Abstract
The opportunistic fungal pathogen Candida albicans damages host cells via its peptide toxin, candidalysin. Before secretion, candidalysin is embedded in a precursor protein, Ece1, which consists of a signal peptide, the precursor of candidalysin and seven non-candidalysin Ece1 peptides (NCEPs), and is found to be conserved in clinical isolates. Here we show that the Ece1 polyprotein does not resemble the usual precursor structure of peptide toxins. C. albicans cells are not susceptible to their own toxin, and single NCEPs adjacent to candidalysin are sufficient to prevent host cell toxicity. Using a series of Ece1 mutants, mass spectrometry and anti-candidalysin nanobodies, we show that NCEPs play a role in intracellular Ece1 folding and candidalysin secretion. Removal of single NCEPs or modifications of peptide sequences cause an unfolded protein response (UPR), which in turn inhibits hypha formation and pathogenicity in vitro. Our data indicate that the Ece1 precursor is not required to block premature pore-forming toxicity, but rather to prevent intracellular auto-aggregation of candidalysin sequences.
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Data availability
The data supporting the findings of this study are available within the paper, its supplementary files and source data files. Correspondence and requests for materials should be addressed to the corresponding authors. Source data are provided with this paper.
References
Bischofberger, M., Iacovache, I. & van der Goot, F. G. Pathogenic pore-forming proteins: function and host response. Cell Host Microbe 12, 266–275 (2012).
Krawczyk, P. A., Laub, M. & Kozik, P. To kill but not be killed: controlling the activity of mammalian pore-forming proteins. Frontiers in Immunology 11, 601405 (2020).
Henry, B. D. et al. Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice. Nat. Biotechnol. 33, 81–88 (2015).
Yakimova, E. T., Yordanova, Z. P., Slavov, S., Kapchina-Toteva, V. M. & Woltering, E. J. Alternaria alternata AT toxin induces programmed cell death in tobacco. J. Phytopathol. 157, 592–601 (2009).
Curtis, M. J. & Wolpert, T. J. The victorin-induced mitochondrial permeability transition precedes cell shrinkage and biochemical markers of cell death, and shrinkage occurs without loss of membrane integrity. Plant J. 38, 244–259 (2004).
Fernández-García, L. et al. Toxin-antitoxin systems in clinical pathogens. Toxins 8, 227 (2016).
Wang, X., Yao, J., Sun, Y.-C. & Wood, T. K. Type VII toxin/antitoxin classification system for antitoxins that enzymatically neutralize toxins. Trends Microbiol. 29, 388–393 (2021).
Peraro, M. D. & van der Goot, F. G. Pore-forming toxins: ancient, but never really out of fashion. Nat. Rev. Microbiol. 14, 77–92 (2016).
Gonzalez, M. R., Bischofberger, M., Pernot, L., van der Goot, F. G. & Freche, B. Bacterial pore-forming toxins: the (w)hole story? Cell. Mol. Life Sci. 65, 493–507 (2008).
Los, F. C., Randis, T. M., Aroian, R. V. & Ratner, A. J. Role of pore-forming toxins in bacterial infectious diseases. Microbiol. Mol. Biol. Rev. 77, 173–207 (2013).
Peschel, A. & Otto, M. Phenol-soluble modulins and staphylococcal infection. Nat. Rev. Microbiol. 11, 667–673 (2013).
Cheung, G. Y., Joo, H. S., Chatterjee, S. S. & Otto, M. Phenol-soluble modulins–critical determinants of staphylococcal virulence. FEMS Microbiol. Rev. 38, 698–719 (2014).
Moyes, D. L. et al. Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature 532, 64–68 (2016).
Mayer, F. L., Wilson, D. & Hube, B. Candida albicans pathogenicity mechanisms. Virulence 4, 119–128 (2013).
Richardson, J. P. et al. Processing of Candida albicans Ece1p is critical for candidalysin maturation and fungal virulence. mBio 9, e02178-17 (2018).
Mogavero, S. et al. Candidalysin delivery to the invasion pocket is critical for host epithelial damage induced by Candida albicans. Cell. Microbiol. 23, e13378 (2021).
Westman, J. et al. Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion. Cell Rep. 38, 110187 (2022).
