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Mucin O-glycans are natural inhibitors of Candida albicans pathogenicity

Abstract

Mucins are large gel-forming polymers inside the mucus barrier that inhibit the yeast-to-hyphal transition of Candida albicans, a key virulence trait of this important human fungal pathogen. However, the molecular motifs in mucins that inhibit filamentation remain unclear despite their potential for therapeutic interventions. Here, we determined that mucins display an abundance of virulence-attenuating molecules in the form of mucin O-glycans. We isolated and cataloged >100 mucin O-glycans from three major mucosal surfaces and established that they suppress filamentation and related phenotypes relevant to infection, including surface adhesion, biofilm formation and cross-kingdom competition between C. albicans and the bacterium Pseudomonas aeruginosa. Using synthetic O-glycans, we identified three structures (core 1, core 1 + fucose and core 2 + galactose) that are sufficient to inhibit filamentation with potency comparable to the complex O-glycan pool. Overall, this work identifies mucin O-glycans as host molecules with untapped therapeutic potential to manage fungal pathogens.

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Fig. 1: Mucins across major mucosal surfaces share a conserved function in attenuating C. albicans virulence in vitro and in vivo.
Fig. 2: Mucin glycans potently inhibit filamentation in a time- and dose-dependent manner.
Fig. 3: Mucin glycans act via Nrg1 to prevent filamentation and hyphal gene expression.
Fig. 4: Mucin glycans downregulate virulence cascades and mediate fungal–bacterial dynamics.
Fig. 5: Native mucins across microbial niches display a plethora of complex glycan structures with regulatory potential.
Fig. 6: Synthetic core 1- and core 2-modified glycans are sufficient to suppress C. albicans filamentation.

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Data availability

High-throughput sequencing data presented here are deposited in the Gene Expression Omnibus (GEO) under accession numbers GSE197249 (Fig. 1) and GSE192826 (Figs. 2 and 3). All raw MS data related to mucin glycan profiles were deposited at GlycoPost under accession number GPST000254 for non-sulfated glycans and accession number GPST000258 for sulfated glycans. Source data are provided with this paper. All other data are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank B. Wang and V.P. Patil for comments on the manuscript. This research was supported by The NIH NIBIB award R01EB017755-04 (OSP 6940725; to K.R.), the MRSEC Program of the National Science Foundation under award DMR-1419807 (to K.R.), the National Science Foundation Career award PHY1454673 (to K.R.), the U.S. Army Research Office under cooperative agreement W911NF-19-2-0026 for the Institute for Collaborative Biotechnologies (to K.R.), the core center grant P30-ES002109 from the National Institute of Environmental Health Sciences (to K.R.), the Toxicology Training Grant support T32-ES007020 (to J.T.), the National Center for Functional Glycomics Grant P41GM103694 (to R.D.C.), the Swiss National Science Foundation grant CRSK-3_196773 (to R.H.) and the NIH NIGMS award R35GM124594 (to C.J.N.) by the Kamangar family in the form of an endowed chair (to C.J.N.). The content is the sole responsibility of the authors and does not represent the views of the funders. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

Author information

Authors and Affiliations

Authors

Contributions

J.T. and K.R. designed the experiments. N.K. performed RNA extraction for the mucin RNA-sequencing and preliminary experiments on the bacterial–fungal coculture. B.S.T. purified glycans. C.J.N., M.G., A.V.A. and T.J.L. designed, performed and analyzed mucin RNA-sequencing experiments. R.H. designed the synthetic approach and synthesized individual glycans. C.Y.K. performed a preliminary glycan analysis. S. Lamont and D.J.W. designed and performed murine experiments. B.B. and M.I. compiled bioinformatic parameters for glycan accession numbers and reporting. J.T. performed the C. albicans experiments. S. Lehoux and R.D.C. performed the preliminary glycan MS experiments. K.A. designed and performed MS of permethylated glycans. J.T., R.H., M.T. and K.R wrote the manuscript.

Corresponding authors

Correspondence to Rachel Hevey or Katharina Ribbeck.

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Competing interests

C.J.N. is a cofounder of BioSynesis, Inc., a company developing diagnostics and therapeutics for biofilm infections. A patent application based on these results has been submitted by Massachusetts Institute of Technology and University of Basel with R.H., J.T. and K.R. as inventors. All other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Mucins are the bioactive components of mucus that suppress adhesion and filamentation in C. albicans independent of growth.

