PMC

Effects of inhibition of EGFR with AG1478 or cetuximab and inhibition of HER2 with AG825 or trastuzumab on epithelial cell endocytosis of C. albicans (A and B) or S. cerevisiae expressing C. albicans ALS3 (C). Results are the mean ± SD of three experiments, each performed in triplicate. *P < 0.01 vs. control.

HER2 functions with EGFR to mediate the endocytosis of C. albicans. (A) Endocytosis of C. albicans hyphae by NIH/3T3 cells expressing human EGFR, HER2, or both EGFR and HER2. (B) Effects of inhibiting EGFR with cetuximab on the endocytosis of C. albicans by NIH/3T3 cells expressing either human HER2 or human HER2 and EGFR. (C) Immunoblots of lysates of NIH/3T3 cells expressing human EGFR, HER2, or both EGFR and HER2. The blots were probed with antibodies that recognized both human and mouse proteins. Results in A and B are the mean ± SD of three experiments, each performed in triplicate. *P < 0.05 compared with control.

Both HER2 and E-cadherin are required for maximal endocytosis of C. albicans. (A) Confocal microscopic images of epithelial cells that had been infected with C. albicans and then stained for HER2 and E-cadherin. A DIC image of the same microscope field is shown. Insets are higher magnification images of the organisms indicated by the large arrows. Smaller solid arrows indicate the accumulation of HER2 and E-cadherin around the hyphae. (Scale bar: 20 μm.) (B) Effects of siRNA knockdown of E-cadherin and trastuzumab inhibition of HER2 on the endocytosis of C. albicans. Results are the mean ± SD of three experiments, each performed in triplicate. *P < 0.01 vs. control; ^†P < 0.01 vs. cells transfected with E-cadherin siRNA and not exposed to trastuzumab. (C) Immunoblot of total epithelial cell lysate showing E-cadherin knockdown.

GW2974 blocks the phosphorylation of EGFR and HER2 and reduces the severity of disease during experimental OPC. (A) Confocal images of phospho-EGFR and -HER2 in the oral epithelium of uninfected mice (Left), mice that were infected with C. albicans and given no drug (Center), and mice that were infected with C. albicans and treated with GW2974 (Right). (B and C) Treatment with GW2974 ameliorates OPC as determined by oral fungal burden (B) and histopathology (C) after 5 d of infection. Results in B are the median ± the interquartile range of combined results of two independent experiments (n = 14). (D) GW2974 inhibits endocytosis of C. albicans by OKF6/TERT-2 oral epithelial cells. Results are the mean ± SD of three experiments, each performed in triplicate. *P = 0.005 compared with control. (Scale bar: A, 20 μm; C, 40 μm.)

C. albicans induces EGFR and HER2 phosphorylation. (A) Time course of tyrosine phosphorylation of EGFR and HER2 in OKF6/TERT-2 cells in response to infection with C. albicans hyphae. (B) Accumulation of phosphorylated EGFR and HER2 around C. albicans cells. (Left) Confocal images of OKF6/TERT-2 cells that were infected with C. albicans and then stained with phospho-specific EGFR and HER2 antibodies. (Right) The same microscope fields imaged by DIC. Insets are higher magnification images of the organisms indicated by the large arrows. Smaller solid arrows indicate the accumulation of phosphorylated EGFR and HER2 around the hyphae. (Scale bar: 20 μm.) (C–E) Specificity of EGFR and HER2 phosphorylation. Immunoblots showing the amount of phosphorylated EGFR and HER2 in OKF6/TERT-2 cells that were exposed for 20 min to medium alone (control) or hyphal or yeast-phase organisms of C. albicans SC5314 (C), the indicated C. albicans mutant strains (D), or S. cerevisiae containing the backbone vector (pADH1) or expressing C. albicans ALS3 (pALS3; E).

Affinity purification of epithelial cell EGFR and HER2 by C. albicans. (A and B) Binding of FaDu cell EGFR to C. albicans. Immunoblots of biotin-labeled FaDu cell surface proteins eluted from hyphae of C. albicans SC5314. Blots were probed with an antibiotin antibody (A) or an anti-EGFR antibody (B). (C and D) C. albicans interacts with both EGFR and HER2 in extracts of epithelial cell membrane proteins. Immunoblots of OKF6/TERT-2 oral epithelial cell proteins eluted from the indicated C. albicans clinical (C) and mutant (D) strains. Blots were probed with antibodies against EGFR and HER2. (E) Als3 binds to EGFR and HER2. Immunoblots of OKF6/TERT-2 cell membrane proteins that had been eluted from S. cerevisiae containing the backbone vector (pADH1) or expressing C. albicans ALS3 (pALS3). (F) Confocal microscopic images of OKF6/TERT-2 epithelial cells that had been infected with C. albicans SC5314 and then stained for EGFR and HER2. The same microscopic field was imaged by differential interference contrast (DIC) optics to show the organisms. Insets are higher magnification images of the organisms indicated by the large arrows. Smaller solid arrows indicate the accumulation of EGFR and HER2 around the hyphae. (Scale bar: 20 μm.)