Ho, J. et al. Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor. Nat. Commun. 10, 2297 (2019).
Verma, A. H. et al. Oral epithelial cells orchestrate innate type 17 responses to Candida albicans through the virulence factor candidalysin. Sci. Immunol. 2, eaam8834 (2017).
Drummond, R. A. et al. CARD9(+) microglia promote antifungal immunity via IL-1beta- and CXCL1-mediated neutrophil recruitment. Nat. Immunol. 20, 559–570 (2019).
Swidergall, M. et al. Candidalysin is required for neutrophil recruitment and virulence during systemic Candida albicans infection. J. Infect. Dis. 220, 1477–1488 (2019).
Chu, H. et al. The Candida albicans exotoxin candidalysin promotes alcohol-associated liver disease. J. Hepatol. 72, 391–400 (2020).
Pekmezovic, M. et al. Candida pathogens induce protective mitochondria-associated type I interferon signalling and a damage-driven response in vaginal epithelial cells. Nat. Microbiol. 6, 643–657 (2021).
Wu, Y. et al. Candida albicans elicits protective allergic responses via platelet mediated T helper 2 and T helper 17 cell polarization. Immunity 54, 2595–2610.e7 (2021).
Li, X. V. et al. Immune regulation by fungal strain diversity in inflammatory bowel disease. Nature 603, 672–678 (2022).
Liao, C. et al. Bacillus thuringiensis Cry1Ac protoxin and activated toxin exert differential toxicity due to a synergistic interplay of cadherin with ABCC transporters in the cotton bollworm. Appl. Environ. Microbiol. 88, e0250521 (2022).
Schmitt, M. J. & Breinig, F. Yeast viral killer toxins: lethality and self-protection. Nat. Rev. Microbiol. 4, 212–221 (2006).
Howard, S. P. & Buckley, J. T. Activation of the hole-forming toxin aerolysin by extracellular processing. J. Bacteriol. 163, 336–340 (1985).
Coburn, P. S. & Gilmore, M. S. The Enterococcus faecalis cytolysin: a novel toxin active against eukaryotic and prokaryotic cells. Cell. Microbiol. 5, 661–669 (2003).
Memariani, H. & Memariani, M. Anti-fungal properties and mechanisms of melittin. Appl. Microbiol. Biotechnol. 104, 6513–6526 (2020).
Keller, M. D. et al. Decoy exosomes provide protection against bacterial toxins. Nature 579, 260–264 (2020).
Almagro Armenteros, J. J. et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 37, 420–423 (2019).
Sircaik, S. et al. The protein kinase Ire1 impacts pathogenicity of Candida albicans by regulating homeostatic adaptation to endoplasmic reticulum stress. Cell. Microbiol. 23, e13307 (2021).
Guillemette, T. et al. Methods for investigating the UPR in filamentous fungi. Methods Enzymol. 490, 1–29 (2011).
Wimalasena, T. T. et al. Impact of the unfolded protein response upon genome-wide expression patterns, and the role of Hac1 in the polarized growth, of Candida albicans. Fungal Genet. Biol. 45, 1235–1247 (2008).
Chaffin, W. L. Effect of tunicamycin on germ tube and yeast bud formation in Candida albicans. J. Gen. Microbiol. 131, 1853–1861 (1985).
Sidrauski, C. & Walter, P. The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell 90, 1031–1039 (1997).
Travers, K. J. et al. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101, 249–258 (2000).
Pincus, D. et al. BiP binding to the ER-stress sensor Ire1 tunes the homeostatic behavior of the unfolded protein response. PLoS Biol. 8, e1000415 (2010).
Jenssen, H., Hamill, P. & Hancock, R. E. Peptide antimicrobial agents. Clin. Microbiol. Rev. 19, 491–511 (2006).
Outram, M. A., Solomon, P. S. & Williams, S. J. Pro-domain processing of fungal effector proteins from plant pathogens. PLoS Pathog. 17, e1010000 (2021).
Ceremuga, M. et al. Melittin—a natural peptide from bee venom which induces apoptosis in human leukaemia cells. Biomolecules 10, 247 (2020).
Senior, A. W. et al. Improved protein structure prediction using potentials from deep learning. Nature 577, 706–710 (2020).
