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Activation of oral epithelial EphA2-EFGR signaling by Candida albicans virulence factors

During oropharyngeal candidiasis (OPC), Candida albicans invades and damages oral epithelial cells, which respond by producing proinflammatory mediators that recruit phagocytes to foci of infection. The ephrin type-A receptor 2 (EphA2) detects β-glucan and plays a central role in stimulating epithelial cells to release proinflammatory mediators during OPC. The epidermal growth factor receptor (EGFR) also interacts with C. albicans and is known to be activated by the Als3 adhesin/invasin and the Ece1/Candidalysin pore-forming toxin. Here, we investigated the interactions among EphA2, EGFR, Als3 and Ece1/Candidalysin during OPC. We found that Als3 and Ece1/Candidalysin function in the same pathway to damage epithelial cells in vitro. They also work together to cause OPC in mice. EGFR and EphA2 constitutively associate with each other as part of a physical complex and are mutually dependent for C. albicans-induced activation. In vitro, either Als3 or Ece1/Candidalysin is required for C. albicans to activate EGFR, sustain EphA2 activation, and stimulate epithelial cells to secrete CXCL8/IL-8 and CCL20. In the mouse model of OPC, Ece1/Candidalysin alone activates EGFR and induces CXCL1/KC and CCL20 production. Ece1/Candidalysin is also necessary for the production of IL-1α and IL-17A independently of Als3 and EGFR. These results delineate the complex interplay between host cell receptors and C. albicans virulence factors during the induction of OPC and the resulting oral inflammatory response. Author summary Oropharyngeal candidiasis occurs when the fungus Candida albicans proliferates in the mouth. The disease is characterized by fungal invasion of the superficial epithelium and a localized inflammatory response. Two C. albicans virulence factors contribute to the pathogenesis of OPC, Als3 which enables the organisms to adhere to and invade host cells and Ece1/Candidalysin which is pore-forming toxin that damages host cells. Two epithelial cell receptors, ephrin type-A receptor 2 (EphA2) and the epidermal growth factor receptor (EGFR) are activated by C. albicans. Here, we show that EphA2 and EGFR form part of complex and that each receptor is required to activate the other. Als3 and Ece1/Candidalysin function in the same pathway to damage epithelial cells. In isolated epithelial cells, both of these virulence factors activate EphA2 and EGFR, and stimulate the production of inflammatory mediators. In the mouse model of OPC, Ece1/Candidalysin elicits of a subset of the oral inflammatory response. Of the cytokines and chemokines induced by this toxin, some require the activation of EGFR while others are induced independently of EGFR. This work provides a deeper understanding of the interactions among C. albicans virulence factors and host cell receptors during OPC.


Abstract 23
During oropharyngeal candidiasis (OPC), Candida albicans invades and damages oral epithelial 24 cells, which respond by producing proinflammatory mediators that recruit phagocytes to foci of 25 infection. The ephrin type-A receptor 2 (EphA2) detects β-glucan and plays a central role in 26 stimulating epithelial cells to release proinflammatory mediators during OPC. The epidermal 27 growth factor receptor (EGFR) also interacts with C. albicans and is known to be activated by the 28 Als3 adhesin/invasin and the Ece1/Candidalysin pore-forming toxin. Here, we investigated the 29 interactions among EphA2, EGFR, Als3 and Ece1/Candidalysin during OPC. We found that Als3 30 and Ece1/Candidalysin function in the same pathway to damage epithelial cells in vitro. They also 31 work together to cause OPC in mice. EGFR and EphA2 constitutively associate with each other 32 as part of a physical complex and are mutually dependent for C. albicans-induced activation. In 33 vitro, either Als3 or Ece1/Candidalysin is required for C. albicans to activate EGFR, sustain EphA2 34 activation, and stimulate epithelial cells to secrete CXCL8/IL-8 and CCL20. In the mouse model 35 of OPC, Ece1/Candidalysin alone activates EGFR and induces CXCL1/KC and CCL20 36 production. Ece1/Candidalysin is also necessary for the production of IL-1α and IL-17A 37 independently of Als3 and EGFR. These results delineate the complex interplay between host 38 cell receptors and C. albicans virulence factors during the induction of OPC and the resulting oral 39 inflammatory response. 40 41 Introduction and is not sufficient to initiate a significant inflammatory response. By contrast, exposure to live 86 C. albicans induces sustained EphA2 activation and a strong inflammatory response [16]. 87 Another proinflammatory epithelial cell stimulus is Candidalysin. This toxin causes an 88 influx of calcium into the epithelial cells, which activates matrix metalloproteases and induces 89 shedding of native EGFR ligands, leading to activation of EGFR signaling and a proinflammatory 90 response by epithelial cells [18]. 91 In the current study, we sought to elucidate how C. albicans infection prolongs EphA2 92 activation and induces a proinflammatory response during OPC. We found that in vitro Als3 and 93 Ece1 function in the same pathway to activate epithelial cell EGFR, which maintains EphA2 94 phosphorylation and stimulates the secretion of CXCL8/IL-8 and CCL20. In mice with acute OPC, 95 pharmacological inhibition of EGFR leads to a reduction in oral fungal burden and impaired 96 inflammatory response during disease progression. Maximal mucosal infection requires Als1 and 97 Als3, which act in the same pathway as Ece1, while the mucosal inflammatory response is largely 98 driven by Ece1. Thus, C. albicans activation of EGFR mediates both fungal invasion of the 99 epithelium and induction of the local inflammatory responses to this fungus. 100 101

