The pgdA Gene Encodes for a PeptidoglycanN-Acetylglucosamine Deacetylase in Streptococcus pneumoniae

Abstract

Analytical work on the fractionation of the glycan strands of Streptococcus pneumoniae cell wall has led to the observation that an unusually high proportion of hexosamine units (over 80% of the glucosamine and 10% of the muramic acid residues) was not N-acetylated, explaining the resistance of the peptidoglycan to the hydrolytic action of lysozyme, a muramidase that cleaves in the glycan backbone. A gene, pgdA, was identified as encoding for the peptidoglycan N-acetylglucosamine deacetylase A with amino acid sequence similarity to fungal chitin deacetylases and rhizobial NodB chitooligosaccharide deacetylases. Pneumococci in which pgdA was inactivated by insertion duplication mutagenesis produced fully N-acetylated glycan and became hypersensitive to exogenous lysozyme in the stationary phase of growth. The pgdA gene may contribute to pneumococcal virulence by providing protection against host lysozyme, which is known to accumulate in high concentrations at infection sites.The unusual complexity and diversity of the cell surface of Streptococcus pneumoniae is apparent both in the capsular structure and in the underlying cell wall structure of this bacterium, and this complexity and diversity may be related to the multiplicity of interactions of this microbe and its human host. S. pneumoniae is capable of producing at least 90 chemically distinct capsular polysaccharides (1). The cell wall of this microorganism contains a teichoic acid of unusually complex chemistry (2, 3) the components of which include ribitol phosphate, galactosamine, trideoxydiaminohexose, and covalently linked phosphocholine residues (4). The peptidoglycan of S. pneumoniae is also unusual because its stem peptides are cross-linked in both a direct and an indirect manner (5). Furthermore, the proportion of distinct linear and branched muropeptides in the peptidoglycan is clonally related (6). The pneumococcal cell wall is a potential target for components of the first line host defense such as lysozyme. In addition, the peptidoglycan and teichoic acid may represent bacterial ligands recognized by the innate immune system of the host.The recent introduction of high resolution analytical techniques and genetic approaches began to shed light on the determinants and biological functions of the pneumococcal cell wall. In this study we describe the identification of a genetic determinant, pgdA, of the first bacterial peptidoglycan GlcNAc deacetylase. We show that the innate activity of this enzyme is responsible for the high proportion of non-acetylated hexosamine residues in the peptidoglycan that appears to play a role in the resistance of S. pneumoniae to the activity of exogenous lysozyme, an enzyme that is known to accumulate in high concentrations at infection sites. EXPERIMENTAL PROCEDURESBacterial Strains, Plasmids, and Growth Media-Cultures of S. pneumoniae R36A, a non-encapsulated laboratory strain from the Rockefeller University collection, were grown in a casein-based semi-synthetic medium (C ϩ Y) containing 1 mg/ml yeast extract (7) o...

Structure and metal-dependent mechanism of peptidoglycan deacetylase, a streptococcal virulence factor

“…SpPgdA Deacetylates GlcNAc3 on the Middle Sugar. SpPgdA has been shown to deacetylate GlcNAc residues in native peptidoglycan (4). Here, GlcNAc 3 is used as a pseudosubstrate, with production of free amine fitting a first-order reaction rate (Fig.…”

“…One example of this is the de-N-acetylation of the GlcNAc residues. Recent work has identified the protein responsible for this deacetylation step in the pathogen Streptococcus pneumoniae (4). S. pneumoniae GlcNAc peptidoglycan deacetylase (SpPgdA) was found to de-N-acetylate GlcNAc sugars in pneumococcal peptidoglycan, with overall deacetylation levels up to 80% (4).…”

Oxidative Stress-induced Peptidoglycan Deacetylase in Helicobacter pylori

“…RHA1, Corynebacterium efficiens, Rhodobacter sphaeroides, and Mycobacterium smegmatis; however, in none of them has this protein been studied. One member of the bacterial polysaccharide deacetylase family proteins that has been well characterized is the peptidoglycan deacetylase (PgdA) from Steptococcus pneumoniae (26). A small region (47 amino acids) at the N terminus of HP310 shows 36% identity with the catalytic C-terminal domain of PgdA protein (Fig.…”

“…2). PgdA protein was shown to be the primary enzyme responsible for deacetylation of the hexosamine residues of the peptidoglycan in S. pneumoniae (26).…”

“…Chemical N-Acetylation of Peptidoglycan-Portions of the peptidoglycan preparations were resuspended by sonication as described above and subjected to N-acetylation, using saturated NaHCO 3 and 5% acetic anhydride essentially as described (26). The N-acetylated peptidoglycan was recovered by centrifugation (14,000 ϫ g, 3 min) and then subjected to lysozyme digestion, borodeuteride reduction, workup, and HPLC analysis as described above.…”