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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2012 Aug 13;109(35):14194–14199. doi: 10.1073/pnas.1117676109

EGFR and HER2 receptor kinase signaling mediate epithelial cell invasion by Candida albicans during oropharyngeal infection

Weidong Zhu a, Quynh T Phan a, Pinmanee Boontheung b,1, Norma V Solis a, Joseph A Loo b,c, Scott G Filler a,d,2
PMCID: PMC3435201  PMID: 22891338

Abstract

The fungus Candida albicans is the major cause of oropharyngeal candidiasis (OPC). A key feature of this disease is fungal invasion of oral epithelial cells, a process that can occur by active penetration and fungal-induced endocytosis. Two invasins, Als3 and Ssa1, induce epithelial cell endocytosis of C. albicans, in part by binding to E-cadherin. However, inhibition of E-cadherin function only partially reduces C. albicans endocytosis, suggesting that there are additional epithelial cell receptors for this organism. Here, we show that the EGF receptor (EGFR) and HER2 function cooperatively to induce the endocytosis of C. albicans hyphae. EGFR and HER2 interact with C. albicans in an Als3- and Ssa1-dependent manner, and this interaction induces receptor autophosphorylation. Signaling through both EGFR and HER2 is required for maximal epithelial cell endocytosis of C. albicans in vitro. Importantly, oral infection with C. albicans stimulates the phosphorylation of EGFR and HER2 in the oral mucosa of mice, and treatment with a dual EGFR and HER2 kinase inhibitor significantly decreases this phosphorylation and reduces the severity of OPC. These results show the importance of EGFR and HER2 signaling in the pathogenesis of OPC and indicate the feasibility of treating candidal infections by targeting the host cell receptors with which the fungus interacts.

The fungus Candida albicans grows on mucosal surfaces as part of the normal human flora. When local or systemic antifungal defense mechanisms are impaired, this organism can overgrow and cause oropharyngeal candidiasis (OPC), leading to significant morbidity in patients with HIV/AIDS, Sjögren syndrome, diabetes mellitus, and head and neck cancers (1, 2). Although OPC can be treated with oral polyene or azole antifungal agents, resistance can develop in immunocompromised patients who receive prolonged therapy (1).

Invasion of oral epithelial cells by C. albicans is central to the pathogenesis of OPC, and this organism can penetrate into oral epithelial cells by two different mechanisms (3). One mechanism is active penetration, in which C. albicans hyphae progressively elongate and physically push their way into epithelial cells (4). The other mechanism is induced endocytosis. In this mechanism, the C. albicans invasins, Als3 and Ssa1, bind to epithelial cell E-cadherin and activate the clathrin-dependent endocytosis machinery (5–7). Importantly, E-cadherin is not the only epithelial cell receptor for C. albicans, because chelation of calcium with EGTA to block cadherin function only reduces epithelial cell endocytosis of C. albicans by 30–40% (Fig. S1).

We set out to identify an additional epithelial cell receptor for C. albicans. We discovered that the EGF receptor (EGFR) and a related protein, HER2, are involved in the host cell interactions of C. albicans and mediate its endocytosis by oral epithelial cells in vitro. Furthermore, we found that C. albicans induces EGFR and HER2 autophosphorylation in the oral epithelium of mice with OPC and that treatment of mice with a dual EGFR and HER2 kinase inhibitor significantly attenuates this disease.

Results

C. albicans Als1 and Ssa1 Bind to Epithelial Cell EGFR and HER2.

To identify an additional epithelial cell receptor for C. albicans, biotinylated cell surface proteins from the FaDu oral epithelial cell line were isolated and then affinity-purified over intact C. albicans hyphae. These proteins were separated by SDS/PAGE and detected by immunoblotting with an antibiotin antibody. A prominent band with a molecular mass of ∼160 kDa was detected (Fig. 1A). By liquid chromatography—tandem MS microsequencing, this band was found to contain EGFR. The presence of EGFR in FaDu cell proteins that had been affinity-purified over C. albicans hyphae was verified by immunoblotting with an anti-EFGR antibody (Fig. 1B). EGFR is frequently overexpressed in oropharyngeal carcinoma cells (8), and the FaDu cell line was obtained from a patient with pharyngeal carcinoma. Therefore, we repeated the immunoblotting experiments using the well-differentiated OKF6/TERT-2 cell line, which was developed by the forced expression of the human telomerase catalytic subunit in oral epithelial cells from a healthy adult (9). Three clinical isolates of C. albicans pulled down EGFR in protein extracts from these cells (Fig. 1C). Based on these results, all subsequent experiments were performed with the OKF6/TERT-2 epithelial cell line.

