Fig 1.
Dectin-1 alone is not sufficient to bind C. neoformans spores.
CHO-K1 cells were engineered to express murine Dectin-1 (Clec-7a) with a C-terminal HA tag. Dectin-1-HA protein expression and localization were verified with an antibody directed against HA and conjugated to Cy3 (red). Visual assays were used to assess binding of heat-killed C. albicans yeast (A) and heat-killed C. neoformans spores (B). Fungal cells were stained with calcofluor white (blue). Cells were evaluated using both light and fluorescence microscopy at 1000X magnification. White bars represent 10 μm. (C) Interactions between CHO cells and live yeast or live spores were quantified in a representative biological replicate (1 of 3) by assessing a minimum of 300 CHO cells (7–10 frames) per condition, and the change in association was compared in the presence or absence of Dectin-1-HA for each fungal cell type using a Fisher’s Exact Test for statistical analysis (bracket values). X-axis shows fungal cell types. Y-axis represents % CHO cells associated with fungi. Black bars represent Dectin-1-HA expressing cells. Gray bars represent cells harboring an empty expression vector (EV).
Fig 2.
Dectin-1 alone does not recognize cryptococcal spores.
(A) Reporter cells expressing mouse FcRγ chain only (FcR), Mincle and FcRγ chain together (Mincle+FcR), MCL and FcRγ chain together (MCL+FcR), Dectin-2 and FcRγ chain together (Dec-2+FcR), and Dectin-1-CD3ζ (Dec-1) were left unstimulated (black bars), stimulated with heat-killed C. albicans yeast (positive control, light gray bars), or stimulated with heat-killed C. neoformans spores (dark gray bars). After 18 hours of co-incubation, ß-galactosidase activity was measured using a colorimetric assay and expressed in absorbance units (AU) on the y-axis. Data shown are the mean ± the standard deviation of duplicate wells of a single experiment and are representative of two independent experiments. Statistical analysis was carried out using a Student’s t-test on two biological replicates that included two technical replicates each. (B) Weak activation of Dectin-2 increased with higher numbers of spores and depended on the presence of FcRγ (Dec-2+FcR). X-axis shows the ratio of fungal cells to reporter cells with C. neoformans spores. Y-axis shows absorbance units (AU). FcR = FcR chain only, Dec-2 = Dectin-2 alone, Dec-2+FcRm = Dectin-2 expressed with an inactive FcRγ mutant. (C) Quantitative association of C. neoformans spores with receptor-expressing cell lines. Histograms showing number of cells associated with Uvitex 2B-labeled spores in each receptor-expressing subline as indicated. X-axes represent a log scale of fluorescence; y-axes show cell number. Bars delineate range of fluorescence indicative of C. neoformans binding; numbers on bars are the percentage of the total number of cells associated with C. neoformans spores.
Fig 3.
Absence of Dectin-1 or Dectin-2 does not alter association of cryptococcal spores with primary murine phagocytes.
(A) Macrophage-enriched bone marrow-derived phagocytes from WT (black bars), Dectin-1 knockout (dark gray bars) and Dectin-2 knockout (light gray bars) mice were co-incubated with live C. neoformans spores or heat-killed C. albicans yeast, and microscopy was used to assess association of the fungal cells with the macrophages. X-axis shows the fungal cell types being assessed. Y-axis shows the percent of fungal-associated phagocytes relative to WT. Statistical analysis was carried out on 3 individual microscopic fields containing approximately 50 macrophages each. P-values are the result of a Student’s t-test. (B) Representative microscopy fields at 400X of heat-killed C. albicans co-incubated with WT and Dectin-1 knockout macrophage-enriched phagocytes (no difference was observed with C. neoformans). (Black bars = 50 μm) (C) Macrophage-enriched bone marrow-derived phagocytes from WT, Dectin-1-/-, Dectin-2-/-, Card-9-/-, FcRγ-/-, Mincle-/- and MR-/- mice were co-incubated with live C. neoformans spores (black bars) or live C. neoformans yeast (light gray bars) and association (binding + phagocytosis) frequencies determined by CFU analysis. X-axis shows the macrophage genotypes. Y-axis shows the percent of introduced fungal cells that were associated with the phagocytes. Data show the mean and standard deviations from a single experiment carried out in triplicate. An ANOVA with a Dunnett’s post-hoc test revealed that phagocyte association with spores was not statistically different from WT for any of the knock out phagocytes tested (p>0.05); results are representative of those obtained from 2–5 individual experiments for each knockout line compared to WT.
Fig 4.
Dectin-1-/- and Dectin-2-/- phagocytes show modest changes in internalization of C. neoformans spores.
Flow cytometry was used to differentiate and assess internalization and binding of C. neoformans spores by DC-enriched bone marrow-derived phagocytes and AMs. (A) Results of flow cytometry analysis using DC-enriched phagocytes shown as FACS plots and (B) bar graphs of spore internalization. DC-enriched phagocytes from WT, Dectin-1-/-, and Dectin-2-/- mice show modest differences in internalization (GFP+CFW-) and binding (GFP+CFW+) of C. neoformans spores. ns = not significant (p = 0.1 by Student's t-test). (C) Results of flow cytometry analysis using AMs shown as FACS plots and (D) bar graphs of spore internalization. AMs from Dectin-1-/- and Dectin-2-/- mice show decreases in the phagocytosis of spores as compared to WT. ns = not significant (p = 0.09 by Student’s t-test). In all cases FACS plots were generated from experiments carried out in triplicate, with the exception of Dectin-2-/- AMs, which were in duplicate. X-axes represent increasing calcofluor white signal. Y-axes represent increasing GFP signal. Bar graphs show average values, and error bars represent the standard error of the mean. X-axes indicate phagocyte background, and y-axes represent percent of phagocytes harboring spores.
Fig 5.
Mice lacking Dectin-1 or Dectin-2 do not show increased susceptibility or resistance to C. neoformans.
Groups of WT (black), Dectin-1-/-(light gray), and Dectin-2-/- (dark gray) mice were infected intranasally with 2.5x105 C. neoformans spores and sacrificed when they became moribund. X-axis shows the number of days post-infection. Survival was tracked daily through the course of the infection. Y-axis shows the percentage of mice surviving in each group. There was no statistical difference between any of the mouse genotypes as assessed by a Log Rank (Mantel-Cox) test, (p > 0.1).
Fig 6.
Model of receptor recognition of C. neoformans spores.
(A) Soluble chimeric Dectin-1-Fc protein binds to β-glucan on C. neoformans spores, whereas Dectin-1 within the context of the cell membrane does not bind spores. This inability to bind could be due to topological interference between components on the spore coat and those on the host membrane and/or general inaccessibility of spore β-1,3-glucan to membrane-bound Dectin-1. In the case of C. albicans, β-1,3-glucan is exposed in such a way that both soluble and membrane-bound Dectin-1 can bind to it and engage downstream signaling. (B) Two overarching models of C. neoformans spore phagocytosis. Numerous Contributors (left): Although none of the receptors tested in these studies contributed significantly to spore phagocytosis, it is possible that many diverse receptors, including those tested, could each mediate weak spore interactions that facilitate phagocytosis. Unidentified Receptor(s) (right): It is possible that one or more currently unidentified or unknown receptors could be responsible for mediating the uptake of spores.