PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides

Abstract

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

Structural variations and roles of rhamnose-rich cell wall polysaccharides in Gram-positive bacteria

“…Thus, as a whole, Rha-CWPS appear to be polyanionic cell wall glycopolymers, similar to WTA. In a few cases, no phosphate group has been identified in the Rha-CWPS structures, but there is a possibility that it could be lost during acidic extraction procedures, as was reported for GAC in S. pyogenes ( 26 ).…”

“…For the sake of simplicity in this text, we will refer to this linear Rha-rich backbone as rhamnan in all cases. The rhamnan polymer is synthesized with a linker unit made of GlcNAc, which may be N -deacetylated to glucosamine (GlcNH 2 ) in certain cases ( 26 ). In addition, a cap structure made of sugar has been suggested by experimental data ( 14 , 27 ) but it is not known at present whether this is a conserved feature of Rha-CWPS.…”

“…Until very recently, though, the chemical structure of the linkage unit had not been experimentally established. Work in S. pyogenes revealed that GAC is linked to peptidoglycan through glucosamine-1-phosphate rather than the expected GlcNAc-P, which explains why GAC is not released from cell walls by mild acid treatment ( 26 ). This GlcN residue is formed by enzymatic N -deacylation of the GlcNAc residue located at the reducing end of the rhamnan chain after its synthesis, and this modification is catalyzed by a de- N -acetylase named PplD.…”

“…Interestingly, inactivation of the de- N -acetylase PplD in S. pyogenes , which deacetylates the linkage GlcNAc, had the inverse effect on bacterial sensitivity to cationic antimicrobial peptides. By producing positively charged GlcN, PplD contributes to the neutralization of the negative charges displayed by GroP, thus increasing protection against cationic antimicrobial peptides ( 26 ).…”

“…These antibodies also promoted opsonophagocytosis by neutrophils in vitro . However, for vaccine design, detailed knowledge of the structure of the immunizing antigen is of prime importance since, for example, antibodies induced against GlcNAc side-chain substituents of GAC could cross-react with human tissue antigens, which would strongly limit vaccine development ( 26 , 106 ).…”