Fig 2: Gab1, 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 a-actin. Data are representative of three independent experiments.

Fig 3: Gab1, 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-1ß secretion. A–D, 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.

Fig 4: Gab1, 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-1a, and IL-1ß, which mediate the subsequent stages of the innate and adaptive immune responses. Figure created with Biorender.com

Fig 5: EGFR 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 a-actin loading control is reused from Fig. 3, A and B).