Treatment with a pan-caspase inhibitor does not prevent increased virulence upon inhibition of NFκB activation.

Treatment with a pan-caspase inhibitor (Z-VAD) had no significant effect on (A) host-cell damage, (B) barrier integrity, or (C) fungal translocation when used alone or in combination with the NFκB inhibitor quinazoline (QNZ). All values are shown as the mean with standard deviation. Host-cell damage (A), barrier integrity (B), and fungal translocation (C) data were compared using a one-way ANOVA with a post-hoc Šidák’s multiple comparisons test. Statistical significance: *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001. (TIF)</p

DMSO vehicle control has no effect on host damage or translocation of <i>C</i>. <i>albicans</i>.

C2BBe1 cells were treated with a DMSO vehicle control and infected with the WT or ece1Δ/Δ C. albicans strains. There were no significant changes in (A) host cell damage or (B) fungal translocation. All values are shown as the mean with standard deviation. (TIF)</p

Source data.

All data used to generate graphs for main text and supplementary figures. (XLSX)</p

Loss of <i>ECE1</i> increases the transcriptional zinc starvation response during infection of IECs.

Fold change in gene expression in C. albicans WT (BWP17) and ece1Δ/Δ strains during co-incubation with C2BBe1 cells for (A) ZRT101, (B) ZRT2, (C) PRA1, (D) ZRT3, and (E) ZRC1. Fold changes are calculated as the normalized gene expression at each time point compared to the respective 0 h control samples. (F) Fold change in normalized gene expression of the ece1Δ/Δ strain compared to WT(BWP17) at 24 h in medium only or during infection of IECs. Gene expression was analyzed by q-RT-PCR and is normalized to ACT1 as a housekeeping gene. All values are shown as the mean with standard deviation. All the ratio data from q-RT-PCR experiments were log-transformed before performing a two-tailed, paired t-test. Statistical significance: *, P ≤ 0.05.</p

Transcriptional response of IECs to infection with non-damaging and non-filamentous <i>C</i>. <i>albicans</i>.

Genes differentially expressed when comparing the non-damaging ece1Δ/Δ and the non-filamentous efg1ΔΔ/cph1ΔΔ strains to their respective WT strains (ece1Δ/Δ compared to WT (BWP17) and efg1ΔΔ/cph1ΔΔ compared to WT (SC5314)). The data are shown as the Log2(fold-change) of infected cells with the different strains compared to uninfected IECs. Asterisks indicate genes with statistically significant differences in expression (DESeq2 p (TIFF)</p

Treatment of IECs with another NFκB inhibitor increases host cell damage, loss of barrier integrity, and fungal translocation.

(A) Inhibition of NFκB activation using the NFκB inhibitor SC75741 at concentrations of either 2.5 or 5 μM increased the damage of WT (BWP17) C. albicans, but not for the ece1Δ/Δ strains. LDH release was adjusted by subtracting the release from uninfected and untreated host cells. (B) NFκB inhibition using either concentration also decreased the barrier integrity during infection with both WT and ece1Δ/Δ strains. (C) Fungal translocation was significantly increased during infection with both WT and ece1Δ/Δ C. albicans upon inhibition of NFκB using both concentrations of SC75741. These results match those obtained with the high-affinity NFκB inhibitor quinazoline. All values are shown as the mean with standard deviation. Host-cell damage (A), barrier integrity (B), and fungal translocation (C) data were compared using a one-way ANOVA with a post-hoc Šidák’s multiple comparisons test. Statistical significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (TIF)</p

Overrepresentation analysis of DEGs.

Enrichment analysis for C. albicans and host DEGs from both the IEC infection time course and mutant comparison RNA sequencing datasets as stated in the Description sheet. (XLSX)</p

Western blot detection of immune signaling proteins for IECs infected with WT and <i>ece1</i>Δ/Δ.

(A) Confluent, differentiated C2BBe1 cells were infected with WT (BWP17) and ece1Δ/Δ C. albicans for 6 h and the protein content was sampled. Proteins involved in the damage response of oral epithelial cells were detected with ACT1 serving as a control. n.d. = not determined. (B) Protein levels normalized to actin. For p38, MKP1, EPHA2, EGFR, and AKT the normalized protein level for the phosphorylated protein is presented relative to the total respective protein level. (TIF)</p

Addition of exogenous zinc alleviates the transcriptional zinc starvation response in the absence of <i>ECE1</i> but does not affect host-cell damage, fungal translocation, or fungal load.

(A) Host-cell damage in the absence or presence of 25 μM exogenous ZnSO4 for the WT mutant (same data presented in Fig 2D) and the ece1Δ/Δ strain. (B) Fungal translocation of the WT(BWP17) and ece1Δ/Δ strains with or without the addition of 25 μM ZnSO4. (C) Relative quantification of fungal gDNA during infection of IECs. All samples are compared to the WT(BWP17) without added zinc. (D) Fold change in normalized gene expression of the ece1Δ/Δ strain compared to WT(BWP17) at 24 h during infection of IECs with addition of 25 μM ZnSO4. Gene expression was normalized to ACT1 as a housekeeping gene. All values are shown as the mean with standard deviation. (TIF)</p

Loss of <i>ECE1</i> has no effect on the transcriptional zinc starvation response in medium only.

Fold change in gene expression in C. albicans WT (BWP17) and ece1Δ/Δ strains during incubation in cell culture medium for (A) ZRT101, (B) ZRT2, (C) PRA1, (D) ZRT3, and (E) ZRC1. All values are shown as the mean with standard deviation. (TIF)</p