Fig. 5. The leu1 mutant is deficient in capsule formation and has attenuated virulence.

(A) Strains were grown in defined low-iron medium at 30°C. Photographs were taken after 24-h incubation and after negative staining using India ink to visualize the capsule. The scale bar indicates 5 −m. (B) Ten female BALB/c mice were infected intranasally with each of the strains indicated, and the survival of the mice was monitored twice per day. The results from the assays indicate that LEU1 is required for full virulence. (C) Distribution of fungal cells in the organs of mice infected by the inhalation method. Organs from mice infected with the wild-type strain, the leu1 mutant or the LEU1 reconstituted strain were collected at time of death, and fungal burdens were monitored in organs by determining colony-forming units (CFU) upon plating on YPD medium. Three mice for each strain were used for the experiments, and horizontal bars in each graph represent an average CFUs. In all organs, differences of fungal burdens between the leu1 mutant and the wild-type, and between the leu1 mutant and the reconstitute strain were statistical significance (p<0.05) except that in blood (p × 0.1599, the leu1 mutant vs. the wild-type; p × 0.052, the leu1 mutant vs. the reconstitute strain).