Candidalysin (CL) activates EGFR. a Phosphorylation: Infection of TR146 cells with WT C. albicans induces EGFR phosphorylation at two distinct tyrosine sites, Y1068 and Y845, while a strain lacking the candidalysin-encoding region (ece1Δ/Δ+ECE1_Δ184–279) does not (left panel). Phosphorylation of EGFR at both sites was also induced following direct candidalysin treatment in a dose-dependent manner (right panel). Data are representative of three biological repeats. Protein lysates were taken at 2 h p.i. for western blot analysis. Solid vertical lines indicate omitted, extraneous portions of blot images. b Internalisation: At 30 min post CL treatment, EGFR is significantly internalised. Data collected from a total of 120,000 cells per group over three independent experiments. Median value indicated, error bars represent min-max data points. Values are shown as a Log transformed ratio of EGFR staining intensity inside the cell to the total cell intensity of EGFR staining. c Murine pEGFR staining: Immune compromised mice were infected with a C. albicans ece1Δ/Δ mutant (lacking ECE1 which encodes the parent protein from which candidalysin is derived) and tongues were harvested at day 1 p.i. Paraffin-embedded, 3 mm tissue sections were prepared and stained for pEGFR (Y1068). The ece1Δ/Δ mutant exhibits reduced capacity to phosphorylate EGFR above a given threshold (threshold set using Image J software), when compared to WT C. albicans infections. Data from 9 (WT) and 11 (ece1Δ/Δ) individual mice obtained over two independent experiments. d Murine pEGFR staining: Immune competent mice were infected with the candidalysin-deficient ece1Δ/Δ+ECE1_Δ184–279 mutant which also exhibited a reduced ability to phosphorylate EGFR at Y1068, when compared to WT infection. Data obtained from one experiment, 5 mice per group. Unpaired T-tests were used to determine statistical significance in (b, c, d). Error bars from (c, d) represent Standard deviation (SD). *p < 0.05, **p < 0.01

Inhibition of EGFR suppresses neutrophil recruitment and enhances mortality in a zebrafish swimbladder model of candidiasis. Candidalysin does not bind EGFR. a Mortality: In the presence of AG1478 EGFR inhibitor, C. albicans-infected fish exhibited enhanced mortality with 70% death by day 3 p.i. (red), whereas all the infected vehicle treated fish survived (blue). b, c Neutrophil recruitment: EGFR inhibition resulted in significantly fewer neutrophils (green) recruited into the infected swimbladder (outlined in purple). Numbers in graph c provide the median neutrophil count per group. d Fungal load: No significant change in fungal burden was observed in the presence of AG1478 inhibitor, as quantified by the number of red pixels, normalised to the area of the swimbladder. Log-rank with a Bonferroni correction was used for (a), Kruskal-Wallis with Dunn’s post-test correction for (c) and Mann-Whitney for (d). Median and interquartile range are plotted in (c, d). *p < 0.05, ***p < 0.001, n.s. p > 0.05. e Candidalysin does not co-localise with EGFR: Confocal images of TR146 cells show that at 2 min post exposure, fluorescently labelled candidalysin-488 (green) can be seen within the cell cytoplasm whilst EGFR (red) remains at the cell surface. At 30 min post toxin exposure, although both EGFR and candidalysin are found within cells, no co-localisation was observed, and staining patterns are distinct for each. Confocal point images have a 91.36 µm x 90.79 µm (left image) and 35.9 µm x 35.9 µm (right image) field of view. Images are representative of three individual experiments. f Surface plasmon resonance response of candidalysin and EGFR domains : SPR signal response for the different domains of EGFR did not reach above 50 RU when interacting with biotinylated-candidalysin (180 s injection of 500 nM of titrant at 10 µL/min flow, 25 °C). Approximate response expected for binding of extracellular or cytoplasmic domains is 900 RU (dotted line) at a Kd of 20 mM