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Mucin O-glycans suppress quorum-sensing pathways and genetic transformation in Streptococcus mutans

Nature Microbiology volume 6, pages 574–583 (2021)Cite this article

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Abstract

Mucus barriers accommodate trillions of microorganisms throughout the human body while preventing pathogenic colonization1. In the oral cavity, saliva containing the mucins MUC5B and MUC7 forms a pellicle that coats the soft tissue and teeth to prevent infection by oral pathogens, such as Streptococcus mutans2. Salivary mucin can interact directly with microorganisms through selective agglutinin activity and bacterial binding2,3,4, but the extent and basis of the protective functions of saliva are not well understood. Here, using an ex vivo saliva model, we identify that MUC5B is an inhibitor of microbial virulence. Specifically, we find that natively purified MUC5B downregulates the expression of quorum-sensing pathways activated by the competence stimulating peptide and the sigX-inducing peptide5. Furthermore, MUC5B prevents the acquisition of antimicrobial resistance through natural genetic transformation, a process that is activated through quorum sensing. Our data reveal that the effect of MUC5B is mediated by its associated O-linked glycans, which are potent suppressors of quorum sensing and genetic transformation, even when removed from the mucin backbone. Together, these results present mucin O-glycans as a host strategy for domesticating potentially pathogenic microorganisms without killing them.

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Fig. 1: Salivary mucin MUC5B reduces the expression of genes in quorum-sensing-associated pathways.
Fig. 2: Mucin O-glycans suppress the expression of quorum-sensing genes that regulate competence, virulence and bacteriocin production.
Fig. 3: Mucin glycans reduce genetic transformation by suppressing competence in a manner that is dependent on sugar identity and independent of signal peptide transport.
Fig. 4: Mucin and mucin glycans reduce natural genetic transformation of S. mutans in ex vivo human saliva.

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Data availability

The high-throughput sequencing data presented in Figs. 1 and 2 are deposited at the Gene Expression Omnibus under accession number GSE163258. All other data are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

Code used for transcriptional analysis is available at GitHub (https://github.com/cwerlang/Smutans-MUC5B-RNASeq).

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Acknowledgements

We thank S. Underhill, S. Hagen, S.-J. Ahn and R. Burne for sharing several genetically modified S. mutans UA159 strains; B. Spellerberg for sharing the PBSU101 plasmid; the staff at the Koch Institute Swanson Biotechnology Center for technical support, specifically A. Leshinsky and H. Amoroso in the Biopolymers and Proteomics core; the staff at the MIT BioMicro Center for technical support, especially S. Levine, N. Kamelamela and A. Stortchevoi; and E. S. Frenkel, B. Turner, B. Wang, C. Wu and the entire Ribbeck group at MIT for discussions. We acknowledge funding support from the MIT Deshpande Center, NIBIB/NIH (no. R01-EB017755), NSF CAREER (no. 1454673), NIH Common Fund (no. U01GM125267), the Amgen Scholars Program, the National Institute of Environmental Health Sciences of the NIH (no. P30-ES002109), the NSF MRSEC Program (no. DMR-14-19807), the US Army Research Office under cooperative agreement W911NF-19-2-0026 for the Institute for Collaborative Biotechnologies, the NSF Graduate Research Fellowship Program (no.1122374) and the NIGMS/NIH Interdepartmental Biotechnology Training Program (no. T32 GM008334).

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Author notes

  1. Wesley G. Chen

    Present address: Johnson & Johnson Innovation LLC, Cambridge, MA, USA

  2. Carly Tymm

    Present address: Department of Data Science, Dana-Farber Cancer Institute, Boston, MA, USA

  3. Cassidy J. Mileti

    Present address: Department of Biomedical Engineering, UC Davis, Davis, CA, USA

  4. Ana C. Burgos

    Present address: Department of Obstetrics and Gynecology, Loyola University Medical Center, Maywood, IL, USA

  5. Kris Kim

    Present address: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA

Authors and Affiliations

  1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Caroline A. Werlang, Wesley G. Chen, Kelsey M. Wheeler, Carly Tymm, Cassidy J. Mileti, Ana C. Burgos, Kris Kim & Katharina Ribbeck

  2. Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA

    Kazuhiro Aoki & Michael Tiemeyer

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Contributions

C.A.W., W.G.C. and K.M.W. generated mucin-related biochemical reagents and protocols. K.A. performed MS analysis. C.A.W., W.G.C., C.T., C.J.M., A.C.B. and K.K. performed transformation and biofilm formation experiments and developed related protocols. C.A.W. isolated RNA and analysed gene expression data. M.T. and K.R. supervised the study. All of the authors contributed to writing and reviewing the manuscript.

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Correspondence to Katharina Ribbeck.

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The authors declare no competing interests.

Additional information

Peer review information Nature Microbiology thanks Suzanne Dawid, Stephen Hagan, Celine Levesque and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 MUC5AC glycan pools are highly similar to MUC5B glycan pools in composition and effects on S. mutans gene expression and phenotypes.

