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Modification of cell wall polysaccharide guides cell division in Streptococcus mutans

Abstract

In ovoid-shaped, Gram-positive bacteria, MapZ guides FtsZ-ring positioning at cell equators. The cell wall of the ovococcus Streptococcus mutans contains peptidoglycan decorated with serotype c carbohydrates (SCCs). In the present study, we identify the major cell separation autolysin AtlA as an SCC-binding protein. AtlA binding to SCC is attenuated by the glycerol phosphate (GroP) modification. Using fluorescently labeled AtlA constructs, we mapped SCC distribution on the streptococcal surface, revealing enrichment of GroP-deficient immature SCCs at the cell poles and equators. The immature SCCs co-localize with MapZ at the equatorial rings throughout the cell cycle. In GroP-deficient mutants, AtlA is mislocalized, resulting in dysregulated cellular autolysis. These mutants display morphological abnormalities associated with MapZ mislocalization, leading to FtsZ-ring misplacement. Altogether, our data support a model in which maturation of a cell wall polysaccharide provides the molecular cues for the recruitment of cell division machinery, ensuring proper daughter cell separation and FtsZ-ring positioning.

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Fig. 1: Modification of SCCs with Glc side chains and GroP.
Fig. 2: Modifications of SCC control S. mutans cell aggregation and morphology.
Fig. 3: Mislocalization of AtlA promotes autolysis of ΔsccH and ΔsccN.
Fig. 4: AtlA binds to the poly(Rha) backbone of SCC.
Fig. 5: Modifications of SCCs guide the positioning of FtsZ- and MapZ-rings.
Fig. 6: A schematic model of cell division in S. mutans.

Data availability

Source data are provided with this paper. All other data generated during this study are included in the article and supplementary information files.

References

  1. 1.

    Brown, S., Santa Maria, J. P. Jr. & Walker, S. Wall teichoic acids of Gram-positive bacteria. Annu. Rev. Microbiol. 67, 313–336 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Higgins, M. L. & Shockman, G. D. Model for cell wall growth of Streptococcus faecalis. J. Bacteriol. 101, 643–648 (1970).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Land, A. D. & Winkler, M. E. The requirement for pneumococcal MreC and MreD is relieved by inactivation of the gene encoding PBP1a. J. Bacteriol. 193, 4166–4179 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Sham, L. T., Tsui, H. C., Land, A. D., Barendt, S. M. & Winkler, M. E. Recent advances in pneumococcal peptidoglycan biosynthesis suggest new vaccine and antimicrobial targets. Curr. Opin. Microbiol. 15, 194–203 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Wheeler, R., Mesnage, S., Boneca, I. G., Hobbs, J. K. & Foster, S. J. Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria. Mol. Microbiol. 82, 1096–1109 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Massidda, O., Novakova, L. & Vollmer, W. From models to pathogens: how much have we learned about Streptococcus pneumoniae cell division? Environ. Microbiol. 15, 3133–3157 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    van Raaphorst, R., Kjos, M. & Veening, J. W. Chromosome segregation drives division site selection in Streptococcus pneumoniae. Proc. Natl Acad. Sci. USA 114, E5959–E5968 (2017).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Fleurie, A. et al. MapZ marks the division sites and positions FtsZ rings in Streptococcus pneumoniae. Nature 516, 259–262 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Holeckova, N. et al. LocZ is a new cell division protein involved in proper septum placement in Streptococcus pneumoniae. mBio 6, e01700–e01714 (2014).