Results 102
The cellular fate of epithelial EphA2 differs depending on the type of stimulation 103 When oral epithelial cells are infected with yeast-phase C. albicans, the fungus germinates and 104 begins to form hyphae within 60 min. Previously, we found that C. albicans infection induces 105 EphA2 autophosphorylation on serine 897 within 15 min of infection and that this phosphorylation 106 is sustained for at least 90 min. By contrast, when oral epithelial cells are exposed to purified β-107 glucan in the form of zymosan or laminarin, EphA2 is phosphorylated for only the first 30 min of 108 contact. At later time points, EphA2 phosphorylation returns to basal levels, even though β-glucan 109 is still present [16]. To investigate how oral epithelial cells respond to prolonged exposure to 110 different EphA2 agonists, we compared their response to the native EphA2 ligand, ephrin A1 111 (EFNA1) and to C. albicans. After 15 min of exposure, both stimuli induced phosphorylation of 112 EphA2 (Fig 1A, S1A Fig). After 60 min of exposure, the two stimuli induced distinctly different 113 responses. In cells exposed to EFNA1, there was minimal phosphorylation of EphA2, and total 114 EphA2 levels declined (Fig 1B,S1B and S1C Fig). In cells exposed to C. albicans, EphA2 115 phosphorylation was sustained and there was no decrease in total EphA2 levels. Thus, exposure 116 to C. albicans not only induces EphA2 phosphorylation, but prevents the subsequent EphA2 117 degradation that normally occurs with prolonged exposure to EFNA1 [19]. 118 119

EGFR activation sustains C. albicans-induced EphA2 phosphorylation 120
EphA2 and EGFR function in the same pathway to mediate the endocytosis of C. albicans by oral 121 epithelial cells in vitro. Also, EphA2 is required for C. albicans to activate EGFR because siRNA 122 knockdown of EphA2 in oral epithelial cells blocks fungal-induced phosphorylation of EGFR [16]. 123 Cross-talk between EphA2 and EGFR has also been observed in other cell types [20,21]. To 124 determine if EGFR is required for sustained activation of EphA2, we tested the effects of inhibiting 125 EGFR on C. albicans-induced EphA2 phosphorylation. When EGFR was either knocked down 126 with siRNA or inhibited with the specific EGFR kinase inhibitor, gefitinib [22], EphA2 127 phosphorylation was transient, occurring within 30 min of infection, but declining to basal levels 128 by 90 min (Fig 1C-1E, S1D-S1F Fig). Therefore, activation of EGFR by C. albicans is required to 129 sustain EphA2 phosphorylation. 130 Cross talk between EphA2 and EGFR may reflect a physical interaction between these 131 two receptors. We investigated this possibility by immunoprecipitation experiments. When EphA2 132 was immunoprecipitated from lysates of uninfected epithelial cells, EGFR was pulled down with it 133 (Fig 1F and 1G). The interaction between EphA2 and EGFR appeared to be constitutive because 134 the amount of EGFR that was associated with EphA2 did not change significantly when the 135 epithelial cells were infected with C. albicans. In reciprocal experiments, we determined that 136 immunoprecipitation of EGFR from infected and uninfected epithelial cells also pulled down 137 similar amounts of EphA2 (Fig 1F and 1G). These results indicate that EphA2 and EGFR likely 138 form part of a complex that enables each receptor to influence the activity of the other. 139 140 C. albicans Als3 and Ece1 are required for prolonged EphA2 activation 141 Two C. albicans virulence factors, the Als3 invasin and the Ece1/Candidalysin toxin, have been 142 shown to be required for activation of EGFR [18,23,24]. To investigate the relationship between 143 Als3 and Ece1/Candidalysin and the stimulation of epithelial cells, we constructed als3Δ/Δ and 144 ece1Δ/Δ single gene deletion mutants and an als3Δ/Δ ece1Δ/Δ double gene deletion mutant. As 145 expected [3,7], when epithelial cells were infected with either the als3Δ/Δ or als3Δ/Δ ece1Δ/Δ 146 mutants, the number of cell-associated C. albicans cells (a measure of adherence) and 147 endocytosed C. albicans cells was significantly reduced relative to the wild-type strain (Fig 2A). 148 By contrast, the ece1Δ/Δ mutant acted similarly to the wild-type parent strain in these assays. 149 Also, both the als3Δ/Δ and ece1Δ/Δ mutants caused significantly less epithelial cell damage than 150 the wild-type strain ( Fig 2B). The als3Δ/Δ ece1Δ/Δ double mutant caused the least amount of 151 epithelial cell damage, which was significantly less than that caused by the als3Δ/Δ single mutant 152 ( Fig 2B). Collectively, these data support the model that Als3-mediated epithelial cell invasion is 153 required for Ece1-induced damage. 154 To determine if Als3 and Ece1 are required for sustained EphA2 activation, we analyzed 155 the phosphorylation of EphA2 and EGFR in response to the als3Δ/Δ and ece1Δ/Δ mutant strains. 156 While infection with both mutant strains induced EphA2 phosphorylation in oral epithelial cells 157 after 30 min, neither mutant strain was able to sustain EphA2 phosphorylation at 90 min 158 ( Fig 2C, S2 Fig). This lack of sustained EphA2 phosphorylation was likely due to the absence of 159 EGFR activation, because neither the als3Δ/Δ nor the ece1Δ/Δ mutant induced significant 160 phosphorylation of EGFR. 161

