FIG 5.

Effect of OA on the intracellular growth of C. neoformans. (A) (a and b) EM of intracellular C. neoformans (Cn) exposed to 0.2 mM OA showing the gathering of host LD (hLD) around the phagosome, in addition to several LD in C. neoformans. Scale bars, 500 nm. (B) TLC analysis of intracellular C. neoformans upon excess OA, showing neutral lipid fractions of cellular extracts of C. neoformans-infected BMDM (a) and C. neoformans isolated from BMDM (b) without (control; −) or with (+) 0.4 mM OA for 4 h. In panel a, OA incubation led to increased TAG and CE formation in BMDM similar to that under uninfected conditions (arrows). In panel b, an increase in TAG was observed in isolated C. neoformans (asterisk). Nonpolar lipid standards are shown in lane 3. MO, methyl oleate; Chol, cholesterol. (C) (a) The survival of C. neoformans in BMDM, which were treated with 0.2 mM and 0.4 mM OA, was determined by CFU counts 7 h and 24 h after phagocytosis. The CFU values at 7 h and 24 h are normalized to those at time zero. Data are the means of triplicates, and error bars are SD. (b) Tables show the absolute numbers per experimental replicate. *, P < 0.01 by two-way ANOVA, multiple-comparison test. (D) BMDM, treated with or without 0.2 mM OA, were infected with C. neoformans. The number of C. neoformans cells which underwent intracellular replication and the time interval between the first and second budding (doubling time) were measured in a 24-h time-lapse movie. The data were obtained from three independent biological experiments. Error bars represent the 95% confidence interval of the mean. **, P < 0.0001 by Fisher's exact test. (E) The start point of C. neoformans replication is not affected by OA. The histogram represents the percentage of cryptococcal cells which underwent first budding at the indicated times after phagocytosis during a 24-h time-lapse movie. Experiments were performed in triplicate. Error bars are SD, with no significant differences observed.