Figure 1.
Adhesion of C. albicans Δals mutants to TR146 oral epithelial cell monolayers.
C. albicans yeast cells (100 cfu) were added to TR146 monolayers and incubated at 37°C for 90 min. Each Δals strain was tested individually and compared to the wild-type strain SC5314 (URA3/URA3) and the CAI4 control (ura3::URA3). After extensive washing, molten (45°C) Sabouraud's dextrose agar was added and the plates were then incubated at 30°C for 24 h for colony development of adhered C. albicans cells. Results were expressed as the percentage of adhered C. albicans cells. Data represent mean values ± SEM and are representative of two independent experiments. * p<0.05 compared to CAI4. (A) % adherence of Δals strains; only Δals3 showed significantly decreased adhesion to TR146 monolayers. (B) The parent strain CAI4 and the Δals3 strain were added to TR146 oral epithelial cells and incubated at 37°C for 90 min. The monolayers were then formalin fixed and morphology was assessed by DIC microscopy at ×400.
Figure 2.
Induction of cell damage by C. albicans Δals mutants.
C. albicans yeasts were added to TR146 monolayers and incubated under standard conditions for 24 h. A fungal:epithelial cell MOI of 0.01 was used. Cell culture medium was collected and assessed for lactate dehydrogenase (LDH) release as a measure of epithelial cell damage induced by C. albicans Δals mutants (A) or the ALS3 reintegrant strain (B). Data represent mean values ± SEM and are representative of three independent experiments. * p<0.05 compared to CAI4. The Δals3 strain was the only one that showed significantly reduced cell damage compared to the control. Reintegration of a wild-type ALS3 allele restored cell damage capability to the strain.
Figure 3.
Cytokine induction by C. albicans Δals mutants.
C. albicans yeasts were added to TR146 epithelial cell monolayers and incubated under standard conditions for 24 h. A fungal:epithelial cell MOI of 0.01 was used. Cell culture medium was collected and cytokine levels measured using a multiplex microbead assay. Data represent mean values ± SEM and are representative of three independent experiments. * p<0.05 compared to CAI4. (A) The Δals3 strain was the only one that showed significantly reduced cytokine production compared to the control. Reintegration of a wild-type ALS3 allele restored the native cytokine production to the strain (B).
Figure 4.
Activation of MKP1 and c-Fos signaling by C. albicans Δals mutants.
C. albicans yeasts were added to TR146 oral epithelial cell monolayers and incubated under standard conditions for 2 h. Results from a fungal:epithelial cell MOI of 10 are shown here. Epithelial cell lysates were separated by SDS-PAGE and Western blotted to detect MKP1, c-Fos or α-actin (positive control). Data are representative of four independent experiments. There was no difference in MKP1 or c-Fos between control C. albicans strains (SC5314 and CAI4) and the Δals mutants.
Figure 5.
Adhesion, cell damage, cytokine production and activation of MKP1 and c-Fos by C. albicans Δals3 at different MOIs.
C. albicans CAI4 yeast cells (200 cfu) and C. albicans Δals3mutant yeast cells (200 cfu - low dose or 300 cfu - high dose) were added to TR146 monolayers and incubated for 90 min to determine the number of colonies and the percentage of adherence (A). C. albicans Δals3 mutant, the parent strain CAI4 and wild-type strain SC5314 were added to TR146 oral epithelial cells under standard culture conditions for 2 h or 24 h at a fungal:epithelial cell MOI ranging between 0.01 and 10. Induction of cell damage assessed by LDH release (B) and the production of cytokines by multiplex microbead assay (luminex) (C) were assayed in the cell culture medium supernatants at 24 h. (D) Induction of MKP1 phosphorylation and c-Fos at 2 h (MOI of 1). Data represent mean values ± SEM and are representative of duplicate experiments. * p<0.05 compared to CAI4.
Figure 6.
Stimulation of oral epithelial cells with NT-Als3 recombinant protein.
Different concentrations of NT-Als3 ranging from 0.3–10 µg/ml and wild-type strain SC5314 were applied to TR146 oral epithelial cells and incubated at 37°C in 5% CO2 for 24 h (A, B) or 2 h (C) as described in Materials and Methods. Cell culture medium was collected and assessed for production of cytokines (A) and LDH release (B). (C) Total protein was isolated and induction of MKP1 phosphorylation and c-Fos assessed. Bands are shown relative to α-actin loading control. SC5314 was used in all assays at a fungal:epithelial cell MOI of 0.01 (A, B) or 10 (C). Data represent mean values ± SEM and are representative of duplicate experiments. * p<0.05; ** p<0.01 compared to SC5314.