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

Abstract

In many Lactobacillales species ( 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, , 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-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 and In conclusion, the elucidation of GAC biosynthesis in reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall.

Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides

“…The observation that the SMUΔsccH mutant could only be rescued from from Zn (II) toxicity and acquisition of hGIIA resistance by expression of the native sccH, suggested that the site of GroP attachment to SRPs might involve the species-specific side chains (Glc vs. GlcNAc), rather than the identical polyrhamnose backbone. Consistent with this hypothesis, the glycerol and phosphate contents in the SRP isolated from two GlcNAc-deficient mutants, 5005ΔgacL and 5005ΔgacI 22 are similar to those in 5005ΔgacH ( Fig. 7d).…”

“…GAC was released from the cell wall by sequential digestion with mutanolysin (Sigma Aldrich) and recombinant PlyC amidase 22 , and partially purified by a combination of size exclusion and ion-exchange chromatography. Mutanolysin digests contained 5 mg/mL of cell wall suspension in 0.1 M sodium acetate, pH 5.5, 2 mM CaCl 2 and 5 U/ml mutanolysin.…”

“…The first five genes in both operons, together with gacG/sccG, are conserved in many streptococcal species and they participate in polyrhamnose backbone synthesis and transport 21 . In GAS, we have demonstrated that gacI, gacJ, gacK and gacL encode the machinery to generate and add the immunodominant GlcNAc side-chain to the polyrhamnose backbone 15,22 . In S. mutans, sccM and sccN are required for immunoreactivity with serotype c-specific antiserum suggesting a function for these genes in Glc side-chain attachment to the polyrhamnose backbone 23 .…”

Streptococcal Lancefield polysaccharides are critical cell wall determinants for human Group IIA secreted phospholipase A2 to exert its bactericidal effects

“…In contrast to dltA and lytR , mutation of gacI , which results in loss of the GAC GlcNAc side chain [ 45 ], increased GAS resistance to hGIIA by approximately 10-fold compared to the parental or gacI -complemented ( gacI* ) strain ( Fig 2C ). The GAC is conserved in all GAS serotypes.…”

“…A recent study demonstrates that GacI is a membrane protein that is required for the intracellular formation of undecaprenyl-P-GlcNAc [ 45 ]. Therefore, loss of GacI could alter membrane composition and fluidity to impact the activity of hGIIA on the membrane.…”