a) Mucus, which is made of gel-forming mucins (with protein names including MUC), covers all non-keratinized epithelial surfaces of the body. This study investigated MUC5B purified from human saliva and MUC2 and MUC5AC purified from porcine intestinal and gastric mucus respectively. b) Native mucus across three major sources decreases adherence to polystyrene wells. Bars indicate mean ± s.e.m. from n = 3 biologically independent replicates using fluorescence images. Significance was assessed using ordinary one-way ANOVA followed by Bonferroni correction for multiple comparisons, ****P < 0.0001. c) C. albicans SC5314 cells were diluted into RPMI medium in the presence or absence of mucus or a mucin-depleted filtrate. C. albicans colony-forming units (CFUs) were determined via serial dilutions on YEPD plates. Data are mean CFU ± s.e.m. from n = 2 biologically independent replicates. d) Fluorescence microscopy images assaying adhesion of wild-type (WT) C. albicans expressing green fluorescent protein (GFP) at 90 min in mucus, mucus-depleted filtrate, or MUC2. Similar results were observed in different fields across three biologically independent replicates. Scale bar, 50 μm. e) The depletion of mucus components leads to increased fungal adherence. Supplementation of mucus filtrates with exogenous purified mucins leads to decreased adhesion. Bars indicate mean ± s.e.m. from n = 3 biologically independent replicates using fluorescence images. Significance was assessed using one-way ANOVA followed by Bonferroni correction for multiple comparisons; ****P < 0.0001.

Source data

Extended Data Fig. 2 Mucin glycan-mediated suppression of virulence pathways is independent of specific experimental conditions, including medium and time point.

a) Quantification of C. albicans morphology in the presence of mucins at 8 h using phase-contrast images from n = 3 (MUC5AC) or n = 4 (Media, MUC5B, MUC2) biologically independent replicates. b) C. albicans SC5314 cells were diluted into the specified medium with or without mucin glycans and cultured at 37 °C. Phase-contrast images of C. albicans were acquired after 8 h in the presence or absence of mucin glycan libraries purified from MUC5AC. Scale bar, 20 μm. c) C. albicans SC5314 cells were diluted into the specified medium with or without mucin glycans and cultured at 37 °C. Phase-contrast images of C. albicans were acquired after 4 h in the presence or absence of mucin glycan libraries purified from MUC5AC. Scale bar, 20 μm. d) Quantification of C. albicans morphology in the presence of mucin glycans in RPMI medium at 8 h using phase-contrast images from n = 6 (CPH1), n = 4 (WT), n = 3 (RAS1) biologically independent replicates. prADH1-CPH1 strain abbreviated as CPH1; RAS1G13V strain abbreviated as RAS1. e) Quantification of C. albicans morphology in the presence of mucin glycans in Spider medium at 8 h using phase-contrast images from n = 3 (WT), n = 4 (EFG1-) or n = 5 (EFG1+) independent biological replicates. PCKpr-efg1-T206E strain abbreviated as EFG1. f) Quantification of C. albicans morphology in the presence of synthesized glycans in RPMI medium at 8 h using phase-contrast images from n = 6 (MG), n = 3 (1, 3, 4, 6) biologically independent replicates. 1, Core 1; 3, Core 1+fucose; 6, Core 2+galactose; 4, Core 1+sialic acid. For b,c, similar results were observed in different fields of view across three biologically independent replicates. For a,d,e,f, the percentage of hyphae was obtained by dividing the number of hyphae by the total number of cells from n > 100 cells. Bars indicate mean ± s.e.m. MG, mucin glycans.

Source data

Extended Data Fig. 3 Filamentation suppression by mucin glycans occurs in a concentration-dependent manner.

C. albicans SC5314 cells were diluted into RPMI medium with or without mucin glycans at the indicated concentrations and cultured at 37 °C for 8 h. Phase-contrast images of C. albicans were acquired after 8 h in the presence or absence of mucin glycan libraries purified from MUC5AC. Similar results were observed in different fields of view across three biologically independent replicates. Scale bar, 20 μm.

Extended Data Fig. 4 Mucin glycans downregulate virulence traits and alter fungal-bacterial dynamics.

a) RNA sequencing data for selected genes belonging to the filamentation pathway that are differentially regulated in the presence of MUC5AC glycans. A complete list of fold-change (FC) values and false discovery rate (FDR)-adjusted P values is provided in Supplementary Table 3. FC data are mean measurements from n = 3 biologically independent replicates. FDR-adjusted P-values were determined using the Benjamini–Hochberg P-value adjustment method. b) qRT-PCR confirms that mucin glycans downregulate the expression of key virulence genes. Exposure to 0.1% MUC5AC glycans decreases the expression of filamentation genes and increases the expression of YWP1 and NRG1 at 8 h. Data are log2-transformed qPCR measurements of relative gene expression normalized to a control gene (ACT1). Data are mean ± s.e.m. from n = 3 biologically independent replicates. c) Phase-contrast images of planktonic cells from biofilms grown in the presence or absence of 0.1% MUC5AC glycans imaged at 24 h. Scale bar, 20 μm. d) Confocal microscopy of C. albicans stained with calcofluor white stain (blue) and P. aeruginosa-mCherry (red) cocultured with or without 0.1% MUC5AC glycans at 24 h. Scale bar, 20 μm. e) Confocal microscopy of Δ/Δnrg1 mutant strain cells stained with calcofluor white stain (blue) and P. aeruginosa-mCherry (red) cocultured with or without 0.1% MUC5AC glycans at 24 h. Scale bar, 20 μm. For c,d,e, similar results were observed in different fields of view across three biologically independent replicates.

