Sulfated glycosaminoglycans are host epithelial cell targets of the Candida albicans toxin candidalysin

Abstract

Candidalysin, a cytolytic peptide produced by the fungal pathogen Candida albicans, is a key virulence factor. However, its host cell targets remain elusive. Here we performed a genome-wide loss-of-function CRISPR screen in the TR146 human oral epithelial cell line and identified that disruption of genes (XYLT2, B3GALT6 and B3GAT3) in glycosaminoglycan (GAG) biosynthesis conferred resistance to damage induced by candidalysin and live C. albicans. Surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin binds to sulfated GAGs, facilitating its enrichment on the host cell surface. Adding exogenous sulfated GAGs or the analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate also inhibited C. albicans invasion and fungal-induced epithelial cell cytokine production. In mice with vulvovaginal candidiasis, topical dextran sulfate administration reduced intravaginal tissue damage and inflammation. Collectively, sulfated GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage.

Extended Data Fig. 1. Cas9-expressing TR146 cells display comparable phenotypes to wild-type TR146 cells in response to wild-type C. albicans SC5314 and candidalysin.

a, Western blotting of 3 clones of TR146 cells that stably expressed the P_{EF_la}-Cas9-B1asticidin construct, b, C. albicans association to and endocytosis by wild-type TR146 cells and the indicated clones of TR146-Cas9 cells, c, Cell survival of TR146 cells in response to candidalysin (30 μM) for 6 h as measured by an XTT assay. d, Survival of TR146 cells and TR146-Cas9 clones as measured by an XTT assay after a 6-h exposure to C. albicans (multiplicity of infection [MOI]=5) or candidalysin (30 μM). Clone 4 was selected for use in the subsequent experiments. Results are mean ± SD of 3 (b, c) or 2 experiments (d), each performed in triplicate (b, c) or duplicate (d).

Extended Data Fig. 2 |. Western blots and immunofluorescence images of gene knockouts and knockdowns.

a, Western blotting of B3gat3, Xylt2, B3galt6, Slc39a9 and Gbf1 in the corresponding CRISPR knockout cells or siRNA knockdown cells. Arrows indicate the target proteins, b, Tyk2 siRNA knockdown does not affect survival of oral epithelial cells after 6-h of candidalysin exposure. The left panel shows a representative Tyk2 western blot, and the middle panel shows the survival (measured by an XTT assay) of epithelial cells exposed to the indicated concentrations of candidalysin. The plots represent the combined results of 3 experiments, each performed in triplicate. The right panels show the concentration of candidalysin that yielded 50% survival (IC₅₀), which was calculated from the data in the corresponding graph in the middle panel. Results are mean ± SD. ns, not significant by the unpaired, two- sided Student’s t test. c, Representative immunofluorescence images of TR146, B3GAT3^−/−, XYLT2^−/− and B3GALT6^−/− cells stained with an anti-heparan sulfate antibody (red) and DAPI (blue). Scale bar: 50μm. d, Flow cytometric analysis of heparan sulfate expression on the surface of TR146 cells and the indicated mutants.

Extended Data Fig. 3 |. Structures of GAGs and GAG analogs used in the experiments.

a, Structure of the naturally occurring GAGS, hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate B (dermatan sulfate) and chondroitin sulfate C. b, Structure of the GAG analogs dextran/dextran sulfate, and alpha-cyclodextrin/sulfated alpha-cyclodextrin.

Extended Data Fig. 4 |. Sulfated GAGs but not carboxylate or non-sulfated GAGs protect epithelial cells from candidalysin-induced damage.

