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

Nature Microbiology volume 9, pages 2553–2569 (2024)Cite this article

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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.

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Fig. 1: Genes identified by the genome-wide CRISPR screen that are required for maximal candidalysin-induced epithelial cell damage.
Fig. 2: Sulfated GAGs and dextrin analogues bind to candidalysin and provide dose-dependent protection to epithelial cells against candidalysin-induced damage.
Fig. 3: GAGs enhance candidalysin polymerization and aggregation.
Fig. 4: Sulfated GAGs mediate epithelial cell invasion, damage and stimulation of oral epithelial cells by live C. albicans.
Fig. 5: Sulfated GAGs mediate stimulation of oral epithelial cells by candidalysin and live C. albicans.
Fig. 6: Dextran sulfate protects vaginal epithelial cells from damage and inhibits pro-inflammatory cytokine production in vitro and during mouse vulvovaginal candidiasis.

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Data availability

The original sequencing data were deposited in NCBI under BioProject PRJNA1081917. All data from this study are presented in the Article and the extended data figures and table. Source data are provided with this paper.

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Acknowledgements

This work was supported in part by grants R01DE026600 (to S.G.F.), R01AI134796 (to B.M.P.), R35GM140846 (to F.N.B.), U01-AI124319 and U19-AI172713 (to M.R.Y.), and S10OD028523 and R21AI156573 (to R.J.L. and F.Z.) from the National Institutes of Health, USA, and 2122027 (to G.M.K.) from the National Science Foundation, USA. J.M. received financial support from the China Scholarship Council award 201906150153 and the UTHSC Center for Pediatric Experimental Therapeutics. K.G.S. acknowledges support from the Research Excellence Program at the University of Missouri. C.M.R. was supported by a Graduate Advancement & Training Education Fellowship from the University of Tennessee—Oak Ridge Innovation Institute. We thank Z.-Q. Koo and Y.-H. Tsai of the Institute of Microbiology and Immunology in Taipei for the detailed protocol for labelling candidalysin with CFSE. The funding agencies had no role in the study design, data collection, data interpretation or preparation of the paper. The elements in our diagrams were adapted from the Servier Medical Art (SMART, https://smart.servier.com/) under CC BY 4.0.

Author information

Authors and Affiliations

  1. Institute for Infection and Immunity, Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA, USA

    Jianfeng Lin, Quynh T. Phan, Norma V. Solis, Hong Liu, Masato Tashiro, Michael R. Yeaman & Scott G. Filler

  2. Pharmaceutical Sciences Program, College of Graduate Health Sciences, University of Tennessee Health Science Center, Memphis, TN, USA

    Jian Miao

  3. Department of Physics and Astronomy, University of Missouri, Columbia, MO, USA

    Katherine G. Schaefer & Gavin M. King

  4. Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, USA

    Charles M. Russell & Francisco N. Barrera

  5. Genome Science and Technology, University of Tennessee, Knoxville, TN, USA

    Robert J. Pyron

  6. Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

    Fuming Zhang, Jonathan S. Dordick & Robert J. Linhardt

  7. Department of Infectious Diseases, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

    Masato Tashiro

  8. David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    Michael R. Yeaman & Scott G. Filler

  9. Division of Infectious Diseases, Department of Medicine, Harbor-UCLA Medical Center, Torrance, CA, USA

    Michael R. Yeaman & Scott G. Filler

  10. Division of Molecular Medicine, Department of Medicine, Harbor-UCLA Medical Center, Torrance, CA, USA

    Michael R. Yeaman

  11. Department of Biochemistry, University of Missouri-Columbia, Columbia, MO, USA

    Gavin M. King

  12. Department of Clinical Pharmacy and Translational Science, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN, USA

    Brian M. Peters

  13. Department of Microbiology, Immunology, and Biochemistry, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, USA

    Brian M. Peters

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  1. Jianfeng Lin
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Contributions

J.L. and S.G.F. initiated and designed the project. S.G.F. secured funding for the project. J.L. carried out the CRISPR–Cas9 screen. J.L. constructed the cell knockouts. M.R.Y. and S.G.F. provided candidalysin, and J.L. performed the candidalysin damage assays, immunofluorescence imaging and immunoblots. Q.T.P. and J.L. did the fungal invasion assays. H.L. and J.L. performed the IL-1β, GM-CSF and CXCL8 ELISAs, flow cytometry and calcium flux assays. M.T. and J.L. did the LDH assays. K.G.S. and G.M.K. performed electron microscopy and AFM imaging. F.Z., J.S.D. and R.J.L. performed the SPR assays and acquired funding. C.M.R., R.J.P. and F.N.B. did the dye release and C-laurdan assays. B.M.P., J.M. and N.V.S. carried out the animal infection experiment and related assays. J.L. and S.G.F. wrote the paper with input, revision and review from all co-authors.

