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
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