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. 2022 Jul 5;11(7):768.
doi: 10.3390/pathogens11070768.

Establishing Minimal Conditions Sufficient for the Development of Titan-like Cells in Cryptococcus neoformans/ gattii Species Complex

Affiliations

Establishing Minimal Conditions Sufficient for the Development of Titan-like Cells in Cryptococcus neoformans/ gattii Species Complex

Mariusz Dyląg et al. Pathogens. .

Abstract

Opportunistic pathogens of the anamorphic genus Cryptococcus are unique considering their virulence factors that in the context of pathogenesis allowed them to achieve evolutionary success. Morphological transformation into giant (Titan) cells is one of the factors contributing to cryptococcosis. Recently established in vitro protocols demonstrate that 5 or 10% fetal bovine serum (FBS) combined with 5% CO2, 37 °C, and sufficiently low cell density, triggers cellular enlargement (Serum protocols). However, the FBS components that promote this morphological transition remain incompletely characterized. In search of minimal conditions necessary for stimulating the formation of Titan cells, we performed a study where we eliminated serum from the protocol (Serum-free protocol) and instead systematically adjusted the amount of glucose, source of nitrogen (ammonium sulfate), and the pH. We found that exposing cells to PBS with adjusted pH to 7.3, and supplemented with 0.05% glucose, 0.025% ammonium sulfate, 0.004% K2HPO4, 0.0035% MgSO4, in the presence of 5% CO2 at 37 °C triggers Titan-like cell formation to the same degree as the previously established protocol that utilized 10% FBS as the sole nutrient source. Titan-like cells obtained according to this Serum-free protocol were characterized by cell body size over ten microns, a single enlarged vacuole, thick cell wall, extensive polysaccharide capsule, and changes in the level of cell ploidy, all currently known hallmarks of Titan cells found in vivo. Strikingly, we found that in both, Serum and Serum-free protocols, an optimal pH for Titan-like cell development is ~7.3 whereas relatively acidic pH (5.5) prevents this morphological transition and promotes robust proliferation, while alkaline pH (~8.0) has a profound growth inhibitory effect. Our study demonstrates a critical role of pH response to the formation of Titan cells and indicates that conditions that allow restricted proliferation in the presence of 5% CO2 are sufficient for this morphological transition to form enlarged cells in Cryptococcus neoformans and Cryptococcus gattii species complex.

Keywords: Titan cells; cryptococcosis; pathogenesis; virulence factors; yeast-like fungi.

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Conflict of interest statement

The authors declare no conflict of interest. The authors alone are responsible for the content and writing of the paper.

Figures

Figure 1
Figure 1
The effect of pH and nutrient level on the formation of Titan cells. (A) A spot test assay was utilized to test the sensitivity to alkaline pH of representative strains (alternative strains of the same species are indicated by numbers and are characterized in detail in Table 2) of the non-neoformans and non-gattii species as compared to four representative strains (underlined) of the C. neoformans/C. gattii species complex. Experiments based on spot test assay were performed in two independent repetitions. (B) The level of pH is critical for the formation of Titan-like cells in C. neoformans (H99) and C. deuterogattii (R265) in a modified "Serum" protocol based on a method developed by Dambuza I.M. et al. [18]. (C) The level of pH (4.5, 5.5, 7.3, and 8.0) influences the percentage of Titan cells obtained in a modified protocol based on the method established by Hommel B. et al. [17]. The formation of Titan-like cells was tested for two representatives of the C. neoformans (strains MD31 and H99), and at least 200 cells were measured for each sample. Differences in cell body diameter between the experimental groups were significant (one-way ANOVA, p = 0.001). (D) Optimal conditions for the development of Titan-like cells were established in a Serum-free protocol. A method developed by Dambuza I.M. et al. has been modified such that the fetal bovine serum (FBS) has been replaced by various levels of nutrients (ammonium sulfate: AS, glucose: G) and pH levels (5.5, 7.3, 8.0), as indicated (see text for more details). Results are shown for C. deuterogattii strain R265 and C. neoformans strain MD31. For each strain and condition, at least 200 cells were measured. Above the plot, for each sample, the percentage of Titan cells (cell body diameter above 10 microns) is indicated. For each of the four versions of the nutrient content, the median cell body diameter at pH = 7.3 was significantly different from the diameter of cells incubated at pH 5.5 or 8.0 (Kruskal-Wallis test, p = 0.001). Scale bars are 20 microns in B and 10 microns in C.
Figure 2
Figure 2
A direct comparison of the percentage of Titan cells obtained in an optimized Serum-free protocol (Low nutr., 0.025% ammonium sulfate, 0.05% glucose) as compared to the method developed by Dambuza I.M. et al. (FBS) [18]. (A) C. neoformans strain MD31 and C. deuterogattii strain R265 were utilized. As controls, cultures grown in a rich YPD medium at logarithmic (Log. Phase, shown in A and C) or stationary phase (Stat. phase, shown in A) of growth were evaluated. For each sample, the cell/capsule diameter for at least 150 cells was measured. Star indicates statistical significance (p = 0.001). (B) Cells evaluated in A were stained with India ink to visualize the capsule. (C) Nuclei of cells evaluated in A were stained with SYTOX® Green and fluorescence flow cytometry was performed to examine ploidy. The Y-axis scale was adjusted in each panel to facilitate the comparison of peaks for each sample. The scale bar in B represents 10 microns.
Figure 3
Figure 3
Analysis of the DNA content in cells grown in YNB medium (control, blue) or cells subject to Serum-free protocol (red). Cells were stained for DNA content with either SYTOX® Green or propidium iodide. Yellow indicates additional control of cells that were not stained for the DNA content. The analysis was performed for C. neoformans MD31 and C. deuterogattii R265 strains. The percentage of the population was normalized to mode.

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