EGFR induces a subset of EphA2-mediated pro-inflammatory responses in oral epithelial 163 cells 164
To investigate the roles of EGFR and EphA2 in stimulating an epithelial cell proinflammatory 165 response in vitro, we used siRNA to knock down EGFR and EphA2 prior to infecting the cells with 166 C. albicans. We found that knockdown of EGFR inhibited the secretion of CXCL8/IL-8 and CCL20 167 similarly to knockdown of EphA2 ( Fig 3A). Although knockdown of EGFR had no effect on the 168 release of IL-1α, and IL-1β, knockdown of EphA2 reduced the secretion of both of these cytokines. 169 Inhibition of EGFR phosphorylation with gefitinib also inhibited the secretion of CXCL8/IL-8 and 170 CCL20, but not IL-1α or IL-1β ( Fig 3B). These results suggest that activation of both EphA2 and 171 EGFR is required for C. albicans to induce epithelial cells to secrete CXCL8/IL-8 and CCL20. 172 They also indicate that EphA2 mediates production of IL-1α and IL-1β independently of EGFR. 173 Next, we investigated the roles of Als3 and Ece1 in stimulating oral epithelial cells in vitro. 174 Infection with the als3Δ/Δ and ece1Δ/Δ single mutants and the als3Δ/Δ ece1Δ/Δ double mutant 175 induced significantly less secretion of CXCL8/IL-8, CCL20, IL-1α, and IL-1β than the wild-type 176 parent strain ( Fig 3C). Of note, the epithelial cell stimulation defect of the als3Δ/Δ ece1Δ/Δ double 177 mutant was no greater than that of either the als3Δ/Δ or ece1Δ/Δ single mutant. Collectively, these 178 results support the model that C. albicans Als3 and Ece1/Candidalysin function in the same 179 pathway to activate EGFR, which prolongs EphA2 phosphorylation and stimulates oral epithelial 180 cells to secrete CXCL8/IL-8 and CCL20. These results also suggest that Als3 and 181 Ece1/Candidalysin induce the production of IL-1α and IL-1β via a mechanism that requires EphA2 182 but is independent of EGFR. 183

184
The response of OKF6/TERT-2 epithelial cells to C. albicans differs significantly from 185

TR146 epithelial cells 186
The above experiments were conducted with OKF6/TERT-2 oral epithelial cells, which were 187 generated by transfecting normal human cells with a constitutively active telomerase [25]. Using 188 the TR146 human buccal squamous cell carcinoma cell line,  induced activation of EGFR is required for the fungus to stimulate oral epithelial cells to secrete 190 multiple cytokines [18]. One difference between their results and those presented here is that 191 EGFR was required for induction of IL-1α and IL-1β in TR146 cells but not OKF6/  To investigate this difference, we compared the interactions of C. albicans with both cell lines. We 193 found that while the adherence of C. albicans to both types of epithelial cells was similar, TR146 194 cells endocytosed over 3-fold more C. albicans cells than did OKF6/TERT-2 cells ( Fig 4A). Also,195 gefitinib modestly inhibited the adherence of C. albicans to TR146 cells, but not OKF6/TERT-2 196 cells, and it inhibited C. albicans endocytosis by TR146 cells by 30% and endocytosis by 197 OKF6/TERT-2 cells by 64%. Thus, in TR146 cells relative to OKF6/TERT-2 cells, EGFR is more 198 important for adherence but less important for inducing the endocytosis of C. albicans. 199 We also used a 51 Cr release assay to compare the susceptibility of the two cell types to 200 damage induced by C. albicans. In a time-course study, C. albicans caused significantly more 201 damage to TR146 cells than to OKF6/TERT-2 cells at all time points (Fig 4C). In fact, after 24 hr 202 of infection, C. albicans-induced damage of TR146 cells was near-maximal, whereas the extent 203 of damage to the OKF6/TERT-2 cells was significantly less ( Fig 4D). Gefitinib had no effect on 204 the extent of C. albicans-induced damage to either cell line. Collectively, these data indicate that 205 TR146 cells are more susceptible to C. albicans-induced damage than OKF6/TERT-2 cells. 206 The extent of EGFR phosphorylation and cytokine production by TR146 cells and 207 OKF6/TERT-2 cells also differed. Uninfected TR146 cells had significantly higher levels of total 208 and phosphorylated EGFR than OKF6/TERT-2 cells (Fig 4E, S3 Fig). Uninfected TR146 cells 209 also secreted over 30-fold more CXCL8/IL-8, 15-fold more CCL20, and 2-fold more IL-1α than 210 OKF6/TERT-2 cells (Fig 4F). The levels of these cytokines and IL-1β significantly increased when 211 the TR146 cells were infected with C. albicans ( Fig 4G). While gefitinib inhibited the production of 212 CXCL8/IL-8 and CCL20 by C. albicans infected TR146 cells, it also decreased the production of 213 IL-1α and IL-1β ( Fig 4G). Thus, EGFR governs C. albicans-induced production of chemokines in 214 both cell lines, but it regulates production of IL-1α and IL-1β only in TR146 cells. 215 216 Inhibition of EGFR decreases EphA2 phosphorylation, oral fungal burden, and the host 217 inflammatory response during OPC 218 To assess the role of EGFR in mediating the host inflammatory response to C. albicans during 219 OPC, we treated immunocompetent mice with gefitinib and then orally inoculated them with 220 C. albicans. Immunocompetent mice were used to avoid potentially confounding effects of 221 immunosuppression on the host inflammatory response. When immunocompetent mice are orally 222 inoculated with C. albicans strain SC5314, infection persists for 2 days, after which the organism 223 is cleared by the host inflammatory response [14,26]. 224 Using flow cytometry and phosphospecific antibodies, we assessed the effects of gefitinib 225 on EGFR and EphA2 phosphorylation in the oral epithelium. As predicted by the in vitro data, 226 treatment with gefitinib reduced the phosphorylation of both EGFR and EphA2 in CD45 -EpCam + 227 cells after 1 d of infection (Fig 5). At this time point, the gefitinib-treated mice had a 3-fold reduction 228 in oral fungal burden compared to untreated mice (Fig 6A), probably due to inhibition of epithelial 229 cell invasion by the drug. 230 To determine the effects of gefitinib on the inflammatory response, we measured whole 231 tongue levels of CXCL1/KC, CCL20, IL-1α and IL-1β. To obtain a more comprehensive view of 232 the effects of gefitinib, we also measured the levels of IL-17A, S100A8, and TNFα. After 1 day of 233 infection, the oral tissues of the gefitinib-treated mice contained significantly less CXCL1/KC, 234 CCL20, IL-1α, IL-1β, IL-17A, and the host defense peptide S100A8 than control mice ( Fig 6B). 235 There were also significantly fewer neutrophils and inflammatory monocytes in the oral tissues of 236 the gefitinib-treated mice (Fig 6C and 6D, S4 Fig). After 2 days of infection, the gefitinib-treated 237 and control mice had similar oral fungal burden (Fig 6A). At this time point, treatment with gefitinib 238 reduced the levels of CXCL1/KC, CCL20, S100A8 and TNFα, but not IL-1α, IL-1β, and IL-17A 239 ( Fig 6E). These results indicate that blocking EGFR reduces a subset of the early inflammatory 240 response to acute OPC in immunocompetent mice. 241 Because gefitinib impaired the host inflammatory response to C. albicans, we investigated 242 its effects on the candidacidal activity of neutrophils and macrophages. We found that gefitinib 243 did not decrease the capacity of human neutrophils, bone marrow (BM) neutrophils isolated from 244 gefitinib-treated mice, or murine BM derived macrophages to kill C. albicans in vitro (S5 Fig). Taken together, these results indicate that while EGFR signaling is required for epithelial cells to 246 mount a pro-inflammatory response to C. albicans, it is dispensable for governing phagocyte 247 killing of the organism. 248