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Perez, A. J. et al. Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae. Proc. Natl Acad. Sci. USA 116, 3211–3220 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Manuse, S. et al. Structure–function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ. Nat. Commun. 7, 12071 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Yamamoto, H., Miyake, Y., Hisaoka, M., Kurosawa, S. & Sekiguchi, J. The major and minor wall teichoic acids prevent the sidewall localization of vegetative dl-endopeptidase LytF in Bacillus subtilis. Mol. Microbiol. 70, 297–310 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Schirner, K., Marles-Wright, J., Lewis, R. J. & Errington, J. Distinct and essential morphogenic functions for wall- and lipo-teichoic acids in Bacillus subtilis. EMBO J. 28, 830–842 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Schlag, M. et al. Role of staphylococcal wall teichoic acid in targeting the major autolysin Atl. Mol. Microbiol. 75, 864–873 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Lunderberg, J. M., Liszewski Zilla, M., Missiakas, D. & Schneewind, O. Bacillus anthracis tagO Is required for vegetative growth and secondary cell wall polysaccharide synthesis. J. Bacteriol. 197, 3511–3520 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Bonnet, J. et al. Nascent teichoic acids insertion into the cell wall directs the localization and activity of the major pneumococcal autolysin LytA. Cell Surf. 2, 24–37 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Mistou, M. Y., Sutcliffe, I. C. & van Sorge, N. M. Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria. FEMS Microbiol. Rev. 40, 464–479 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    St Michael, F. et al. Investigating the candidacy of the serotype specific rhamnan polysaccharide based glycoconjugates to prevent disease caused by the dental pathogen Streptococcus mutans. Glycoconj. J. 35, 53–64 (2018).

    Google Scholar 

  19. 19.

    Coligan, J. E., Kindt, T. J. & Krause, R. M. Structure of the streptococcal groups A, A-variant and C carbohydrates. Immunochemistry 15, 755–760 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    McCarty, M. Variation in the group-specific carbohydrate of group A streptococci. II. Studies on the chemical basis for serological specificity of the carbohydrates. J. Exp. Med. 104, 629–643 (1956).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Edgar, R. J. et al. Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides. Nat. Chem. Biol. 15, 463–471 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Rush, J. S. et al. The molecular mechanism of N-acetylglucosamine side-chain attachment to the Lancefield group A carbohydrate in Streptococcus pyogenes. J. Biol. Chem. 292, 19441–19457 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Brown, T. A. Jr. et al. A hypothetical protein of Streptococcus mutans is critical for biofilm formation. Infect. Immun. 73, 3147–3151 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Shibata, Y., Kawada, M., Nakano, Y., Toyoshima, K. & Yamashita, Y. Identification and characterization of an autolysin-encoding gene of Streptococcus mutans. Infect. Immun. 73, 3512–3520 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Ahn, S. J. & Burne, R. A. The atlA operon of Streptococcus mutans: role in autolysin maturation and cell surface biogenesis. J. Bacteriol. 188, 6877–6888 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Yoshimura, G. et al. Identification and molecular characterization of an N-acetylmuraminidase, Aml, involved in Streptococcus mutans cell separation. Microbiol Immunol. 50, 729–742 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Yamashita, Y. et al. A novel gene required for rhamnose-glucose polysaccharide synthesis in Streptococcus mutans. J. Bacteriol. 181, 6556–6559 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Shibata, Y., Yamashita, Y., Ozaki, K., Nakano, Y. & Koga, T. Expression and characterization of streptococcal rgp genes required for rhamnan synthesis in Escherichia coli. Infect. Immun. 70, 2891–2898 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Zorzoli, A. et al. Group A, B, C, and G Streptococcus Lancefield antigen biosynthesis is initiated by a conserved alpha-d-GlcNAc-beta-1,4-l-rhamnosyltransferase. J. Biol. Chem. 294, 15237–15256 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Li, Y. et al. MapZ forms a stable ring structure that acts as a nanotrack for FtsZ treadmilling in Streptococcus mutans. ACS Nano. 12, 6137–6146 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Neuhaus, F. C. & Baddiley, J. A continuum of anionic charge: structures and functions of d-alanyl-teichoic acids in Gram-positive bacteria. Microbiol. Mol. Biol. Rev. 67, 686–723 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Yu, Y. et al. Systematic hydrogen-bond manipulations to establish polysaccharide structure–property correlations. Angew. Chem. Int. Ed. Engl. 58, 13127–13132 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Yu, Y. & Delbianco, M. Conformational studies of oligosaccharides. Chemistry 26, 9814–9825 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Merzlyak, E. M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4, 555–557 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Pedelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Catalao, M. J., Figueiredo, J., Henriques, M. X., Gomes, J. P. & Filipe, S. R. Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria. PLoS ONE 9, e113796 (2014).

