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Receptor-kinase EGFR-MAPK adaptor proteins mediate the epithelial response to Candida albicans via the cytolytic peptide toxin, candidalysin

Open AccessPublished:August 26, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102419
      Candida albicans (C. albicans) is a dimorphic commensal human fungal pathogen that can cause severe oropharyngeal candidiasis (oral thrush) in susceptible hosts. During invasive infection, C. albicans hyphae invade oral epithelial cells (OECs) and secrete candidalysin, a pore-forming cytolytic peptide that is required for C. albicans pathogenesis at mucosal surfaces. Candidalysin is produced in the hyphal invasion pocket and triggers cell damage responses in OECs. Candidalysin also activates multiple MAPK-based signaling events that collectively drive the production of downstream inflammatory mediators that coordinate downstream innate and adaptive immune responses. The activities of candidalysin are dependent on signaling through the epidermal growth factor receptor (EGFR). Here, we interrogated known EGFR-MAPK signaling intermediates for their roles mediating the OEC response to C. albicans infection. Using RNA silencing and pharmacological inhibition, we identified five key adaptors, including growth factor receptor-bound protein 2 (Grb2), Grb2-associated binding protein 1 (Gab1), Src homology and collagen (Shc), SH2-containing protein tyrosine phosphatase-2 (Shp2), and casitas B-lineage lymphoma (c-Cbl). We determined that all of these signaling effectors were inducibly phosphorylated in response to C. albicans. These phosphorylation events occurred in a candidalysin-dependent manner and additionally required EGFR phosphorylation, matrix metalloproteinases (MMPs), and cellular calcium flux to activate a complete OEC response to fungal infection. Of these, Gab1, Grb2, and Shp2 were the dominant drivers of ERK1/2 activation and the subsequent production of downstream innate-acting cytokines. Together, these results identify the key adaptor proteins that drive the EGFR signaling mechanisms that underlie oral epithelial responses to C. albicans.

      Keywords

      Abbreviations:

      AMP (antimicrobial peptide), Clys (candidalysin), MMP (matrix metalloproteinase), OEC (oral epithelial cell), PFT (pore-forming toxin), qPCR (quantitative PCR)
      Fungal infections are highly underappreciated contributors of morbidity and mortality in humans, but to date, there are no vaccines to any pathogenic fungi (
      • Brown G.D.
      • Denning D.W.
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      Hidden killers: human fungal infections.
      ). Candida species are a dominant cause of both superficial and life-threatening fungal infections in humans, with Candida albicans being the most prevalent. Though normally a benign member of the oral, vaginal, and gut commensal microbiota, disturbances caused by environmental cues or a compromised immune system result in the overgrowth of C. albicans, leading to superficial infections of the mucous membranes, in particularly the oral mucosa, that can significantly lower quality of life and affect millions of individuals annually (
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      Immunity to fungal infections.
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      Innate immunity to mucosal Candida infections.
      ).
      An essential feature of C. albicans pathogenesis is the formation of hyphae, which cause physical damage to the mucosal epithelium, thereby inducing expression and secretion of innate immune factors such as antimicrobial peptides, alarmins, and proinflammatory cytokines and chemokines (
      • Pellon A.
      • Sadeghi Nasab S.D.
      • Moyes D.L.
      New insights in Candida albicans innate immunity at the mucosa: toxins, epithelium, metabolism, and beyond.
      ). During infection, C. albicans hyphae secrete candidalysin (Clys) , a cytolytic peptide that is critical for pathogenesis at mucosal surfaces (
      • Moyes D.L.
      • Wilson D.
      • Richardson J.P.
      • Mogavero S.
      • Tang S.X.
      • Wernecke J.
      • et al.
      Candidalysin is a fungal peptide toxin critical for mucosal infection.
      ). Clys is encoded by ECE1, which is highly expressed in hyphae, though not required for filamentation. Clys is generated by processing of its parent protein Ece1p by the kexin proteases (
      • Liu J.
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      • Sansevere E.A.
      • Allert S.
      • Barker K.S.
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      A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis.
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      Processing of Candida albicans ece1p is critical for Candidalysin maturation and fungal virulence.
      ). Clys secretion triggers epithelial cell damage and activation of a mitogen-activated protein kinase (MAPK)–based signaling pathway that leads to secretion of inflammatory mediators and cell survival signals (
      • Mogavero S.
      • Sauer F.M.
      • Brunke S.
      • Allert S.
      • Schulz D.
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      Candidalysin delivery to the invasion pocket is critical for host epithelial damage induced by Candida albicans.
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      Candida albicans-induced epithelial damage mediates translocation through intestinal barriers.
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      Candidalysin triggers epithelial cellular stresses that induce necrotic death.
      ,
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      • Kotowicz N.K.
      • Coleman B.M.
      • Green M.J.
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      The Candida albicans toxin candidalysin mediates distinct epithelial inflammatory responses through p38 and EGFR-ERK pathways.
      ). The epidermal growth factor receptor (EGFR) is key for mediating the host immune response against several microbial pathogens (
      • Ho J.
      • Moyes D.L.
      • Tavassoli M.
      • Naglik J.R.
      The role of ErbB receptors in infection.
      ), including C. albicans (
      • Ho J.
      • Yang X.
      • Nikou S.A.
      • Kichik N.
      • Donkin A.
      • Ponde N.O.
      • et al.
      Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.
      ,
      • Swidergall M.
      • Solis N.v.
      • Millet N.
      • Huang M.Y.
      • Lin J.
      • Phan Q.T.
      • et al.
      Activation of EphA2-EGFR signaling in oral epithelial cells by Candida albicans virulence factors.
      ,
      • Solis N.v.
      • Swidergall M.
      • Bruno V.M.
      • Gaffen S.L.
      • Filler S.G.
      The aryl hydrocarbon receptor governs epithelial cell invasion during oropharyngeal candidiasis.
      ,
      • Zhu W.
      • Phan Q.T.
      • Boontheung P.
      • Solis N.v.
      • Loo J.A.
      • Fillera S.G.
      EGFR and HER2 receptor kinase signaling mediate epithelial cell invasion by Candida albicans during oropharyngeal infection.
      ,
      • Ho J.
      • Wickramasinghe D.N.
      • Nikou S.-A.
      • Hube B.
      • Richardson J.P.
      • Naglik J.R.
      Candidalysin is a potent trigger of alarmin and antimicrobial peptide release in epithelial cells.
      ). However, the downstream signals that orchestrate epithelial responses to this fungus are still incompletely understood.
      EGFR, also known as Her1 or ErbB1, belongs to a family of receptor tyrosine kinases (RTKs) that regulate cell survival, proliferation, apoptosis, differentiation, and development (
      • Wee P.
      • Wang Z.
      Epidermal growth factor receptor cell proliferation signaling pathways.
      ). Dysregulation of EGFR is also associated with tumorigenesis (
      • Sigismund S.
      • Avanzato D.
      • Lanzetti L.
      Emerging functions of the EGFR in cancer.
      ,
      • Shin D.M.
      • Ro J.Y.
      • Hong W.K.
      • Hittelman W.N.
      Dysregulation of epidermal growth factor receptor expression in premalignant lesions during head and neck tumorigenesis.
      ). EGFR autophosphorylation is a proximal event that initiates downstream signaling, resulting in recruitment of various adaptors and ultimately activating downstream signaling cascades including MAPK, phosphatidylinositol 3-kinase/Akt/Protein Kinase B (PI3K/Akt), and signal transducers and activators of transcription (STATs). Multiple microbes are known to exploit EGFR during pathogenesis, using adaptor activation and phosphorylation to infiltrate host cells and evade the immune system (
      • Ho J.
      • Moyes D.L.
      • Tavassoli M.
      • Naglik J.R.
      The role of ErbB receptors in infection.
      ,
      • Mimuro H.
      • Suzuki T.
      • Tanaka J.
      • Asahi M.
      • Haas R.
      • Sasakawa C.
      Grb2 is a key mediator of Helicobacter pylori CagA protein activities.
      ,
      • Mölleken K.
      • Becker E.
      • Hegemann J.H.
      The Chlamydia pneumoniae invasin protein Pmp21 recruits the EGF receptor for host cell entry.
      ). Additionally, a role for these adaptors in driving immunity to microbes has also emerged (
      • Rajaram M.V.S.
      • Arnett E.
      • Azad A.K.
      • Guirado E.
      • Ni B.
      • Gerberick A.D.
      • et al.
      M. tuberculosis-initiated human mannose receptor signaling regulates macrophage recognition and vesicle trafficking by FcRγ-chain, Grb2, and SHP-1.
      ).
      In the present study, we sought to elucidate the role of MAPK signaling and EGFR adaptors in the oral epithelial cell (OEC) response to C. albicans. We found that growth factor receptor–bound protein 2 (Grb2), Grb2-associated binding protein 1 (Gab1), Src homology and collagen (Shc), SH2 containing protein tyrosine phosphatase-2 (Shp2, also PTPN11), and casitas B-lineage lymphoma (c-Cbl) are all required to mediate an EGFR-dependent response to C. albicans via Clys. EGFR kinase inhibitors negatively impact this response, which also requires matrix metalloproteinases and cellular calcium flux. Accordingly, we illuminate the underlying mechanisms of how early EGFR-mediated epithelial responses are orchestrated by the novel fungal toxin Clys, with implications for defining the host response to the medically important fungus C. albicans.