Bader, O., Krauke, Y. & Hube, B. Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans, C. glabrata, Saccharomyces cerevisiae and Pichia pastoris. BMC Microbiol. 8, 116 (2008).
Liu, J. et al. A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis. PLoS Pathog. 17, e1009884 (2021).
Ropars, J. et al. Gene flow contributes to diversification of the major fungal pathogen Candida albicans. Nat. Commun. 9, 2253 (2018).
Stephens, M., Smith, N. J. & Donnelly, P. A new statistical method for haplotype reconstruction from population data. Am. J. Hum. Genet. 68, 978–989 (2001).
Stephens, M. & Scheet, P. Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. Am. J. Hum. Genet. 76, 449–462 (2005).
Rupniak, H. T. et al. Characteristics of four new human cell lines derived from squamous cell carcinomas of the head and neck. J. Natl Cancer Inst. 75, 621–635 (1985).
Toth, R. et al. Different Candida parapsilosis clinical isolates and lipase deficient strain trigger an altered cellular immune response. Front. Microbiol. 6, 1102 (2015).
Lockhart, S. R. et al. Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. Clin. Infect. Dis. 64, 134–140 (2017).
Zarnowski, R., Sanchez, H. & Andes, D. R. Large-scale production and isolation of Candida biofilm extracellular matrix. Nat. Protoc. 11, 2320–2327 (2016).
Zarnowski, R. et al. Coordination of fungal biofilm development by extracellular vesicle cargo. Nat. Commun. 12, 6235 (2021).
Zarnowski, R. et al. A common vesicle proteome drives fungal biofilm development. Proc. Natl Acad. Sci. USA 119, e2211424119 (2022).
Richardson, J. P. et al. Candidalysins are a new family of cytolytic fungal peptide toxins. mBio 13, e0351021 (2022).
Li, C. et al. FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnol. 11, 92 (2011).
Walther, A. & Wendland, J. An improved transformation protocol for the human fungal pathogen Candida albicans. Curr. Genet. 42, 339–343 (2003).
Wächtler, B., Wilson, D., Haedicke, K., Dalle, F. & Hube, B. From attachment to damage: defined genes of Candida albicans mediate adhesion, invasion and damage during interaction with oral epithelial cells. PLoS ONE 6, e17046 (2011).
von der Haar, T. Optimized protein extraction for quantitative proteomics of yeasts. PLoS ONE 2, e1078 (2007).
Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50, D439–D444 (2021).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Acknowledgements
We thank D. Schulz, J. Mantke, S. Wisgott, N. Jablonowski, D. Poulakis and N. Schuck for excellent technical support throughout the project; L. Dally, T. Narashvili, A. Gille and H. May for help and support with mutant generation and characterization; and S. Brunke and G. Lackner for profound discussions. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project Hu 528/20-1 to R.M., S.A. and B.H.; the DFG Priority Program 2225 Exit Strategies of extracellular pathogens to L.K., B.H. and T.G. (project numbers 446404928 and 446371725); the Cluster of Excellence 2051 ‘Balance of the Microverse’, DFG project number 390713860 to A.A.B. and B.H.; the DFG Collaborative Research Center (CRC)/Transregio 124 ‘FungiNet’, project number 210879364 (project A1, Z2) to A.A.B., C.V., O.K. and T.K.; the Leibniz Association Campus InfectoOptics SAS-2015-HKI-LWC to B.H. and A.K.; the Wellcome Trust (215599_Z_19_Z) to S.M. and B.H.; the German Federal Ministry of Education and Research (BMBF) within the funding programme Photonics Research Germany, Leibniz Center for Photonics in Infection Research (LPI), subproject LPI-BT2, contract number 13N15705 to B.H. and V.T.; J.R.N. was supported by grants from the Wellcome Trust (214229/Z/18/Z) and the National Institutes of Health (DE022550). The National Institutes of Health (R01AI073289) supported D.R.A. and R.Z. The Agence Nationale de Recherche (ANR-10-LABX-62-IBEID) and the Swiss National Science Foundation (Sinergia CRSII5_173863 /1) supported work in Cd’E’s laboratory. The Federal Ministry for Education and Research (BMBF: https://www.bmbf.de/), Germany, Project FKZ 01K12012 ‘RFIN – RNA-Biologie von Pilzinfektionen’ supported M.G.B. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