a, Protection of oral epithelial cells from damage caused by a 6-h exposure to 30 μM candidalysin provided by alginate, b, Protection from damage caused by a 6-h exposure to 30 (μM candidalysin provided by dextran and dextran sulfate (10kD, 40kD and 500kD) (left) or dextran (40kD, 90kD and 500kD) (right). c, Protection from damage caused by a 6-h exposure to 70 μM candidalysin provided by 100 μg/ml of dextran, dextran sulfate, α--cyclodextrin, sulfated α-cyclodextrin, heparin, and heparan sulfate. d, Protection from damage caused by a 24-h exposure to 70 μM candidalysin provided by 100 μg/ml of dextran, and dextran sulfate. e, Representative surface plasmon resonance sensorgrams showing the effects of heparin, heparan sulfate, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, dextran and dextran sulfate on the interaction of candidalysin with heparin on a biosensor chip. f, Combined results of 3 independent experiments showing the inhibitory effects of the various GAGs or GAG analogs on the interaction of candidalysin with heparin. Results in a-d are mean ± SD of 3 independent experiments, each performed in triplicate. Protection was determined using an XTT assay. P values were calculated using the one-way ANOVA with Dunnett’s multiple comparisons test (c, d, and f).

Extended Data Fig.5 |. Candidalysin interacts with GAGs and their analogs.

a, Transmission electron microscopy (TEM, top left) and atomic force microcopy (AFM, bottom left) images of candidalysin on a solid substrate with or without dextran, dextran sulfate and heparan sulfate. Candidalysin (3 μM) was incubated with dextran, dextran sulfate, or heparan sulfate (50 μg/mL) for 30 rnin before TEM imaging, and candidalysin (333 nM) was incubated with dextran, dextran sulfate, or heparan sulfate (5 μg/mL ) for AFM imaging. The right panel shows the quantification of the AFM images in terms of loops per μm² for each treatment. Scale bar: 200 nm. b, Diagram illustrating the C-laurdan assay, c, Time course of the effects of dextran sulfate (50μg/mL) on the GP score in the C-laurdan assay in the absence of candidalysin. Data are the mean ± SD of 3 experiments, d, Dextran sulfate (50 μg/mL) reduces the rate of candidalysin-induced membrane damage of l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) unilamellar vesicles. Representative time course (left). Combined results from 4 experiments (right). t₅₀ values represent the time at which 50% membrane leakage occurred. Results are mean ± SD. The data were analyzed using an unpaired, two-sided Student’s test with significant p-values shown in comparison to the control, e, Comparison of the effect of candidalysin and CFSE-labeled candidalysin on the survival of oral epithelial cells after a 6-h exposure to 10, 30 and 90 uM candidalysin. Survival was measured using an XTT assay. Results are mean ± SD of two experiments, each performed in triplicate, f, Colocalization analysis of heparan sulfate and CFSE-candidalysin on TR146 cells. Overlap coefficient R and Pearson’s coefficient R(r) were generated by the Olympus microscopy software CellSens. Results are median (min to max) of 3 independent experiments, each quantifying 4 independent images, g, Damage to wild-type TR146 and B3GAT3^−/−‘ cells caused by the C. albicans ecelΔ/Δ mutant (MOI = 5) after 5 h of infection, as measured by an LDH assay. Results are mean ± SD of three experiments, each performed in triplicate, h, i, Effects of 100 μg/ml dextran and dextran sulfate on the number of cell-associated and endocytosed cells of the C. albicans ecelΔ/Δ mutant. The average number of organisms per high-power field that were associated with and endocytosed by TR146 cells were 8.81 ± 1.81 and 2.27 ± 1.31, respectively. Results are mean ± SD of three independent experiments, each performed with a single replicate (a, c, d) or in triplicate (e-i). P values were calculated using the unpaired, two-sided Student’s t-test (d, g) and one way ANOVA with Dunnett’s test for multiple comparisons (a, e, h, i).

Extended Data Fig.6 |. Effects of dextran and dextran sulfate on stimulation of B3GAT3^−/− cells by candidalysin and live C. albicans.

