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J Biol Chem. 2017 Nov 24;292(47):19441-19457. doi: 10.1074/jbc.M117.815910. Epub 2017 Oct 11.

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

Author information

1
From the Department of Molecular and Cellular Biochemistry and.
2
Division of Cardiovascular Medicine and the Gill Heart Institute, University of Kentucky, Lexington, Kentucky 40536 and.
3
the Department of Medical Microbiology, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands.
4
From the Department of Molecular and Cellular Biochemistry and nkorotkova@uky.edu.

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.

KEYWORDS:

Streptococcus pyogenes (S. pyogenes); carbohydrate biosynthesis; cell wall; glycosyltransferase; lipid intermediate; polysaccharide

PMID:
29021255
PMCID:
PMC5702681
DOI:
10.1074/jbc.M117.815910
[Indexed for MEDLINE]
Free PMC Article

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