Recognition of Yeast by Murine Macrophages Requires Mannan but Not Glucan

Expression of yEmRFP does not affect the rate or length of hyphal formation. The kinetics and extent of hyphal formation were examined in wild-type strain BWP17 or yEmRFP-overexpressing strain SKY38. Yeast cells were grown to early logarithmic stage (1 to 2 OD₆₀₀ units/ml), seeded on glass coverslips, and induced to form hyphae by the addition of 20% serum and a temperature shift to 37°C. Coverslips were removed at the indicated times and viewed by light or fluorescence microscopy. It should be noted that these panels do not represent overlapping images from the same field.

Phagocytosis of red fluorescent C. albicans by J774 macrophages in real time. Red fluorescent C. albicans cells (strain SKY38) were added to J774 macrophages grown on coverslips in multiwell plates. Yeast cells were added at an MOI of ∼1 yeast cell per macrophage, and at various times after addition of yeast cells, CW (1 μg/ml) and PSA-FITC (20 μg/ml) were added to the culture medium to stain the surface of noningested yeast cells and macrophages, respectively. Cells were viewed by fluorescence microscopy directly, without washing, using RFP, GFP, and 4′,6-diamidino-2-phenylindole filter sets. Arrows denote RFP-positive internalized yeast cells that are inaccessible to staining with CW.

Yeast cells are ingested more efficiently than hyphal cells by J774 macrophages. Competition assays were performed with various combinations of S. cerevisiae, C. glabrata, and C. albicans yeast or hyphal cells (preincubated for 20 min with 20% serum at 37°C to induce hyphae). Equal numbers of yeast cells expressing yEmRFP or yEGFP were mixed together and added to J774 macrophages at an MOI of 5. After 40 min, CW was added and red or green cells were scored as being in or out of macrophages. (A) Graphic representation of quantitative competition assays between nontagged C. albicans yeast cells (strain SKY51) and those expressing yEGFP (strain SKY43) that were mixed with RFP-expressing C. albicans yeast cells (strain SKY38), with S. cerevisiae yeast cells (strain SEY6210 plus pADH-yEGFP), or with C. albicans hyphal cells (strain SKY38). Sc, Ca, and Cg, S. cerevisiae, C. albicans, and C. glabrata, respectively. The percent internalization ratio was calculated by scoring yeast cells as red or green and as CW positive (outside) or CW negative (inside), as described in Materials and Methods. Representative single-focal-plane images of these assays are shown in panel B, with arrows highlighting single macrophages that have taken up both red and green yeast cells at various ratios.