Fig. 6

CgCKB1 and CgCKB2 are essential to virulence in vivo. (A) 200 mg/kg cyclophosphamide was intraperitoneally injected per ICR mouse (5–6 weeks old, 24–26 g) on day − 3 and every fourth day later. Then mice were infected with 1 × 10⁸ WT, Δckb1 or Δckb2 cells in 200 µL in 0.9% (w/v) saline (n = 12). Mice in the agonal stage were humanely euthanized by cervical dislocation. Experiments were terminated on day 14. Analysis by the Gehan-Breslow-Wilcoxon test indicated that in contrast to WT, the virulence of CgCKB1 and CgCKB2 null mutants was significantly attenuated (p < 0.05). (B)(C)(D) Fungal burden assays were performed using ICR mice (n = 6). ICR mice were immunosuppressed with 200 mg/kg cyclophosphamide on day − 3 and were further injected with 5 × 10⁷ yeast cells through the tail veil on day 0. Organs were harvested, weighed and mechanically homogenized 3 days post-infection. Tissue homogenates were diluted, plated onto YPD agar, and incubated at 30℃ for 2 days. CFUs were calculated and analyzed. Error bars show standard deviation. Comparisons were performed using one-way ANOVA followed by Tukey test, and asterisks indicate statistically significant differences (**: p < 0.01; ***: p < 0.001, ****: p < 0.0001). CBS138 was the used as the WT and the knockout strains were generated using CBS138 as the parent; (E) Representative HE (Hematoxylin-Eosin) and PAS (Periodic Acid-Schiff) stained sections of kidneys from immunosuppressed ICR mice on day 3 post-infection. Magnification was ×20. PAS-positive yeast cells were indicated by red arrowhead. Data are representative of at least three replicates.