Fig. 1.

Fig. 1.

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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.)

EGFR frequently forms a heterodimer with HER2 (10). Using an anti-HER2 antibody to probe immunoblots of epithelial cell proteins that had been eluted from C. albicans hyphae, we determined that the organism also interacts with HER2 (Fig. 1C). Furthermore, coimmunoprecipitation experiments showed that EGFR formed a complex with HER2 in OKF6/TERT-2 cells in both the presence and absence of C. albicans (Fig. S2). Therefore, this organism interacts with both members of the EGFR–HER2 complex.

Next, we investigated whether the two known C. albicans invasins, Als3 and Ssa1, were required for interaction with EGFR and HER2. Hyphae of the poorly endocytosed als3Δ/Δ (5) and ssa1Δ/Δ (Fig. S3A) single mutants bound only weakly to both of these proteins in the affinity purification assay (Fig. 1D). Furthermore, binding to both EGFR and HER2 was restored by reintegrating WT copies of ALS3 and SSA1 into the als3Δ/Δ and ssa1Δ/Δ mutants, respectively. As expected, hyphae of the als3Δ/Δ ssa1Δ/Δ double mutant also bound very weakly to EGFR and HER2. Therefore, Als3 and Ssa1 are necessary for C. albicans to interact with these epithelial cell proteins.

To determine if Als3 and Ssa1 interact with EGFR and HER2, we tested strains of Saccharomyces cerevisiae that expressed C. albicans ALS3 or SSA1. S. cerevisiae expressing CaALS3 was endocytosed by OKF6/TERT-2 epithelial cells. However, S. cerevisiae expressing CaSSA1 was unable to adhere to these cells sufficiently to induce endocytosis, although this strain was endocytosed by FaDu epithelial cells and endothelial cells (Fig. S3B). Because we found that Ssa1 is necessary but not sufficient to induce endocytosis by OKF6/TERT-2 cells, we focused subsequent studies on Als3.

In the affinity purification assay, S. cerevisiae expressing CaALS3 bound to both EGFR and HER2, whereas the control strains of S. cerevisiae did not (Fig. 1E). These data suggest that Als3 interacts with EGFR and HER2.

To ascertain if EGFR and HER2 interact with C. albicans in intact epithelial cells, we examined the distribution of the two proteins in infected OKF6/TERT-2 cells by confocal microscopy. We observed that EGFR and HER2 accumulated together around the same C. albicans hyphae (Fig. 1F). This accumulation was specific for these proteins, because another epithelial cell surface protein, intercellular adhesion molecule 1 (ICAM-1), did not accumulate around the organism (Fig. S4). Collectively, these results suggest that C. albicans interacts with EGFR and HER2 in both epithelial cell membrane extracts and intact epithelial cells.

C. albicans Stimulates Tyrosine Phosphorylation of EGFR and HER2.

When EGFR and HER2 are activated, they are autophosphorylated on specific tyrosine residues (11, 12). We investigated whether the interaction of C. albicans with EGFR and HER2 stimulated the phosphorylation of these two proteins. Time course studies revealed that WT C. albicans hyphae induced phosphorylation of both EGFR and HER2, which began within 10 min of infection, peaked at 20 min, and remained above basal levels for at least 40 min (Fig. 2A). Consistent with these results, phosphorylated EGFR and HER2 were observed to accumulate around C. albicans hyphae in intact epithelial cells (Fig. 2B).

Fig. 2.

Fig. 2.

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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).

Yeast-phase cells of the WT strain induced minimal tyrosine phosphorylation of EGFR and HER2, showing that activation of these receptors is hyphal-specific (Fig. 2C). Furthermore, hyphae of the poorly endocytosed als3Δ/Δ and ssa1Δ/Δ single mutants as well as hyphae of the als3Δ/Δ ssa1Δ/Δ double mutant induced less phosphorylation of EGFR and HER2 than the WT strain (Fig. 2D). Finally, phosphorylation of EGFR and HER2 was induced by infection with S. cerevisiae expressing CaALS3 but not the control strain containing the backbone vector (Fig. 2E). Collectively, these results suggest that the hyphal-specific invasin Als3 interacts with and activates EGFR and HER2.

Inhibition of EGFR and HER2 Reduces Epithelial Cell Endocytosis of C. albicans.

