Mucin glycans attenuate the virulence of Pseudomonas aeruginosa in infection

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Abstract

A slimy, hydrated mucus gel lines all wet epithelia in the human body, including the eyes, lungs, and gastrointestinal and urogenital tracts. Mucus forms the first line of defence while housing trillions of microorganisms that constitute the microbiota1. Rarely do these microorganisms cause infections in healthy mucus1, suggesting that mechanisms exist in the mucus layer that regulate virulence. Using the bacterium Pseudomonas aeruginosa and a three-dimensional (3D) laboratory model of native mucus, we determined that exposure to mucus triggers downregulation of virulence genes that are involved in quorum sensing, siderophore biosynthesis and toxin secretion, and rapidly disintegrates biofilms—a hallmark of mucosal infections. This phenotypic switch is triggered by mucins, which are polymers that are densely grafted with O-linked glycans that form the 3D scaffold inside mucus. Here, we show that isolated mucins act at various scales, suppressing distinct virulence pathways, promoting a planktonic lifestyle, reducing cytotoxicity to human epithelia in vitro and attenuating infection in a porcine burn model. Other viscous polymer solutions lack the same effect, indicating that the regulatory function of mucin does not result from its polymeric structure alone. We identify that interactions with P. aeruginosa are mediated by mucin-associated glycans (mucin glycans). By isolating glycans from the mucin backbone, we assessed the collective activity of hundreds of complex structures in solution. Similar to their grafted counterparts, free mucin glycans potently regulate bacterial phenotypes even at relatively low concentrations. This regulatory function is likely dependent on glycan complexity, as monosaccharides do not attenuate virulence. Thus, mucin glycans are potent host signals that ‘tame’ microorganisms, rendering them less harmful to the host.

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Fig. 1: Native whole mucus suppresses virulence traits in the opportunistic pathogen P. aeruginosa.
Fig. 2: Mucins are sufficient to attenuate P. aeruginosa virulence in vitro and in vivo.
Fig. 3: The virulence systems suppressed by mucin are downstream of multiple distinct regulatory cascades, and regulation of these systems is independent of bacterial motility and aggregation.
Fig. 4: Complex O-linked glycans are the major regulatory component of MUC5AC.

Data availability

High-throughput sequencing data presented in Figs. 1 and 4 are deposited in the Gene Expression Omnibus (GEO) under accession number GSE136097. All other data that support the findings of this study are available from the corresponding author on reasonable request.

References

  1. 1.

    Schroeder, B. O. Fight them or feed them: how the intestinal mucus layer manages the gut microbiota. Gastroenterol. Rep. 7, 3–12 (2019).

  2. 2.

    Sadikot, R. T., Blackwell, T. S., Christman, J. W. & Prince, A. S. Pathogen-host interactions in Pseudomonas aeruginosa pneumonia. Am. J. Respir. Crit. Care Med. 171, 1209–1223 (2005).

  3. 3.

    Parsek, M. R. & Singh, P. K. Bacterial biofilms: an emerging link to disease pathogenesis. Annu. Rev. Microbiol. 57, 677–701 (2003).

  4. 4.

    Co, J. Y. et al. Mucins trigger dispersal of Pseudomonas aeruginosa biofilms. NPJ Biofilms Microbiomes 4, 23 (2018).

  5. 5.

    Wagner, E., Wheeler, K. M. & Ribbeck, K. Mucins and their role in shaping the functions of mucus barriers. Annu. Rev. Cell Dev. Biol. 34, 189–215 (2018).

  6. 6.

    Toyofuku, M. et al. Quorum sensing regulates denitrification in Pseudomonas aeruginosa PAO1. J. Bacteriol. 189, 4969–4972 (2007).

  7. 7.

    Rohmer, L., Hocquet, D. & Miller, S. I. Are pathogenic bacteria just looking for food? Metabolism and microbial pathogenesis. Trends Microbiol. 19, 341–348 (2011).

  8. 8.

    Jimenez, P. N. et al. The multiple signaling systems regulating virulence in Pseudomonas aeruginosa. Microbiol. Mol. Biol. Rev. 76, 46–65 (2012).

  9. 9.

    Balasubramanian, D., Schneper, L., Kumari, H. & Mathee, K. A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence. Nucleic Acids Res. 41, 1–20 (2013).

  10. 10.

