Fig 1.
C. neoformans APE4 is required for growth and cell size maintenance under temperature and nutrient stress.
Serial dilutions of wild type (KN99α), mutant (ape4) and reconstituted (ape4+APE4) strains were spotted on solid rich medium (YPD) followed by incubation at 30°C and 37°C for 48 hours (A). The yeast cell size was determined at different temperatures (30°C and 37°C) and media conditions [liquid rich medium (YPD) and synthetic medium (SD) without ammonium sulfate (-N) and amino acids (-AA), A and B, respectively]. One -way ANOVA, Tukey's test for multiple comparisons (P < 0.01** and P<0.0001****).
Fig 2.
Ape4 is required for C. neoformans capsule production.
The wild type (KN99α), ape4, and ape4+APE4 strains where inoculated in CO2-independent medium and capsule production was evaluated after 72 hours of incubation at 30°C and 37°C. Scale bar represents 10 μm.
Fig 3.
Reduced phospholipase activity in the ape4 mutant is demonstrated by reduced precipitation zone on egg yolk agar medium compared to control strains.
(The scale represents 1 cm).
Fig 4.
Cell wall integrity and osmotic stress were altered in the ape4 mutant compared to wild type (KN99α) and reconstituted (ape4+APE4) strains.
The cell wall integrity was tested on YPD plates at 30°C, supplemented with 0.5% Congo Red (A) and the effect of osmotic stress was assessed on YPD plates supplemented with 0.75M and 1.5 M of NaCl (B) and KCl (C).
Fig 5.
C. neoformans sensitivity to amphotericin B and fluconazole.
The wild type (KN99α), mutant (ape4) and the reconstituted (ape4+APE4) strains were subjected to E-test analysis (Biomerieux, cat. 510818) with fluconazole and amphotericin B at 30°C. The ape4 mutant strains is more sensitive to fluconazole (A), whereas no difference among the tested strains is evident for amphotericin B. ANOVA (t-Student's and Scott Knout, p <0.05).
Fig 6.
The ape4 mutant demonstrates reduced survival in co-culture with J774A.1 macrophages after 24 hours of co-cultivation.
This graph represents four independent experiments. The asterisk denotes statistically significant difference (P <0.0001) relative to both wild-type and reconstituted strains at a CI of 95% by Tukey’s post-test following one-way ANOVA. Error bars represent 95% CIs for each group of data.
Fig 7.
Ape4 is required for full C. neoformans virulence in a murine model.
The wild type (KN99α), mutant (ape4) and the reconstituted (ape4+APE4) strains were inoculated by nasal inhalation in C57BL/6 mice. Survival was followed during the course of the infection up to 40 days. p value was <0.001 for the comparisons between ape4, and wild type and ape4+APE4.
Fig 8.
Effect of high temperature on APE4 expression pattern.
Yeast cells grown in rich medium (YPD) were washed and transferred to fresh rich medium (A), and synthetic dextrose supplemented with nitrogen source (SD + N) (B) and synthetic dextrose without nitrogen source (SD—N) (C) and incubated at 30°C and 37°C for 2 hours. All values were statistically validated by ANOVA, p< 0.05(*) and p≤ 0.01(**).
Fig 9.
Influence of different nitrogen sources on C. neoformans growth.
The wild type (KN99α), mutant (ape4) and the reconstituted (ape4+APE4) strains were cultured in rich medium (YPD) (A), synthetic dextrose (SD) supplemented with ammonium sulfate and amino acids (B), L-proline (C) or uric acid (D). All assays were performed at 30°C in triplicates up to 72 hours. Values were statistically validated by ANOVA (Bonferroni post-test, p<0.05 GraphPad Prism program 5).
Fig 10.
The effect of a non-preferred carbon source on the ape4 mutant growth.
The growth pattern of the wild type (KN99α), mutant (ape4) and reconstituted (ape4+APE4) strains was evaluated in defined medium (SD) supplemented with nitrogen source and galactose (2%) at 30°C for up to 72 hours. All assays were performed in triplicate and values were statistically validated (ANOVA, Bonferroni post-test, p<0.05).
Fig 11.
GFP-Ape4 sub-cellular localization.
The C. neoformans Ape4 protein was fused to GFP (GFP-Ape4) to show its cellular localization during the yeast growth (KN990α) in rich (YPD) and defined medium without nitrogen source (SD—NS) at 30°C and 37°C. (A) Yeast cells images were captured by Differential Interference Contrast (DIC) microscopy, (B) FM4-64 demonstrates endocytic vesicles, and (C) epifluorescent microscopy demonstrates cell localization of GFP-Ape4. (D) Merged images of FM4-64 and GFP-Ape4. Arrows indicate FM4-64 stained vesicles, GFP labeled Ape4 and co-localization of both. All images were processed using the Zen 2011 software (Zeiss). Scale bar represent 5 μm.
Fig 12.
Effect of high temperature on the expression of C. neoformans autophagy-related genes in rich medium (YPD).
Elevated temperature (37°C) results in greater than a two-fold increase in expression of 13 out of 21 genes reported to be involved in the autophagy process when cells are growing in rich medium. All fold values were calculated from triplicate assays and were statistically validated by ANOVA (p< 0.05).
Fig 13.
C. neoformans autophagy-related genes respond to lack of nitrogen at the permissive temperature (30°C).
Fifteen of twenty-one genes tested had an increase in their expression pattern above two-fold when the yeast cells were subjected to nitrogen starvation. All fold values were calculated from triplicate assays and were statistically validated by ANOVA (p< 0.05).
Fig 14.
Effect of nitrogen deprivation and high temperature (37°C) on the expression of C. neoformans autophagy-related genes.
High temperature in combination with limiting nitrogen source induced the expression pattern above two-fold for 18 out of 21 autophagy-related genes predicted for C. neoformans. All fold values were calculated from triplicate assays and were statistically validated by ANOVA (p< 0.05).