Article | Published:

Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides

Abstract

Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. Here we identify gacH, a gene in the Streptococcus pyogenes group A carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA-secreted phospholipase A2. Subsequent structural and phylogenetic analysis of the GacH extracellular domain revealed that GacH represents an alternative class of glycerol phosphate transferase. We detected the presence of glycerol phosphate in the GAC, as well as the serotype c carbohydrate from Streptococcusmutans, which depended on the presence of the respective gacH homologs. Finally, nuclear magnetic resonance analysis of GAC confirmed that glycerol phosphate is attached to approximately 25% of the GAC N-acetylglucosamine side-chains at the C6 hydroxyl group. This previously unrecognized structural modification impacts host–pathogen interaction and has implications for vaccine design.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

Illumina sequencing reads from the Tn-seq analysis were deposited in the NCBI Sequence Read Archive under the accession number SRP150081. The Tn-seq data, analyses and pipeline for the Tn-seq analyses are accessible under the DOI number 10.5281/zenodo.2541163 in GitHub as the following link: https://doi.org/10.5281/zenodo.2541163. Atomic coordinates and structure factors of the reported crystal structures have been deposited in the Protein Data Bank with accession codes 5U9Z (apo eGacH) and 6DGM (GroP•eGacH complex). All data generated during this study are included in the article, and supplementary information files or will be available from the corresponding authors upon reasonable request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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).

  2. 2.

    Weidenmaier, C. & Peschel, A. Teichoic acids and related cell-wall glycopolymers in Gram-positive physiology and host interactions. Nat. Rev. Microbiol. 6, 276–287 (2008).

  3. 3.

    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).

  4. 4.

    Huang, D. H., Rama Krishna, N. & Pritchard, D. G. Characterization of the group A streptococcal polysaccharide by two-dimensional 1H-nuclear-magnetic-resonance spectroscopy. Carbohydr. Res. 155, 193–199 (1986).

  5. 5.

    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).

  6. 6.

    Van der Beek, S. L. et al. A Streptococcus and defines a new class of monomeric dTDP-4-dehydrorhamnose reductases (RmlD). Mol. Microbiol. 98, 946–962 (2015).

  7. 7.

    Tsuda, H., Yamashita, Y., Shibata, Y., Nakano, Y. & Koga, T. Genes involved in bacitracin resistance in Streptococcus mutans. Antimicrob. Agents Chemother. 46, 3756–3764 (2002).

  8. 8.

    De, A. et al. Deficiency of RgpG causes major defects in cell division and biofilm formation, and deficiency of LytR-CpsA-Psr family proteins leads to accumulation of cell wall antigens in culture medium by Streptococcus mutans. Appl. Environ. Microbiol. 83, e00928 (2017).

  9. 9.

    Nagata, E. et al. Serotype-specific polysaccharide of Streptococcus mutans contributes to infectivity in endocarditis. Oral Microbiol. Immunol. 21, 420–423 (2006).

  10. 10.

    Henningham, A. et al. Virulence role of the GlcNAc side chain of the Lancefield cell wall carbohydrate antigen in non-M1-serotype group A Streptococcus. mBio 9, e02294 (2018).

  11. 11.

    van Sorge, N. M. et al. The classical Lancefield antigen of group A Streptococcus is a virulence determinant with implications for vaccine design. Cell Host Microbe 15, 729–740 (2014).

  12. 12.

    Kabanova, A. et al. Evaluation of a group A Streptococcus synthetic oligosaccharide as vaccine candidate. Vaccine 29, 104–114 (2010).

  13. 13.

    Sabharwal, H. et al. group A Streptococcus (GAS) carbohydrate as an immunogen for protection against GAS infection. J. Infect. Dis. 193, 129–135 (2006).

  14. 14.

    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).

  15. 15.

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

  16. 16.

    Le Breton, Y. et al. Essential genes in the core genome of the human pathogen Streptococcus pyogenes. Sci. Rep. 5, 9838 (2015).