Next, we used specific inhibitors of EGFR and HER2 to determine the functional significance of C. albicans-induced activation of these receptors. Both the selective EGFR kinase inhibitor, AG1478 (13), and the anti-EGFR monoclonal antibody, cetuximab, significantly reduced the endocytosis of C. albicans (Fig. 3 A and B). The endocytosis of this organism was also significantly reduced by the selective HER2 tyrosine kinase inhibitor, AG825 (14), and the anti-HER2 monoclonal antibody, trastuzumab. Neither AG1478 nor AG825 influenced C. albicans growth rate (mean doubling times in the presence of AG1748 and AG825 were 99% ± 3% and 103% ± 17% of control, respectively) or germination on epithelial cells, indicating that these inhibitors acted on the epithelial cells and not the organism. Importantly, the combination of cetuximab plus trastuzumab did not inhibit endocytosis more than either monoclonal antibody alone (Fig. 3B). These results suggest that the interaction of C. albicans with the EGFR–HER2 heterodimer induces epithelial cells to endocytose this organism.

Fig. 3.

Fig. 3.

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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.

Als3 Is Sufficient to Induce EGFR- and HER2-Mediated Endocytosis.

To determine whether EGFR and HER2 mediated endocytosis induced by Als3, we tested the strain of S. cerevisiae that expressed CaALS3. We found that both cetuximab and trastuzumab inhibited the endocytosis of this strain (Fig. 3C). These antibodies also inhibited the endocytosis of latex beads coated with the recombinant N terminus of Als3 (Fig. S5). Collectively, these data suggest that Als3 induces endocytosis by directly binding to and activating epithelial cell EGFR and HER2.

EGFR and HER2 Function Cooperatively to Induce Endocytosis.

Next, we investigated whether activation of EGFR with its natural ligand, EGF, could increase the endocytosis of WT C. albicans and/or overcome the endocytosis defect of the als3Δ/Δ null mutant. Although incubating epithelial cells with EGF induced strong phosphorylation of EGFR, it did not stimulate phosphorylation of HER2 (Fig. S6A). Thus, EGFR can be activated independently of HER2 in this cell line. Furthermore, addition of EGF to the epithelial cells along with WT C. albicans did not increase the endocytosis of this organism (Fig. S6B). In fact, the highest concentration of EGF actually inhibited the endocytosis of C. albicans, probably by reducing the amount of surface-exposed EGFR (15). Moreover, activation of EGFR with exogenous EGF did not rescue the endocytosis defect of the als3Δ/Δ null mutant. These results suggest that activation of EGFR alone is not sufficient to induce the endocytosis of C. albicans.

To further dissect the roles of EGFR and HER2 in mediating the endocytosis of C. albicans, we used NIH/3T3 murine fibroblastoid cell lines that heterologously expressed human EGFR, HER2, or both proteins (16). Expression of EGFR alone did not increase endocytosis of C. albicans above basal levels (Fig. 4A). In contrast, expression of HER2 alone resulted in a significant increase in C. albicans endocytosis, and expression of HER2 plus EGFR did not further increase this process. Thus, the presence of HER2 is essential for mediating the endocytosis of C. albicans.

Fig. 4.

Fig. 4.

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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.

Although binding to EGFR alone was insufficient to induce endocytosis of C. albicans by NIH/3T3 cells, the finding that inhibition of EGFR reduced C. albicans endocytosis by oral epithelial cells suggested that EGFR may function cooperatively with HER2. To investigate this possibility, we treated NIH/3T3 cells with cetuximab to inhibit EGFR. As expected, this antibody significantly reduced the endocytosis of C. albicans by NIH/3T3 cells expressing both human EGRF and HER2 (Fig. 4B). However, it also inhibited the endocytosis of C. albicans by NIH/3T3 cells that expressed only human HER2. Therefore, we examined the levels of EGFR and HER2 expression in the NIH/3T3 cells by immunoblotting with antibodies that recognized both murine and human EGFR and HER2. We found that untransfected NIH/3T3 cells contained low levels of murine EGFR and HER2. Although the cells that expressed human EGFR contained very little murine HER2, the cells that expressed human HER2 still contained detectable murine EGFR. Therefore, our finding that cetuximab inhibited the endocytosis of C. albicans by these cells indicates that the murine EGFR formed a functional heterodimer with human HER2. Collectively, these results show that C. albicans interaction with the EGFR–HER2 heterodimer is sufficient to induce the endocytosis of this organism.

Relationship Between HER2 and E-Cadherin.