      Results

      C. albicans activates EGFR-associated adaptors through secreted Clys

      In establishment of oropharyngeal candidiasis (oral thrush), OECs respond to the pore-forming toxin (PFT) Clys secreted by C. albicans hyphae. OEC responses require EGFR signals, which leads to the release of antifungal proinflammatory mediators that ultimately coordinate an effective host response (
      • Pellon A.
      • Sadeghi Nasab S.D.
      • Moyes D.L.
      New insights in Candida albicans innate immunity at the mucosa: toxins, epithelium, metabolism, and beyond.
      • Nikou S.A.
      • Kichik N.
      • Brown R.
      • Ponde N.O.
      • Ho J.
      • Naglik J.R.
      • et al.
      Candida albicans interactions with mucosal surfaces during health and disease.
      ). However, the underlying mechanisms of the early EGFR molecular events activated in response to C. albicans and Clys remain incompletely characterized. Accordingly, TR146 buccal epithelial cells were infected with a C. albicans (BWP17 + CIp30, parental strain) at a multiplicity of infection of 10 for 2 h, a time point known to be optimal for EGFR activation (
      • Ho J.
      • Yang X.
      • Nikou S.A.
      • Kichik N.
      • Donkin A.
      • Ponde N.O.
      • et al.
      Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.
      ). Notably, all proteins known to interact with EGFR were inducibly phosphorylated following infection with C. albicans including Gab1 (Y659), Grb2 (p-Tyrosine), Shc (Y317), Shp2 (Y580), and c-Cbl (Y774) (Fig. 1A).
      Figure thumbnail gr1
      Figure 1Candidalysin is necessary and sufficient to activate Gab1, Grb2, Shc, Shp2, and c-Cbl adaptors in human oral epithelial cells. A, candidalysin (Clys) activates Gab1, Grb2, Shc, Shp2, and c-Cbl adaptors in human TR146 oral epithelial cells (OECs). Immunoblot showing phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl in response to 2 h infection of TR146 OECs with the indicated strains of C. albicans. B, Clys is sufficient to activate Gab1, Grb2, Shc, Shp2, and c-Cbl. Immunoblot showing phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl following stimulation with Clys at 30 min and 2 h in OECs. Blots are representative of three independent experiments.
      Since filamentation is required for C. albicans–induced epithelial responses (
      • Moyes D.L.
      • Runglall M.
      • Murciano C.
      • Shen C.
      • Nayar D.
      • Thavaraj S.
      • et al.
      A biphasic innate immune MAPK response discriminates between the yeast and hyphal forms of candida albicans in epithelial cells.
      ), we asked whether Clys was required for adaptor activation. Mutant C. albicans strains lacking the Clys parent gene ECE1 (ece1Δ/Δ) or with a deletion of the region that encodes Clys (ece1Δ/Δ+ECE1Δ184–279) failed to activate Gab1, Shc, Shp2, and c-Cbl (Fig. 1A). However, both mutant strains lacking Clys were able to induce phosphorylation of Grb2, though to a lesser extent than the parental and revertant strains, suggesting Clys is not the sole driver of Grb2 phosphorylation. To determine whether Clys alone could activate this panel of adaptors, TR146 cells were stimulated with Clys over a time course of 2 h. We treated cells with a strongly lytic concentration (70 μM) that triggers membrane damage, calcium flux, and cytokine release, an intermediate sublytic concentration (15 μM) that activates epithelial signal transduction through MAPK, p38/MKP-1, and c-Fos and the production of immune regulatory cytokines, and a non-lytic concentration (3 μM) that induces low level secretion of selected cytokines (Moyes et al., 2016). Phosphorylation of Gab1 at Y659 was detectable within 30 min of exposure to all concentrations of Clys, with phosphorylation levels increased strongly in response to higher concentrations of Clys (70 or 15 μM) at 2 h post treatment (Fig. 1B). Lower concentrations of Clys that do not cause cell lysis (3 μM) also induced pGab1 Y659 at 2 h post treatment. In contrast, treatment with 70 or 15 μM Clys induced Grb2 phosphorylation within 30 min, which was sustained until 2 h. Increased phosphorylation levels were only observed in cells stimulated with 3 μM Clys following 2 h of treatment. Phosphorylation of Shc Y317 was observed within 30 min following treatment with all concentrations of Clys, with a dose-dependent response observed at 30 min and 2 h post stimulation. Together, these data suggest that there is differential temporal organization of adaptors in response to C. albicans and Clys, which appears to be an early, coordinated event.
      Unexpectedly, the kinetics of Shp2 phosphorylation in response to Clys differed from Gab1, Grb2, and Shc, as phosphorylation of Shp2 Y580 was seen only after 2 h of treatment with 70 or 15 μM Clys, with negligible changes in phosphorylation observed with 3 μM (Fig. 1B). These data suggest either that Shp2 may play a role in the response against Clys when a significant amount of epithelial damage has been induced or slower kinetics of Shp2 activity in comparison to the other adaptors. Treatment of TR146 cells with 70 or 15 μM Clys triggered strong c-Cbl pY774 activation that was sustained at 30 min and 2 h post stimulation (Fig. 1B). Only very modest changes were observed with 3 μM Clys. Collectively, these data show that Clys can induce phosphorylation of adaptors in TR146 epithelial cells in a time- and dose-dependent manner, with distinct activation profiles for adaptors in response to lytic versus sublytic concentrations of Clys.