  17. 17.

    van Hensbergen, V. P. et al. Streptococcal Lancefield polysaccharides are critical cell wall determinants for human Group IIA secreted phospholipase A2 to exert its bactericidal effects. PLoS Pathog. 14, e1007348 (2018).

  18. 18.

    Weiss, J. P. Molecular determinants of bacterial sensitivity and resistance to mammalian Group IIA phospholipase A2. Biochim. Biophys. Acta 1848, 3072–3077 (2015).

  19. 19.

    Ong, C. L., GillenC. M., Barnett, T. C., Walker, M. J. & McEwan, A. G. An antimicrobial role for zinc in innate immune defense against group A Streptococcus. J. Infect. Dis. 209, 1500–1508 (2014).

  20. 20.

    Graham, M. R. et al. Virulence control in group A Streptococcus by a two-component gene regulatorysystem: global expression profiling and in vivo infection modeling. Proc. Natl Acad. Sci. USA 99, 13855–13860 (2002).

  21. 21.

    Lu, D. et al. Structure-based mechanism of lipoteichoic acid synthesis by Staphylococcus aureus LtaS. Proc. Natl Acad. Sci. USA 106, 1584–1589 (2009).

  22. 22.

    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).

  23. 23.

    Campeotto, I. et al. Structural and mechanistic insight into the Listeria monocytogenes two-enzyme lipoteichoic acid synthesis system. J. Biol. Chem. 289, 28054–28069 (2014).

  24. 24.

    Fischer, W., Laine, R. A. & Nakano, M. On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. II Structures of glycerophosphoglycolipids. Biochim. Biophys. Acta 528, 298–308 (1978).

  25. 25.

    Karatsa-Dodgson, M., Wormann, M. E. & Grundling, A. In vitro analysis of the Staphylococcus aureus lipoteichoic acid synthase enzyme using fluorescently labeled lipids. J. Bacteriol. 192, 5341–5349 (2010).

  26. 26.

    Kennedy, E. P., Rumley, M. K., Schulman, H. & Van Golde, L. M. Identification of sn-glycero-1-phosphate and phosphoethanolamine residues linked to the membrane-derived oligosaccharides of Escherichia coli. J. Biol. Chem. 251, 4208–4213 (1976).

  27. 27.

    Marino, J. P. et al. Three-dimensional triple-resonance 1H, 13C, 31P experiment: sequential through-bond correlation of ribose protons and intervening phosphorus along the RNA oligonucleotide backbone. J. Am. Chem. Soc. 116, 6472–6473 (1994).

  28. 28.

    Heymann, H., Manniello, J. M. & Barkulis, S. S. Structure of streptococcal cell walls. V. Phosphate esters in the walls of group A Streptococcus pyogenes. Biochem. Biophys. Res. Commun. 26, 486–491 (1967).

  29. 29.

    Emdur, L. I., Saralkar, C., McHugh, J. G. & Chiu, T. H. Glycerolphosphate-containing cell wall polysaccharides from Streptococcus sanguis. J. Bacteriol. 120, 724–732 (1974).

  30. 30.

    Prakobphol, A., Linzer, R. & Genco, R. J. Purification and characterization of a rhamnose-containing cell wall antigen of Streptococcus mutans B13 (serotype d). Infect. Immun. 27, 150–157 (1980).

  31. 31.

    Pritchard, D. G., Michalek, S. M., McGhee, J. R. & Furner, R. L. Structure of the serotype f polysaccharide antigen of Streptococcus mutans. Carbohydr. Res. 166, 123–131 (1987).

  32. 32.

    Pritchard, D. G., Gregory, R. L., Michalek, S. M. & McGhee, J. R. Characterization of the serotype e polysaccharide antigen of Streptococcus mutans. Mol. Immunol. 23, 141–145 (1986).

  33. 33.