E-cadherin functions as an epithelial cell receptor for C. albicans (5). Therefore, we investigated the relationship between HER2 and E-cadherin in mediating the endocytosis of C. albicans. We found that, in intact epithelial cells, both HER2 and E-cadherin accumulated around the same C. albicans hyphae (Fig. 5A). Next, we examined the relative contributions of HER2 and E-cadherin to the endocytosis of C. albicans. As expected (5), siRNA knockdown of E-cadherin significantly decreased C. albicans endocytosis (Fig. 5 B and C). However, the anti-HER2 antibody, trastuzumab, caused a significantly greater reduction in endocytosis than the E-cadherin siRNA. Furthermore, the combination of trastuzumab and E-cadherin siRNA did not inhibit endocytosis more than trastuzumab alone (P = 0.56). Therefore, E-cadherin and HER2 likely act in the same pathway to mediate the endocytosis of C. albicans.

Fig. 5.

Fig. 5.

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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.

Inhibition of EGFR and HER2 Ameliorates Disease in the Mouse Model of OPC.

To determine if C. albicans interacts with EGFR and HER2 in vivo, we used the mouse model of OPC. We found that oral infection with C. albicans induced marked tyrosine phosphorylation of EGFR and HER2 in the oral mucosa (Fig. 6A). Furthermore, treatment of mice with GW2974, an orally bioavailable dual EGFR and HER2 kinase inhibitor (17), greatly reduced this phosphorylation and caused a 33-fold reduction in oral fungal burden compared with control animals that did not receive this drug (Fig. 6B). The beneficial effects of GW2974 were confirmed by histopathologic analysis, which revealed that the tongues of GW2974-treated mice had only small foci of infection that contained rare hyphae (Fig. 6C). In contrast, the tongues of the control mice had larger lesions with numerous hyphae. As expected, GW2974 also inhibited the endocytosis of C. albicans by oral epithelial cells in vitro (Fig. 6D), and it had no effect on C. albicans growth rate or hyphal formation in vitro (mean doubling time in the presence of GW2974 was 94% ± 7% of control). These results indicate that signaling through EGFR and HER2 is important for C. albicans to cause OPC.

Fig. 6.

Fig. 6.

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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.)

Discussion

Invasion of oral epithelial cells is an important step in the pathogenesis of OPC. Here, we report that EGFR and HER2 function cooperatively as epithelial cell receptors for the C. albicans Als3 invasin. This conclusion is supported by our findings that C. albicans hyphae as well as S. cerevisiae expressing CaALS3 interacted with both EGFR and HER2, stimulating their phosphorylation and inducing endocytosis. In addition, blocking either EGFR or HER2 inhibited the endocytosis of C. albicans, and blocking both EGFR and HER2 simultaneously did not further decrease C. albicans endocytosis. Finally, NIH/3T3 cells expressing human HER2 combined with either murine or human EGFR endocytosed C. albicans efficiently.

Importantly, although EGFR signaling seemed to be required for induction of C. albicans endocytosis, it was not sufficient to mediate this process alone. For example, treatment of epithelial cells with EGF, which induced the phosphorylation of EGFR but not HER2, did not increase the endocytosis of either WT C. albicans or the als3Δ/Δ mutant. Also, NIH/3T3 cells that expressed EGFR but not HER2 endocytosed C. albicans very poorly. Collectively, these data support a model in which C. albicans Als3 interacts with the EGFR-HER2 complex and activates EGFR-HER2 signaling, and thereby, it induces epithelial cells to endocytose the organism. The Ssa1 invasin is also required for C. albicans to activate EGFR-HER2, although it is not yet clear whether this invasin binds to this complex directly.

EGFR and HER2 have been previously found to be important for the interactions of viruses and bacteria with host cells. For example, EGFR mediates the uptake of cytomegalovirus by monocytes and influenza A virus by epithelial cells (18, 19). Also, HER2 signaling stimulates endothelial cell invasion by Neisseria meningitidis and neuronal demyelination in response to Mycobacterium leprae (20, 21). However, a unique finding with C. albicans is that EGFR and HER2 function together to mediate the endocytosis of this organism.