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Rosenow, M. A., Huffman, H. A., Phail, M. E. & Wachter, R. M. The crystal structure of the Y66L variant of green fluorescent protein supports a cyclization–oxidation–dehydration mechanism for chromophore maturation. Biochemistry 43, 4464–4472 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Dufour, D. & Levesque, C. M. Cell death of Streptococcus mutans induced by a quorum-sensing peptide occurs via a conserved streptococcal autolysin. J. Bacteriol. 195, 105–114 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Templeton, D. W., Quinn, M., Van Wychen, S., Hyman, D. & Laurens, L. M. Separation and quantification of microalgal carbohydrates. J. Chromatogr. A 1270, 225–234 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Kojima, N., Araki, Y. & Ito, E. Structural studies on the linkage unit of ribitol teichoic acid of Lactobacillus plantarum. Eur. J. Biochem. 148, 29–34 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Lehrman, M. A. & Gao, N. Alternative and sources of reagents and supplies of fluorophore-assisted carbohydrate electrophoresis (FACE). Glycobiology 13, 1G–3G (2003).

    PubMed  PubMed Central  Google Scholar 

  42. 42.

    Chaturvedi, S. K., Ma, J., Brown, P. H., Zhao, H. & Schuck, P. Measuring macromolecular size distributions and interactions at high concentrations by sedimentation velocity. Nat. Commun. 9, 4415 (2018).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Schuck, P. Size–distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606–1619 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Brautigam, C. A. Calculations and publication-quality illustrations for analytical ultracentrifugation data. Methods Enzymol. 562, 109–133 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Stafford, W. F. & Braswell, E. H. Sedimentation velocity, multi-speed method for analyzing polydisperse solutions. Biophys. Chem. 108, 273–279 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank S.-J. Ahn (University of Florida) for the kind gift of anti-AtlA antibodies, J. Abranches (University of Florida) for providing S. mutans serotype e, f and k strains; J. F. Timoney (University of Kentucky) and J. Huebner (von Hauner Children’s Hospital, LMU) for providing S. equi and E. faecalis, respectively; J. M. Bosken and E. D. Hall (University of Kentucky) for the use of the Thermo Fisher Scientific GC–MS instrument and C. Velez-Ortega (University of Kentucky) for access to a Leica SP8 confocal microscope. This work was supported by National Institutes of Health (NIH) grants (nos. R01 DE028916 from the National Institute of Dental and Craniofacial Research (NIDCR) and R01 AI143690 from the National Institute of Allergy and Infectious Diseases to N.K., R01 GM094363 from the National Institute of General Medical Sciences to A.B.H. and R01 DC014658 from the NIDCD to G.I.F.), Tenovus Scotland large research grant (no. T17/17) and University of Dundee Wellcome Fund (grant no. 105606/Z/14/Z) to S.A.C. and H.C.D., and the Wellcome and Royal Society grant (no. 109357/Z/15/Z) to H.C.D.. SEM was performed at the Electron Microscopy Center, which belongs to the National Science Foundation NNCI Kentucky Multiscale Manufacturing and Nano Integration Node, supported by ECCS-1542174. Carbohydrate composition analysis at the Complex Carbohydrate Research Center was supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, US Department of Energy grant (no. DE-FG02-93ER20097). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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S.Z., C.T.C., J.S.R., S.A.C., A.E.Y., A.B.H., N.M.v.S., H.C.D., G.I.F., K.V.K. and N.K. designed the experiments. S.Z., C.T.C., J.S.R., C.W.K., S.A.C., A.E.Y., H.C.D., K.V.K. and N.K. performed functional and biochemical experiments. S.Z. and G.I.F. performed microscopy analysis. N.K., K.V.K. and N.M.v.S. constructed plasmids and isolated mutants. S.Z., C.T.C., J.S.R., S.A.C., A.E.Y., A.B.H., H.C.D., K.V.K. and N.K. analyzed the data. N.K. wrote the manuscript with contributions from all authors. All authors reviewed the results and approved the final version of the manuscript.

