Candida albicans beta-glucan exposure is controlled by the fungal CEK1-mediated mitogen-activated protein kinase pathway that modulates immune responses triggered through dectin-1

Abstract

Innate immunity to Candida albicans depends upon the recognition of molecular patterns on the fungal cell wall. However, the masking of major components such as beta-glucan seems to be a mechanism that fungi have evolved to avoid immune cell recognition through the dectin-1 receptor. Although the role of C. albicans mitogen-activated protein kinase (MAPK) pathways as virulence determinants has been established previously with animal models, the mechanism involved in this behavior is largely unknown. In this study we demonstrate that a disruption of the C. albicans extracellular signal-regulated kinase (ERK)-like 1 (CEK1)-mediated MAPK pathway causes enhanced cell wall beta-glucan exposure, triggering immune responses more efficiently than the wild type, as measured by dectin-1-mediated specific binding and human dendritic cell (hDC)- and macrophage-mediated phagocytosis, killing, and activation of intracellular signaling pathways. At the molecular level, the disruption of CEK1 resulted in altered spleen tyrosine kinase (Syk), Raf-1, and ERK1/2 activations together with IkappaB degradation on hDCs and increased dectin-1-dependent activator protein 1 (AP-1) activation on transfected cells. In addition, concurring with these altered pathways, we detected increased reactive oxygen species production and cytokine secretion. In conclusion, the CEK1-mediated MAPK pathway is involved in beta-glucan exposure in a fungal pathogen, hence influencing dectin-1-dependent immune cell recognition, thus establishing this fungal intracellular signaling route as a promising novel therapeutic target.

FIG. 1.

Candida albicans-MAPK signal transduction pathways and cell wall morphology of C. albicans wild-type CAF2 and the cek1 deletion mutant. (A) The main elements of MAPK signal transduction pathways in C. albicans are schematized, and the physiological function of each pathway is indicated. The CEK1-mediated MAPK pathway is highlighted. (B and C) Transmission electron micrographs of the cell wall of wt strain CAF2 (B) and the cek1 deletion mutant (C), which lacks Cek1 MAPK. Scale bar, 100 nm.

FIG. 2.

Deletion of the CEK1-mediated MAPK pathway results in cell wall β-glucan exposure and increased dectin-1 recognition. (A) Representative flow cytometry analysis of β-glucan exposure on live C. albicans wt CAF2 and cek1 deletion mutants. Light-gray-filled histograms correspond to control antibody (Ab), dark-gray-filled histograms correspond to anti-β-glucan MAb or soluble dectin-1-Fc (sDectinFc), and the empty histogram represents heat-killed positive-control yeast cells. Statistical analysis of these data is shown below and represents the mean fluorescence intensity (MFI) ± SD (after the subtraction of control Ab MFI). **, P < 0.001. (B) Confocal immunofluorescence analysis of individual yeast cells showing representative differential interference contrast (DIC) (Nomarski) microscopy and fluorescence images overlaid. Fungi were stained alternatively with anti-β-glucan MAb or sDectinFc followed by Alexa-488-labeled secondary Abs. (C) Confocal immunofluorescence analysis of individual yeast cells stained as described above (B) showing a tridimensional reconstruction of z-stack fluorescence images. Arrows indicate stronger-stained patches corresponding to bud scars. For CAF2 yeast cells, DIC and fluorescence image overlays are also shown.

FIG. 3.

Dectin-1-dependent binding to C. albicans is enhanced on cek1 mutants. (A) Binding of wt CAF2 or cek1 mutant yeast cells to wt K562 and transfected K562-dectin-1 cells. Cells were incubated with FITC-labeled C. albicans strains (10 yeast cells:1 cell) and analyzed by flow cytometry. Bars represent percentages of cells with bound fungi (means ± SD for eight independent experiments). Statistical analyses compared untreated K562 cell binding to wt CAF2 with binding to the cek1 mutant (solid arrows) and also untreated K562 cell binding to the cek1 mutant with MGD3 MAb- or laminarin (LAM)-pretreated cell binding to this mutant (dotted arrows). **, P < 0.001. The inset at the top shows flow cytometry expression levels of dectin-1 on wt or stably transfected K562 cells (filled histogram, control MAb; empty histogram, anti-dectin-1 MAb). The inset below shows a representative C. albicans cek1 mutant binding profile for K562-dectin-1 cells (light-gray-filled histogram) and blocking profiles with MGD3 (gray empty histogram)- or laminarin (black empty histogram)-pretreated cells. (B) Comparative binding of K562-dectin-1 stably transfected cells to cek1 and cek2 deletion mutants and the corresponding wt strains, CAF2 and RM100, respectively. Differences between data were not significant, unless otherwise indicated. *, P < 0.05.