E-cadherin is another epithelial cell receptor for Als3 and Ssa1 (5, 6). Studies with CHO cells that express human E-cadherin show that binding of Als3 to this receptor is sufficient to induce endocytosis (5). Importantly, CHO cells do not express detectable HER2 (22), indicating that E-cadherin can mediate endocytosis in the absence of this second receptor. However, NIH/3T3 cells do not express E-cadherin (23). Thus, our finding that ectopic expression of human HER2 in these cells is sufficient to induce C. albicans endocytosis suggests that HER2, combined with EGFR, can function independently of E-cadherin. Importantly, the combination of HER2 inhibition plus E-cadherin knockdown did not decrease endocytosis more than inhibition of HER2 alone. These results suggest that HER2 and E-cadherin function in the same pathway to mediate the endocytosis of C. albicans.

C. albicans is similar to the bacterium Listeria monocytogenes in that both organisms invade epithelial cells by binding to E-cadherin and activating the clathrin-dependent endocytic pathway (7, 24). Our current data show that EGFR and HER2 function as receptor tyrosine kinases that function along with E-cadherin to mediate the endocytosis of C. albicans. Analogously, the met tyrosine kinase also participates in host cell endocytosis of Listeria (25). Thus, binding to both E-cadherin and a receptor tyrosine kinase seems to represent a common process by which microbial pathogens subvert host signaling pathways to invade epithelial cells.

Because EGFR and HER2 play key roles in the development of multiple types of cancer, the development of small-molecule inhibitors of EGFR and HER2 is the focus of intense investigation (26). The availability of GW2974, an orally bioavailable inhibitor of EGFR and HER2 kinase activity, provided a unique opportunity to evaluate the role of these receptors in the pathogenesis of OPC. Using the mouse model of OPC, we discovered that C. albicans infection stimulated the phosphorylation of EGFR and HER2 in the oral mucosa. Moreover, treatment with GW2974 inhibited this phosphorylation and significantly reduced oral fungal burden. These results strongly suggest that signaling through EGFR and HER2 is a key step in the development of OPC. They also indicate that epithelial cell endocytosis of C. albicans is important for the pathogenesis of this disease. We speculate that the residual organisms present in the oral mucosa of mice treated with GW2974 may have invaded by the paracellular route and/or active penetration (4, 27).

In summary, EGFR and HER2 function together as epithelial cell receptors for C. albicans both in vitro and in vivo. Of importance, inhibiting EGFR and HER2 signaling ameliorates experimental OPC, suggesting that blocking epithelial cell invasion of C. albicans by targeting its host receptors is an effective therapeutic strategy against OPC. A potential advantage of such an approach is that, because it does not alter the growth of the organism, it would be less likely to induce resistance, which is a significant problem when conventional antifungal prophylaxis is administered for a prolonged period (1).

Materials and Methods

Fungal Strains and Cell Lines.

The strains of C. albicans and S. cerevisiae used in the experiments (listed in Table S1) were grown as described previously (5, 28). The FaDu (American Type Culture Collection) and OKF6/TERT-2 oral epithelial cell lines (provided by Jim Rheinwald, Dana-Farber/Harvard Cancer Center, Boston, MA) (9) were grown as described (5, 9). OKF6/TERT-2 cells were transfected with random control siRNA (Qiagen) or E-cadherin siRNA (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. The NIH/3T3 cell lines expressing EGFR, HER2, or EGFR and HER2 were provided by Nadege Gaborit (Institut de Recherche en Cancérologie de Montpellier, Montpellier, France) and grown as outlined (16).

Isolation and Identification of Epithelial Cell Membrane Proteins.

Epithelial cell membrane proteins that bound to C. albicans or S. cerevisiae were isolated and affinity-purified as previously outlined (5, 28–30). Proteins eluted from the fungi were separated by SDS/PAGE and detected by immunoblotting with antibodies against biotin (clone BN-32; Sigma-Aldrich), EGFR (sc-03; Santa Cruz Biotechnology), and HER2 (clone 3B5; Santa Cruz Biotechnology). To identify epithelial cell proteins that bound to C. albicans, FaDu cell proteins were extracted with 7 M urea/2 M thiourea in PBS with Ca2+ and Mg2+ and protease inhibitors (Sigma-Aldrich). The lysates were separated by SDS/PAGE, and a band with a molecular mass of ∼160 kDa was excised. In-gel protein reduction, alkylation, trypsin digestion, and peptide extraction for sequencing were accomplished manually using standard protocols (31). Protein identification was performed by liquid chromatography—tandem MS. The MS/MS spectra were searched against the Swiss-Prot protein sequence database using Mascot (Matrix Science).

Confocal Microscopy.