      Gab1, Grb2, Shc, Shp2, and c-Cbl activation requires EGFR activation, matrix metalloproteinases, and calcium flux

      While EGFR and the Ephrin family member, EphA2, play critical roles in mediating the innate epithelial response to Clys and C. albicans (
      • Ho J.
      • Yang X.
      • Nikou S.A.
      • Kichik N.
      • Donkin A.
      • Ponde N.O.
      • et al.
      Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.
      ,
      • Swidergall M.
      • Solis N.v.
      • Millet N.
      • Huang M.Y.
      • Lin J.
      • Phan Q.T.
      • et al.
      Activation of EphA2-EGFR signaling in oral epithelial cells by Candida albicans virulence factors.
      ), it is unknown whether the activation of corresponding adaptors in response to hyphae relies exclusively on EGFR. To determine whether EGFR drives Clys-induced adaptor activation, we analyzed the effects of the EGFR-specific kinase inhibitors on the activation of Gab1, Grb2, Shc, Shp2, and c-Cbl. Treatment of TR146 cells with EGFR inhibitors gefitinib (used at a final concentration of 1 μM) or PD153035 (0.5 μM) reduced C. albicans–induced phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl at 2 h post infection (Fig. 2A). Similarly, inhibition of EGFR either abolished or reduced phosphorylation of all adaptors in response to Clys (70 or 15 μM), indicating that EGFR activation is indeed required for Clys-induced adaptor activity (Fig. 2B).
      Figure thumbnail gr2
      Figure 2Candidalysin-induced adaptor activation requires EGFR and MMP activity. Inhibition of EGFR activity suppresses (A) C. albicans- and (B) candidalysin (Clys)-induced adaptor activation. Use of Gefitinib or PD153035 EGFR TK inhibitors suppresses WT C. albicans–induced and Clys-induced activation of Gab1, Grb2, Shc, Shp2, and c-Cbl. C, MMP inhibition suppresses candidalysin-induced adaptor activity. Pretreatment of TR146 cells with Marimastat (10 or 1 μM, designated M10 or M1) suppressed Clys-induced phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl. Protein lysates were isolated 2 h post stimulation. Blots are representative of two (Grb2) or three (Gab1, Shc, Shp2, and c-Cbl) independent experiments. (Note: because data from other experiments presented in this article were originally run on the same gel, the α-actin loading control is reused in , AC). MMP, matrix metalloproteinase.
      Matrix metalloproteinases (MMPs) are zinc-dependent metalloendopeptidases that cleave and release EGF family proligands, activating EGFR signaling (
      • Ho J.
      • Yang X.
      • Nikou S.A.
      • Kichik N.
      • Donkin A.
      • Ponde N.O.
      • et al.
      Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.
      ). Pretreatment of TR146 OECs with the pan-MMP inhibitor Marimastat (10 or 1 μm) suppressed phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl upon Clys stimulation (Fig. 2C) and infection with C. albicans (Fig. S1). Consistent with this, a Clys-deficient C. albicans strain (ece1Δ/Δ+ECE1Δ184–279), failed to phosphorylate Gab1, Shc, and c-Cbl, though induced very modest levels of phosphorylation for Grb2 and Shp2. Additionally, a blocking anti-EGFR mAb (Cetuximab, 10 μg/mL) reduced/abolished the phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl in response to Clys and C. albicans infection (Fig. 3), demonstrating that Clys-induced activation of adaptors is mediated by MMPs, which in turn mediate ligand-induced activation of EGFR. Previously, calcium influx induced by Clys activity was also shown to contribute to EGFR phosphorylation (
      • Ho J.
      • Yang X.
      • Nikou S.A.
      • Kichik N.
      • Donkin A.
      • Ponde N.O.
      • et al.
      Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.
      ). Accordingly, pretreatment of TR146 cells with the calcium chelator, BAPTA-AM (30 or 10 μM), also suppressed Clys-induced and C. albicans–induced EGFR adaptor activation (Fig. S2). These data show that Clys secreted by C. albicans activates EGFR-related adaptors and downstream signaling programs in OECs via a mechanism that requires calcium influx, MMP activity, and EGFR ligands.
      Figure thumbnail gr3
      Figure 3EGFR ligand binding is required for C. albicans and candidalysin-induced adaptor activation. Pretreatment of TR146 cells with the anti-EGFR mAb, Cetuximab suppressed phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl following (A) C. albicans infection or (B) candidalysin (Clys) stimulation. Blots are representative of two (Grb2) or three (Gab1, Shc, Shp2, and c-Cbl) independent experiments. (Note: because data from other experiments presented in this article were originally run on the same gel, the α-actin loading control is reused from , A and B).

      EGFR adaptors mediate Clys-induced MAPK signaling responses but are dispensable for cell survival and damage protection

      Clys permeabilizes target cell membranes, resulting in damage (release of lactate dehydrogenase (LDH)) and the activation of PI3K/Akt/mTOR and MAPK (MKP1/c-Fos) signaling pathways (
      • Moyes D.L.
      • Wilson D.
      • Richardson J.P.
      • Mogavero S.
      • Tang S.X.
      • Wernecke J.
      • et al.
      Candidalysin is a fungal peptide toxin critical for mucosal infection.
      ,
      • Moyes D.L.
      • Shen C.
      • Murciano C.
      • Runglall M.
      • Richardson J.P.
      • Arno M.
      • et al.
      Protection against epithelial damage during Candida albicans infection is mediated by PI3K/Akt and mammalian target of rapamycin signaling.
      ). Notably, Akt/mTOR is important in mediating cell survival and protection against damage (
      • Moyes D.L.
      • Shen C.
      • Murciano C.
      • Runglall M.
      • Richardson J.P.
      • Arno M.
      • et al.
      Protection against epithelial damage during Candida albicans infection is mediated by PI3K/Akt and mammalian target of rapamycin signaling.
      ), and MAPK is required for induction of immune responses (
      • Moyes D.L.
      • Wilson D.
      • Richardson J.P.
      • Mogavero S.
      • Tang S.X.
      • Wernecke J.
      • et al.
      Candidalysin is a fungal peptide toxin critical for mucosal infection.
      ) during epithelial C. albicans infection. Furthermore, Gab1, Grb2, and Shp2 adaptors can mediate the cell survival pathway (
      • Mattoon D.R.
      • Lamothe B.
      • Lax I.
      • Schlessinger J.
      The docking protein Gab1 is the primary mediator of EGF-stimulated activation of the PI-3K/Akt cell survival pathway.
      ).
      To determine whether this panel of EGFR adaptors can modulate these cellular responses, TR146 cells were transfected with siRNAs for Gab1, Grb2, Shc, Shp2, and c-Cbl for 48 h and stimulated with C. albicans or Clys for 2 h. LDH release from TR146 cells was unaffected by knockdown of all adaptors following treatment with C. albicans (Fig. S3A) or Clys (70 or 15 μM) (Fig. S3B). Accordingly, a Clys null strain (ece1Δ/Δ+ECE1Δ184-279) caused no apparent change in epithelial integrity. Assessment of the Akt/mTOR pathway similarly showed no change in phosphorylation upon adaptor knockdown (Fig. S3C). Thus, these EGFR adaptors do not appear to be required to protect epithelial cells from damage or for cell survival in response to C. albicans infection and Clys. In contrast, siRNA knockdown of Gab1 and Grb2 had a marked impact on c-Fos protein levels and suppressed phosphorylation of MKP1/DUSP1 in response to C. albicans and Clys (Fig. 4, A and B). Knockdown of Shc had only a minor impact on c-Fos and MKP1/DUSP1 activation, and the effects of Shp2 or c-Cbl knockdown via siRNA were negligible.
      Figure thumbnail gr4
      Figure 4Gab1, Grb2, and Shc adaptors mediate candidalysin-induced MAPK signalling responses. Knockdown of Gab1, Grb2, or Shc suppresses c-Fos expression, MKP1/DUSP1 and ERK1/2 phosphorylation. Following siRNA transfection, cells were infected with the indicated strains (A) or stimulated with candidalysin (B) and lysates were collected after 2 h for Western blotting for total c-Fos, phospho-MKP1/DUSP1, phospho-ERK1/2, phospho-p38, phospho-SAPK/JNK, or α-actin. Data are representative of three independent experiments.
      To better understand how Gab1 and Grb2 activate c-Fos and MKP1/DUSP1, we next assessed the three major branches of the MAPK signaling cascade, ERK1/2, p38 and SAPK/JNK. In response to C. albicans, knockdown of Gab1 and Grb2 had a marked effect on ERK1/2 phosphorylation but not p38 or SAPK/JNK (Fig. 4, A and B). While Grb2 knockdown also showed a decrease in pERK1/2 in response to Clys, Gab1 knockdown showed a modest increase in ERK1/2, p38, and SAPK/JNK phosphorylation. As expected, a Clys-null strain (ece1Δ/Δ+ECE1Δ184-279) did not trigger MAPK phosphorylation. Thus, Gab1 and Grb2 are required for MAPK activation during C. albicans infection, but the Gab1 activation pattern differs when responding to exogenously-applied synthetic Clys.
      Impairing Shp2 via siRNA had no detectable effect on C. albicans and Clys-induced signals; however, a caveat is that Shp2 knockdown was inefficient (60%, Fig. S4). Therefore, we also addressed this question by treating TR146 cells with SHP099 HCL, an allosteric inhibitor that selectively and potently targets Shp2. Cells were pretreated with SHP099 HCL (10 or 5 μM) for 1 h and then assessed for cell damage, MAPK and Akt/mTOR pathway activation after infection. Similar to observations with Shp2 siRNA, LDH release and mTOR phosphorylation from TR146 OECs was unaffected by Shp2 inhibition (Fig. S5). However, treatment with SHP099 HCL suppressed C. albicans–induced and Clys-induced levels of c-Fos, pMKP1/DUSP1, and pERK1/2, though not pSAPK/JNK or pp38 (Fig. 5, A and B). Thus, in addition to a key role for Gab1 and Grb2 adaptors, Shp2 also contributes to ERK1/2 activation in response to C. albicans and Clys.
      Figure thumbnail gr5
      Figure 5Shp2 mediates candidalysin-induced MAPK signaling responses. Inhibition of Shp2 activity suppresses C. albicans–induced and candidalysin (Clys)-induced c-Fos, pMKP1/DUSP1, and ERK1/2 activation. TR146 cells were pretreated with the Shp2 inhibitor, SHP099 HCL, for 1 h. Cells were infected with the indicated strains (A) or stimulated with Clys (B) and protein lysates isolated at 2 h for Western blotting for total c-Fos, phospho-MKP1/DUSP1, phospho-ERK1/2, phospho-p38, phospho-SAPK/JNK, or α-actin. Data are representative of three independent experiments.