C. albicans adhesins/invasins and Ece1/Candidalysin function together during OPC 250
To investigate the roles Als3 and Ece1/Candidalysin in stimulating the host response during 251 oropharyngeal candidiasis, we orally infected immunocompetent mice with C. albicans als3Δ/Δ 252 and ece1Δ/Δ mutants. After 1 day of infection, mice inoculated with the als3Δ/Δ mutant had 253 reduced oral fungal burden relative to those infected with the wild-type strain, and mice inoculated 254 with the ece1Δ/Δ mutant had an even greater decrease in fungal burden ( Fig 7A). After 2 days of 255 infection, the fungal burden of mice infected with the als3Δ/Δ mutant was similar to that of mice 256 infected with the wild-type strain, whereas the fungal burden of mice infected with the ece1Δ/Δ 257 mutant remained significantly lower ( Fig 7A). We also determined that C57BL/6 mice infected with 258 the ece1Δ/Δ mutant constructed in either strain SC5314 or BWP17 had reduced oral fungal 259 burden (S6 Fig). Thus, in the acute oropharyngeal candidiasis model, Als3 is necessary for 260 maximal infection at day 1, whereas Ece1/Candidalysin is required for maximal infection at both 261 days 1 and 2. 262 C. albicans must first adhere to host cells in order to invade them. C. albicans Als1 is 263 structurally similar to Als3 and it can function as both an adhesin and invasin [3,27]. We reasoned 264 that the presence of Als1 in als3Δ/Δ mutant could enable the organism to adhere to and invade 265 the oral epithelium in the absence of Als3. To test this hypothesis, we constructed als1Δ/Δ, 266 als1Δ/Δ als3Δ/Δ, and als1Δ/Δ als3Δ/Δ ece1Δ/Δ mutants. In vitro, the als1Δ/Δ als3Δ/Δ mutant had 267 significantly reduced adherence to OKFG/TERT-2 cells compared to the als3Δ/Δ mutant, 268 indicating that Als1 and Als3 make independent contributions to C. albicans adherence (S7A Fig). 269 Because the als3Δ/Δ single mutant was endocytosed so poorly by oral epithelial cells and caused 270 so little host cell damage, deletion of ALS1 did not reduce these interactions further (S8B and 271