a, Western blot results of epidermal growth factor receptor (EGFR), extracellular regulated kinasel/2 (ERK1/2) and the c-Fos transcription factor in B3GAT3^−/− epithelial cells induced by wild-type C. albicans SC5314 or the ecelΔ/Δ mutant (MOI = 5), or candidalysin (10 μM) with or without dextran (DX) and dextran sulfate (DS) (100 μg/ml) at the indicated time points. Shown are representative results of 3 independent experiments. b, The effects of dextran (DX) and dextran sulfate (DS) (100 μg/ml) on the production of CXCL8 (top), IL-1β (middle) and GM-CSF (bottom) by B3GAT3^−/− cells incubated for 6 h with wild-type C. albicans (MOI=5) or candidalysin (10 μM) (left panel) or infected with the C. albicans ecelΔ/Δ mutant (right panel). Results are mean ± SD of 3 experiments, each performed in duplicate or triplicate. c, Effects of dextran and dextran sulfate (100 μg/ml) on the production of CXCL8, IL-1β, and GM-CSF by TR146 cells infected with the ecelΔ/Δ mutant (right, MOI=5) for 6 hours. P values were calculated using the one-way ANOVA with Dunnett’s multiple comparisons test (b-c).

Extended Data Fig. 7 |. Dextran sulfate protects vaginal epithelial cells from damage and inhibits pro-inflammatory cytokine production in CD-1 mice with vulvovaginal candidiasis.

a-d, CD-1 mice were treated with either dextran sulfate or vehicle alone intravaginally prior to vaginal inoculation with C. albicans SC5314 and daily thereafter. After 3 days of infection, the concentration of adenylate kinase (a measure of host cell damage) (a), neutrophils (PMN) (b), IL-1β (c), and fungal colony forming units (CFU) (d) in the vaginal lavage fluid was determined. Results in (a-d) are the mean ± SD of 5 mice per experimental group in a single experiment. P values were calculated using the student’s t-test (a-d).

Fig. 1│. Genes identified by the genome-wide CRISPR screen that are required for maximal candidalysin-induced epithelial cell damage.

a, Schematic diagram of the genome-wide CRISPR screen used to identify genes required for susceptibility to candidalysin-induced epithelial cell damage and scatterplot of the results. The shape of the symbols indicates the number of sgRNAs targeting each gene that were significantly enriched in the screen. The sigmaFC gene rank for each gene was calculated by adding the fold changes of all sgRNAs that target that gene, multiplied by the number of sgRNAs that showed significant enrichment. The top 7 enriched genes (sigmaFC > 6) are labeled in the plot. P values were calculated using the α-RRA and STARS, and adjusted for multiple hypothesis tests using the Benjamini-Hochberg FDR procedure. b, Effects of CRISPR deletion (B3GAT3, XYLT2, B3GALT6, SLC39A9, EMP1), pharmacologic inhibition (Tyk2), and siRNA knockdown (GBF1) on the survival of oral epithelial cells after 6 h of candidalysin exposure. The left panels show the survival (measured by an XTT assay) of epithelial cells exposed to the indicated concentrations of candidalysin. The plots represent the combined results of 3–4 experiments, each performed in triplicate. The right panels show the concentration of candidalysin that yielded 50% survival (IC₅₀), which was calculated from the data in the corresponding graph in the left panels. Results are mean ± SD. K/O, knockout; P values were calculated using the unpaired, two-sided Student’s t test.

Fig. 2│. Sulfated glycosaminoglycans (GAGs) and dextrin analogues bind to candidalysin and provide dose-dependent protection to epithelial cells against candidalysin-induced damage.

a, Diagram illustrating the enzymes that catalyze the biosynthesis of the tetra-saccharide linker present in all GAGs. The genes that encode the enzymes are denoted under the monosaccharide, and the ones in purple are those identified by the CRISPR screen. b, Protection from damage caused by a 6-h exposure to 30 μM candidalysin provided by the indicated GAGs as measured by an XTT assay. The curves were generated from the data of 3 independent experiments, each performed in triplicate. Percent protection was calculated as the normalized increase in host cell survival relative to control cells treated with candidalysin alone. c, Surface plasmon resonance (SPR) sensorgram of the interaction of candidalysin with heparin immobilized on the biosensor chip. The concentration of candidalysin used to generate each sensorgram is indicated on the graph. The binding kinetics (k_on, association rate constant; k_off, dissociation rate constant; t_1/2, half-life; and K_D=k_on/k_off, binding equilibrium dissociation constant) were calculated from the global fitting of the five concentrations using a 1:1 Langmuir binding model. d, Protection from damage caused by a 6-h exposure to 30 μM candidalysin provided by dextran, dextran sulfate, α-cyclodextrin (α-CD) or sulfated α-cyclodextrin measured by an XTT assay. The curves were generated from the data of 3 independent experiments. e, Structure of heparin showing the location of the sulfate groups. f, Representative surface plasmon resonance sensorgrams showing the effects of 2-O-desulfated heparin (2-Des hep), 6-O-desulfated heparin (6-Des hep), and N-desulfated heparin (N-Des hep) on the interaction of candidalysin with heparin on a biosensor chip. g, Combined results of 3 independent experiments showing the inhibitory effects of the various desulfated heparins on the interaction of candidalysin with heparin. Results are mean ± SD. P values were calculated using the one-way ANOVA with Dunnett’s multiple comparisons test.