      Gab1, Grb2, and Shp2 are required for production of inflammatory effectors in response to Clys

      A characteristic endpoint of Clys activity and EGFR activation is the production of inflammatory cytokines and chemokines. Using siRNA knockdown, we evaluated the role of this panel of EGFR adaptors in driving the production of G-CSF, GM-CSF, IL-6, IL-1α, and IL-1β from TR146 cells 24 h after C. albicans or Clys treatment. Knockdown of Gab1, Grb2, or Shc reduced secretion of GM-CSF, G-CSF, and IL-1α in response to C. albicans (Figs. 6, A, B and S6A) and Clys (Fig. S6, B, C, and E). Notably, only Gab1 or Grb2 knockdown reduced release of IL-1β and IL-6 in response to C. albicans (Fig. 6, C and D), suggesting that distinct pathways dictate adaptor-mediated cytokine responses during fungal signaling on OECs. Interestingly, knockdown of c-Cbl appeared to increase the release of G-CSF in response to C. albicans and GM-CSF and IL-6 in response to Clys (Figs. 6A, S6, C and D), possibly suggesting a negative regulatory role for c-Cbl in immune signaling. The Shp2 inhibitor, SHP099 HCL, impaired G-CSF, GM-CSF, IL-1α, and IL-β secretion in response to C. albicans treatment (Figs. 6E, S7, A, C, and D). Similarly, Shp2 inhibition significantly reduced G-CSF, GM-CSF, and IL-β release in response to Clys (Fig. S7, E, F, and H). Interestingly, Shp2 inhibition resulted in a significant increase in IL-6 release in response to Clys (Fig. 6F).
      Figure thumbnail gr6
      Figure 6Gab1, Grb2, Shc, and Shp2 adaptors mediate C. albicans–induced and candidalysin-induced cytokine secretion in oral epithelial cells. Knockdown of Gab1, Grb2, or Shc or inhibition of Shp2 suppresses G-CSF, GM-CSF, IL-6, and IL-1β secretion. AD, following siRNA knockdown or (E and F) 1 h pretreatment with the Shp2 inhibitor SHP099 HCL, cells were infected with the indicated strains or stimulated with candidalysin and supernatants collected after 24 h. Cytokines were evaluated by Luminex. Data show the mean (±SD) of three replicates. Significance was assessed by one way ANOVA with Bonferroni’s multiple comparisons test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
      OECs respond to Clys-induced tissue injury in part by inducing damage-associated immune responses, including release of alarmins and antimicrobial peptides (AMPs) such as β-defensins (
      • Ho J.
      • Wickramasinghe D.N.
      • Nikou S.-A.
      • Hube B.
      • Richardson J.P.
      • Naglik J.R.
      Candidalysin is a potent trigger of alarmin and antimicrobial peptide release in epithelial cells.
      ). To investigate the role of these adaptors in AMP induction, we incubated TR146 cells with Clys following siRNA knockdown of adaptors or inhibition of Shp2 and evaluated DEFB4A mRNA (encoding BD2). Adaptor knockdown or inhibition had no significant effect on gene expression in response to Clys (Fig. S8, A and B). As Clys does not stimulate S100 protein release (
      • Ho J.
      • Wickramasinghe D.N.
      • Nikou S.-A.
      • Hube B.
      • Richardson J.P.
      • Naglik J.R.
      Candidalysin is a potent trigger of alarmin and antimicrobial peptide release in epithelial cells.
      ), their expression was not assessed. Overall, these data indicate that Gab1, Grb2, Shc, and Shp2 adaptors but not c-Cbl play pivotal roles in mediating cytokine but not alarmin innate responses to fungal infection.