S8C Fig) 272
After 1 day of oral infection, mice infected with the als1Δ/Δ als3Δ/Δ mutant had a slight 273 reduction in oral fungal burden relative to mice infected with the wild-type strain, but this difference 274 was not statistically significant ( Fig 7B). At this time point, the fungal burden of mice infected with 275 ece1Δ/Δ single mutant and the als1Δ/Δ als3Δ/Δ ece1Δ/Δ triple mutant was significantly reduced. 276 After 2 days of infection, the fungal burden of mice infected with the als1Δ/Δ als3Δ/Δ double 277 mutant was decreased to the same extent as mice infected with either the ece1Δ/Δ single mutant 278 or the als1Δ/Δ als3Δ/Δ ece1Δ/Δ triple mutant ( Fig 8B). These data suggest that adherence and 279 invasion mediated by Als1 and Als3 combined with host cell damage caused by 280 Ece1/Candidalysin play key roles in inducing acute OPC. 281 To determine the contributions of Als1, Als3 and Ece1/Candidalysin to the induction and 282 maintenance of the oral innate immune response, we determined the levels of inflammatory 283 mediators in the infected tongues after 1 and 2 days of infection. Three different patterns of 284 response were observed. The levels of IL-1β were similar in mice infected with all strains of C. 285 albicans, indicating that the production of this cytokine is independent of Als1, Als3 and 286 Ece1/Candidalysin (Fig 8). The levels of many inflammatory mediators, such as IL-1α, CXCL1/KC 287 CCL20, IL-17A, and S100A8 were decreased to the same extent in mice infected with either the 288 ece1Δ/Δ mutant or the als1Δ/Δ als3Δ/Δ ece1Δ/Δ mutant, suggesting that the production of these 289 mediators is dependent on Ece1/Candidalysin but independent of Als1 and Als3. The levels of 290 TNFα were significantly reduced only in mice infected with the als1Δ/Δ als3Δ/Δ ece1Δ/Δ mutant, 291 signifying that Als1, Als3, and Ece1/Candidalysin function cooperatively to induce the production 292 of this cytokine. Thus, while the production of some inflammatory mediators during OPC is largely 293 driven by Ece1/Candidalysin, Als1 and Als3 contribute to the production of TNFα. 294 295

