Article Figures & Data
Figures
- FIG 1
Yeast-form cells of the cho1Δ/Δ mutant exhibit increased exposure of β(1-3)-glucan. Secondary immunofluorescence with an anti-β(1-3)-glucan primary antibody and a Cy3-conjugated secondary antibody reveals that the cho1Δ/Δ mutant has greater β(1-3)-glucan exposure than the other strains. Strains were grown to log phase in YPD at 30°C. WT, wild type.
- FIG 2
The cho1Δ/Δ mutant has increased binding to the Dectin1 receptor. (A) C. albicans strains grown overnight in YPD were stained with sDectin-1–Fc and a fluorescently labeled secondary antibody, showing that the cho1Δ/Δ mutant exhibits greater staining than all of the other strains. (B) Flow cytometry reveals that the cho1Δ/Δ mutant (solid red line) has greater binding to the Dectin-1 receptor than the other strains. (C) A graph of the relative mean staining intensity of each sample reveals that the cho1Δ/Δ mutant exhibits significantly greater staining with sDectin-1–Fc than the other strains. *, P < 0.05.
- FIG 3
Hyphal formation by the cho1Δ/Δ mutant is not significantly impaired in serum or host tissue. (A) C. albicans strains were incubated in 100% human serum and observed by time-lapse microscopy on thermally regulated glass slides. After 3 h, hyphal length was measured as an indicator of growth over time. (B) C. albicans strains expressing GFP were stained with Alexa Fluor dye during yeast-form growth. Stained yeast cells were then injected into mice via the tail vein, and after 6 h, the mice were sacrificed and their kidneys were removed and then homogenized and viewed by microscopy. Hyphae expressing GFP indicate new growth that occurred within mouse tissue. (C) Percentages of yeast cells inoculated in vivo that generated hyphae in mouse kidneys. A minimum of 24 cells were analyzed per strain. WT, wild type.
- FIG 4
The hyphal form of the cho1Δ/Δ mutant has increased exposure of β(1-3)-glucan and binding by the Dectin-1 receptor. Cells were grown as hyphae in human serum. (A) Secondary immunofluorescence analysis was performed with an anti-β(1-3)-glucan antibody. Increased exposure of β(1-3)-glucan in the cho1Δ/Δ mutant and the psd1Δ/Δ psd2Δ/Δ double mutant appears to localize at malformed hyphal tips (indicated by arrows). Staining was performed after formaldehyde fixation. (B) In the absence of formaldehyde fixation, only the cho1Δ/Δ mutant revealed strong staining with either anti-β(1-3)-glucan antibody or sDectin-1–Fc. These probes exhibited overlapping staining patterns in the hyphal cell wall. Top row, bright-field optics; middle row, staining with anti-β(1-3)-glucan antibody and a Cy3 (red)-conjugated secondary antibody; bottom row, staining with sDectin-1–Fc and a Cy2 (green)-conjugated secondary antibody. WT, wild type.
- FIG 5
There are no differences in killing by phagocytes or defensins. (A) RAW264.7 macrophages and C. albicans cells were coincubated for 6 h, and C. albicans was plated to measure the number of viable CFU. C. albicans strains mock challenged with macrophages were used to calculate the percentages of C. albicans cells killed. (B) Human neutrophils were tested for the ability to kill the different strains by an approach similar to that used for panel A, except that they were coincubated for only 2 h. (C) The cho1Δ/Δ mutant was incubated with β-defensin or mock challenged, and then the viable CFU were counted. WT, wild type.
- FIG 6
The cho1Δ/Δ mutant elicits TNF-α from macrophages in a Dectin-1-dependent manner. A TNF-α-specific ELISA was used to measure TNF-α elicited from RAW-BLUE macrophages in response to a C. albicans challenge after 4 h. Pretreatment of RAW-BLUE macrophages with an anti-Dectin-1 neutralizing antibody reveals that the TNF-α response is Dectin-1 mediated. *, P < 0.0001. WT, wild type.
Tables
- TABLE 1
C. albicans strains used in this study
Strain Parent Genotype Source or reference SC5314 Clinical isolate Prototrophic wild type 43 YLC337 SC5314 cho1Δ/cho1Δ 7 YLC344 SC5314 cho1Δ/cho1Δ::CHO1-SAT1 7 YLC280 SC5314 psd1Δ/psd1Δ 7 YLC294 SC5314 psd1Δ/psd1Δ::PSD1-SAT1 7 YLC375 SC5314 psd2Δ/psd2Δ psd1Δ/psd1Δ 7 SED022 YLC337 cho1Δ/Δ::CHO1 with SAT1 flipped out This study SED010 SC5314 Wild type with PENO1-GFP-SAT1 This study SED013 YLC337 cho1Δ/cho1Δ mutant with PENO1-GFP-SAT1 This study SED016 YLC280 psd1Δ/psd1Δ mutant with PENO1-GFP-SAT1 This study SED019 YLC375 psd2Δ/Δ psd1Δ/psd1Δ mutant with PENO1-GFP-SAT1 This study SED024 SED022 cho1Δ/cho1Δ::CHO1 mutant with PENO1-GFP-SAT1 This study - TABLE 2
Hyphae of the cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ mutants are malformed
Strain No. of cells with deformity Total no. of cells % with deformity SC5314 0 37 0.00 cho1Δ/Δ mutant 11 23 47.83 cho1::CHO1 mutant 0 13 0.00 psd1Δ/Δ mutant 0 29 0.00 psd1::PSD1 mutant 0 19 0.00 psd1Δ/Δ psd2Δ/Δ double mutant 11 21 52.38
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Fig. S1. The stationary-phase yeast form of the cho1Δ/Δ mutant exhibits increased exposure of β(1-3)-glucan. Fig. S2. β(1-3)-Glucan and Dectin-1 costaining of yeast reveals overlapping staining patterns. Fig. S3. Hyphae costain similarly with β(1-3)-glucan antibody and sDectin-1-Fc. Fig. S4. The cho1Δ/Δ mutant elicits TNF-α from RAW 264.7 macrophages in a Dectin-1-dependent manner.
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- Supplemental file 1 -