FIG. 4.

Phagocytosis and killing of C. albicans by human monocyte-derived dendritic cells (hDCs) and macrophages (hMΦs). (A and B) Confocal microscopy images of hDCs (A) and hMΦs (B) showing extracellular yeast cells (blue), extracellular and internalized yeast cells (green), and actin (red). The images shown are not representative of the results but of the method used to quantify them. (C and D) The fungal phagocytic indices of hDCs (C) and hMΦs (D) exposed to wt CAF2 and the cek1 deletion mutant were measured by differential immunofluorescence labeling. The phagocytic index was assessed by counting the number of internalized yeast cells per 100 phagocytes (at least 10 fields for each immunofluorescence carried out in duplicate). Data for phagocytic assays were collected in at least three independent experiments. *, P < 0.05; **, P < 0.001. (E and F) Killing of wt CAF2 and cek1 strains by hDCs (E) and hMΦs (F) after 4 h of coincubation (1 yeast cell:20 phagocytes). The killing percentage for each strain was expressed as the percent reduction of CFU from hDC- or hMΦ-yeast cocultures versus simultaneous cultures containing yeast cells without phagocyte cells. Data are expressed as means ± SD for at least five independent experiments with different donors. Statistical analyses compared the phagocytic index of hDCs or hMΦs for CAF2 versus the cek1 mutant (solid arrows) and the phagocytic index after treatment or nontreatment of primary cells with MAb MGD3 for the cek1 mutant (dotted arrows).

FIG. 5.

Deletion of CEK1 triggers Syk, ERK, and Raf-1 phosphorylation eliciting IκB degradation on hDCs and induces dectin-1-dependent AP-1 activation on transfected cells. (A) Western blot of cell lysates from hDCs cocultured with live C. albicans strains (10 yeast cells:1 cell) blotted with anti-phospho-Syk, -ERK, and -Raf-1 Abs versus anti-total Syk, -ERK, and -Raf-1 Abs, respectively. The bottom shows IκB degradation assayed by blotting with anti-IκB-α Ab. Below, flow cytometry histograms show dectin-1 expression levels of hDCs from three representative donors (D1 to D3) (filled histogram, control Ab; empty histogram, MGD3 MAb). (B) Luciferase activity of HEK293T cells transiently transfected with the indicated plasmids after incubation with zymosan (ZYM) or C. albicans UV-inactivated yeast cells. Luciferase activity was calculated as firefly/Renilla luciferase activities and normalized versus the control (untreated). Bars represent relative light units (RLU) as means ± SD for five independent experiments, each carried out in duplicate. Statistical analyses compared the luciferase activity induction of HEK293T-dectin-1-transfected cells exposed to the wt versus the mutant strain. *, P < 0.05. The inset shows the flow cytometry expression profile of HEK293T-dectin-1-transfected cells (filled histogram, control Ab; empty histogram, dectin-1 staining).

FIG. 6.

The C. albicans cek1 mutant enhances respiratory burst and dectin-1-dependent cytokine secretion. (A and B) ROS secretion analysis of hMΦs (A) and hDCs (B) cocultured with UV-inactivated strains (10 yeast cells:1 cell). Asterisks indicate significant differences between untreated and treated cells (square brackets) or between cek1 mutants and wt strain CAF2 (*, P < 0.05; **, P < 0.001; ***, P < 0.0005). ROS intracellular production is represented as the means ± SD for at least five independent experiments. (C) Mean levels of TNF-α secretion by hDCs stimulated with zymosan (ZYM) or UV-inactivated or heat-killed C. albicans strains (50 yeast cells:1 hDC). Data are representative of data from nine independent healthy donors (means ± SD). (D) Mean levels of TNF-α (6 h) and IL-10 (18 h) in stimulated hDCs (10 yeast cells:1 hDC) in the presence or absence of anti-human dectin-1 MAb (MGD3). ELISA results for test conditions were averaged for six independent healthy donors and are expressed as means ± SD (pg/ml). Data are not significant unless otherwise indicated.