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R.M., A.K. and S.A. designed and performed most of the experimental work, analysed most of the data, wrote and edited the manuscript. M.H., T.H., S.M. and D.Y. performed further laboratory work. T.K. performed LC–MS analyses. C.M., M.-E.B. and C.d’E. performed sequence alignments and corresponding data analyses, and designed the corresponding figures. S.G. and T.H. performed peptide aggregation and membrane permeabilization experiments. C.N. and T.G. analysed the peptide aggregation and membrane permeabilization data and wrote the corresponding parts of the manuscript. E.L.P., S.L. and J.P.R. performed Orbit e16 experiments and analysed the corresponding data. V.T. contributed to the experimental design and data analysis of the western blots. R.Z. produced fEVs. C.V. and M.G.B. were involved in the design and execution to the EV protection assays and hEV production. S.M. and L.K. were involved in the experimental design, wrote and edited parts of the manuscript. L.K., J.P.R., M.G.B., C.N., T.G., O.K., T.K., A.A.B. and D.R.A. contributed to editing the manuscript. J.R.N. acquired funding and was involved in the experimental design of the Orbit e16 experiments, writing and editing of the manuscript. B.H. conceived and designed the study, acquired funding, was involved in experimental design, and wrote and edited the manuscript.
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Extended data
Extended Data Fig. 1 Synthetic P2-CaL and P3-P4 fusion peptides are non-toxic.
a, Damage of oral epithelial cells upon treatment with 40 µM synthetic CaL, synthetic fusion peptides P2-CaL and P3-P4, or a combination of CaL and the respective single peptide for 24 h. Epithelial damage was assessed by quantification of extracellular LDH. Dotted line represents CaL-induced host cell damage (100 %). Statistical analysis was performed with one-way ANOVA and Dunnett’s multiple comparisons test based on the absorbance data, relative to the CaL-treated sample, ***p = 0.001, ****p < 0.0001, n = 4 (biological replicates), more statistical parameters are listed in the Source Data Extended Data Fig. 1, mean ± s.d. b, Scheme of the fusion peptides with respective peptide sequence. The fusion peptide P2-CaL ends with K instead of KR, mimicking Kex1 processing of the mature peptide. In fusion peptide P3-P4, the P3 sequence ends with KR to keep the internal Kex2 processing site, whereas P4 ends with K to mimic the Kex1-processed mature peptide.
Extended Data Fig. 2 Susceptibility of different Candida species to candidalysin.
a-e, Growth of a, Candida glabrata (ATCC2001) b, Candida tropicalis (DSM4959) c, Candida parapsilosis (GA1)50 d, Candida auris (B8441)51 e, Candida haemulonii (CBS 5149 T) with or without addition of 70 µM synthetic candidalysin (CaL) or synthetic melittin (Mel). Yeast growth was monitored for 24 h by measuring the absorbance (600 nm) every 30 min. a-e n = 3 (biological replicates), mean ± s.d.
Extended Data Fig. 3 Schematic of how Ece1 modification (NCEP modifications or deletions) promotes ER stress and an unfolded protein response.
Left: In Wt C. albicans cells, transcription of native ECE1 is followed by translation and folding of Ece1 in the ER, transport to the Golgi apparatus and sequential processing by Kex2 and Kex1 proteases, enabling candidalysin and NCEP secretion. Right: In NCEP mutants, the modified Ece1 structure promotes misfolding which triggers ER stress. Ece1 modifications hinder correct Kex2/1 processing leading to abolished or impaired secretion of candidalysin. The figures were drawn using pictures from Servier Medical Art by Servier, licensed under a Creative Commons Attribution 3.0 Unported License.
