The Globular C1q Receptor Is Required for Epidermal Growth Factor Receptor Signaling during Candida albicans Infection

Abstract

During oropharyngeal candidiasis, Candida albicans activates the epidermal growth factor receptor (EGFR), which induces oral epithelial cells to endocytose the fungus and synthesize proinflammatory mediators. To elucidate EGFR signaling pathways that are stimulated by C. albicans, we used proteomics to identify 1,214 proteins that were associated with EGFR in C. albicans-infected cells. Seven of these proteins were selected for additional study. Among these proteins, WW domain-binding protein 2, Toll-interacting protein, interferon-induced transmembrane protein 3 (IFITM3), and the globular C1q receptor (gC1qR) were found to associate with EGFR in viable oral epithelial cells. Each of these proteins was required for maximal endocytosis of C. albicans, and all regulated fungus-induced production of interleukin-1β (IL-1β) and/or IL-8, either positively or negatively. gC1qR was found to function as a key coreceptor with EGFR. Interacting with the C. albicans Als3 invasin, gC1qR was required for the fungus to induce autophosphorylation of both EGFR and the ephrin type A receptor 2. The combination of gC1qR and EGFR was necessary for maximal endocytosis of C. albicans and secretion of IL-1β, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by human oral epithelial cells. In mouse oral epithelial cells, inhibition of gC1qR failed to block C. albicans-induced phosphorylation, and knockdown of IFITM3 did not inhibit C. albicans endocytosis, indicating that gC1qR and IFITM3 function differently in mouse versus human oral epithelial cells. Thus, this work provides an atlas of proteins that associate with EGFR and identifies several that play a central role in the response of human oral epithelial cells to C. albicans infection. IMPORTANCE Oral epithelial cells play a key role in the pathogenesis of oropharyngeal candidiasis. In addition to being target host cells for C. albicans adherence and invasion, they secrete proinflammatory cytokines and chemokines that recruit T cells and activated phagocytes to foci of infection. It is known that C. albicans activates EGFR on oral epithelial cells, which induces these cells to endocytose the organism and stimulates them to secrete proinflammatory mediators. To elucidate the EGFR signaling pathways that govern these responses, we analyzed the epithelial cell proteins that associate with EGFR in C. albicans-infected epithelial cells. We identified four proteins that physically associate with EGFR and that regulate different aspects of the epithelial response to C. albicans. One of these is gC1qR, which is required for C. albicans to activate EGFR, induce endocytosis, and stimulate the secretion of proinflammatory mediators, indicating that gC1qR functions as a key coreceptor with EGFR.

Keywords: Candida albicans; endocytosis; epidermal growth factor receptor; host defense; oral epithelial cells.

FIG 1

Proximity ligation assays (PLA) showing the physical association of the epidermal growth factor receptor (EGFR) with WW domain-binding protein 2 (WBP2), Toll-interacting protein (TOLLIP), interferon-induced transmembrane protein 3 (IFITM3), and the globular C1q receptor (gC1qR) in the OKF6/TERT-2 oral epithelial cell line. (A) Confocal microscopic images of epithelial cells that had been either incubated with medium alone (top) or infected with C. albicans (bottom) for 90 min. Red spots indicate the regions where the indicated proteins associate with EGFR. Arrows indicate the accumulation of the proteins around C. albicans hyphae. Results are representative of three independent experiments. Scale bar, 25 μm. (B) Quantitative analysis of the images to determine the percentage of C. albicans cells with spots indicating the colocalization of EGFR with WBP2, TOLLIP, IFITM3, or gC1qR. Results are the mean ± SD from three independent experiments. Ca, C. albicans; Orgs, organisms.

FIG 2

Functional analysis of proteins that interact with EGFR. Oral epithelial cells were transfected with EGFR (A), WBP2 (B), TOLLIP (C), IFITM3 (D), and gC1qR (E) siRNA. For each siRNA, the extent of protein knockdown and its effects on the number of cell-associated organisms, the number of endocytosed organisms, IL-1β secretion, and IL-8 secretion were determined. The graphs show the mean ± SD from three independent experiments, each performed in triplicate. The data were analyzed using one-way analysis of variance with Dunnett’s test for multiple comparisons. Ca, C. albicans; Ctrl, control; ns, not significant; Orgs/HPF, organisms per high-power field; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Surface-expressed gC1qR mediates the endocytosis of C. albicans. (A) Effects of two different anti-gC1qR monoclonal antibodies on the endocytosis of C. albicans by oral epithelial cells. (B) Effects of the anti-gC1qR antibody 74.5.2 and the EGFR kinase inhibitor gefitinib on the endocytosis of C. albicans by oral epithelial cells. (C and D) Endocytosis of C. albicans by NIH/3T3 cells expressing human gC1qR and/or human EGFR. (C) Additive effects of EGFR and gC1qR on endocytosis. (D) Effects of inhibiting surface-expressed gC1qR with the anti-gC1qR antibody 74.5.2 on endocytosis. Results are the mean ± SD from three independent experiments, each performed in triplicate. The data were analyzed using one-way analysis of variance with Dunnett’s test for multiple comparisons. ns, not significant; Orgs/HPF, organisms per high-power field; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