      Discussion

      The oral epithelium coordinates the early antifungal response to infection through the release of cytokines, chemokines, and other inflammatory mediators that recruit leukocytes, infiltrating neutrophils, and inflammatory monocytes to combat infection. Interactions between epithelial cells and C. albicans are critical for host defense mechanisms to mucosal infection. The discovery of the C. albicans–derived PFT, Clys, was a paradigm shift in understanding how hyphae promote pathogenesis, simultaneously causing target cell damage but also activating innate immune responses that are essential to limit control of this commensal microbe. This discovery led to a myriad of studies revealing how Clys activity interfaces with the host immune system and balances commensalism with pathogenicity (
      • Liu J.
      • Willems H.M.E.
      • Sansevere E.A.
      • Allert S.
      • Barker K.S.
      • Lowes D.J.
      • et al.
      A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis.
      ,
      • Richardson J.P.
      • Mogavero S.
      • Moyes D.L.
      • Blagojevic M.
      • Krüger T.
      • Verma A.H.
      • et al.
      Processing of Candida albicans ece1p is critical for Candidalysin maturation and fungal virulence.
      ,
      • Mogavero S.
      • Sauer F.M.
      • Brunke S.
      • Allert S.
      • Schulz D.
      • Wisgott S.
      • et al.
      Candidalysin delivery to the invasion pocket is critical for host epithelial damage induced by Candida albicans.
      ,
      • Allert S.
      • Förster T.M.
      • Svensson C.M.
      • Richardson J.P.
      • Pawlik T.
      • Hebecker B.
      • et al.
      Candida albicans-induced epithelial damage mediates translocation through intestinal barriers.
      ,
      • Blagojevic M.
      • Camilli G.
      • Maxson M.
      • Hube B.
      • Moyes D.L.
      • Richardson J.P.
      • et al.
      Candidalysin triggers epithelial cellular stresses that induce necrotic death.
      ,
      • Ho J.
      • Yang X.
      • Nikou S.A.
      • Kichik N.
      • Donkin A.
      • Ponde N.O.
      • et al.
      Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.
      ,
      • Verma A.H.
      • Richardson J.P.
      • Zhou C.
      • Coleman B.M.
      • Moyes D.L.
      • Ho J.
      • et al.
      Oral epithelial cells orchestrate innate type 17 responses to Candida albicans through the virulence factor candidalysin.
      ,
      • Verma A.H.
      • Zafar H.
      • Ponde N.O.
      • Hepworth O.W.
      • Sihra D.
      • Aggor F.E.Y.
      • et al.
      IL-36 and IL-1/IL-17 drive immunity to oral candidiasis via parallel mechanisms.
      ,
      • Westman J.
      • Plumb J.
      • Licht A.
      • Yang M.
      • Allert S.
      • Naglik J.R.
      • et al.
      Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion.
      ,
      • Wu Y.
      • Zeng Z.
      • Guo Y.
      • Song L.
      • Weatherhead J.E.
      • Huang X.
      • et al.
      Candida albicans elicits protective allergic responses via platelet mediated T helper 2 and T helper 17 cell polarization.
      ,
      • Swidergall M.
      • Khalaji M.
      • Solis N.v.
      • Moyes D.L.
      • Drummond R.A.
      • Hube B.
      • et al.
      Candidalysin is required for neutrophil recruitment and virulence during systemic Candida albicans infection.
      ,
      • Drummond R.A.
      • Swamydas M.
      • Oikonomou V.
      • Zhai B.
      • Dambuza I.M.
      • Schaefer B.C.
      • et al.
      CARD9 + microglia promote antifungal immunity via IL-1β- and CXCL1-mediated neutrophil recruitment.
      )
      Clys activates the EGFR and MAPK signaling pathways (
      • Moyes D.L.
      • Wilson D.
      • Richardson J.P.
      • Mogavero S.
      • Tang S.X.
      • Wernecke J.
      • et al.
      Candidalysin is a fungal peptide toxin critical for mucosal infection.
      • Ho J.
      • Yang X.
      • Nikou S.A.
      • Kichik N.
      • Donkin A.
      • Ponde N.O.
      • et al.
      Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.
      ), but how these pathways are controlled remained incompletely defined. The EGFR adaptor proteins play a critical role in facilitating signaling between upstream cell surface RTKs, such as EGFR, as well as downstream cellular signaling cascades that are fundamental for a diverse array of cellular processes (
      • Lemmon M.A.
      • Schlessinger J.
      Cell signaling by receptor tyrosine kinases.
      ). We show that not only does OEC infection with C. albicans activate the Gab1, Grb2, Shc, Shp2, and c-Cbl adaptors but that this activity is mainly driven by Clys. While Clys-deficient strains reduced phosphorylation of Grb2 in OECs compared to Clys-producing strains, there were still residual levels of phosphorylation remaining, suggesting that Clys is not the only driver of Grb2 activity during infection. This may be due to interactions between fungal proteins on the surface of invading hyphae and host epithelial cell receptors, EphA2, EGFR, and globular C1q receptor (gC1qR), which are known to mediate numerous proinflammatory responses to C. albicans (
      • Swidergall M.
      • Solis N.v.
      • Millet N.
      • Huang M.Y.
      • Lin J.
      • Phan Q.T.
      • et al.
      Activation of EphA2-EGFR signaling in oral epithelial cells by Candida albicans virulence factors.
      ,
      • Swidergall M.
      • Solis N.v.
      • Lionakis M.S.
      • Filler S.G.
      EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans.
      ,
      • Phan Q.T.
      • Lin J.
      • Solis N.v.
      • Eng M.
      • Swidergall M.
      • Wang F.
      • et al.
      The globular C1q receptor is required for epidermal growth factor receptor signaling during Candida albicans infection.
      ).
      The distinct temporal activity of these EGFR adaptors suggests they may mediate the early, coordinated response of OECs to ensure appropriate signaling during fungal infection. Notably, no studies have implicated EGFR adaptor activation in response to fungal infection and very few in bacterial infections (
      • Mimuro H.
      • Suzuki T.
      • Tanaka J.
      • Asahi M.
      • Haas R.
      • Sasakawa C.
      Grb2 is a key mediator of Helicobacter pylori CagA protein activities.
      ,
      • Mölleken K.
      • Becker E.
      • Hegemann J.H.
      The Chlamydia pneumoniae invasin protein Pmp21 recruits the EGF receptor for host cell entry.
      ,
      • Sun H.
      • Shen Y.
      • Dokainish H.
      • Holgado-Madruga M.
      • Wong A.
      • Ireton K.
      Host adaptor proteins Gab1 and CrkII promote InIB-dependent entry of Listeria monocytogenes.
      ). In addition to EGFR, Gab1, Grb2, Shc, Shp2, and c-Cbl are essential for signaling responses of RTKs, including EphA2, c-Met, and other Her-family members (
      • Batzer A.G.
      • Rotin D.
      • Ureña J.M.
      • Skolnik E.Y.
      • Schlessinger J.
      Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor.
      ,
      • Holgado-Madruga M.
      • Emlet D.R.
      • Moscatello D.K.
      • Godwin A.K.
      • Wong A.J.
      A Grb2-associated docking protein in EGF- and insulin-receptor signalling.
      ,
      • Xiang Y.P.
      • Xiao T.
      • Li Q.G.
      • Lu S.S.
      • Zhu W.
      • Liu Y.Y.
      • et al.
      Y772 phosphorylation of EphA2 is responsible for EphA2-dependent NPC nasopharyngeal carcinoma growth by Shp2/Erk-1/2 signaling pathway.
      ). The near abolishment of adaptor activation in response to EGFR inhibition confirmed EGFR is a critical receptor for adaptor protein activation and downstream responses to C. albicans via Clys. Previously, we reported that inhibition of EGFR reduces MAPK signaling and subsequent immune responses during infection (
      • Ho J.
      • Yang X.
      • Nikou S.A.
      • Kichik N.
      • Donkin A.
      • Ponde N.O.
      • et al.
      Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.
