Abstract
Streptococcus pyogenes or Group A Streptococcus (GAS) causes numerous diseases in humans, ranging from minor skin and throat infections, to life-threatening systemic conditions such as bacteremia, necrotizing fasciitis, and toxic-shock syndrome. GAS synthesizes a key antigenic surface polymer — the Lancefield group A Carbohydrate (GAC). GAC is attached to peptidoglycan and consists of a polyrhamnose polymer, with N-acetylglucosamine (GlcNAc) side chains, that is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report we demonstrate that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphoryl-undecaprenol (GlcNAc-P-P-Und) produced by GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by GacI. The GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Furthermore, our data suggests that GlcNAc-P-Und is used by the membrane glycosyltransferase GacL to transfer GlcNAc from GlcNAc-P-Und to the polyrhamnose polysaccharide. In addition, GacJ, a small membrane protein, forms a complex with GacI and significantly stimulates its catalytic activity. We show that GlcNAc modification of polyrhamnose protects GAS from amidase-induced lysis. Thus, our study significantly expands our understanding of the biosynthesis of GAS cell wall polysaccharide and points to the functional importance of polysaccharide modifications in protection of peptidoglycan from lytic enzymes.
IMPORTANCE In many species of Lactobacillales peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited study. The important human pathogen, Streptococcus pyogenes, synthesizes a cell wall-associated polyrhamnose polysaccharide with N-acetylglucosamine (GlcNAc) side chains. The GlcNAc side chain is an important virulence determinant used by bacteria to evade the host innate immune defense. Here we identify the molecular mechanism of polyrhamnose modification with GlcNAc in S. pyogenes. We show that GacI synthesizes GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) aided by GacJ. GlcNAc-P-Und is apparently used by GacL to transfer GlcNAc to polyrhamnose. We demonstrate that GacI homologs perform a similar function in Streptococcus agalactiae and Enterococcus faecalis. Thus, the elucidation of polysaccharide biosynthesis in S. pyogenes enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall.