Candidalysin triggers epithelial cellular stresses that induce necrotic death

Abstract

Candida albicans is a common opportunistic fungal pathogen that causes a wide range of infections from superficial mucosal to hematogenously disseminated candidiasis. The hyphal form plays an important role in the pathogenic process by invading epithelial cells and causing tissue damage. Notably, the secretion of the hyphal toxin candidalysin is essential for both epithelial cell damage and activation of mucosal immune responses. However, the mechanism of candidalysin-induced cell death remains unclear. Here, we examined the induction of cell death by candidalysin in oral epithelial cells. Fluorescent imaging using healthy/apoptotic/necrotic cell markers revealed that candidalysin causes a rapid and marked increase in the population of necrotic rather than apoptotic cells in a concentration dependent manner. Activation of a necrosis-like pathway was confirmed since C. albicans and candidalysin failed to activate caspase-8 and -3, or the cleavage of poly (ADP-ribose) polymerase. Furthermore, oral epithelial cells treated with candidalysin showed rapid production of reactive oxygen species, disruption of mitochondria activity and mitochondrial membrane potential, ATP depletion and cytochrome c release. Collectively, these data demonstrate that oral epithelial cells respond to the secreted fungal toxin candidalysin by triggering numerous cellular stress responses that induce necrotic death. TAKE AWAYS: Candidalysin secreted from Candida albicans causes epithelial cell stress. Candidalysin induces calcium influx and oxidative stress in host cells. Candidalysin induces mitochondrial dysfunction, ATP depletion and epithelial necrosis. The toxicity of candidalysin is mediated from the epithelial cell surface.

Figure 1.. Cytotoxicity profile of candidalysin-treated epithelial cells.

Fluorescence microscopy images of TR146 oral epithelial cells exposed to the indicated concentrations of candidalysin for 3 h, 6 h and 24 h. Staurosporine and Triton X-100 were used as positive controls to induce apoptosis and necrosis, respectively. Cells were stained with Hoechst 33342 (blue: live cells), FITC-conjugated Annexin V (green: apoptotic cells), and EthD-III (red/yellow: necrotic cells). Data are representative of 3 independent experiments. Images were taken with a fluorescence microscope at 100x magnification. Scale bars = 500 μm.

Figure 2.. Cleavage or activation of apoptotic caspases and PARP in oral epithelial cells in response to C. albicans and candidalysin.

(A,B) TR146 oral epithelial cells were incubated with candidalysin (70, 30, 15 and 3 μM) or live C. albicans (wild type and mutant strains) at a MOI of 0.01. As a positive control, 0.1 μM staurosporine was used to induce apoptosis. Total protein was isolated and analysed by SDS-PAGE and western blotting for the presence of procaspase/caspase-8 (A), pro-caspase/caspase-3 (B). (C) Caspase-8 and caspase-3 activity was also quantified by luminescence assay. Data represent caspase-8 and caspase-3 activity relative to the untreated control. Error bars represent mean values ± SEM of 3 independent experiments. *P < 0.05; one-way ANOVA. (D) Cells were treated as in (A-B), total protein was isolated and analysed by SDS-PAGE and western blotting for the presence of PARP.

Figure 3.. Candidalysin induces loss of metabolic activity, depletion of intracellular ATP and accumulation of intracellular ROS.

(A) TR146 oral epithelial cells were treated with candidalysin (3, 15, 30, 70 μM) for 3 h and assayed for metabolic activity by MTT assay. (B) Time-course experiments (0.5–6 h) of TR146 oral epithelial cells treated with candidalysin at 70 or 15 μM and assayed for metabolic activity by MTT. (C) TR146 oral epithelial cells were treated with candidalysin (3, 15, 30, 70 μM) for 3 h and assayed for intracellular ATP by luminescence assay. (D) Time-course experiments (0.5–6 h) of TR146 oral epithelial cells treated with candidalysin at 70 or 15 μM and assayed for intracellular ATP by luminescence assay. Triton X-100 (1%) (TX) was used as a positive control. Data are expressed as a percentage of metabolic activity or intracellular ATP in comparison with the untreated control. Error bars represent mean values ± SEM of 3 independent experiments. (E) To assess the production of intracellular ROS, TR146 oral epithelial cells were pre-incubated with DCFH-DA for 1 h and subsequently incubated with candidalysin (70, 15 and 3 μM). Fluorescence was measured in real time over 30 min. Data are expressed as relative fluorescence units (RFU). (F) RFU data at 10, 20 and 30 min are plotted as histograms of mean ± SEM of ROS production corresponding to 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; one-way ANOVA.

Figure 4.. Candidalysin induces loss of mitochondrial membrane potential and cytochrome c release.

(A) Epithelial cells were pre-labelled with Rhodamine 123 (green) and incubated with candidalysin/control peptide in the presence or absence of calcium then visualized by confocal microscopy in the presence of DAPI (cyan). Images are maximum projections representative of ≥ 30 fields from 3 independent experiments. Scale bars = 10 μm. (B) Following C. albicans invasion, TR146 cells pre-labeled with Rhodamine 123 (green) were stained 5 min with 10 μg mL⁻¹ calcofluor white (to label C. albicans; white) and visualized by confocal microscopy. Diffuse green images indicate a reduction in mitochondrial potential. Images are central optical slices representative of ≥ 15 fields from 2 separate experiments. The white solid box within XY optical slice indicates location of accompanying XZ and YZ reconstructions (right). XZ and XY images of invading C. albicans indicate the upper and lower bounds of epithelial plasma membrane, visualized by brightfield microscopy, with dotted white lines. Scale bars = 5 μm. (C) Candidalysin exerts its effects while localized at the plasma membrane of epithelial cells. After incubation with candidalysin-Alexa 647, TR146 cells were visualized by confocal microscopy. Candidalysin-Alexa 647 shown in white. The location of the plasma membrane, visualized by brightfield microscopy, is indicated by a dotted yellow line. Left image is a central XY slice; yellow dotted crosshairs within image indicate the locations of accompanying XZ (horizontal line) and YZ (vertical line) slices (displayed to the right). Images are optical slices representative of ≥ 10 fields from 2 separate experiments. Scale bars = 5 μm. (D, E) TR146 oral epithelial cells were incubated with 30 μM candidalysin or live C. albicans (wild type and mutant strains) at a MOI of 0.01. Staurosporine (0.1 μM) was used as positive control. Cytochrome c was quantified in exhausted culture medium by ELISA. Data are expressed as fold change of cytochrome c levels comparison to the untreated control. Error bars represent mean values ± SEM of 3 independent experiments. Significant differences relative to the untreated control: * P < 0.05; one-way ANOVA.