Fig. 3│. GAGs enhance candidalysin polymerization/aggregation.

a, Atomic force microscopic images of supported DOPC lipid bilayers exposed to 333 nM candidalysin with or without 5 μg/ml dextran, dextran sulfate and heparan sulfate (upper panel), and quantification of the number of pores per μm² for each treatment (lower panel). Scale bar: 200 nm. Results are mean ± SD of 3 experiments. b, Effects of incubating candidalysin in 1 μM C-laurdan for 4 h on the generalized polarization (GP) score (left panel). The effects of dextran sulfate on the GP scores of 0.2 μM and 0.5 μM candidalysin (right panel). Results are mean ± SD of 3 independent experiments. c, d, Co-localization of candidalysin with heparan sulfate on the surface of wild-type TR146 (c) or GAG-deficient B3GAT3^−/− (d) epithelial cells incubated with CSFE-labeled candidalysin (10 μM) for the indicated times. Arrows indicate representative candidalysin aggregates. Scale bar: 20 μm. e, Quantification of the number of candidalysin aggregates per microscopic field on the indicated cells after 20 min exposure to CFSE-candidalysin. Results are mean ± SD of 3 random microscopic fields per experiment from 3 independent experiments. f, Interaction of CFSE-candidalysin with a 1:1 mixture of TR146 and GAG-deficient B3GAT3^−/− cells for 20 min. Arrows indicate representative candidalysin aggregates. The TR146 cells stain for heparan sulfate (red) and are marked with asterisks. Scale bar: 20 μm. g, Quantification of the candidalysin aggregates per TR146 or B3GAT3^−/− cell in the mixed population of TR146/B3GAT3^−/− cells. Results are mean ± SD of 5 random fields per experiment from 3 independent experiments. h, Effects of calcium ionophore A23187 (1 μM) or candidalysin (10 μM) on the levels of free intracellular calcium in TR146 and B3GAT3^−/− cells as determined by flow cytometry. 0–90s represents the baseline levels and data collection were resumed at 120s after treatment. Results are mean ± SD of 4 independent experiments. P values were calculated using the one-way ANOVA with the Dunnett’s multiple comparisons test (a, b), and by the unpaired, two-sided Student’s t test (e, g).

Fig. 4│. Sulfated GAGs mediate epithelial cell invasion, damage, and stimulation of oral epithelial cells by live C. albicans.

a, b, C. albicans association with (a) and endocytosis by (b) wild-type and B3GAT3^−/−, XYLT2^−/−, and B3GALT6^−/− mutant epithelial cells. The average number of organisms per high-power field that were associated with and endocytosed by TR146 cells were 12.84 ± 4.97 and 2.91 ± 1.27, respectively. Results are the mean ± SD of 4 experiments, each performed in triplicate. c, Protection against damage caused by 5 h of infection with live wild-type C. albicans at a multiplicity of infection (MOI) of 5 in B3GAT3^−/−, XYLT2^−/−, and B3GALT6^−/− mutant cells relative to wild-type TR146 epithelial cells, as measured by an XTT assay. Results are mean ± SD of 3 experiments, each performed in replicates of 4. d, Effects of dextran and dextran sulfate (100 μg/ml) on damage to TR146 and B3GAT3^−/− cells caused by 5-h infection with live C. albicans SC5314 at an MOI of 5, as measured by an LDH release assay. Results are the mean ± SD of 5 experiments, each performed in triplicate. e, f, Effects of dextran and dextran sulfate (100 μg/ml) on wild-type C. albicans association with (e) and endocytosis by (f) the indicated epithelial cells. The average number of organisms per high-power field that were associated with and endocytosed by TR146 cells were 9.64 ± 2.68 and 1.88 ± 0.56, respectively. Results are the mean ± SD of 4 experiments, each performed in triplicate. P values were calculated using the unpaired, two-tailed Student’s test (a-c) or one-way ANOVA with Dunnett’s multiple comparisons test (d-f).