    Van Der Reijden, W. A., Veerman, E. C. I. & Nieuw Amerongen, A. V. Rheological properties of commercially available polysaccharides with potential use in saliva substitutes. Biorheology 31, 631–642 (1994).

  11. 11.

    Jin, C. et al. Structural diversity of human gastric mucin glycans. Mol. Cell. Proteom. 16, 743–758 (2017).

  12. 12.

    Karlsson, N. G., Nordman, H., Karlsson, H., Carlstedt, I. & Hansson, G. C. Glycosylation differences between pig gastric mucin populations: a comparative study of the neutral oligosaccharides using mass spectrometry. Biochem. J. 326, 911–917 (1997).

  13. 13.

    Holmen Larsson, J. M., Thomsson, K. A., Rodriguez-Pineiro, A. M., Karlsson, H. & Hansson, G. C. Studies of mucus in mouse stomach, small intestine, and colon. III. Gastrointestinal Muc5ac and Muc2 mucin O-glycan patterns reveal a regiospecific distribution. Am. J. Physiol. Gastrointest. Liver Physiol. 305, G357–G363 (2013).

  14. 14.

    Cummings, R. D. & Pierce, J. M. The challenge and promise of glycomics. Chem. Biol. 21, 1–15 (2014).

  15. 15.

    Xia, B., Royall, J. A., Damera, G., Sachdev, G. P. & Cummings, R. D. Altered O-glycosylation and sulfation of airway mucins associated with cystic fibrosis. Glycobiology 15, 747–775 (2005).

  16. 16.

    Nakano, M., Saldanha, R., Göbel, A., Kavallaris, M. & Packer, N. H. Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells. Mol. Cell. Proteom. 10, M111.009001 (2011).

  17. 17.

    Lee, S. H. et al. Core2 O-glycan structure is essential for the cell surface expression of sucrase isomaltase and dipeptidyl peptidase-IV during intestinal cell differentiation. J. Biol. Chem. 285, 37683–37692 (2010).

  18. 18.

    Yamada, K. & Kinoshita, M. Comparative studies on the structural features of O-glycans between leukemia and epithelial cell lines. Proteome Res. 8, 521–537 (2009).

  19. 19.

    Varki, A. Biological roles of glycans. Glycobiology 27, 3–49 (2017).

  20. 20.

    Ventre, I. et al. Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc. Natl Acad. Sci. USA 103, 171–176 (2006).

  21. 21.

    Basu Roy, A. & Sauer, K. Diguanylate cyclase NicD-based signalling mechanism of nutrient-induced dispersion by Pseudomonas aeruginosa. Mol. Microbiol. 94, 771–793 (2014).

  22. 22.

    Landry, R. M., An, D., Hupp, J. T., Singh, P. K. & Parsek, M. R. Mucin–Pseudomonas aeruginosa interactions promote biofilm formation and antibiotic resistance. Mol. Microbiol. 59, 142–151 (2006).

  23. 23.

    Secor, P. R., Michaels, L. A., Ratjen, A., Jennings, L. K. & Singh, P. K. Entropically driven aggregation of bacteria by host polymers promotes antibiotic tolerance in Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 115, 10780–10785 (2018).

  24. 24.

    Cattoir, V. et al. Transcriptional response of mucoid Pseudomonas aeruginosa to human respiratory mucus. mBio 3, e00410-12 (2012).

  25. 25.

    Duan, K. & Surette, M. G. Environmental regulation of Pseudomonas aeruginosa PAO1 las and Rhl quorum-sensing systems. J. Bacteriol. 189, 4827–4836 (2007).

  26. 26.

    Arora, S. K., Ritchings, B. W., Almira, E. C., Lory, S. & Ramphal, R. The Pseudomonas aeruginosa flagellar cap protein, FliD, is responsible for mucin adhesion. Infect. Immun. 66, 1000–1007 (1998).

  27. 27.

    Chua, S. L. et al. In vitro and in vivo generation and characterization of Pseudomonas aeruginosa biofilm-dispersed cells via c-di-GMP manipulation. Nat. Protoc. 10, 1165–1180 (2015).

  28. 28.

    Frenkel, E. S. & Ribbeck, K. Salivary mucins protect surfaces from colonization by cariogenic bacteria. Appl. Environ. Microbiol. 81, 332–338 (2015).

  29. 29.

    Kavanaugh, N. L., Zhang, A. Q., Nobile, C. J., Johnson, A. D. & Ribbeck, K. Mucins suppress virulence traits of Candida albicans. mBio 5, e01911-14 (2014).