Extended Data Fig. 4 The ER stressor tunicamycin inhibits hyphal growth.
a+b, Growth of a C. albicans Wt strain at 30 °C (a) and 37 °C (b) in YPD supplemented with tunicamycin (2 µg/mL (Wt + T)). Growth was monitored for 24 h by measuring the absorbance (600 nm) every 30 min. Data were normalized following subtraction of the first measurement (baseline), biological replicates n = 3, mean ± s.d.c+d, Hyphal length of Wt C. albicans with tunicamycin treatment (2 µg/mL) after 6 h (c, mean Wt hyphal length: 125.6 µm ± 9.4 µm) or microcolony diameter of the Wt with tunicamycin treatment (2 µg/mL) after 24 h (d, mean Wt microcolony diameter: 404.4 µm ± 21.10 µm). Hyphal length and microcolony diameter were determined microscopically by measuring at least 50 hyphae or 20 microcolonies per replicate. Statistical analysis was performed with a paired t-test, two-tailed, on the raw µm values, c, **p = 0.0015 and d, ***p = 0.0009 n = 3, mean ± s.d.
Extended Data Fig. 5 Altered hyphal formation, UPR dynamics, and UPR quantification in response to Ece1 modifications.
Ece1 sequence variations lead to reduced hypha formation but do not affect hypha formation per se, and ER stress induced by Ece1 modification is ameliorated over time. a, Filamentation of Wt (n = 18), ece1Δ/Δ (n = 9), ece1Δ/Δ+ECE1 (n = 9) complemented strain, a mutant strain lacking the candidalysin-encoding sequence only (ΔP3), NCEP deletion mutants, and scrambled peptide mutants after 6 h (hyphal length, black, n = 3) or 24 h (microcolony diameter, grey, n = 3). Black dotted line represents Wt-level after 6 h of hypha induction, grey dotted line represents Wt-level after 24 h of hypha induction, n represents biological replicates. Hyphal length and late stage filamentation were determined microscopically measuring at least 50 hyphae or 20 microcolonies per replicate (scr-all 24 h ≥ 12). Analysis was performed with two-way ANOVA and a Šídák’s multiple comparisons test of every 6 h value relative to the respective 24 h value. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, ns = non-significant, more statistical parameters are listed in the Source Data Extended Data Fig. 5, mean ± s.d. Values for the 6 h time point are also presented in Fig. 4b as % of the Wt and shown here as absolute values for comparison against the size of late stage filamentation size. b+c, Analysis and quantification of HAC1 mRNA splicing as a read-out for ER stress and UPR induction. Activation of a UPR was determined by visualization of HAC1 mRNA splicing by RT-qPCR after culturing the indicated C. albicans strains for b, 20 min and 24 h, and c, 1 h and 2 h under yeast- and/or hypha-inducing conditions. A band at 97 bp shows HAC1 mRNA splicing, indicating ER stress and UPR activation Y = yeast growth conditions (30 °C, 180 rpm shaking); H = hyphal growth conditions (37 °C, 5 % CO2, plastic surface); positive control: Wt treated with tunicamycin (2 µg/mL (Wt + T)). One representative replicate from 3 biological repeats per time point is shown. Quantification of HAC1 mRNA splicing is presented as the percentage of spliced HAC1 mRNA from total HAC1 mRNA level quantified with Bio1D from Vilber Lourmat. b, n = 3 and c, n = 4. A graphical representation of quantified HAC1 mRNA splicing for all replicates including standard deviation and statistics is presented in Supplementary Fig. 2. In c, 2 h Wt+T and Wt bands derived from the same gel were cropped and swapped in position to maintain a consistent sample order. Cropped regions are indicated in the Extended_Data_Figure_5_1h_2h_all_replicates_source_data_gels file, with a frame.
Extended Data Fig. 6 Candidalysin secretion is impaired in NCEP mutants.
a, Representative pictures of candidalysin (CaL) staining for each tested strain. CaL was stained by anti-CaL nanobody VHH CAL-F1 (green)3 and extracellular fungal regions by Concanavalin A-AlexaFluor 647 (blue) after infecting TR146 oral epithelial cells for 4 h. Green arrows indicate CaL staining, white scale bars represent 10 µm. Quantification of the mean fluorescence intensity of each strain is presented in Supplementary Fig. 3. ece1Δ/Δ, ΔP3, scrP3, and scr-all lack a native candidalysin epitope. b, The numbers of CaL-positive (green) or CaL-negative (grey) hyphae were determined and the percentage of CaL-positive or CaL-negative hypha was calculated. At least 50 hyphae from two independent replicates were analyzed per mutant (except the scr-all mutant (hyphae ≥ 36) which was predominantly yeast cells, and the scr7 mutant (hyphae ≥ 47)), n = 2 biological replicates, bar presents mean. c, Invasion of NCEP mutants into oral epithelial cells presented as invasion rate (%). Invasion was determined microscopically after differential staining of intra-and extracellular fungal parts for at least 50 hyphae per strain and replicate (n = 3). Statistical analysis was performed with one-way ANOVA and a Dunnett’s multiple comparisons test relative to the Wt sample, mean ± s.d. *p ≤ 0.05, **p ≤ 0.01, ****p < 0.0001, more statistical parameters are listed in the Source Data Extended Data Fig. 6.