Fig. 5│. Sulfated GAGs mediate stimulation of oral epithelial cells by candidalysin and live C. albicans.

a, Simplified model of epithelial immune signaling in response to C. albicans and candidalysin (left panel). Immunoblot analysis of the effects of dextran (DX, 100 μg/ml) and dextran sulfate (DS, 100 μg/ml) on the phosphorylation of epidermal growth factor receptor (EGFR), extracellular regulated kinase1/2 (ERK1/2) and the C-Fos transcription factor in TR146 cells induced by wild-type C. albicans SC5314 or the ece1Δ/Δ mutant at MOI of 5, or candidalysin (10 μM) at the indicated time points (right panel). Shown are representative results of 3 independent experiments. b-d, Effects of dextran and dextran sulfate (100 μg/ml) on the production of CXCL8 (b), IL-1β (c) and GM-CSF (d) by TR146 cells infected with C. albicans SC5314 (MOI=5, top) or incubated with candidalysin (10 μM, bottom) for 6 h. Results in (b-d) are mean ± SD of 3 experiments, each performed in triplicate or replicates of 4. P values were calculated using the one-way ANOVA with Dunnett’s multiple comparisons test (b-d).

Fig. 6│. Dextran sulfate protects vaginal epithelial cells from damage and inhibits pro-inflammatory cytokine production in vitro and during murine vulvovaginal candidiasis.

a, Survival of A431 vulvar epithelial cells after incubation with 30 μM candidalysin for 6 h (left). Protection from candidalysin-induced damage by dextran and dextran sulfate (right). Results are mean ± SD of 3–4 experiments, each performed in triplicate. b, Effects of dextran and dextran sulfate (100 μg/ml) on the production of IL-1β and CXCL8 by vaginal epithelial cells incubated with C. albicans (MOI=5) or candidalysin (10 μM) for 24 h. Results are mean ± SD of 3 experiments, each performed in duplicate or triplicate. c-g, C57BL/6 mice were administered either dextran sulfate or vehicle alone intravaginally prior to inoculation with C. albicans and once daily thereafter. After 3 days of infection, the concentration of adenylate kinase (a measure of host cell damage) (c), neutrophils (PMN) (d), IL-1β (e), and fungal colony forming units (CFU) (f) in the vaginal lavage fluid was determined. Results in c-f are the mean ± SD 2 experiments, one that used 3 mice per condition and the other that used 4 mice per condition. g, Representative images of hematoxylin and eosin (H&E) stained histological sections of vaginal tissue day3 post-infection. The scale bar represents 200 μm in full images and 50 μm in magnified images in the lower right. h, A proposed working model of how cell surface GAGs facilitate C. albicans and candidalysin -induced cell damage and the protective effects of exogenous dextran sulfate. Free candidalysin binds to cell surface GAGs such as heparan sulfate or chondroitin sulfate (1). Binding to cell surface GAGs enhances the aggregation and polymerization of candidalysin (2), which facilitates pore formation on the host cell plasma membrane (3), leading to host cell damage. Exogenous dextran sulfate causes aggregation of candidalysin extracellularly. C. albicans cells adhere to and invade epithelial cells to cause cell damage. Exogenous dextran sulfate binds to C. albicans, reducing adherence to and invasion of host cells. P values were calculated using the unpaired, two-sided Student’s t-test (a, c-f) or one-way ANOVA with Dunnett’s multiple comparisons test (b).