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Candidalysin is a fungal peptide toxin critical for mucosal infection

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Abstract

Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name ‘Candidalysin’ for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

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Figure 1: ECE1 is required for epithelial activation and C. albicans infection.
Figure 2: Ece1-III62–93 is the active region of Ece1p and is required for TR146 cell activation and mucosal C. albicans infection.
Figure 3: Ece1-III62–93 functions as a cytolytic peptide toxin.
Figure 4: Ece1-III62–92K functions as a cytolytic peptide toxin that activates and damages epithelial cells.

Change history

  • 01 April 2016

    A label in Fig. 3b of the PDF file initially published online was corrupted and was replaced.

  • 07 April 2016

    A missing citation to the Extended Data Tables was corrected in the HTML on 7 April 2016.

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Acknowledgements

We thank S. Gaffen, B. Klein, C. Hertweck, A. Tucker, J. Green and S. Challacombe for comments on the manuscript. For experimental assistance, we thank S. Bevan and D. Andersson (calcium assays), D. Nayar (histology), D. Rahman and M. Mistry (murine model), M. Nilan (zebrafish model), S. Groth (FRET spectroscopy), N. Gebauer (Impedance experiments), D. Schulz (kex1∆/∆ strain) and our colleagues for supplying fungal mutant strains. This work was supported by grants from the Medical Research Council (MR/J008303/1, MR/M011372/1), Biotechnology & Biological Sciences Research Council (BB/J015261/1), FP7-PEOPLE-2013-Initial Training Network (606786) to J.R.N.; Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology (097377/Z/11/Z) to J.R.N. and D.W.; Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (102549/Z/13/Z) to D.W.; Deutsche Forschungsgemeinschaft CRC/TR124 FungiNet Project C1 and Z2, Deutsche Forschungsgemeinschaft SPP 1580 (Hu 528/17-1) and CSCC, German Federal Ministry of Education and Health (BMBF) 01EO1002 to B.Hu.; Cluster of Excellence ‘Inflammation at interfaces’ and Deutsche Forschungsgemeinschaft SPP1580 project GU 568/5-1 to T.G.; National Institutes of Health (R15AI094406) and the Burroughs Wellcome Fund to R.T.W.

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Authors and Affiliations

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Contributions

D.L.M., J.P.R., S.X.T., M.R., C.M., M.B., S.I.I. and N.K. performed signalling, transcription factor, calcium and cytokine assays, and murine work; D.W., S.H., S.M., T.M.F., B.He., L.K. A.H., O.B. and O.Ku. created fungal strains and performed fluorescent microscopy, adhesion, invasion, gene expression and damage assays; R.L.G. and R.T.W. performed zebrafish experiments; J.W. and T.G. performed biophysical analysis with artificial membranes; J.R. performed whole patch clamp analysis; G.V. performed electron microscopy; S.T. performed histological analysis; S.M., T.L., T.K. and O.Kn. performed LC-MS analyses; J.R.N., B.Hu., D.L.M., J.P.R. and D.W. wrote the paper; J.R.N., B.Hu. and E.C. supervised the project.

Corresponding author

Correspondence to Bernhard Hube.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 C. albicans ECE1 expression and phenotypic effects of ECE1 gene deletion.

a, Relative expression (vs t = 0) of ECE1 in C. albicans wild type over time after addition of yeast cells to TR146 epithelial cells as measured by RT-qPCR. b, Imaging confirmation of ECE1 expression over time within C. albicans wild-type strain. C. albicans cells expressing GFP under the control of the ECE1 5′ intragenic region, containing the ECE1 promoter, were grown on TR146 epithelial cells and stained with calcofluor white (CFW, post-permeabilization) to show cell wall chitin and Alexa-Fluor-647-labelled concanavalin A (ConA, pre-permeabilization) to show carbohydrates. A composite image showing CFW, ConA, GFP and the brightfield (BF) image is shown. c, Scanning electron micrographs (top panels, 5 h) and light microscopy (bottom panels, 24 h) showing no gross abnormalities in hypha formation between C. albicans wild-type (BWP17 + CIp30), ECE1-deletion (ece1Δ/Δ) and ECE1 re-integrant (ece1Δ/Δ + ECE1) strains after infection of TR146 epithelial cells. d, No difference in adhesion of C. albicans wild-type, ece1Δ/Δ and ece1Δ/Δ + ECE1 strains to TR146 epithelial cells after 60 min. e, No difference in invasion of C. albicans wild-type, ece1Δ/Δ and ece1Δ/Δ + ECE1 strains into TR146 epithelial cells after 3 h. f, Fluorescence staining of C. albicans wild-type and ece1Δ/Δ hyphae invading through TR146 epithelial cells. Fungal cells are stained with calcofluor white (CFW, post-permeabilization) and Alexa-Fluor-647-labelled concanavalin A (ConA, pre-permeabilization) to show cell wall chitin and carbohydrates, respectively, and to distinguish between invading hyphae (only stained after permeabilization) and non-invading hyphae (stained both pre- and post-permeabilization). Levels of chitin and β-glucan are comparable in both strains. White arrows indicate invasion into epithelial cells. Data shown are representative (b, c, f) or the mean (a, d, e) of three biological replicates. Error bars show ± s.e.m.