gC1qR is required for production of proinflammatory mediators by oral epithelial cells in response to C. albicans infection. (A to F) Oral epithelial cells were infected with C. albicans in the presence of the anti-gC1qR antibody 74.5.2 or gefitinib for 8 h, after which the concentration of the indicated inflammatory mediators in the medium was analyzed by ELISA (A and B) or Luminex cytometric bead array (C to F). Results are the mean ± SD from three independent experiments, each performed in duplicate. The data were analyzed using one-way analysis of variance with Dunnett’s test for multiple comparisons. Ca, C. albicans; Ctrl, control; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

Interactions of gC1qR and EGFR with C. albicans. (A and B) Effects of the anti-gC1qR antibody 74.5.2 and gefitinib on the phosphorylation of EGFR and EphA2 in response to 90-min infection with C. albicans. (A) Representative immunoblots. (B) Densitometric analysis of three immunoblots, such as the ones shown in panel A. (C and D) Effects of anti-gC1qR and anti-EGFR antibodies on binding of gC1qR and EGFR to C. albicans hyphae. (C) Representative immunoblots. (D) Densitometric analysis of four immunoblots, such as the ones shown in panel C. Results are the mean ± SD from 3 to 4 independent experiments. (E and F) Representative confocal images of the proximity ligation assay (PLA) showing the association of gC1qR with EGFR around hyphae of C. albicans wild-type, als1Δ/Δ, and ece1Δ/Δ strains, but not the als3Δ/Δ mutant (E). (F) Quantitative analysis of the images such as the one in panel E to determine the percentage of hyphae of the indicated C. albicans strains, with spots indicating the colocalization of EGFR with gC1qR. Results are the mean ± SD from three independent experiments. The numerical data were analyzed using one-way analysis of variance with Dunnett’s test for multiple comparisons. Ca, C. albicans; Ctrl, control; ns, not significant; Orgs, organisms; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

Inhibition of gC1qR has no significant effect on the outcome of oropharyngeal candidiasis. (A) Effects of the indicated anti-gC1qR antibodies on the endocytosis of C. albicans by primary mouse oral epithelial cells. Results are the mean ± SD from three independent experiments, each performed in triplicate. The data were analyzed using one-way analysis of variance with Dunnett’s test for multiple comparisons. (B and C) Effects of the anti-gC1qR antibody 74.5.2 and gefitinib on the outcome of oropharyngeal candidiasis after 1 day of infection as shown by oral fungal burden (B) and oral myeloperoxidase (MPO) content (C). Results are the median ± interquartile range from two independent experiments, each with 4 to 5 mice per group. The data were analyzed using the Kruskal-Wallis test. ns, not significant; Orgs/HPF, organisms per high-power field; **, P < 0.01; ***, P < 0.001.

FIG 7

Mouse and human oral epithelial cells respond differently to C. albicans. (A and B) Effects of the anti-gC1qR antibody 74.5.2 on the phosphorylation of EGFR in primary human oral epithelial cells in response to 90-min infection with C. albicans. (A) Representative immunoblots. (B) Densitometric analysis of four immunoblots, such as the ones shown in panel A. (C) The anti-gC1qR antibody 74.5.2 inhibits the endocytosis of C. albicans by primary human oral epithelial cells. (D and E) Effects of the anti-gC1qR antibody 74.5.2 and gefitinib on the phosphorylation of EGFR in primary mouse oral epithelial cells in response to 90-min infection with C. albicans. (D) Representative Western blots. (E) Densitometric analysis of four Western blots, such as the ones shown in panel D. (F) Effects of siRNA knockdown of IFITM3 on primary human oral epithelial cells. Results are the mean ± SD from three independent experiments, each performed in triplicate. The data were analyzed using one-way analysis of variance with Dunnett’s test for multiple comparisons. Ca, C. albicans; ns, not significant; Orgs/HPF, organisms per high-power field; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 8

Diagram of the functional interaction of C. albicans Als3 with gC1qR and EGFR in human oral epithelial cells. Als3 interacts either directly or indirectly with both gC1qR and EGFR, leading to the activation of EGFR and subsequent induction of endocytosis and secretion of proinflammatory mediators.