against fungal data bases were carried out. T: Titan-like cells (cells incubated in TCM at 104 cells/mL); S (cells of regular size, incubated in TCM at 106 cells/mL).
Table 2.
Genes significantly repressed (>2.0-fold and p<0.05) at 7 or 18h during the formation of titan-like cells.
Functional assignments were based on Gene Ontology. For unassigned genes, additional Blastp comparisons against fungal data bases were carried out. T: Titan-like cells (cells incubated in TCM at 104 cells/mL); S (cells of regular size, incubated in TCM at 106 cells/mL).
Fig 12.
Phenotype of the mutant lacking calnexin (cne1).
Pictures of the WT (KN99 strain,A) and a cne1 mutant (lacking calnexin, CNAG_02500, B) in liquid Sabouraud medium. The scale bars denote 20 μm. (C) Measurement of cell body size of the WT strain (KN99) and the mutant lacking calnexin. The cells were incubated in liquid Sabouraud at 30°C or TCM (overnight) at 37°C with 5% CO2.
Fig 13.
Effect of iron chelation on cryptococcal morphology.
The effect of iron chelation on cell body size was observed by the supplementation of TCM with the iron chelator Bathophenanthroline disulfonic acid (BPS) in TCM cultures inoculated at 104 cells/mL cultures (A) and 106 cells/mL cultures (B). The cells were incubated at 37°C overnight with 5% CO2 without shaking. CT, control cells without BPS.
Table 3.
List of strains used.