    Vickery, C. R., Wood, B. M., Morris, H. G., Losick, R. & Walker, S. Reconstitution of Staphylococcus aureus lipoteichoic acid synthase activity identifies Congo red as a selective inhibitor. J. Am. Chem. Soc. 140, 876–879 (2018).

  34. 34.

    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).

  35. 35.

    Peschel, A. et al. Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. J. Biol. Chem. 274, 8405–8410 (1999).

  36. 36.

    Falagas, M. E., Rafailidis, P. I. & Matthaiou, D. K. Resistance to polymyxins: mechanisms, frequency and treatment options. Drug Resist. Updat. 13, 132–138 (2010).

  37. 37.

    Djoko, K. Y., Ong, C. L., Walker, M. J. & McEwan, A. G. The role of copper and zinc toxicity in innate immune defense against bacterial pathogens. J. Biol. Chem. 290, 18954–18961 (2015).

  38. 38.

    Ong, C. L., Walker, M. J. & McEwan, A. G. Zinc disrupts central carbon metabolism and capsule biosynthesis in Streptococcus pyogenes. Sci. Rep. 5, 10799 (2015).

  39. 39.

    McDevitt, C. A. et al. A molecular mechanism for bacterial susceptibility to zinc. PLoS Pathog. 7, e1002357 (2011).

  40. 40.

    Buckland, A. G. & Wilton, D. C. Inhibition of secreted phospholipases A2 by annexin V. Competition for anionic phospholipid interfaces allows an assessment of the relative interfacial affinities of secreted phospholipases A2. Biochim. Biophys. Acta 1391, 367–376 (1998).

  41. 41.

    Koprivnjak, T., Peschel, A., Gelb, M. H., Liang, N. S. & Weiss, J. P. Role of charge properties of bacterial envelope in bactericidal action of human group IIA phospholipase A2 against Staphylococcus aureus. J. Biol. Chem. 277, 47636–47644 (2002).

  42. 42.

    Hunt, C. L., Nauseef, W. M. & Weiss, J. P. Effect of d-alanylation of (lipo)teichoic acids of Staphylococcus aureus on host secretory phospholipase A2 action before and after phagocytosis by human neutrophils. J. Immunol. 176, 4987–4994 (2006).

  43. 43.

    Carapetis, J. R., Steer, A. C., Mulholland, E. K. & Weber, M. The global burden of group A streptococcal diseases. Lancet Infect. Dis. 5, 685–694 (2005).

  44. 44.

    Goldstein, I., Rebeyrotte, P., Parlebas, J. & Halpern, B. Isolation from heart valves of glycopeptides which share immunological properties with Streptococcus haemolyticus group A polysaccharides. Nature 219, 866–868 (1968).

  45. 45.

    Ayoub, E. M. & Dudding, B. A. Streptococcal group A carbohydrate antibody in rheumatic and nonrheumatic bacterial endocarditis. J. Lab. Clin. Med. 76, 322–332 (1970).

  46. 46.

    Kirvan, C. A., Swedo, S. E., Heuser, J. S. & Cunningham, M. W. Mimicry and autoantibody-mediated neuronal cell signaling in Sydenham chorea. Nat. Med. 9, 914–920 (2003).

  47. 47.

    Le Breton, Y. et al. Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection. PLoS Pathog. 13, e1006584 (2017).

  48. 48.

    Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

  49. 49.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

  50. 50.

    Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68 (1989).

  51. 51.

    Trevino, J., LiuZ., Cao, T. N., Ramirez-Pena, E. & Sumby, P. RivR is a negative regulator of virulencefactor expression in group A Streptococcus. Infect. Immun. 81, 364–372 (2013).

  52. 52.

    Ghomashchi, F. et al. Preparation of the full set of recombinant mouse- and human-secreted phospholipases A2. Methods Enzymol. 583, 35–69 (2017).

  53. 53.

