FIGURE 1.

Absence of zinc transporter influences the outcome of Cryptococcus gattii from Acanthamoeba castellanii. (A) Cells of A. castellanii (1 × 10⁵) were incubated with WT, Δzip1 or Δzip1::ZIP1 C. gattii strains (1 × 10⁶ cells) in PYG medium for 3 or 24 h in 96-well plates to allow phagocytosis. The wells were washed with PBS and A. castellanii cells were lysed. Yeast CFU count was assessed in YPD agar. The phagocytosis index was calculated using the total number of CFUs by the number of protozoan cells. Data are shown as mean ± SD. The asterisks denote statistically significant differences between the Δzip1 and the WT or Δzip1::ZIP1 conditions (^∗p < 0.05; ^∗∗∗∗p < 0.001 as revealed by Student’s t-test). (B) Influence of zinc on the phagocytosis was accessed by culturing cells of A. castellanii (1 × 10⁵) with WT, Δzip1 or Δzip1::ZIP1 C. gattii strains (1 × 10⁶ cells) in PYG medium added of ZnCl₂ (10 μM) for 24 h in 96-well plates. After washing, A. castellanii cells were lysed and yeast CFU count was assessed in YPD agar. Data are shown as mean ± SD. The asterisks denote statistically significant differences between the Δzip1 and the WT or Δzip1::ZIP1 conditions (^∗p < 0.05; as revealed by Student’s t-test). (C) Fluorescence microscopy of WT and Δzip1 cells stained with Calcofluor White (Blue) and WGA (green) to access the surface structure of cryptococcal cells. Scale bars = 10 μm. (D) IPR assays were performed by incubating A. castellanii cells with WT or Δzip1 C. gattii strains in PYG medium for 3 h in 96-well plates. After extensive washing to remove non-associated yeast cells, cells were incubated with medium added or not of ZnCl₂ (10 μM). Interaction was allowed for a further 24 h, at 30°C. The A. castellanii cells were washed with PBS, lysed, and the yeast CFU count was performed in YPD agar. Data are shown as mean ± SD. The asterisks denote statistically significant differences between the conditions (^∗∗p < 0.01 as revealed by Student’s t-test).