NFκB activation limits ECE1-dependent damage and paracellular translocation.

(A) Inhibition of NFκB activation using the high affinity NFκB activation inhibitor quinazoline (QNZ) increases the damage potential of C. albicans wildtype, but not for the non-filamentous efg1ΔΔ/cph1ΔΔ strain or the non-damaging ece1Δ/Δ strain towards C2BBe1 cells after 24 h. LDH release was adjusted by subtracting the release from uninfected and untreated host cells. (B) Inhibition of NFκB activation increases fungal translocation of C. albicans after 24 h across intestinal epithelial cells independent of damage potential. (C) Blocking NFκB activation significantly increases the breakdown of barrier integrity after 24 h of the intestinal epithelial cell layer for efg1ΔΔ/cph1ΔΔ and ece1Δ/Δ, with a similar but not significant effect on both WT strains. (D) E-cadherin protein levels normalized to GAPDH and presented relative to levels in untreated C2BBe1 cells. QNZ treatment further increased degradation of E-cadherin during infection with WT (SC5314) and ece1Δ/Δ. (E) Fluorescent labeling of E-cadherin during C. albicans infection with and without QNZ treatment (scale bar = 50 μm). Inhibition of NFκB activation decreased the organization of E-cadherin at IEC borders upon infection with the ece1Δ/Δ and both WT strains. Representative pictures for each strain and treatment condition are shown from 3 biological replicates with median score values in the inset for each strain and condition. All values are shown as the mean with standard deviation. Host-cell damage (A), fungal translocation (B), and barrier integrity (C) data were compared using a one-way ANOVA. Statistical significance for host-cell damage (A) and fungal translocation (B) data was determined with a post-hoc Tukey’s multiple comparisons test, while significance for the barrier integrity (C) data was determined using a post-hoc Šidák’s multiple comparisons test. Statistical significance: **, P ≤ 0.01; ****, P ≤ 0.0001.