      ). Interestingly, our results show that some, but not all, adaptors orchestrate the downstream mechanisms of epithelial immunity to infection. Recent work has demonstrated that Grb2 is associated with EGFR in response to C. albicans infection (
      • Phan Q.T.
      • Lin J.
      • Solis N.v.
      • Eng M.
      • Swidergall M.
      • Wang F.
      • et al.
      The globular C1q receptor is required for epidermal growth factor receptor signaling during Candida albicans infection.
      ), and sustained activation of both EGFR and EphA2 by Clys and the hyphal adhesin, Als3, is required for proinflammatory responses during C. albicans infection (
      • Swidergall M.
      • Solis N.v.
      • Millet N.
      • Huang M.Y.
      • Lin J.
      • Phan Q.T.
      • et al.
      Activation of EphA2-EGFR signaling in oral epithelial cells by Candida albicans virulence factors.
      ). However, unlike Clys, recombinant Als3 does not induce cell damage, activate c-Fos/MKP1/DUSP1, or promote cytokine responses (
      • Murciano C.
      • Moyes D.L.
      • Runglall M.
      • Tobouti P.
      • Islam A.
      • Hoyer L.L.
      • et al.
      Evaluation of the role of Candida albicans agglutinin-like sequence (ALS) proteins in human oral epithelial cell interactions.
      ), which are downstream of EGFR signaling, suggesting Als3 does not directly activate these EGFR adaptors. Additionally, it should be noted that an ALS3-deficient strain is poor at forming the invasion pocket, which is required to enable efficient delivery and activity of Clys (
      • Mogavero S.
      • Sauer F.M.
      • Brunke S.
      • Allert S.
      • Schulz D.
      • Wisgott S.
      • et al.
      Candidalysin delivery to the invasion pocket is critical for host epithelial damage induced by Candida albicans.
      ). Together, these data indicate that Als3 does not directly activate EGFR adaptors and that its role in driving epithelial signaling and/or immune responses is probably due to its role in facilitating the formation of the invasion pocket to permit efficient secretion of Clys, which activates these processes.
      Clys induces EGFR activation by activating MMPs, which cleave EGFR proligands that subsequently bind EGFR, and inducing calcium influx (
      • Moyes D.L.
      • Wilson D.
      • Richardson J.P.
      • Mogavero S.
      • Tang S.X.
      • Wernecke J.
      • et al.
      Candidalysin is a fungal peptide toxin critical for mucosal infection.
      ,
      • Ho J.
      • Yang X.
      • Nikou S.A.
      • Kichik N.
      • Donkin A.
      • Ponde N.O.
      • et al.
      Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.
      ). Various MMPs activate EGFR in response to a diverse array of stimuli (
      • Prenzel N.
      • Zwick E.
      • Daub H.
      • Leserer M.
      • Abraham R.
      • Wallasch C.
      • et al.
      EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF.
      ,
      • Zhang Q.
      • Adiseshaiah P.
      • Reddy S.P.
      Matrix metalloproteinase/epidermal growth factor receptor/mitogen-activated protein kinase signaling regulate fra-1 induction by cigarette smoke in lung epithelial cells.
      ). Additionally, MMP activation and subsequent cleavage and release of EGFR ligands are involved in the activation of EGFR in response to Helicobacter pylori and Clostridium difficile (
      • Keates S.
      • Sougioultzis S.
      • Keates A.C.
      • Zhao D.
      • Peek R.M.
      • Shaw L.M.
      • et al.
      cag+ Helicobacter pylori induce transactivation of the epidermal growth factor receptor in AGS gastric epithelial cells.
      ,
      • Na X.
      • Zhao D.
      • Koon H.W.
      • Kim H.
      • Husmark J.
      • Moyer M.P.
      • et al.
      Clostridium difficile toxin B activates the EGF receptor and the ERK/MAP kinase pathway in human colonocytes.
      ). The role of MMPs in infectious diseases, such as mycobacterial infection and Paracoccidioides brasiliensis infection, has also been reported (
      • Taylor J.L.
      • Hattle J.M.
      • Dreitz S.A.
      • Troudt J.L.M.
      • Izzo L.S.
      • Basaraba R.J.
      • et al.
      Role for matrix metalloproteinase 9 in granuloma formation during pulmonary Mycobacterium tuberculosis infection.
      ,
      • Nishikaku A.S.
      • Ribeiro L.C.
      • Molina R.F.S.
      • Albe B.P.
      • Cunha C.D.S.
      • Burger E.
      Matrix metalloproteinases with gelatinolytic activity induced by Paracoccidioides brasiliensis infection.
      ). We now demonstrate that MMPs mediate antifungal immune responses via EGFR through the adaptors described herein.
      Clys is the first PFT identified in a human fungal pathogen. Like analogous toxins in other organisms, Clys causes a rapid influx of intracellular calcium, thought to be a pivotal step in the activation of downstream signaling events (
      • Bouillot S.
      • Reboud E.
      • Huber P.
      Functional consequences of calcium influx promoted by bacterial pore-forming Toxins.
      ). Calcium influx triggered by bacterial PFTs is also a key factor in the induction of cellular responses that limit infection and promote survival (
      • Hsuan S.L.
      • Kannan M.S.
      • Jeyaseelan S.
      • Prakash Y.S.
      • Sieck G.C.
      • Maheswaran S.K.
      Pasteurella haemolytica A1-derived leukotoxin and endotoxin induce intracellular calcium elevation in bovine alveolar macrophages by different signaling pathways.
      ,
      • Laestadius Å.
      • Richter-Dahlfors A.
      • Aperia A.
      Dual effects of Escherichia coli α-hemolysin on rat renal proximal tubule cells.
      ). We found that chelation of calcium reduced the activation of adaptors in response to C. albicans and Clys. As such, our results demonstrate a role for calcium flux induced by Clys in inducing EGFR adaptor activation and epithelial immune responses during C. albicans infection.
      Clys induces cell stress in OECs, with the release of alarmins, ATP, and AMPs (
      • Ho J.
      • Wickramasinghe D.N.
      • Nikou S.-A.
      • Hube B.
      • Richardson J.P.
      • Naglik J.R.
      Candidalysin is a potent trigger of alarmin and antimicrobial peptide release in epithelial cells.
      ). Our findings reveal that these adaptors play a negligible role in expression of a key host-protective AMP, DEFB4A (human BD-2). Interestingly, Shc activation has been associated with cellular stress responses (
      • Migliaccio E.
      • Giogio M.
      • Mele S.
      • Pelicci G.
      • Reboldi P.
      • Pandolfi P.P.
      • et al.
      The p66(shc) adaptor protein controls oxidative stress response and life span in mammals.
      ), and Shp2 is critical in regulating the stress response to aryl hydrocarbon receptor activation and calcium dynamics in mast cells (
      • Wang H.C.
      • Zhou Y.
      • Huang S.K.
      SHP-2 phosphatase controls aryl hydrocarbon receptor-mediated ER stress response in mast cells.
      ). Though this aspect of adaptor activation via cellular stress was not assessed here, stress-induced calcium flux is a key biological response to bacterial PFTs, and it is likely that Clys induces stress-associated signals in OECs during C. albicans infection, which may activate EGFR adaptors to mediate this specific response to Clys.
      This work establishes roles for multiple EGFR-related adaptors in the response to Clys secreted from C. albicans. Notably, four of these proteins, Gab1, Grb2, Shc, and Shp2, were required for G-CSF and GM-CSF secretion (Figs. 6, A, B, S7, A, E, and F). The secretion of IL-1α was reduced when Gab1 or Grb2 were knocked down via siRNA (Fig. S6A) or when the activity of Shp2 was inhibited chemically (Fig. S7C). In a similar manner, IL-1β and IL-6 secretion were reduced upon siRNA knockdown of Gab1 but not Grb2 in response to infection or Clys treatment (Fig. 6D). Inhibition of Shp2 effectively reduced IL-1β but not IL-6. These distinct roles for each adaptor in mediating the secretion of IL-1α, IL-1β, and IL6 indicate a potentially extensive and complex network of regulation of these cytokines in response to infection (Fig. 7) (Table 1).
