Fig. 4

Candidalysin-induced release of EREG and EPG contribute to subsequent EGFR-mediated signalling. Release of EREG (a), EPG (b) and NRGs 2, 3 and 4 (c–e) was observed following treatment of candidalysin to TR146 cells, in a dose-dependent manner with rapid onset (within 15 min). AREG was also detected in candidalysin-treated cell supernatants, but the release was gradual and accumulated over 6 h (f). At 24 h p.i. epiregulin (EREG) (g) and epigen (EPG) (h) were induced by candidalysin-expressing (WT and ece1Δ/Δ+ECE1 (black bars)) but not candidalysin-deficient (ece1Δ/Δ and ece1Δ/Δ+ECE1_Δ184–279 (grey bars)) C. albicans strains. While amphiregulin (AREG) was induced by all fungal strains tested, diminished potency was observed by those unable to express candidalysin (i). Increasing concentrations of EREG, EPG or EREG + EPG, were used to stimulate TR146 oral epithelial cells. j Phosphorylation of EGFR (at Y1068 and Y845 sites) and MKP1 proteins occurred in a dose-dependent manner in response to EREG and EREG + EPG, but not EPG alone. Induction of c-Fos by ligand stimulation was not dose-dependent (j). The effects of ligand exposure are not comparable to that of 70 µM candidalysin. While a dose-dependent trend of IL-6 (m), GM-CSF (n) and G-CSF (o) induction is observed in response to increasing concentrations of all ligand combinations, the changes are not statistically significant. IL-1α (k) and IL-1β (l) are not induced by ligand exposure. One-way ANOVA followed by a Bonferroni multiple comparison’s test was used to calculate statistical significance between groups. Graphs are an average of 3 (a–g, i–o) or 2 (h) independent experiments; one-way ANOVA with Bonferroni multiple comparison’s test used to assess statistical significance between samples from the same timepoint. Error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001