  30. 30.

    Caldara, M. et al. Mucin biopolymers prevent bacterial aggregation by retaining cells in the free-swimming state. Curr. Biol. 22, 2325–2330 (2012).

  31. 31.

    Lieleg, O., Lieleg, C., Bloom, J., Buck, C. B. & Ribbeck, K. Mucin biopolymers as broad-spectrum antiviral agents. Biomacromolecules 13, 1724–1732 (2012).

  32. 32.

    Huang, Y., Mechref, Y. & Novotny, M. V. Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis. Anal. Chem. 73, 6063–6069 (2001).

  33. 33.

    Packer, N. H., Lawson, M. A., Jardine, D. R. & Redmond, J. W. A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj. J. 15, 737–747 (1998).

  34. 34.

    Chen, F.-T. A. & Evangelista, R. A. Profiling glycoprotein N-linked oligosaccharide by capillary electrophoresis. Electrophoresis 19, 2639–2644 (1998).

  35. 35.

    Guttman, A. Analysis of monosaccharide composition by capillary electrophoresis. J. Chromatogr. A 763, 271–277 (1997).

  36. 36.

    Taniguchi, T. et al. N-Glycosylation affects the stability and barrier function of the MUC16 mucin. J. Biol. Chem. 292, 11079–11090 (2017).

  37. 37.

    Afgan, E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 46, W537–W544 (2018).

  38. 38.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

  39. 39.

    Caballero, A., Thibodeaux, B., Marquart, M., Traidej, M. & O’Callaghan, R. Pseudomonas keratitis: protease IV gene conservation, distribution, and production relative to virulence and other Pseudomonas proteases. Investig. Ophthalmol. Vis. Sci. 45, 522–530 (2004).

  40. 40.

    Howe, T. R. & Iglewski, B. H. Isolation and characterization of alkaline protease-deficient mutants of Pseudomonas aeruginosa in vitro and in a mouse eye model. Infect. Immun. 43, 1058–1063 (1984).

  41. 41.

    Dumas, Z., Ross-Gillespie, A. & Kümmerli, R. Switching between apparently redundant iron-uptake mechanisms benefits bacteria in changeable environments. Proc. R. Soc. B 280, 20131055 (2013).

  42. 42.

    Junker, L. M. & Clardy, J. High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development. Antimicrob. Agents Chemother. 51, 3582–3590 (2007).

  43. 43.

    Roy, S. et al. Mixed-species biofilm compromises wound healing by disrupting epidermal barrier function. J. Pathol. 233, 331–343 (2014).

  44. 44.

    Borlee, B. R. et al. Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix. Mol. Microbiol. 75, 827–842 (2010).

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Acknowledgements

We thank S. Lory for comments and Life Science Editors for editing assistance. This research was supported by NIBIB/NIH grant R01 EB017755-04 (OSP 6940725), the National Science Foundation Career award PHY-1454673, funding from the Deshpande Center for Technological Innovation and the MRSEC Program of the National Science Foundation under award DMR-1419807 (to K.R.), NIH grant P41GM103694 (to R.D.C.) and NIEHS/NIH grant P30-ES002109. D.J.W. is supported by NIH grants R01AI34895 and R01AI097511. This material is based on research supported by the National Science Foundation Graduate Research Fellowship under grant no. 1745302 and the MIT/NIGMS Biotechnology Training Program grant 5T32GM008334-28 (to K.M.W.). G.C.-O. is supported by the Early Postdoc Mobility Fellowship of the Swiss National Science Foundation (grant no. P2ZHP3_164844).

Author information

K.M.W., G.C.-O., B.S.T., S.D.-N., J.Y.C., D.J.W., R.D.C. and K.R. designed the experiments. K.M.W., G.C.-O., B.S.T., S.D.-N., J.Y.C. and S.L. conducted experiments. All of the authors analysed the data and contributed to writing the manuscript.

Correspondence to Katharina Ribbeck.

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Supplementary Information

Supplementary Figs. 1–14, Supplementary Tables 4, 8 and 9, legends for Supplementary Tables 1–9 and Supplementary References.

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Wheeler, K.M., Cárcamo-Oyarce, G., Turner, B.S. et al. Mucin glycans attenuate the virulence of Pseudomonas aeruginosa in infection. Nat Microbiol (2019) doi:10.1038/s41564-019-0581-8

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