Discussion 296
Oral epithelial cells play a central role in orchestrating the host defense against C. albicans 297 during OPC [1,11,28,29]. Our current data show that in oral epithelial cells, EphA2 and EGFR 298 form part of a complex and that EGFR activation mediates a subset of proinflammatory response 299 to this fungus. Previously, we found that EphA2 activation is required for C. albicans to stimulate 300 EGFR [16]. Here, we determined that activation of EGFR is in turn necessary for C. albicans to 301 sustain activation of EphA2, both in vitro and in vivo. Thus, these two receptors function 302 interdependently to mediate the epithelial cell response to C. albicans. These data also indicate 303 that the initiation of a proinflammatory response in oral epithelial cells requires two signals, one 304 induced by EphA2 binding to fungal β-glucans and the other induced by EGFR stimulated by 305 fungal invasins and Ece1/Candidalysin. 306 EphA2 and EGFR are known to interact in epithelial cell cancers, especially those that 307 have become resistant to EGFR inhibitors, in which siRNA knockdown of EphA2 restores 308 sensitivity to pharmacological EGFR inhibition [30,31]. Interestingly, treatment of malignant cell 309 lines with soluble EFNA1 has the same effect as EphA2 siRNA, presumably because EFNA1 310 induces EphA2 endocytosis and subsequent degradation. Our data indicate that C. albicans 311 activates EphA2 differently than EFNA1 because binding of C. albicans stabilizes EphA2 and 312 prevents its degradation. The sustained EphA2 protein levels likely contribute to the prolonged 313 EphA2 signaling induced by C. albicans infection. 314 We found that C. albicans activation of EGFR resulted in the production of 315 proinflammatory mediators by two different oral epithelial cell lines. Recently, it has been reported 316 that Ece1/Candidalysin activates EGFR and stimulates the TR146 oral epithelial cell line to 317 produce Il-1α, IL-1β, IL-6, G-CSF, and GM-CSF [18]. In the current work, knockdown of EGFR 318 with siRNA or inhibition of EGFR with gefitinib significantly reduced C. albicans-induced 319 production of CXCL8/IL-8 and CCL20 by both TR146 cells and OKF6-TERT2 cells. However, 320 inhibition of EGFR only decreased production of Il-1α and IL-1β by TR146 cells, but not by OKF6-321 TERT2 cells. It was also determined that TR146 cells had higher basal levels of total and 322 phosphorylated EGFR than OKF6-TERT2 cells, which may explain why cytokine production by 323 TR146 cells was more sensitive to EGFR inhibition. Nevertheless, the result with both oral 324 epithelial cells lines indicate that C. albicans activates EGFR, which induces the production of 325 proinflammatory mediators. 326 The current data indicate a functional interaction between Als3 and Ece1/Candidalysin. 327 Consistent with previous reports [3,7], we found that both the als3Δ/Δ mutant and the ece1Δ/Δ 328 mutant were defective in damaging oral epithelial cells in vitro. Also, these two mutants were 329 similarly impaired in their capacity to stimulate epithelial cells to secrete proinflammatory 330 mediators. Strikingly, deletion of ALS3 in the ece1Δ/Δ mutant did not lead to a further reduction 331 in epithelial cell damage and production of proinflammatory mediators. These results indicate that 332 Als3 and Ece1/Candidalysin function in the same pathway to damage epithelial cells and stimulate 333 them to secrete proinflammatory mediators in vitro. A potential explanation for the functional 334 linkage between Als3 and Ece1/Candidalysin is that when C. albicans hyphae are endocytosed 335 via Als3, secreted Ece1/Candidalysin is contained within the endocytic vacuole, where it 336 accumulates to sufficiently high concentration that it damages the epithelial cell and activates 337 EGFR to induce the production of inflammatory mediators. This possibility is supported by our 338 previous finding that inhibiting the endocytosis of C. albicans by treating epithelial cells with 339 cytochalasin D also blocks fungal-induced damage [32]. 340 The central role of EGFR in mediating the host inflammatory response was demonstrated 341 in the immunocompetent mouse model of OPC. Treatment of mice with gefitinib inhibited C. 342 albicans-induced phosphorylation of both EphA2 and EGFR in the oral epithelial cells, and 343 reduced the tissue levels of CXCL1/KC, CCL20, S100A8 after both 1 and 2 days of infection. As 344 a result, the accumulation of neutrophils and inflammatory monocytes in the oral tissues was 345 dramatically decreased. In the gefitinib-treated mice, the levels of IL-1α, IL-1β, IL-17A, and TNFα 346 were only reduced at a single time point, suggesting that the production of these cytokines may 347 be governed independently of EGFR. 348 Inhibition of EGFR has different effects in different models of mucosal infection. Recently, 349 Ho et al. [18] reported that in the zebrafish swimbladder model of mucosal candidiasis, gefitinib 350 treatment impaired the accumulation of neutrophils and led to enhanced mortality, even though it 351 had no effect on fungal burden. Our findings demonstrate that in immunocompetent mice, gefitinib 352 has different effects; although it reduces cytokine production and accumulation of phagocytes, it 353 also decreases oral fungal burden. We and others have found previously that EGFR inhibition 354 also reduces oral fungal burden in steroid-immunosuppressed mice [5,18]. As inhibition of EGFR 355 reduces C. albicans endocytosis by oral epithelial cells in vitro, it is probable that the decrease in 356 oral fungal burden by EGFR inhibition is due to impaired epithelial cell invasion in vivo. 357 When we tested the virulence of the C. albicans mutants with deletions in ALS1, ALS3,358 and/or ECE1, we found that some of the in vitro data were recapitulated in mice. After 2 days of 359 infection, mice inoculated with the ece1Δ/Δ mutant, the als1Δ/Δ als3Δ/Δ mutant, and the als1Δ/Δ 360 als3Δ/Δ ece1Δ/Δ mutant all had a similar reduction in oral fungal burden. This finding parallels 361 the reduced epithelial cell damage induced by these strains in vitro and indicates that the 362 combination of Als1 and Als3 function along with Ece1/Candidalysin to induce oral infection. It 363 was notable that although the als1Δ/Δ als3Δ/Δ mutant had extremely impaired adherence to and 364 invasion of oral epithelial cells In vitro, its virulence defect in mice was less severe. A likely 365 explanation for this result is that other adhesins and invasins such as Hwp1 [33] and Ssa1 [4] 366 may compensate for the absence of Als1 and Als3 in vivo. It is also possible that the als1Δ/Δ 367 als3Δ/Δ mutant may invade oral epithelial cells in vivo by a receptor-independent mechanism, 368 such as by active penetration [2]. 369 The mouse data indicated that different subsets of the host inflammatory response are 370 induced by different combinations of Als1, Als3, and Ece1/Candidalysin. Mice infected with the 371 ece1Δ/Δ mutant had reduced levels of CXCL1/KC and CCL20 in their oral tissues, similar to mice 372 that had been treated with gefitinib. Although mice infected with the als1Δ/Δ als3Δ/Δ ece1Δ/Δ 373 mutant had a comparable reduction in these chemokines, animals infected with the als1Δ/Δ 374 als3Δ/Δ had wild-type levels. Collectively, these results suggest that Ece1/Candidalysin is 375 necessary for C. albicans to active EGFR in vivo, which stimulates the production of CXCL1/KC 376 and CCL20. These results also indicate that in vivo, Als1 and Als3 are dispensable for the 377 induction of this aspect of the inflammatory response. 378 The data with the mutant strains of C. albicans showed that Als1, Als3, and 379 were not reduced in mice treated with gefitinib, they were consistently decreased only in mice that 385 were infected with the als1Δ/Δ als3Δ/Δ ece1Δ/Δ mutant. These data suggest that the induction of 386 TNFα production is independent of EGFR signaling, but dependent on the combined activity of 387 Als1, Als3, and Ece1/Candidalysin. 388 The current results indicate that the capacity of Ece1/Candidalysin to induce IL-1β 389 production is dependent on the anatomic site of infection. We found that mice infected with the 390 ece1Δ/Δ mutant strains had wild-type levels of IL-1β in their oral tissues, indicating that production 391 of this cytokine is independent of Ece1/Candidalysin. In the mouse model of disseminated 392 candidiasis, Ece1/Candidalysin is dispensable for IL-1β production in the kidneys [34], but 393 essential for IL-1β production in the brains [35]. Thus, C. albicans infection induces IL-1β 394 production in the oral mucosa by a different mechanism than in the brain. 395 A paradoxical finding was that although mice infected with the ece1Δ/Δ mutant had a 396 markedly reduced inflammatory response, they also had reduced oral fungal burden. This 397 reduction in oral fungal burden was observed in two strains of mice infected with independent 398 ece1Δ/Δ mutants and has also been reported in the immunosuppressed mouse model of OPC 399 [7]. Because of the decreased inflammatory response induced by the ece1Δ/Δ mutant, it would 400 have been expected that this strain would be able to proliferate unimpeded in the oral cavity. For 401 example, when mice are infected intravenously with an ece1Δ/Δ mutant, there is significantly 402 reduced production of proinflammatory cytokines and accumulation of neutrophils in the brain, 403 and this dampened inflammatory response leads to increased brain fungal burden [35]. One 404 potential explanation for the reduced oral fungal burden in mice infected with the ece1Δ/Δ mutant 405 is that even in the absence of Ece1/Candidalysin, there is still some inflammatory response,

