The molecular mechanism of N-acetylglucosamine side-chain attachment to the Lancefield group A carbohydrate in Streptococcus pyogenes

Jeffrey S. Rush, Rebecca J. Edgar, Pan Deng, Jing Chen, Haining Zhu, Nina M. Van Sorge, Andrew J. Morris, Konstantin V. Korotkov, X. Natalia Korotkova

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

In many Lactobacillales species (i.e. lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, Streptococcus pyogenes, synthesizes a key antigenic surface polymer, the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with N-acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry, and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphoryl-undecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltransferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function in Streptococcus agalactiae and Enterococcus faecalis. In conclusion, the elucidation of GAC biosynthesis in S. pyogenes reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall.

Original languageEnglish
Pages (from-to)19441-19457
Number of pages17
JournalJournal of Biological Chemistry
Volume292
Issue number47
DOIs
StatePublished - Nov 24 2017

Bibliographical note

Funding Information:
This work was supported in part by National Institutes of Health Grants R21AI113253 from the NIAID (to N. K.), R01GM102129 (to J. S. R.), and 1S10OD021753 (to A. J. M.) and by the Center of Biomedical Research Excellence (COBRE) pilot grant (to K. V. K., N. K., and J. S. R.) supported by National Institutes of Health Grant P30GM110787 from the NIGMS. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Funding Information:
1Supported by Vidi Grant 91713303 from the Netherlands Organization for Scientific Research (NWO).

Funding Information:
This work was supported in part by National Institutes of Health Grants R21AI113253 from the NIAID (to N. K.), R01GM102129 (to J. S. R.), and 1S10OD021753 (to A. J. M.) and by the Center of Biomedical Research Excellence (COBRE) pilot grant (to K. V. K., N. K., and J. S. R.) supported by National Institutes of Health Grant P30GM110787 from the NIGMS. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 1 Supported by Vidi Grant 91713303 from the Netherlands Organization for Scientific Research (NWO). We thank Dr. Charles J. Waechter for encouragement and helpful discussions. Carbohydrate composition analysis at the Complex Carbohydrate Research Center was supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, United States Department of Energy Grant DE-FG02-93ER20097 to Parastoo Azadi.

Funding Information:
ogy and pathogenesis. The polyrhamnose core of GAC is modified with GlcNAc in an ~2:1 ratio (3, 7) of rhamnose to GlcNAc. Collectively, the results of our study suggest a molecular mechanism of GAC biosynthesis in which rhamnan polymer is assembled at the cytoplasmic face of the plasma membrane, translocated to the cell surface, and modified by GlcNAc on the outer side of the membrane as illustrated in Fig. 9. We report that a lipid carrier, GlcNAc-P-P-Und, synthesized by GAS GacO, is a potential acceptor for initiation of rhamnan backbone biosynthesis. We speculate that the next step of rhamnan biosynthesis involves the action of the GacB, GacC, GacG, and GacF glycosyltransferases. The lipid-anchored polyrhamnose is then translocated across the membrane by the ABC transporter encoded by gacD and gacE. This hypothesis is supported by studies of rhamnan biosynthesis in S. mutans (13, 31, 32).

Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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