Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
[Preprint]. 2023 Feb 23:2023.02.23.529756.
doi: 10.1101/2023.02.23.529756.

Candida albicans stimulates the formation of a multi-receptor complex that mediates epithelial cell invasion during oropharyngeal infection

Quynh T Phan  1 , Norma V Solis  1 , Max V Cravener  2 , Marc Swidergall  1   3 , Jianfeng Lin  1 , Manning Y Huang  4 , Hong Liu  1 , Shakti Singh  1 , Ashraf S Ibrahim  1   3 , Massimiliano Mazzone  5   6 , Aaron P Mitchell  2 , Scott G Filler  1   3
Affiliations

Candida albicans stimulates the formation of a multi-receptor complex that mediates epithelial cell invasion during oropharyngeal infection

Quynh T Phan et al. bioRxiv. .

Update in

Abstract

Fungal invasion of the oral epithelium is central to the pathogenesis of oropharyngeal candidiasis (OPC). Candida albicans invades the oral epithelium by receptor-induced endocytosis but this process is incompletely understood. We found that C. albicans infection of oral epithelial cells induces c-Met to form a multi-protein complex with E-cadherin and the epidermal growth factor receptor (EGFR). E-cadherin is necessary for C. albicans to activate both c-Met and EGFR and to induce the endocytosis of C. albicans . Proteomics analysis revealed that c-Met interacts with C. albicans Hyr1, Als3 and Ssa1. Both Hyr1 and Als3 were required for C. albicans stimulation of c-Met and EGFR in oral epithelial cells in vitro and for full virulence during OPC in mice. Treating mice with small molecule inhibitors of c-Met and EGFR ameliorated OPC, demonstrating the potential therapeutic efficacy of blocking these host receptors for C. albicans .