Fungal strains 428
The C. albicans strains used in this study are listed in S1 NatR transformants were PCR genotyped to verify deletion of ECE1 as described above. 445 The second set of C. albicans mutants was constructed in strain SC5314 using the 446 transient CRISPR-Cas9 system [37] combined with the Nat flipper approach [39,40]. Briefly, the 447 gRNA and Cas9 constructs were amplified from vector pV1093 (S3 Table)

PCR. 462
For the experiments, the C. albicans cells were grown for 18 h in YPD broth in a shaking 463 incubator at 30°C. The fungal cells were harvested by centrifugation, washed twice with 464 phosphate-buffered saline (PBS), and counted using a hemacytometer. 465

Oral epithelial cells 466
The OKF6/TERT-2 immortalized human oral epithelial cell line was kindly provided by J. Immunoblotting 488 OKF6/TERT-2 cells or TR146 cells in 24-well tissue culture plates were switched to supplement 489 free KSF medium or DMEM/F12 medium, respectively for 1 h and then infected with 1 × 10 6 C. 490 albicans yeast for various times as described previously [16]. Next, the cells were rinsed with cold 491 HBSS containing protease and phosphatase inhibitors, detached from the plate with a cell 492 scraper, and collected by centrifugation. After boiling the cells in sample buffer, the resultant 493 lysate was separated by SDS-PAGE, and phosphorylation was detected by immunoblotting with 494 specific antibodies against pEphA2 (#6347, Cell Signaling) and pEGFR (#2234, Cell Signaling). 495 Next, the blot was stripped, and the total amount of each protein was detected by immunoblotting 496 with antibodies against EphA2 (D4A2, Cell Signaling), EGFR (#4267, Cell Signaling), and β-actin 497 (#a5441, Sigma). Each experiment was performed at least 3 times. 498 499 Immunoprecipitation 500 OKF6/TERT-2 cells were grown in 75 cm 2 flasks to confluency, switched to KSF medium without 501 supplements for 3 h, and then infected with 1x10 8 C. albicans yeast. After 30 or 90 min. 502 OKF6/TERT-2 were washed with ice-cold cold PBS (with Mg 2+ , and Ca 2+ ), scraped from the 503 flasks, and lysed with 100 µl ice-cold 5.8% octyl β-D-glucopyranoside (0479-5g; VWR) in the 504 present of protease/phosphatase inhibitors. Whole cells lysates were precleared with 20µl of 505 protein A/G plus (sc-2003; Santa Cruz Biotechnology) at 4°C for 30minutes. The bead-protein 506 mix was centrifuged at 3000 rpm for 30 sec at 4°C and supernatants were collected. 2 μg of anti-507 EGFR antibody (sc-101; Santa Cruz Biotechnology) or anti-EphA2 antibody (#6347, Cell 508 Signaling) was added to 500 µg of protein and incubated on a rotator at 4°C for 2 hours. 25µl of 509 protein A/G plus was added to each immunoprecipitation sample and incubated for an additional 510 hour at 4°C. Samples were pelleted at 3000 rpm for 30 sec, and washed 3 times in 500 µl of ice-511 cold 1.5% octyl β-D-glucopyranoside. Proteins were eluted with 30 µl of 2X SDS buffer and then 512 heated at 90°C for 5 minutes. Samples were centrifuged at 3000 rpm for 30 sec after which the 513 supernatants were collected, separated by SDS-PAGE, and analyzed as described above. 514

Measurement of epithelial cell endocytosis 515
The endocytosis of C. albicans by oral epithelial cells was quantified as described previously [44]. 516 OKF6/TERT-2 or TR146 oral epithelial cells were grown to confluency on fibronectin-coated 517 circular glass coverslips in 24-well tissue culture plates and then infected for 120 min with 518 2×10 5 yeast-phase C. albicans cells per well, after which they were fixed, stained, and mounted 519 inverted on microscope slides. The coverslips were viewed with an epifluorescence microscope, 520 and the number of endocytosed organisms per high-power field was determined, counting at least 521 100 organisms per coverslip. Each experiment was performed at least 3 times in triplicate. 522

Cytokine and chemokine measurements in vitro 523
Cytokine levels in culture supernatants were determine as previously described [16]. Briefly 524 OKF6/TERT-2 cells or TR146 cells in a 96-well plate were infected with C. albicans at a multiplicity 525 of infection of 5. After 8 h of infection, the medium above the cells was collected, clarified by 526 centrifugation and stored in aliquots at -80 °C. The concentration of inflammatory cytokines and 527 chemokines in the medium was determined using the Luminex multipex assay (R&D Systems). 528

Epithelial cell damage 529
The effects of gefitinib on extent of epithelial cell damage caused by C. albicans was determined 530 by our previously described 51 Cr release assay [24]. OKF6/TERT-2 cells and TR146 cells in a 24-531 well plate were loaded with 51 Cr overnight. The next day, they were incubated with gefitinib or 532 diluent and then infected with C. albicans at a multiplicity of infection of 10. At various time points, 533 the medium above the epithelial cells was collected and the epithelial cells were lysed with 534 RadiacWash (Biodex). The amount of 51 Cr released into the medium and remaining in the cells 535 was determined with a gamma counter, and the percentage of 51 Cr released in the infected cells 536 we compared to the release by uninfected epithelial cells. The experiment was performed 3 times 537 in triplicate. 538