Extended Data Figure 2 C. albicans Ece1p is critical for mucosal virulence in vivo.

a, Fungal burdens recovered from the tongues of mice infected with C. albicans wild-type (BWP17 + CIp30) (number of mice(n) = 13), ECE1-deletion (ece1Δ/Δ) (n = 20) and ECE1 re-integrant (ece1Δ/Δ + ECE1) (n = 24) strains after a 2-day oropharyngeal infection. b, Average percentage of the entire tongue epithelium area infected in different groups of mice infected with the different C. albicans strains. c, Confocal imaging of 4-day post-fertilization (dpf) mpo-gfp transgenic zebrafish swimbladders infected with C. albicans wild-type (BWP17 + CIp30 + dTomato), ECE1-deletion (ece1Δ/Δ + dTomato) and ECE1 re-integrant (ece1Δ/Δ + ECE1 + dTomato) strains for 24 h. C. albicans cells appear red while neutrophils appear green. Red dots outline the swimbladder. Images are composites of maximum projections in the red and green channels (25 slices each, approximately 100 μm depth) with (left) or without (right) a single slice in the DIC channel overlay. Scale bars represent 100 μm. d, Confocal imaging of 4 dpf zebrafish swimbladders infected with C. albicans wild-type (BWP17 + CIp30 + dTomato), ECE1-deletion (ece1Δ/Δ + dTomato) and ECE1 re-integrant (ece1Δ/Δ + ECE1 + dTomato) strains for 24 h stained with the fluorescent exclusion dye Sytox Green. C. albicans cells appear red and damaged epithelial cells appear green. White dots outline the pronephros and red dots outline the swimbladder. Images are composites of maximum projections in the red and green channels (25 slices each, approximately 100 μm depth) with (left) or without (right) a single slice in the DIC channel overlay. High magnification images of the white boxes are shown. Scale bars (bottom right) represent 100 μm (low magnification) and 30 μm (high magnification). Data shown are the mean (a, b) or representative (c, d) of at least three biological replicates. Error bars show ± s.e.m. Data were analysed by Mann–Whitney U-test. ***P < 0.001.

Extended Data Figure 3 Ece1-III62–93 is the active region of Ece1p.

a, Amino acid sequence of Ece1p and a schematic of the protein, indicating the signal peptide (SP), lysine-arginine motifs (KR) at the C terminus of each peptide, and the processed peptides (Ece1-I–VIII) produced by Kex2p cleavage. b, Amino acid sequences of the processed peptides (Ece1-I–VIII) produced by Kex2p cleavage. ce, Induction of GM-CSF (c), IL-1α (d) and IL-6 (e) secreted after stimulation of TR146 epithelial cells for 24 h with varying concentrations of Ece1-III62–93 (1.5–70 μM). f, Phosphorylation of MKP-1 and c-Fos production after 2 h treatment of TR146 epithelial cells with 15 μM of Ece1-III62–85 (hydrophobic region), Ece1-III86–93 (hydrophillic region), Ece1-III62–85 and Ece1-III86–93 together, or Ece1-III62–93 alone. g, Induction of G-CSF secretion after 24 h treatment of TR146 epithelial cells with 15 μM of Ece1-III62–85, Ece1-III86–93, Ece1-III62–85 and Ece1-III86–93 together, or Ece1-III62–93 alone. h, Fold change induction of LDH release after 24 h treatment of TR146 epithelial cells with 70 μM of Ece1-III62-85, Ece1-III86-93, Ece1-III62–85 and Ece1-III86–93 together, or Ece1-III62–93 alone. i, Induction of p-MKP-1 and c-Fos 2 h post-infection (p.i.) with the indicated C. albicans strains (MOI = 10). j, c-Fos DNA binding induction 3 h p.i. with indicated C. albicans strains (MOI = 10). k, G-CSF secretion and l, LDH release 24 h p.i. with indicated C. albicans strains (MOI = 0.01). Data shown are representative (f, i) or the mean (ce, g, h, jl) of three biological replicates. Error bars show ± s.e.m. Data were analysed by one-way ANOVA (ce, g, h, kl) or t-test (j). *P < 0.05, **P< 0.01, ***P < 0.001 (compared with vehicle control). For gel image, see Supplementary Fig. 1.