Source data

Extended Data Fig. 5 Analysis of background ions detected in full MS indicates that the mucin glycan pools do not contain detectable peptide contaminants.

O-glycan pools released (Methods) from mucin glycoproteins (MUC5B is shown as an example) were permethylated and analyzed by NSI-MS following porous graphitized carbon (PGC) clean-up. a) Full MS profile of a mixture of permethylated MUC5B O-glycans. Magnified view of lower mass range (m/z = 200-430) is highlighted in subsequent panels. b) Ions detected in range m/z = 200-430 following injection of vehicle (pure methanol) to demonstrate solvent background. c) Ions detected in range m/z = 200-430 following injection of permethylated MUC5B O-glycans (magnification of spectrum in panel a). Note largest peaks in this mass range are less than 5% of the signal intensity of the largest peaks in panel a. d) Ions detected in range m/z = 200-430 following injection of permethylated N-linked glycans released from a tryptic digest of HEK cell glycoproteins by PNGaseF and subsequent Sep-Pak C18 clean-up, which is unlikely to contain any peptide contamination. The low-abundance contaminant peaks detected in the HEK N-glycan preparation are at m/z values equivalent to those detected in the mucin O-glycan preparation and are, therefore, not derived from contaminant peptides or glycopeptides produced by hydrazinolysis. Y-axis is shown as absolute intensity.

Supplementary information

Supplementary Information

Supplementary Tables 1-4 and Note 1.

Reporting Summary

Supplementary Data 1

Complete fold change values and FDRs for RNA-sequencing experiments in MUC5AC, MUC5B and MUC2. Fold change values are all relative to C. albicans grown in medium alone. Experiments were performed in RPMI medium alone (n = 3) or supplemented with 0.5% MUC5AC (n = 3), MUC5B (n = 3) or MUC2 (n = 3) at 8 h and with the SC5314 strain grown under shaking conditions. Read counts were fitted to a linear model with an empirical Bayes approach, and differential analysis was determined using a two-sided moderated t-test. P values were adjusted for multiple comparisons using Benjamini–Hochberg correction to obtain FDR-adjusted P values (P < 0.05).

Supplementary Data 2

Complete results of pathway analyses of shared differentially expressed genes across mucin types. Overrepresentation of biological pathways in cells grown in the presence of mucins was assessed using one-sided Fisher’s exact test followed by a Benjamini–Hochberg procedure for multiple corrections to obtain FDR-adjusted P values (P < 0.05) for differentially expressed genes from n = 3 (no mucin treatment), n = 3 (MUC5AC treated), n = 3 (MUC5B treated) and n = 3 (MUC2 treated) biologically independent replicates.

Supplementary Data 3

Complete fold change values and FDRs for RNA-sequencing experiments in MUC5AC glycans at 8 h. Fold change values are relative to C. albicans SC5314 grown in medium alone. Experiments were performed in RPMI medium alone (n = 3) or supplemented with 0.1% MUC5AC glycans (n = 3) at 8 h under shaking conditions. Read counts were fitted to a negative binomial model, and differential analysis was determined using the two-sided Wald test. P values were adjusted for multiple comparisons using Benjamini–Hochberg correction to obtain FDR-adjusted P values (P < 0.05).

Supplementary Data 4

Complete results of functional enrichment analyses identify key virulence pathways among downregulated genes in MUC5AC glycans. Significance of enrichment was calculated using a two-sided Mann–Whitney U-test followed by a Benjamini–Hochberg procedure for multiple corrections to obtain FDR-adjusted P values (P < 0.05) for mean log2-transformed fold change values (Supplementary Table 3) from n = 3 biologically independent replicates.

Supplementary Data 5

Complete fold change values and FDRs for RNA-sequencing experiments in MUC5AC glycans at 2 h in wild type. Fold change values are relative to C. albicans SC5314 grown in medium alone. Experiments were performed in RPMI medium supplemented in the presence or absence of 0.1% MUC5AC (n = 2) at 2 h with the SC5314 strain grown under shaking conditions. Read counts were fitted to a negative binomial model, and differential analysis was determined using the two-sided Wald test. P values were adjusted for multiple comparisons using Benjamini–Hochberg correction to obtain FDR-adjusted P values (P < 0.05).

Supplementary Data 6

Complete fold change values and FDRs for RNA-sequencing experiments in MUC5AC glycans at 2 h in Δ/Δnrg1 mutants. Fold change values are relative to C. albicans grown in medium alone. Experiments were performed in RPMI medium supplemented in the presence or absence of 0.1% MUC5AC (n = 2) at 2 h grown under shaking conditions. Read counts were fitted to a negative binomial model, and differential analysis was determined using the two-sided Wald test. P values were adjusted for multiple comparisons using Benjamini–Hochberg correction to obtain FDR-adjusted P values (P < 0.05).

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Takagi, J., Aoki, K., Turner, B.S. et al. Mucin O-glycans are natural inhibitors of Candida albicans pathogenicity. Nat Chem Biol 18, 762–773 (2022). https://doi.org/10.1038/s41589-022-01035-1

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