Glycosylation of Wall Teichoic Acid in Staphylococcus aureus by TarM

Abstract

Wall teichoic acid (WTA) glycopolymers are major constituents of cell envelopes in Staphylococcus aureus and related Gram-positive bacteria with important roles in cell wall maintenance, susceptibility to antimicrobial molecules, biofilm formation, and host interaction. Most S. aureus strains express polyribitol phosphate WTA substituted with D-alanine and N-acetylglucosamine (GlcNAc). WTA sugar modifications are highly variable and have been implicated in bacteriophage susceptibility and immunogenicity, but the pathway and enzymes of staphylococcal WTA glycosylation have remained unknown. Revisiting the structure of S. aureus RN4220 WTA by NMR analysis revealed the presence of canonical polyribitol phosphate WTA bearing only ␣-linked GlcNAc substituents. A RN4220 transposon mutant resistant to WTA-dependent phages was identified and shown to produce altered WTA, which exhibited faster electrophoretic migration and lacked completely the WTA ␣-GlcNAc residues. Disruption of a gene of unknown function, renamed tarM, was responsible for this phenotype. Recombinant TarM was capable of glycosylating WTA in vitro in a UDP-GlcNAc-dependent manner, thereby confirming its WTA GlcNAc-transferase activity. Deletion of the last seven amino acids from the C terminus abolished the activity of TarM. tarM-related genes were found in the genomes of several WTA-producing bacteria, suggesting that TarM-mediated WTA glycosylation is a general pathway in Gram-positive bacteria. Our study represents a basis for dissecting the biosynthesis and function of glycosylated WTA in S. aureus and other bacteria.

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

“…These glycosyltransferases catalyze the addition either α- or β-linked GlcNAc, respectively, to the repeating phospho-ribitol unit. 29,34 Using these strains, we tested the ability of the panel of four antibodies to bind S. aureus that displayed WTA containing either α- or β-linked GlcNAc. Flow cytometry analysis showed that three antibodies, 4462, 4497 and 6078, bound the S. aureus USA300 ΔtarM , but not the S. aureus USA300 ΔtarS strain (Figure 2A-C).…”

Studies of the Genetics, Function, and Kinetic Mechanism of TagE, the Wall Teichoic Acid Glycosyltransferase in Bacillus subtilis 168

“…Previous efforts in characterizing wall teichoic acid glycosyltransferases have employed partially purified enzymes and uncharacterized membrane acceptors to follow enzyme activity (18,19). We purified recombinant B. subtilis 168 TagE to homogeneity and developed an HPLC-based assay to examine the wall teichoic acid glycosyltransferase activity of the enzyme with clean synthetic substrates in vitro.…”

“…Disruption of this gene led to wall teichoic acid that completely lacked N-acetylglucosamine. The wall teichoic acid glycosyltransferase activity of TarM was subsequently confirmed by demonstration that crude extracts containing recombinant enzyme catalyzed the transfer of N-acetylglucosamine onto an uncharacterized membrane acceptor in vitro (18). Glaser and Burger (19) conducted the first and only in vitro study of poly(glycerol phosphate) polymer glycosylation in B. subtilis nearly 50 years ago.…”

“…A similar approach involving phage-resistant mutants was recently used to identify the wall teichoic acid glycosyltransferase in S. aureus. An elegant transposon mutagenesis screen for resistance to phage 80 led to the isolation of tarM (18). Disruption of this gene led to wall teichoic acid that completely lacked N-acetylglucosamine.…”

Glycoepitopes of Staphylococcal Wall Teichoic Acid Govern Complement-mediated Opsonophagocytosis via Human Serum Antibody and Mannose-binding Lectin

“…The molecular pathways of WTA glycosylation in S. aureus were recently elucidated (24,25). Two WTA glycosyltransferases, TarM and TarS, are responsible for modifying WTA with ␣-GlcNAc and ␤-GlcNAc, respectively.…”