Mouse model of oropharyngeal candidiasis 539
Male, 6 week old BALB/c mice were purchased from Taconics and C57BL/6J mice were 540 purchased from Jackson Laboratories. OPC was induced in mice as described previously [24, 541 45]. Starting on day -2 relative to infection, the mice were randomly assigned to receive gefitinib 542 or no treatment. Gefitinib was administered by adding the drug to the powdered chow diet at a 543 final concentration of 200 parts-per-million. For inoculation, the animals were sedated, and a swab 544 saturated with 2 × 10 7 C. albicans cells was placed sublingually for 75 min. Mice were sacrificed 545 after 1 and 2 days of infection. The tongues were harvested, weighed, homogenized and 546 quantitatively cultured. The researchers were not blinded to the experimental groups because the 547 endpoints (oral fungal burden, cytokine levels, and leukocyte numbers) were an objective 548 measure of disease severity. 549 550

Cytokine and chemokine measurements in vivo 551
To determine the whole tongue cytokine and chemokine protein concentrations, the mice were 552 orally infected with C. albicans as above. After 1 and 2 days of infection, the mice were sacrificed, 553 and their tongues were harvested, weighed and homogenized. The homogenates were cleared 554 by centrifugation and the concentration of inflammatory mediators was measured using a 555 multiplex bead array assay (R&D Systems) as previously described [16,46]. 556

Flow cytometry 557
To detect phosphorylation of EphA2, and EGFR in the tongue of C. albicans infected mice, the 558 mice were orally infected with C. albicans as above. After 1 of infection, the mice were sacrificed, 559 and their tongues were harvested, fixed in 3% PFA for 30 min. After washing a single cell 560 suspension was prepared as described above. Single cells were permeabilized with methanol for 561 10 min, washed and stained with anti-pEphA2 (#6347, Cell Signaling) or anti-pEGFR (#2234, Cell 562 Signaling) antibodies over night at 4°C. The next day the cells were further stained with anti-rabbit 563 The number of phagocytes in the mouse tongues were characterized as described 566 elsewhere [47]. Briefly, mice were orally infected with C. albicans as described above. After 1 d 567 of infection, the animals were administered a sublethal anesthetic mix intraperitoneally. The 568 thorax was opened, and a part of the rib cage removed to gain access to the heart. The vena 569 cava was transected and the blood was flushed from the vasculature by slowly injecting 10 mL 570 PBS into the right ventricle. The tongue was harvested and cut into small pieces in 100 µL of ice-571 cold PBS. 1 mL digestion mix (4.8 mg/ml Collagenase IV; Worthington Biochem, and 200 μg/ml 572 DNase I; Roche Diagnostics, in 1x PBS) was added after which the tissue was incubated at 37°C 573 for 45 min. The resulting tissue suspension was then passed through a 100 μm cell strainer. The Ly6G (1A8, BioLegend), CD11b (M1/70; eBioscience), and CD90.2 (30-H12; BioLegend). After 579 washing with FACS buffer, the cell suspension was stained with a LIVE/DEAD fluorescent dye 580 (7-AAD; BD Biosciences) for 10 min. The stained cells were analyzed on a 2-laser LSRII flow 581 cytometer (BD Biosciences), and the data were analyzed using FACS Diva (BD Biosciences) and 582 FlowJo software (Treestar). Only single cells were analyzed, and cell numbers were quantified 583 using PE-conjugated fluorescent counting beads (Spherotech). 584

Phagocyte killing assays 585
The effects of gefitinib on neutrophil killing of C. albicans were determined by our previously 586 described method [24]. To study human cells, neutrophils were isolated from the blood of healthy 587 volunteers and incubated with gefitinib or diluent in RPMI 1640 medium plus 10% fetal bovine 588 serum for 1 h at 37°C. Next, the neutrophils were mixed with an equal number of serum-opsonized 589 C. albicans cells. After a 3 h incubation, the neutrophils were lysed by sonication, and the number 590 of viable C. albicans cells was determined by quantitative culture. 591 To study bone marrow-derived neutrophils and macrophages (BMDMs), bone marrow 592 cells from BALB/c mice (Taconics) were flushed from femurs and tibias using sterile RPMI 1640 593 medium supplemented with 10% fetal bovine serum (FBS) and 2 mM EDTA onto a 50 ml screw 594 top Falcon tube fitted with a 100 μm filter [48]. Mouse neutrophils were purified from bone marrow 595 cells using negative magnetic bead selection according to the manufacturer's instructions 596 (MojoSort, BioLegend). These neutrophils had > 90% purity and > 90% viability as determined by 597 flow cytometry. To isolate BMDMs, 6x10 6 bone marrow cells per 75 cm 2 were seeded in RPMI 598 1640 supplemented with 20% FBS, 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM Glutamine, 599 and 25 ng/ml rHu M-CSF (PeproTech). After 7 days, the BMDMs were treated with gefitinib or the 600 diluent and then incubated with serum-opsonized C. albicans cells (multiplicity of infection 1:20) 601 for 3 h. Next, the BMDMs were scraped, lysed by sonication, and the number of viable C. albicans 602 cells was determined by quantitative culture. 603

Statistics 604
At least three biological replicates were performed for all in vitro experiments unless otherwise 605 indicated. Data were compared by non-parametric Mann-Whitney, or non-parametric one-way 606 Kruskal-Wallis test followed by Dunn's post-hoc using GraphPad Prism (v. 8) software. P values 607 < 0.05 were considered statistically significant. 608

Data Availability 609
All relevant data are within the manuscript and its Supporting Information files. 610 611