Masking of β(1-3)-glucan in the cell wall of Candida albicans from detection by innate immune cells depends on phosphatidylserine

Abstract

The virulence of Candida albicans in a mouse model of invasive candidiasis is dependent on the phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE). Disruption of the PS synthase gene CHO1 (i.e., cho1Δ/Δ) eliminates PS and blocks the de novo pathway for PE biosynthesis. In addition, the cho1Δ/Δ mutant's ability to cause invasive disease is severely compromised. The cho1Δ/Δ mutant also exhibits cell wall defects, and in this study, it was determined that loss of PS results in decreased masking of cell wall β(1-3)-glucan from the immune system. In wild-type C. albicans, the outer mannan layer of the wall masks the inner layer of β(1-3)-glucan from exposure and detection by innate immune effector molecules like the C-type signaling lectin Dectin-1, which is found on macrophages, neutrophils, and dendritic cells. The cho1Δ/Δ mutant exhibits increases in exposure of β(1-3)-glucan, which leads to greater binding by Dectin-1 in both yeast and hyphal forms. The unmasking of β(1-3)-glucan also results in increased elicitation of TNF-α from macrophages in a Dectin-1-dependent manner. The role of phospholipids in fungal pathogenesis is an emerging field, and this is the first study showing that loss of PS in C. albicans results in decreased masking of β(1-3)-glucan, which may contribute to our understanding of fungus-host interactions.

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.