Extended Data Figure 4 Ece1-III62–93 is required for C. albicans mucosal infection.

a, Fungal burdens recovered from the tongues of mice infected with C. albicans wild-type (BWP17 + CIp30) (number of mice (n) = 13), ECE1-deletion (ece1Δ/Δ) (n = 20), ECE1 re-integrant (ece1Δ/Δ + ECE1) (n = 24) and Ece1-III62–93 deletion (ece1Δ/Δ +ECE1Δ184–279) (n = 10) strains after 2-day oropharyngeal infection. b, Average percentage of the entire tongue epithelium area infected in different groups of mice infected with the different C. albicans strains. c, Confocal imaging of 4 dpf zebrafish swimbladders infected with C. albicans Ece1-III62–93 deletion (ece1Δ/Δ + ECE1Δ184–279 + dTomato) and ECE1 re-integrant (ece1Δ/Δ + ECE1 + dTomato) strains for 24 h stained with the fluorescent exclusion dye Sytox Green. C. albicans cells appear red and damaged cells appear green. White dots outline the pronephros and red dots outline the swimbladder. Images are composites of maximum projections in the red and green channels (25 slices each, approximately 100 μm depth) with (left) or without (right) a single slice in the DIC channel overlay. Scale bars (bottom right) represent 100 μm. Data shown are the mean (a, b) or representative (c), of at least three biological replicates. Error bars show ± s.e.m. Data were analysed by Mann–Whitney U-test. **P < 0.01, ***P < 0.001.

Extended Data Figure 5 Ece1-III62–93 is a cytolytic α-helical peptide.

a, Circular dichroism spectra showing the α-helical conformation of Ece1-III62–93 in buffer (100 mM KCl, 5 mM HEPES, pH 7). Increasing the temperature from 25 °C to 40 °C did not affect the stability of the α-helical structure. b, Diagram to illustrate the amphipathic nature of Ece1-III62–93 (residues 62–78, left panel; residues 79–93, right panel). Residues with hydrophobic or polar/charged side chains are displayed with a blue and white background, respectively. Modified from output generated in PEPWHEEL (http://emboss.bioinformatics.nl/cgi-bin/emboss/pepwheel). c, Förster resonance energy transfer (FRET) experiments show the intercalation of Ece1-III62–93 into lipid liposomes (10 μM) composed of DOPC in the absence or presence of cholesterol. Peptide titration of Ece1-III62–93 to liposomes showed slightly enhanced intercalation for pure DOPC. d, Ece1-III62–93 induced the permeabilization of planar lipid membranes composed of DOPC. The graph shows heterogeneous and transient lesions leading finally to a rupture of the membrane. Ece1-III62–93 concentration was 0.125 μM. e, Induction of p-MKP-1 and c-Fos 2 h in TR146 cells post stimulation (p.s.) with Ece1-III62–93KR or Ece1-III62–93AA. f, Secretion of G-CSF from TR146 cells 24 h p.s. with Ece1-III62–93KR or Ece1-III62–93AA. Data shown are representative (ae) or mean (f) of at least three biological replicates. Error bars show ± s.e.m. Data were analysed by one-way ANOVA (f). *P < 0.05, **P < 0.01, ***P < 0.001 (compared with vehicle control). For gel images, see Supplementary Fig. 1.

Extended Data Figure 6 Schematic of the role of Ece1-III in C. albicans infection of epithelial cells.

ah, During early stage infection of the mucosal surface by C. albicans, Ece1-III (red α-helix) is secreted into the invasion pocket created by the invading hypha (a), Sub-lytic concentrations of Ece1-III trigger epithelial signal transduction through MAPK, p38/MKP-1 and c-Fos (b), resulting in the production of immune regulatory cytokines (c), As the severity of the infection increases, Ece1-III accumulates (d), and once lytic concentrations are reached, causes membrane damage and the release of lactate dehydrogenase from the host epithelium (e), concomitant with calcium influx (f). Epithelial signal transduction is maintained (g), and additionally induces the release of damage associated cytokines, such as IL-1α (h). Ece1-III may also have activity on the epithelial surface outside of the invasion pocket and on neighbouring cells not in contact with hyphae if Ece1-III is produced in sufficient concentrations.

Extended Data Table 1 C. albicans strains used in this study
Extended Data Table 2 C. albicans mutant strains constructed and used in this study
Extended Data Table 3 LC-MS/MS analysis of C. albicans Ece1-III
Extended Data Table 4 Oligonucleotide primers used in this study

Supplementary information

Supplementary Figure 1

This file contains uncropped scans of the source gels with size markers indicated. (PDF 199 kb)

Supplementary Table

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Moyes, D., Wilson, D., Richardson, J. et al. Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature 532, 64–68 (2016). https://doi.org/10.1038/nature17625

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