      Figure thumbnail gr7
      Figure 7Gab1, Grb2, Shc, and Shp2 adaptors mediate candidalysin-induced signaling events that trigger oral epithelial cytokine responses. C. albicans infections are initiated by increased fungal burden, associated hypha formation, and secretion of the pore-forming toxin candidalysin (Clys). When accumulated at high concentrations, Clys interacts with an epithelial cell membrane to form pore-like structures that result in membrane damage, alarmin release, and calcium influx. These events lead to activation of matrix metalloproteinases (MMPs) and the cleavage/release of epidermal growth factor receptor (EGFR) ligands. EGFR activation leads to the recruitment and activation of the Gab1, Grb2, Shc, Shp2, and c-Cbl adaptor proteins which in turn mediate MKP1/DUSP1 and c-Fos activation via the MAPK-ERK1/2 signaling pathway. Cumulatively, this leads to induction of GM-CSF and G-CSF, as well as IL-6, IL-1α, and IL-1β, which mediate the subsequent stages of the innate and adaptive immune responses. Figure created with Biorender.com
      Table 1Summary of EGFR-MAPK adaptor knockdown and inhibition phenotypes
      CytokineAdaptor knocked down/inhibited
      Gab1Grb2ShcShp2c-Cbl
      G-CSF↓ ↓↓ ↓↓ ↓↓ ↓↑ ↑
      GM-CSF↓ ↓↓ ↓↓ ↓↓ ↓-
      IL-6↓ ↓↓ ↓-↑ ↑-
      IL-1⍺↓ ↓↓ ↓
      IL-1β↓ ↓--
      ↓ ↓ Significant reduction.
      ↓ Reduced.
      - No change.
      ↑ Increased.
      ↑ ↑ Significant increase.
      Few studies show that Gab1, Grb2, or Shp2 mediate such specific cytokine responses. However, activation of Gab1 and Shp2 can transmit signals to the ERK1/2 signaling cascade to activate the cytokine receptor, gp130 (
      • Takahashi-Tezuka M.
      • Yoshida Y.
      • Fukada T.
      • Ohtani T.
      • Yamanaka Y.
      • Nishida K.
      • et al.
      Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase.
      ), and activation of Shc, Grb2, and Shp2 mediate MAPK activation via the G-CSF receptor (
      • Ward A.C.
      • Monkhouse J.L.
      • Hamilton J.A.
      • Csar X.F.
      Direct binding of Shc, Grb2, SHP-2 and p40 to the murine granulocyte colony-stimulating factor receptor.
      ). Both G-CSF and GM-CSF are required for the resolution of C. albicans infections (
      • Whitney P.G.
      • Bär E.
      • Osorio F.
      • Rogers N.C.
      • Schraml B.U.
      • Deddouche S.
      • et al.
      Syk signaling in dendritic cells orchestrates innate resistance to systemic fungal infection.
      ,
      • Basu S.
      • Quilici C.
      • Zhang H.H.
      • Grail D.
      • Dunn A.
      Mice lacking both G-CSF and IL-6 are more susceptible to Candida albicans infection: critical role of neutrophils in defense against Candida albicans.
      ). The decrease in ERK1/2 phosphorylation in combination with G-CSF and GM-CSF reduction upon Gab1 and Grb2 knockdown suggests that both adaptor proteins control the OEC immune response to Clys, which may subsequently mediate neutrophil mobilization during C. albicans infection in vivo.
      EGFR adaptor activation and the OEC response to fungal infection are not well understood. Our data show that ERK1/2 is the major pathway activated by the EGFR adaptors, rather than p38 or SAPK/JNK, in agreement with work showing that Clys activates p38 independently of EGFR and ERK1/2 and that the EGFR-ERK1/2-c-Fos pathway is downstream of p38 (
      • Nikou S.A.
      • Zhou C.
      • Griffiths J.S.
      • Kotowicz N.K.
      • Coleman B.M.
      • Green M.J.
      • et al.
      The Candida albicans toxin candidalysin mediates distinct epithelial inflammatory responses through p38 and EGFR-ERK pathways.
      ). During infection with C. albicans, MAPK signaling is regulated by MKP1/DUSP1, with ERK1/2 mediating a negative feedback loop to coordinate the innate immune response (
      • Moyes D.L.
      • Runglall M.
      • Murciano C.
      • Shen C.
      • Nayar D.
      • Thavaraj S.
      • et al.
      A biphasic innate immune MAPK response discriminates between the yeast and hyphal forms of candida albicans in epithelial cells.
      ). These data suggest a dual role for ERK1/2 signaling in response to C. albicans and Clys (i) as a mediator of epithelial immune responses and (ii) a regulator of MAPK signaling responses. Interestingly, we only observed a partial reduction in c-Fos total protein when Gab1, Grb2, Shc, or Shp2 were knocked down or inhibited during fungal exposure, suggesting that additional unidentified factors can promote OEC responses against C. albicans and Clys.
      Although Gab1, Grb2, and Shc mediate cytokine responses in response to C. albicans and Clys, knockdown of c-Cbl showed either no effect or an increase in G-CSF, GM-CSF, and IL-1β release. Additionally, inhibition of Shp2 resulted in an increase in IL-6 secretion, most notable with Clys. This suggests a role for c-Cbl and Shp2 as possible negative regulators of EGFR signaling and immune induction. Shp2 has been shown to promote bacterial clearance following post-influenza Staphylococcus aureus pneumonia (
      • Ouyang W.
      • Liu C.
      • Pan Y.
      • Han Y.
      • Yang L.
      • Xia J.
      • et al.
      SHP2 deficiency promotes Staphylococcus aureus pneumonia following influenza infection.
      ) and orchestrate macrophage function against pulmonary bacterial infection (
      • Zhao L.
      • Xia J.
      • Li T.
      • Zhou H.
      • Ouyang W.
      • Hong Z.
      • et al.
      Shp2 deficiency impairs the inflammatory response against Haemophilus influenzae by regulating macrophage polarization.
      ), but its role in fungal infections is largely undefined. However, Shp2 is known to negatively regulate IL-6 release in macrophages in response to TLR ligands (
      • An H.
      • Zhao W.
      • Hou J.
      • Zhang Y.
      • Xie Y.
      • Zheng Y.
      • et al.
      SHP-2 phosphatase negatively regulates the TRIF adaptor protein-dependent type I interferon and proinflammatory cytokine production.
      ); thus, we anticipate that Shp2 has a similar function during OEC infections. The c-Cbl adaptor is important in EGFR trafficking and regulation (
      • de Melker A.A.
      • van der Horst G.
      • Calafat J.
      • Jansen H.
      • Borst J.
      C-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route.
      ,
      • Grøvdal L.M.
      • Stang E.
      • Sorkin A.
      • Madshus I.H.
      Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation.
      ); notably, ubiquitination of EGFR by c-Cbl is implicated in clathrin-independent endocytosis of EGFR and downstream signaling (
      • Sigismund S.
      • Algisi V.
      • Nappo G.
      • Conte A.
      • Pascolutti R.
      • Cuomo A.
      • et al.
      Threshold-controlled ubiquitination of the EGFR directs receptor fate.
      ). Additionally, though the p38 MAPK pathway is pivotal in EGFR endocytosis (
      • Perez Verdaguer M.
      • Zhang T.
      • Paulo J.A.
      • Gygi S.
      • Watkins S.C.
      • Sakurai H.
      • et al.
      Mechanism of p38 MAPK-induced EGFR endocytosis and its crosstalk with ligand-induced pathways.
      ), little is known about the role of p38 in EGFR cellular localization in response to infection. It is likely that the complex dynamics of EGFR trafficking during fungal infection impacts EGFR signaling and consequently the OEC immune response.
      In summary, these data indicate that the EGFR- and MAPK-associated adaptors Gab1, Grb2, Shc, Shp2, and c-Cbl play distinct roles in regulating OEC responses to C. albicans infections (Fig. 7). Though these adaptors are key mediators supporting microbial infection, relatively few studies have evaluated them as regulators of an epithelial immune response. As such, it is likely that EGFR-associated adaptors act as sentinel regulators of epithelial immune responses during mucosal infections.