Highlights: c-Met is an oral epithelial cell receptor for Candida albicans C. albicans infection causes c-Met and the epidermal growth factor receptor (EGFR) to form a complex with E-cadherin, which is required for c-Met and EGFR function C. albicans Hyr1 and Als3 interact with c-Met and EGFR, inducing oral epithelial cell endocytosis and virulence during oropharyngeal candidiasis Dual blockade of c-Met and EGFR ameliorates oropharyngeal candidiasis.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.. C. albicans activates c-Met in oral epithelial cells.
(A) Confocal microscopic images of OKF6/TERT-2 oral epithelial cells infected with C. albicans SC5314 for 20 min. c-Met, the epidermal growth factor receptor (EGFR), and E-cadherin were detected by indirect immunofluorescence using specific antibodies. Arrows point to the organisms in the magnified insets. Scale bar 10 μm. (B and C) Immunoblot analysis showing the time course of the phosphorylation of c-Met and EGFR in oral epithelial cells induced by C. albicans germ tubes (B). Densitometric analysis of 4 immunoblots (C). Data are mean ± SD. (D and E) Knockdown of c-Met with siRNA (D) or inhibition of c-Met signaling with SG523 (E) in oral epithelial cells inhibits the endocytosis of C. albicans. (F) Stimulation of oral epithelial cells with recombinant hepatocyte growth factor (HGF) enhances the endocytosis of C. albicans. Data in (D-F) are the mean ± SD of three experiments, each performed in triplicate. **p < 0.01, ****p < 0.0001, ns; not significant (two-tailed Student’s t test [D and E] or one-way ANOVA with Sidak’s multiple comparisons test [F]).
Fig. 2.
Fig. 2.. Functional interactions among c-Met, EGFR, and E-cadherin during the endocytosis of C. albicans.
(A-C) Immunoblot analysis showing the effects of the EGFR inhibitor gefitinib and the c-Met inhibitor SGX523 on C. albicans-induced phosphorylation of c-Met and EGFR in oral epithelial cells after 20 min of infection. Representative immunoblot (A), densitometric analysis of 4 immunoblots showing the phosphorylation of EGFR (B) and c-Met (C). Data are mean ± SD. (D) Effects of SGX523 and gefitinib on the endocytosis of C. albicans by oral epithelial cells. (E and F) Endocytosis of C. albicans by NIH/3T3 cells that expressed human c-Met (E) or human c-Met, human EGFR, and human Her2 (F). (G-I) Knockdown of E-cadherin by siRNA inhibits the phosphorylation of c-Met and EGFR in oral epithelial cells infected with C. albicans. Representative immunoblot (G). Densitometric analysis of 5 immunoblots showing the phosphorylation of c-Met (H) and EGFR (I). Results are mean ± SD. J and K) Effects of inhibiting c-Met (J) and EGFR (K) in combination with siRNA knockdown of E-cadherin on the endocytosis of C. albicans by oral epithelial cells. Results in (D-F, J, and K) are the mean ± SD of three experiments, each performed in triplicate. *p < 05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant (one-way ANOVA with Sidak’s multiple comparisons test [B-D, H-K] or two-tailed Student’s t test [E and F]).
Fig. 3.
Fig. 3.. C. albicans induces c-Met, EGFR, and E-cadherin to form a multiprotein complex.
(A-C) Proximity ligation assay showing the interaction of c-Met with E-cadherin, EGFR with E-cadherin, and c-Met with EGFR in oral epithelial cells with and without 20-min infection with C. albicans. Confocal microscopic images (A). Scale bar 10 μm. Signal counts (B). Proximity ligation assay showing the interaction of c-Met with E-cadherin, EGFR with E-cadherin, and c-Met with EGFR in oral epithelial cells with and without 20-min infection with C. albicans. (A) Confocal microscopic images. Scale bar 10 μm. (B) Signal counts. (C-E) Co-immunoprecipitation experiments in oral epithelial cells transfected with control or E-cadherin siRNA and then infected with C. albicans for 20 min. (C) Representative immunoblots of proteins obtained by immunoprecipitation with an anti-c-Met antibody. (D and E) Densitometric analysis of 5 immunoblots. Results are mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant (two-tailed Student’s t test [B] or one-way ANOVA with Sidak’s multiple comparisons test [D and E]).
Fig. 4.
Fig. 4.. Hyr1 interacts with c-Met and EGFR.
(A-C). C. albicans germ tubes stimulate phosphorylation of c-Met and EGFR after 20 min of infection. Representative immunoblots (A). Densitometric analysis of 4 immunoblots showing the phosphorylation of c-Met (B) and EGFR (C) induced by C. albicans yeast and hyphae. (D-F) An als3 Δ/Δ ssa1Δ/Δ mutant does not induce phosphorylation of c-Met and EGFR. Representative immunoblots (D). Densitometric analysis of 4 immunoblots showing the phosphorylation of c-Met (E) and EGFR (F) induced by the indicated strains of C. albicans. (G) Far Western blot showing proteins from the indicated C. albicans morphotypes and strains that were recognized by recombinant c-Met. Arrow indicates the protein band. (H-J) Hyr1 is required for maximal phosphorylation of c-Met and EGFR. Representative immunoblots (H). Densitometric analysis of 4 immunoblots showing the phosphorylation of c-Met (I) and EGFR (J) induced by the indicated strains of C. albicans. (K) Hyr1 is required for maximal endocytosis of C. albicans. Results in (B, C, E, F, I-K) are mean ± SD. WT, wild type; *p < 0.05, **p < 0.01, ***p < 0.001, ****p, 0.0001, ns; not significant (one-way ANOVA with Sidak’s multiple comparisons test).
Fig. 5.
Fig. 5.. Interactions of c-Met with Als3.
(A-C) Both Hyr1 and Als3 are required for phosphorylation of c-Met and EGFR. Representative immunoblots from oral epithelial cells infected with the indicated strains of C. albicans (A). Densitometric analysis of 5 immunoblots showing the phosphorylation of c-Met (B) and EGFR (C). (D) Epithelial cell endocytosis of the indicated strains of C. albicans. (E) Damage to oral epithelial cells caused by the indicated strains of C. albicans. (F) Invasion of live and fixed epithelial cells by the wild-type (WT) hyr1Δ/Δ mutant strains. (G-J) Induction of epithelial cell secretion of IL-1α (G), IL-1β (H), IL-8 (I) and GM-CSF (J) by the indicated strains of C. albicans. Results are mean ± SD. WT, wild type; ns, not significant; *p < 0.05, **p < 0.01, ***p, 0.001, ****p < 0.0001 (one-way ANOVA with Sidak’s multiple comparisons test).
Fig. 6.
Fig. 6.. Hyr1 and Als3 mediate virulence during OPC.
(A) Hyr1 and Als3 are required for C. albicans to resist killing by human neutrophils. Neutrophils were infected with the indicated C. albicans strains constructed in the SC5314 strain background. Results are mean ± SD of neutrophils from 3 donors, tested in triplicate. (B) The hyr1Δ/Δ mutant is resistant to neutrophil killing, even in the absence of c-Met. Killing of wild-type and hyr1Δ/Δ mutant strains in the SN250 strain background by neutrophils from Mrp8;Metfl/fl mice, which have a neutrophil-specific deletion in c-Met, and their littermates. Results are mean ± SD of neutrophils from 2 experiments performed in triplicate. (C and D) Oral fungal burden of immunocompetent mice infected with the indicated C. albicans strains for 1 (C) and 2 (D) days. (E and F) Effects of phagocyte depletion on susceptibility to OPC, as determined by oral fungal burden after 1 (E) and 2 (F) days of infection. (G-H) Oral fungal burden after 5 d of infection of mice that had been immunosuppressed with cortisone acetate and treated with gefitinib and/or SGX523 (G). Histopathology of the tongues (H). Scale bar 200 μm. Data are the mean ± SD combined results from 2 independent experiments, each using 4 mice per C. albicans strain (C-F) or 8 mice per condition in one experiment and 5 mice per condition in the other (G). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Sidak’s multiple comparisons test)
See this image and copyright information in PMC

References

    1. Berberi A., Noujeim Z., and Aoun G. (2015). Epidemiology of oropharyngeal candidiasis in human immunodeficiency virus/acquired immune deficiency syndrome patients and CD4+ counts. J Int Oral Health 7, 20–23. - PMC - PubMed
    1. Cawson R.A., and Rajasingham K.C. (1972). Ultrastructural features of the invasive phase of Candida albicans. Br J Dermatol 87, 435–443. - PubMed
    1. Churin Y., Al-Ghoul L., Kepp O., Meyer T.F., Birchmeier W., and Naumann M. (2003). Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response. J Cell Biol 161, 249–255. - PMC - PubMed
    1. Dalle F., Wachtler B., Coralie L., Holland G., Bannert N., Wilson D., Labruere C., Bonnin A., and Hube B. (2010). Cellular interactions of Candida albicans with human oral epithelial cells and enterocytes. Cell Microbiol 12, 248–271. - PubMed
    1. Dickson M.A., Hahn W.C., Ino Y., Ronfard V., Wu J.Y., Weinberg R.A., Louis D.N., Li F.P., and Rheinwald J.G. (2000). Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 20, 1436–1447. - PMC - PubMed

Publication types