Extended Data Fig. 7 Synthetic candidalysin aggregates in aqueous solution.
Candidalysin aggregation was determined in aqueous solution as the count rate of aggregates in a concentration range between 0.05 µM and 100 µM by DLS. Statistical analysis was performed with an unpaired, two-tailed t-test relative to the lowest CaL concentration (0.05 µM) with a statistically significant change starting at 1 µM. n = 3 experiments, ** p = 0.0072, mean ± s.d., more statistical parameters are listed in the Source Data Extended Data Fig. 7.
Extended Data Fig. 8 Candidalysin is the driver of ER stress and UPR in NCEP mutants.
a, Schematic view of NCEP mutants produced for this experiment: 1. Deletion of P3 in combination with P5; 2. Deletion of P3 in combination with P7; 3. Three copies of P3 replacing P5 and P7; b, Damage of TR146 oral epithelial cells 24 h post infection with Wt (n = 6), ece1Δ/Δ (n = 6), ece1Δ/Δ+ECE1 complemented strain, a mutant strain lacking the candidalysin-encoding sequence only (ΔP3), mutants lacking the CaL-encoding sequence in combination with P5 or P7 (ΔP3 + 5, ΔP3 + 7), and a mutant harbouring three copies of CaL (TripleP3) (n = 3). Data are presented as % damage relative to the Wt. Host cell damage was assessed by quantification of extracellular LDH. Statistical analysis was performed with a Mixed-effects analysis and a Dunnett’s multiple comparisons test, two tailed, relative to the Wt sample on raw absorbance values, ***p ≤ 0.001, ****p ≤ 0.0001, n represent biological replicates, uninfected n = 6. c, Hyphal length of the indicated C. albicans strains after 6 h of hypha induction. Data are presented as % Wt hyphal length (mean Wt = 104.5 µm ± 23.9 µm). Hyphal length was determined microscopically measuring at least 50 hyphae per replicate. Statistical analysis was performed with one-way ANOVA and a Dunnett’s multiple comparisons test relative to the hyphal length of the Wt sample (µm). ****p ≤ 0.0001, Wt, ece1Δ/Δ and ece1Δ/Δ+ECE1 n = 8, ΔP3, ΔP3 + 7 and TripleP3 n = 3, ΔP3 + 5 n = 5, n represents biological replicates. d, Filamentation of indicated C. albicans strains after 6 h (hyphal length, black) or 24 h (microcolony diameter, grey). Black dotted line represents Wt-level after 6 h (104 µm ± 23.9 µm) of hypha induction, grey dotted line represents Wt-level after 24 h (340.6 µm ± 80.7 µm) of hypha induction. Hyphal length and late stage filamentation were determined microscopically measuring at least 50 hyphae or 20 microcolonies per strain and replicate. Analysis was performed with two-way ANOVA and a Šídák’s multiple comparisons test of every 6 h value relative to the respective 24 h value. ****p ≤ 0.0001, ns = non-significant, Wt, ece1Δ/Δ and ece1Δ/Δ+ECE1 n = 8, ΔP3, ΔP3 + 7 and TripleP3 n = 3, ΔP3 + 5 n = 5, n represents biological replicates. Some of the values for the 6 h time point are also presented in Fig. 4b as % of the Wt and are shown here as absolute values for comparison against the size of late stage filamentation. e, Dynamics of hypha formation of indicated C. albicans strains was measured from 1.5 h to 4 h every 30 min on plastic at 37 °C 5 % CO2 (n = 3 biological replicates, ≥ 20 hyphae/replicate were tracked over time, except Triple P3: n = 2 for 1.5 h and 2 h, therefore no s.d.). f, Analysis of HAC1 mRNA splicing as a read-out for ER stress and UPR induction. Activation of the UPR was determined by visualization of HAC1 mRNA splicing by RT-qPCR after culturing the respective C. albicans strains for 3 h under yeast- or hypha-inducing conditions. A band at 97 bp shows HAC1 splicing, indicating ER stress and UPR activation. Quantification of HAC1 mRNA splicing is given as percentage (mean out of 3 biological replicates, quantified with Bio1D from