The accumulation of epithelial cell EGFR, HER2, and E-cadherin around C. albicans hyphae was visualized using a minor modification of our previously described methods (5, 29). OKF6/TERT-2 cells were infected with 2 × 105 hyphae of the various C. albicans strains. After 20 min, the cells were fixed in methanol or 3% paraformaldehyde (wt/vol), blocked with 5% goat serum (vol/vol), and incubated with antibodies against total and phospho-specific EGFR and HER2 (EGFR-p-Tyr1068, catalog #2234; HER2-p-Tyr1248, catalog #2244; Cell Signaling) or E-cadherin followed by the appropriate secondary antibodies that had been labeled with either AlexaFluor 488 or AlexaFluor 568. The cells were then imaged by confocal microscopy, and the final images were generated by stacking optical sections along the z axis. The organisms were visualized by DIC imaging.

Detection of EGFR and HER2 Phosphorylation.

OKF6/TERT-2 cells in six-well tissue culture plates were infected with 5.25 × 106 C. albicans or S. cerevisiae cells for various times. Next, the cells were rinsed with cold HBSS containing protease and phosphatase inhibitor mixtures and removed from the plate with a cell scraper. The cells were collected by centrifugation and boiled in sample buffer. The lysates were subjected to SDS/PAGE, and phosphorylated EGFR and HER2 were detected by immunoblotting with phospho-specific EGFR and HER2 antibodies. The blots were then stripped, and total EGFR and HER2 were detected by immunoblotting.

Measurement of Host Cell Endocytosis.

The endocytosis of C. albicans and S. cerevisiae by oral epithelial cells and NIH/3T3 cells was quantified as described previously (5, 28). The OKF6/TERT-2 cells were incubated with either 2 × 105 yeast-phase C. albicans cells or 1 × 106 S. cerevisiae cells per well for 150 min. The NIH/3T3 cells were incubated with 105 C. albicans hyphae per well for 45 min. To determine the effects of the inhibitors on endocytosis, the host cells were incubated with cetuximab (Erbitux; ImClone Systems) or trastuzumab (Herceptin; Genentech) at 10 μg/mL. Control epithelial cells were incubated in a similar concentration of mouse IgG. AG1478 was used at 2 μM (32), AG825 was used at 10 μM (14), and GW2974 was used at 10 μM (33). Control cells were incubated in a similar concentration of diluent (0.8% DMSO for AG1478 and AG825 or 2% ethanol for GW2974). The inhibitors were added to the host cells 45 min before the fungal cells or beads, and they remained in the medium for the entire incubation period.

Murine Model of OPC.

The animal studies were approved by the Animal Care and Use Committee at the Los Angeles Biomedical Research Institute. The effect of GW2974 on the severity of OPC was determined using a minor modification of our standard mouse model (34, 35). Male BALB/C mice were fed either powdered mouse chow or chow containing 200 μg GW2974 per 1 g starting on day −2 relative to infection (17). Cortisone acetate (3 mg/mouse) was administered s.c. on days −1, 1, and 3. For inoculation, the mice were sedated, and a swab saturated with 106 C. albicans cells was placed sublingually for 75 min; 5 d later, the mice were killed, and their tongue and attached tissues were harvested and divided by two. One-half was weighed, homogenized, and quantitatively cultured. The other one-half was processed for histopathology.

To detect phosphorylation of EGFR and HER2, the excised tongues were snap frozen in Optimal Cutting Temperature (OCT); 6-μm-thick sections were cut with a cryostat, dried overnight, and then fixed with cold acetone. The cryosections were rehydrated in PBS and then blocked with PBS containing 5% goat serum and 0.1% Tween 20. They were stained with EGFR-p-Tyr1068 and HER2-pTyr877 (catalog #2241; Cell Signaling) primary antibodies, rinsed, stained with an AlexaFluor 488-labeled goat secondary antibody, and imaged by confocal microscopy. To enable comparison of fluorescence intensity between slides, the same image acquisition settings were used for each experiment.

Statistical Analyses.

In vitro data were evaluated using the unpaired Student t test and presented as mean ± SD. Oral fungal burden data were analyzed with the Wilcoxon Rank Sum test and presented as median ± interquartile range. P values ≤ 0.05 were considered significant.

Supplementary Material

Supporting Information
supp_109_35_14194__index.html (1.1KB, html)

Acknowledgments

We thank Pengnimol Hay and David Villarreal for technical assistance. This work was supported in part by National Institutes of Health Grant R01DE017088.

Footnotes

Conflict of interest statement: S.G.F. holds equity in NovaDigm Therapeutics, Inc.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1117676109/-/DCSupplemental.

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