MUC5AC glycans were isolated from comercially available pig gastric mucin (Millipore Sigma) using beta-elimination51. MUC5AC was purified from pig stomachs as described previously48,50. Sigma PGM is pig gastric mucin purchased from Millipore Sigma that has been dialysed (100 kDa cut-off) and lyophilized. a, A comparison on the most abundant glycan structures (at least 1% of the pool) in MUC5B and MUC5AC (n = 1). b, MUC5AC glycans are relatively enriched for O-GalNAc core-2 structures and display less fucose than MUC5B. c, RNA-Seq was used to profile the effects of 0.1% MUC5AC glycans on gene expression in S. mutans in parallel with studies on MUC5B mucin and MUC5B glycans (n = 2). d, The expression profiles of MUC5B glycans and MUC5AC glycans have a Pearson’s correlation coefficient of 0.93, indicating that they induce highly similar changes in S. mutans global gene expression. e, MUC5AC glycans have a concentration-dependent influence on comS expression. Nonlinear antagonist binding best-fit curves shown (IC50 = 0.05, HillSlope = -6.5, R2 = 0.51). f, RT-qPCR evaluation of S. mutans gene expression after 2 hours incubation in CM with 0.1 wt% of each supplement. We see that all mucins and mucin glycans downregulate the expression of key quorum sensing genes (n > 4, for full data see Source Data). Interestingly, released mucin glycans (both MUC5B and MUC5AC) have stronger effects than intact mucin proteins (MUC5B and MUC5AC polymers). g, Mucins (MUC5B and MUC5AC) and mucin glycans (MUC5B and MUC5AC) reduce transformation frequency of S. mutans at 0.1 wt% in CMedia. While MUC5AC glycans reduce biofilm formation at 0.1 wt% in CMedia, MUC5B glycans did not. h, Growth profiles in CMedia are not altered when supplemented with 0.1 wt% of various monosaccharides. KEY: pig gastric mucin (PGM), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and N-acetylneuraminic acid (Neu5Ac). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS = not significant. For f, data are means, and significance was assessed using unpaired t tests with the Benjamini-Hochberg correction87. For g, data are geometric means ± geometric standard deviations, and significance was assessed using the Kruskal-Wallis test followed by an uncorrected Dunn’s test, which does not assume a Gaussian distribution.

Extended Data Fig. 2 MUC5B reduces transformation and biofilm formation of S. mutans across various media conditions.

a, MUC5B reduces transformation frequency in multiple media conditions: CM (25%Todd-Hewitt), 100%Todd-Hewitt, 100%Todd-Hewitt + 5% Bacto Yeast Extract. b, MUC5B reduces transformation rates in CM with or without 1% sucrose supplementation to promote biofilm formation. c, MUC5B prevents biofilm formation in CM with and without 1% sucrose supplementation, as observed previously22. d, MUC5B reduces transformation efficiency in CM supplemented with 1-10 μM CSP. e, MUC5B reduces transformation in DM supplemented with XIP and (f) CM supplemented with XIP. XIP is less effective at inducing competence in CM compared to DM. *P < 0.05, **P < 0.01. Data are geometric mean ± geometric standard deviation, and significance was assessed using nonparametric Mann-Whitney tests.

Extended Data Fig. 3 MUC5B reduces transformation rates in ΔcomC, ΔcomE, and ΔcomS strains.

a, S. mutans UA159 ΔcomC, ΔcomDE, and ΔcomE18 have natural transformation rates similar to wildtype, and supplementation with 1 μM CSP does not alter transformation rates in ΔcomE. MUC5B is able to induce the same reduction of transformation rates in ΔcomC and ΔcomE strains as in the wildtype, with and without supplemental CSP. b, We confirm that the ΔcomS31 strain was not transformable. However, when supplemented with 10 μM XIP, the ΔcomS knockout shows transformation rates similar to wildtype. Here, we see that MUC5B mucin and MUC5AC glycans reduce transformation rates in the ΔcomS knockout supplemented with 10 μM XIP. *P < 0.05, **P < 0.01, ***P < 0.001, NS = not significant, ND = not detected. Data are geometric means ± geometric standard deviations, and significance was assessed using nonparametric Mann-Whitney tests.

Extended Data Fig. 4 MUC5B prevents biofilm formation and transformation in multiple Streptococcus mutans strains.

a, MUC5B reduces biofilm formation of multiple strains of S. mutans (UA159, SJ, and 28BE3) in CM. MUC5B has no significant effect on biofilm formation of S. sobrinus 6715 (S. sob). b, MUC5B reduces transformation of S. mutans strain 28BE3 in CM. **P < 0.01, NS = not significant. Data are geometric means ± geometric standard deviations, and significance was assessed using nonparametric Mann-Whitney tests.

Supplementary information

Supplementary Information

Supplementary Table 3 and Figs. 1 and 2.

Reporting Summary

Supplementary Table 1

Transcriptional analysis of samples treated with 0.1% MUC5B mucin, 0.1% MUC5B glycans and 0.1% MUC5AC glycans.

Supplementary Table 2

Pathway enrichment analysis.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

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Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

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Werlang, C.A., Chen, W.G., Aoki, K. et al. Mucin O-glycans suppress quorum-sensing pathways and genetic transformation in Streptococcus mutans. Nat Microbiol 6, 574–583 (2021). https://doi.org/10.1038/s41564-021-00876-1

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