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.
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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.
Nina M. van Sorge supported by Vidi Grant 91713303 from the Netherlands Organization for Scientific Research (NWO).
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.
This article contains supplemental Figs. S1–S6, Tables S1–S3, and file S1.
Rush, Jeffrey; Edgar, Rebecca J.; Deng, Pan; Chen, Jing; Zhu, Haining; van Sorge, Nina M.; Morris, Andrew J.; Korotkov, Konstantin V.; and Korotkova, Natalia, "The Molecular Mechanism of N-Acetylglucosamine Side-Chain Attachment to the Lancefield Group A Carbohydrate in Streptococcus pyogenes" (2017). Molecular and Cellular Biochemistry Faculty Publications. 133.