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Correspondence to Natalia Korotkova.

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Peer review information Nature Chemical Biology thanks Rut Carballido-Lopez 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 Modifications of SCC control S. mutans cell aggregation and morphology.

a, Sedimentation phenotype of S. mutans strains after overnight growth in THY broth. b, Sedimentation phenotype of sacculi purified from S. mutans strains. Sacculi were resuspended in PBS (OD600 of 8), and allowed to sediment for 72 hours at 4 °C. c, DIC images of S. mutans strains taken from mid-log growth phase. Blue arrows denote small round cells. Scale bar is 1 µm. Representative images from at least three independent experiments are shown in a, b and c.

Extended Data Fig. 2 Fluorescent microscopy of S. mutans intact cells and sacculi labeled by AtlABSP-GFP.

a, Binding of AtlABSP-GFP to the intact WT, ΔsccH, ΔsccH:psccH, ΔsccN and ΔsccN:psccN bacterial cells. b, Binding of AtlABSP-GFP to the sacculi purified from WT, ΔsccH and ΔsccH:psccH. In a and b, yellow and blue arrows show polar and equatorial sites labeled by AtlABSP-GFP, respectively. Scale bar is 1 µm in a and b. The experiments depicted in a and b were performed independently three times and yielded the same results.

Extended Data Fig. 3 Gating Strategy for Flow Cytometry (Fig. 4b).

In this sample gating, E. coli expressing polyrhamnose on the cell surface and its parental strain (E. coli without polyrhamnose) were gated based on the presence of fluorescent signal (AtlABSP-GFP). Bacterial gating occurred at the GFP/SCC density plot omitting signals derived from bacteria stained with GFP alone (GFP). For flow cytometric analysis, at least 10,000 events were collected. Experiments were performed independently three times and yielded the same results. Dot plots of representative results are shown.

Extended Data Fig. 4 S. mutans WT produces SCCs with different degrees of GroP modification.

a, Ion exchange chromatography of SCCs purified from ΔsccH. b, Ion exchange chromatography of SCCs purified from WT S. mutans. SCC material was loaded onto Toyopearl DEAE-650M and eluted with a NaCl gradient (0-0.5 M). Fractions were analyzed for Rha and Glc contents by anthrone assay and total phosphate (Pi) content by malachite green assay following digestion with perchloric acid. c, Composition analysis of minor and major fractions from b. Fractions were pooled, concentrated, and desalted by spin column and analyzed by GC-MS to determine the Rha and Glc concentrations. The concentration of Glc is presented relative to the Rha concentration. Data are means ± S.D., n = 3 biologically independent experiments. P values were calculated by a two-tailed t-test. d, SDS-PAGE analysis of ANDS-labeled SCCs extracted from S. mutans mutants. In a, b and d, the experiments were performed at least three times and yielded the same results. Data from one representative experiment are shown.

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Supplementary Figs. 1–18, Supplementary Tables 1–6, Supplementary References.

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Source Data Fig. 3b

Uncropped SDS–PAGE gel corresponding to Fig 3b.

Source Data Extended Data Fig. 4d

Uncropped SDS–PAGE gel corresponding to Extended Data Fig 4d.

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Zamakhaeva, S., Chaton, C.T., Rush, J.S. et al. Modification of cell wall polysaccharide guides cell division in Streptococcus mutans. Nat Chem Biol 17, 878–887 (2021). https://doi.org/10.1038/s41589-021-00803-9

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