Corresponding author

Correspondence to Scott G. Filler.

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Extended data

Extended Data Table 1 Sequences of oligonucleotides used for guide RNAs
Full size table

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, Immunoblots of 3 clones of TR146 cells that stably expressed the PEF-1α-Cas9-Blasticidin construct. b, C. albicans association with and endocytosis by wild-type TR146 cells and the indicated clones of TR146-Cas9 cells. c, Survival of TR146 cells in response to a 6-h exposure to 30 μM candidalysin 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).

Source data

Extended Data Fig. 2 Immunoblots and immunofluorescence images of gene knockouts and knockdowns.

a, Immunoblots for B3gat3, Xylt2, B3galt6, Slc39a9 and Gbf1 in the corresponding CRISPR knockout cells or siRNA knockdown cells. Arrows indicate the target proteins. Numbers indicate molecular mass. 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 immunoblot. 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 panel shows 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. P values were calculated using 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) from 3 independent experiments. Scale bar: 50μm. d, Flow cytometric analysis of heparan sulfate expression on the surface of TR146 cells and the indicated mutants.

Source data

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 carboxylated or non-sulfated GAGs protect epithelial cells from candidalysin-induced damage.

a, Alginate does not protect oral epithelial cells from damage caused by a 6-h exposure to 30 μM candidalysin. b, Protection from damage caused by a 6-h exposure to 30 μM candidalysin provided by dextran and dextran sulfate of the indicated molecular masses. 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, b, and f are mean ± SD of 3 independent experiments, and results in c and d are mean ± SD of 3-4 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).

Source data

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

a, Transmission electron microscopic (TEM, top left) and atomic force microscopic (AFM, bottom left) images of candidalysin on a solid substrate with or without dextran, dextran sulfate and heparan sulfate. Scale bar: 200 nm. The right panel shows the number of loops per μm2 in the AFM images from 3 independent experiments. b, Diagram illustrating the C-laurdan assay. c, Time course of the effects of dextran sulfate (50 μg/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 in POPC vesicles. Representative time course (left). Combined results from 4 experiments (right). The t50 represents the time when 50% membrane leakage occurred. Results are mean ± SD of 3 experiments. e, Comparison of the effects of a 6-h exposure to the indicated concentrations of candidalysin and CFSE-candidalysin on the survival (measured by an XTT assay) of TR146 cells. 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 software CellSens. Results are mean ± SD of 3 independent experiments, each quantifying 4 independent images. g, Damage (measure by an LDH assay) to TR146 and B3GAT3-/- cells caused by the C. albicans ece1Δ/Δ mutant (MOI = 5) after 5 h of infection. 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 ece1Δ/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. 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).

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

a, Immunoblot showing the phosphorylation of the epidermal growth factor receptor (EGFR), extracellular regulated kinase1/2 (ERK1/2) and c-Fos transcription factor in B3GAT3-/- epithelial cells induced by wild-type C. albicans SC5314 (MOI = 5), the ece1Δ/Δ mutant, or candidalysin (10 μM) with or without dextran (DX) and dextran sulfate (DS) (100 µg/ml) at the time points indicated on the left. 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 ece1Δ/Δ mutant (right panel). Results are mean ± SD of 3-4 experiments, each performed in replicates of 4. 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 ece1Δ/Δ mutant (right, MOI = 5) for 6 h. Results are mean ± SD of 3 experiments, each performed in replicates of 4. P values were calculated using the one-way ANOVA with Dunnett’s multiple comparisons test (b-c).

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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 wild-type 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 unpaired, two-sided student’s t-test (a-d).

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Uncropped images for Fig. 5 and Extended Data Fig. 6.

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Supplementary Table 1

Genes identified in the CRISPR screen. P values were calculated using α-RRA and STARS, and adjusted for multiple hypothesis tests using the Benjamini–Hochberg FDR procedure.

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Lin, J., Miao, J., Schaefer, K.G. et al. Sulfated glycosaminoglycans are host epithelial cell targets of the Candida albicans toxin candidalysin. Nat Microbiol 9, 2553–2569 (2024). https://doi.org/10.1038/s41564-024-01794-8

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