Vilber Lourmat, mean values, standard deviations and statistics are provided in Supplementary Fig. 2). Y = yeast growth conditions (30 °C, 180 rpm shaking); H = hyphal growth conditions (37 °C, 5 % CO2, plastic surface); positive control: Wt treated with tunicamycin (2 µg/mL (Wt + T)). One representative replicate from 3 biological repeats is shown. g, Western blot analysis of hyphal lysates (3 h hyphal induction, plastic, 37 °C and 5 % CO2) indicates the accumulation of TripleP3 Ece1 polyprotein. Quantification was performed using Image Studio Lite Version 5.2. and normalization was performed based on total protein staining (n = 3 biological replicates). Statistical analysis was performed with one-way ANOVA and a Dunnett’s multiple comparisons test relative to the Wt sample. h, Representative pictures of candidalysin staining are shown for each mutant strain and in comparison, for the Wt and ece1Δ/Δ+ECE1 complemented strain. Candidalysin was stained by anti-candidalysin nanobody VHH CAL-F1 (green)16 and extracellular fungal regions by Concanavalin A-AlexaFluor 647 (blue) after infecting TR146 oral epithelial cells for 4 h. White scale bars represent 10 µm. As expected, CaL antigens were not detected in mutants lacking native CaL sequences (ΔP3 + 5, ΔP3 + 7). Quantification of the mean fluorescence intensity of each strain is presented in Supplementary Fig. 3. i, The number of CaL-positive (green) or CaL-negative (grey) hyphae was determined and the percentage of CaL-positive or CaL-negative hypha was calculated. At least 50 hyphae were analyzed per mutant (except TripleP3: n ≥ 40, predominantly yeast cells) and replicate after fluorescent staining, n = 2 biological replicates. k, Invasion of NCEP mutants into oral epithelial cells given as invasion rate in percentage. Invasion was determined microscopically after differential staining of intra-and extracellular fungal parts for at least 50 hyphae per strain and replicate. Statistical analysis was performed with one-way ANOVA and a Dunnett’s multiple comparisons test relative to the Wt sample (n = 3, ***p ≤ 0.001). b-k, Data for Wt, ece1Δ/Δ, ece1Δ/Δ+ECE1 complemented strain, and the mutant strain lacking the candidalysin-encoding sequence only (ΔP3) have already been displayed in Fig. 3, Fig. 4b+c, Extended Data Fig. 5a, and Extended Data Fig. 6 and are shown here for comparison. All panels, except i, mean ± s.d., and more statistical parameters can be found in the Source Data Extended Data Fig. 8.
Supplementary information
Supplementary Information
Supplementary Figs. 1–3.
Supplementary Tables
Supplementary Tables 1–4.
Supplementary Data 1
ECE1 haplotype mapping for 296 C. albicans isolates.
Supplementary Data 2
LC–MS/MS results
Supplementary Data 3
Alphafold prediction of modified Ece1 sequences.
Supplementary Data 4
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Supplementary Data 5
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Gels 20 min, 24 h timepoints Extended Data Fig. 5b.
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Gels 1 h, 2 h timepoints Extended Data Fig. 5c.
Source Data Extended Data Fig. 6
Statistical source data.
Source Data Extended Data Fig. 7
Statistical source data.
Source Data Extended Data Fig. 8
Statistical source data.
Source Data Extended Data Fig. 8
Gels 3h timepoint Fig. 8f.
Source Data Extended Data Fig. 8
Western blot Fig. 8g.
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Müller, R., König, A., Groth, S. et al. Secretion of the fungal toxin candidalysin is dependent on conserved precursor peptide sequences. Nat Microbiol 9, 669–683 (2024). https://doi.org/10.1038/s41564-024-01606-z
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DOI: https://doi.org/10.1038/s41564-024-01606-z