    Le Breton, Y. & McIver, K. S. Genetic manipulation of Streptococcus pyogenes (the group A Streptococcus, GAS). Curr. Protoc. Microbiol. 30, Unit 9D 3 (2013).

  54. 54.

    Kabsch, W. Xds. Acta Crystallogr. 66, 125–132 (2010).

  55. 55.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. 66, 213–221 (2010).

  56. 56.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. 66, 486–501 (2010).

  57. 57.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. 66, 12–21 (2010).

  58. 58.

    Wagner, R. & Berger, S. Gradient-selected NOESY-A fourfold reduction of the measurement time for the NOESY Experiment. J. Magn. Reson. 123, 119–121 (1996).

  59. 59.

    Willker, W., Leibfritz, D., Kerssebaum, R. & Bermel, W. Gradient selection in inverse heteronuclear correlation spectroscopy. Magn. Reson. Chem. 31, 287–292 (1993).

  60. 60.

    Kellogg, G. W. Proton-detected hetero-TOCSY experiments with application to nucleic acids. J. Magn. Reson. 98, 176–182 (1992).

Download references

Acknowledgements

This work was supported by the Center of Biomedical Research Excellence (COBRE) Pilot Grant (to N.K., K.V.K. and J.S.R.) supported by NIH grant No. P30GM110787 from the National Institute of General Medical Sciences (NIGMS); NIH grant No. R56AI135021 from the National Institute of Allergy and Infectious Diseases (NIAID) (to N.K.); VIDI grant No. 91713303 from the Netherlands Organization for Scientific Research (NWO) (to N.M.vS. and V.P.vH.); the Swedish Research Council (Nos. 2013–4859 and 2017–03703) and The Knut and Alice Wallenberg Foundation (to G.W.); NIH grant No. P30GM127211 from the NIGMS and NIH grant No. 1S10OD021753 (to A.J.M.); the National Health and Medical Research Council of Australia (to M.J.W.); grants from CNRS, ANR (MNaims No. ANR-17-CE17-0012-01) and FRM (No. SPF20150934219) (to G.L.); NIH grant No. AI047928 from NIAID (to K.S.M. and Y.L.B.); and NIH grant No. AI094773 (to N.M.E.S. and A.T.B.). 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) to P.A. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31–109-Eng-38 and NIH grants Nos. S10_RR25528 and S10_RR028976. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, NIGMS including No. P41GM103393. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

Author information

A.R., P.D., Y.L.B., K.S.M., A.G.M., A.J.M., G.L., M.J.W., J.S.R., K.V.K., G.W., N.M.vS. and N.K. designed the experiments. R.J.E., V.P.vH., A.R., A.T., J.S.R., K.V.K., G.W. and N.K. performed functional and biochemical experiments. K.V.K. carried out X-ray crystallography and structure analysis. A.R. and G.W. performed NMR studies. P.D. and A.J.M. performed MS analysis. V.P.vH., N.K. and K.V.K. constructed plasmids and isolated mutants. R.J.E., V.P.vH., A.R., P.D., Y.L.B., N.M.E.S., A.T.B., K.S.M., A.G.M., A.J.M., M.J.W., J.S.R., K.V.K., G.W., N.M.vS. and N.K. analyzed the data. N.M.vS. and N.K. wrote the manuscript with contributions from all authors. All authors reviewed the results and approved the final version of the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Nina M. van Sorge or Natalia Korotkova.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–17, Supplementary Tables 1–7 and Supplementary Note

  2. Reporting Summary

Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.

About this article

Fig. 1: GacH homologs are required for hGIIA bactericidal activity against GAS and S.mutans.
Fig. 2: Deletion of gacI and gacH renders GAS susceptible to Zn2+.
Fig. 3: Structure of eGacH.
Fig. 4: GacH and SccH modify their respective glycopolymers with sn-Gro-1-P.
Fig. 5: NMR analysis confirms the presence of GroP on the C6 GlcNAc hydroxymethyl group of GAC.