      Experimental procedures

      C. albicans strains and Clys

      C. albicans strains were previously described (
      • Moyes D.L.
      • Wilson D.
      • Richardson J.P.
      • Mogavero S.
      • Tang S.X.
      • Wernecke J.
      • et al.
      Candidalysin is a fungal peptide toxin critical for mucosal infection.
      ) and were maintained on yeast extract peptone dextrose agar and cultured in yeast extract peptone dextrose at 30 °C. Fungal cells were harvested by centrifugation, washed twice with PBS, and diluted in appropriate media. The Clys peptide (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNK; also termed Ece1-III62-92K) was synthesized by Peptide Protein Research Ltd and stored frozen at 3.021 mM (10 mg/ml) in sterile water.

      Cell culture

      TR146 human buccal epithelial squamous cell carcinoma cells (
      • Rupniak H.T.
      • Rowlatt C.
      • Lane E.B.
      • Steele J.G.
      • Trejdosiewicz L.K.
      • Hill B.T.
      • et al.
      Characteristics of four new human cell lines derived from squamous cell carcinomas of the head and neck.
      ) were obtained from the European Collection of Authenticated Cell Cultures (ECACC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) nutrient mixture F-12 HAM (DMEM/F-12) (Gibco) supplemented with 15% fetal bovine serum (Gibco) and 1% (v/v) penicillin–streptomycin) (Sigma–Aldrich). Serum-free DMEM/F-12 was used to replace normal growth medium 24 h before and during cell stimulations.

      Reagents

      Inhibitors were from Santa Cruz Biotechnology (Gefitinib), Selleckchem (PD153035, SHP099 HCL, BAPTA-AM), and Tocris Bioscience (Marimastat) and were reconstituted in dimethyl sulfoxide. Cetuximab was a gift from Guy’s Hospital Cancer Centre. TR146 cells were incubated with inhibitors for 1 h prior to C. albicans infection or Clys treatment. Antibodies sources were as follows: Cell Signaling Technology: pGab1 Y659 (#12745S), Gab1 (#3232S), Grb2 (#3972S), pShc Y317 (#2431S), Shc (#2432S), pShp2 Y580 (#3703S), Shp2 (#3752S), pc-Cbl Y774 (#3555S), c-Cbl (#8447S), pMKP1/DUSP1 S359 (#2857S), c-Fos (#2250S), pp44/42 MAPK (ERK1/2) T202/Y204 (#4370S), pp38 MAPK T180/Y182 (#4511S), pSAPK/JNK T183/Y185 (#4668S), pAkt S473 (#4060S), pMTOR S2448 (#5536S) and β-Tubulin (#2146S); Millipore: mouse α-actin (#MAB1501); Jackson ImmunoResearch: goat antimouse IgG (#115-035-062), goat anti-rabbit IgG (#111-035-144) horseradish peroxidase.

      RNA silencing

      RNAi primers were from Thermo Fisher Scientific. TR146 cells were transfected with 5 or 10 nM of Gab1 (s5463), Grb2 (s226232), Shc (s12813), Shp2 (s11525), c-Cbl (s2476), or a negative control siRNA (Silencer Negative Control No. 2 siRNA) using HiPerfect Transfection Reagent (Qiagen) per manufacturers’ instructions. The extent of knockdown was verified after 48 h by immunoblotting or quantitative PCR (qPCR).

      qPCR

      RNA was isolated with RNeasy Mini Kits (Qiagen). Complementary DNA was synthesized using iScript cDNA synthesis Kit (Bio-Rad). Real-time qPCR was performed with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on a CFX Opus 96 real-time PCR instrument (Bio-Rad), normalized to GAPDH. Primers were from QuantiTect Primer Assays (Qiagen).

      Immunoblotting

      TR146 cells were incubated with the indicated fungal strains or treated with Clys (70, 15, or 3 μM) for the indicated time points. Cells were washed with PBS and lysed with radioimmunoprecipitation assay buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS, 20 mM EDTA) containing protease and phosphatase inhibitors (Sigma–Aldrich). Cells were manually detached, and lysates incubated on ice for 30 min. Five to ten micrograms of protein extracts were separated by SDS-PAGE on Bolt 4% to 12% Bis–Tris precast gels (Bio-Rad) using Immobilon chemiluminescent substrate (Millipore). Images were visualized on an Odyssey Infrared Imaging System (LI-COR).

      Immunoprecipitation

      TR146 cells were serum starved and infected with C. albicans at an multiplicity of infection of 10 or treated with Clys (70, 15, or 3 μM). For inhibitor experiments, cells were pretreated 1 h prior to infection or stimulation. Cells were washed with PBS and lysed with buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 5% glycerol) supplemented with protease and phosphatase inhibitors (Sigma–Aldrich) for 30 min. Approximately 100 μl of total lysate was saved for use as loading controls and the remainder was precleared with 25 μl of Protein A/G magnetic beads (Pierce, Thermo Fisher). Lysates (250 μg) were immunoprecipitated with phosphotyrosine Ab-magnetic beads (#8095S, Cell Signaling Technology) or control IgG (#5873, Cell Signaling Technology).

      Quantification of cell damage (LDH assay)

      A Cytox 96 nonradioactive cytotoxicity assay kit (Promega) was used to measure the activity of lactate dehydrogenase (LDH) in culture supernatants as described (
      • Ho J.
      • Yang X.
      • Nikou S.A.
      • Kichik N.
      • Donkin A.
      • Ponde N.O.
      • et al.
      Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor.
      ,
      • Ho J.
      • Wickramasinghe D.N.
      • Nikou S.-A.
      • Hube B.
      • Richardson J.P.
      • Naglik J.R.
      Candidalysin is a potent trigger of alarmin and antimicrobial peptide release in epithelial cells.
      ) according to the manufacturer’s instructions, and recombinant L-Lactic Dehydrogenase from porcine heart (Sigma–Aldrich) was used to generate a standard curve.

      Cytokine secretion

      Cytokine levels in culture supernatants were assessed by Luminex assay (Bio-techne) and a Bioplex-200 machine (Bio-Rad). Bioplex manager 6.1 software (Bio-Rad) was used to determine analyte concentrations.

      Statistics

      Data were analyzed by ANOVA with Bonferroni’s multiple comparisons test using GraphPad Prism (v. 8) software (GraphPad Software Inc). p values < 0.05 were considered significant.

      Data availability

      All data on adaptor phosphorylation, inhibition and knockdown assays, and cytokine expression are contained within this article.

      Supporting information

      This article contains supporting information.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank Bernhard Hube for providing fungal strains and members of the Centre for Host-Microbiome Interactions at King’s College London and members of the Division of Rheumatology and Clinical Immunology at the University of Pittsburgh for helpful suggestions.

      Author contributions

      D. L. M, J. H., S. L. G., and J. R. N. conceptualization; N. O. P. methodology; N. O. P., L. L., A. T., O. W. H., and D. N. W. investigation; N. O. P. writing–original draft; S. L. G., J. P. R., and J. N. writing–review & editing; J. P. R., D. L. M., and J. R. N. supervision; J. R. N., project administration; S. L. G., D. L. M., and J. R. N. funding acquisition.

      Funding and additional information

      S. L. G was supported by the National Institutes of Health (NIH, DE022550). J. R. N. was supported by the Wellcome Trust (214229_Z_18_Z), the NIH (DE022550), and the National Institute for Health and Care Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London and/or the NIHR Clinical Research Facility ( IS-BRC-1215-20006 ). D. L. M was supported by the UKRI-BBSRC (BB/S016899/1). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      Supporting information

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