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Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides


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.

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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: 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.

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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.

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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.