Fig 1.
β-glucan is masked in yeast and exposed in germ-tubes.
Representative images of C. albicans cells stained with Dectin-1-Fc and secondary antibody conjugated to FITC. Hyphae were induced at 37°C for indicated hours and in indicated media. Yeast cells were cultured at 30°C in same medium and for same length of time as hyphae. A fixed time of exposure was used in image acquisitions for all pictures. Experiments were repeated three times, The scale bar represents 10μM. Mean fluorescence intensities per area were quantitated by ImageJ. p Values were calculated using one-way ANOVA with Tukey post hoc analysis (***p<0.001, **p<0.01, *p<0.05).
Fig 2.
Eng1 reduces β-glucan exposure in the yeast form of C albicans.
(a) Depicted regulation of ENG1 expression in yeast and hyphae. (b) Expression levels of ENG1 mRNA in yeast or hyphae grown in YPD for 1 h, quantitated by qPCR. ENG1 transcript level was normalized with ACT1 transcript level. (c) Representative images of C. albicans cells stained with Dectin-1-Fc and secondary antibody conjugated to FITC. Yeast cells were cultured in YPD for 6 hours. The scale bar represents 10μM. Mean fluorescence intensities per area were quantitated by ImageJ. (d) The levels of TNFα in the supernatant of BMDM stimulated with fixed C. albicans yeast form by a MOI of 1:3. (e) Representative images of hyphae stained with Dectin-1-Fc and secondary antibody conjugated to FITC. Hyphae were induced in RPMI for 5 hours. Mean fluorescence intensities were quantitated by ImageJ. The scale bar represents 10μM. (f) The levels of TNFα in the supernatant of BMDM stimulated with WT, eng1 or eng1/ENG1 live yeast by a MOI of 1:1. Experiments were repeated at least three times. p Values were calculated using ANOVA with Tukey post hoc analysis (****p<0.0001; ***p<0.001, **p<0.01, *p<0.05).
Fig 3.
Regulation of β-glucan exposure in yeast by Eng1 and Ywp1.
Representative images of C. albicans yeast cells stained with Dectin-1-Fc and secondary antibody conjugated to FITC. The scale bar represents 10μM. Mean fluorescence intensities per area were quantitated by ImageJ. p Values were calculated using ANOVA with Tukey post hoc analysis (***p<0.001, **p<0.01).
Fig 4.
Down-regulation of ENG1 during cell chain formation is associated with increased β-glucan exposure (a) Transcript levels of ENG1 in fluconazole/caspofungin-treated or untreated cells. (b) The levels of TNFα in the supernatant of BMDM stimulated with fixed yeast cells by a MOI of 1:3. Cells were cultured in YPD with 10ug/mL fluconazole for overnight or 0.06ug/mL Caspofungin for 3 hours. (c) Representative images of yeast form cells stained with Dectin-1-Fc and secondary antibody conjugated to FITC. WT and eng1 supernatant were collected and filtered from saturated overnight culture of WT or eng1 yeast cells. Mean fluorescence intensities per area were quantitated by ImageJ. p Values were calculated using ANOVA with Tukey post hoc analysis (****p<0.0001; *** p<0.001, ** p<0.01, * p<0.05).
Fig 5.
Carbon sources regulated β-glucan exposure through Eng1.
(a) Representative images of C. albicans yeast cells stained with Dectin-1-Fc and secondary antibody conjugated to FITC. Cells from the overnight cultures were diluted to fresh medium containing 2% glucose alone or 1% lactate plus 1% glucose as sole carbon sources, and grown for a further 5 hours. (b) Representative images of C. albicans yeast cells stained with Dectin-1-Fc and secondary antibody conjugated to FITC. Cells were first cultured to log phase to avoid hyphae induction in the later GlcNAc culture. Then cells were transferred to fresh medium containing 2% glucose or GlcNAc as sole carbon sources, and grown for a further 5 hours. Experiment was repeated three times. Fluorescence intensities per area were quantitated by ImageJ. p Values were calculated using ANOVA with Tukey post hoc analysis (** p<0.01, * p<0.05).
Fig 6.
The eng1 mutant was hypo-virulent in male and hyper-virulent in female.
(a) Survival of C57BL/6 male and female mice after intravenous inoculation with 1 × 105 yeast phase cells of the indicated strains of C. albicans. Experiments were done twice; n = 5 for each time. (b) Survival of C57BL/6 and Dectin-1-/- female mice after intravenous inoculation with 1 × 105 yeast phase cells of the indicated strains of C. albicans. Experiments were done twice; n = 3 for each time. p Values were calculated with Gehan-Breslow-Wilcoxon (**p<0.01; * p<0.05).
Fig 7.
(a) Fungal burden of the kidney, brain, spleen, and liver of mice at 1-day and 4-day postinfection after inoculation with 1 × 105 eng1 deletion mutant or eng1/ENG1 complemented C. albicans yeast. Results are median ± interquartile range with 8 mice per strain. Experiments were done twice; n = 3 for the first time and n = 5 for the second time. p Values were calculated with Mann-Whitney test (*p<0.05). (b) Representative images of kidney histology slides of PAS-stained paraffin sections from infected kidneys at day 4 post infection. Sections from two sets of kidneys were analyzed. Circled area in 2x image is shown in the 40x image bellow. Scale bars in 2x and 40x are 500 μM and 25 μM, respectively. Arrows indicate yeast-like cells. (c) Relative cytokine/chemokine levels at 1-day and 4-day post-infection. (d) Relative MPO levels at 1-day and 4-day post-infection. p Values were calculated with Unpaired t-test (**p<0.01; * p<0.05).