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Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. These structures have also gained interest as vaccine antigens, in particular for the human pathogens Group A Streptococcus (GAS) and Streptococcus mutans. Streptococcal cell wall glycopolymers are considered to be functional homologues of wall teichoic acids but surprisingly lack the biologically-relevant and characteristic anionic charge. Here we identify gacH, a gene of unknown function in the GAS Group A Carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA secreted phospholipase A2. To understand the underlying mechanism of these phenotypes, we determined the structure of the extracellular domain of GacH and discover that it represents a new family of glycerol phosphate (GroP) transferases. Importantly, we demonstrate the presence of GroP in both the GAC and the homologous Serotype c Carbohydrate (SCC) from S. mutans, which is conferred by gacH and sccH products, respectively. NMR analysis of GAC released from cell wall by non-destructive methods reveals that approximately 30% of the GAC GlcNAc side-chains are modified by GroP at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design.Graphical abstract

Carbon-13 NMR spin-lattice relaxation rates and NOES are reported for a-and y-cyclodextrin in solution at two temperatures and four magnetic fileds, 4.7,6.3,9.4, and 11.8 T. In addition, transverse relaxation rates are reported at 9.4 T. The data are interpreted using the "model-free" approach of Lipari and Szabo. All the ring carbons are considered to be equivalent in the dynamic sense, while the exocyclic hydroxymethyl groups are treated separately. The multiple-field relaxation of the ring carbons can be reproduced using a truncated two-parameter model, not explicitly including the local, rapid motion correlation time. The global correlation times, on the order of a few nanoseconds, and high order parameters are obtained for the ring carbons. Several procedures for fitting the hydroxymethyl carbon data are described and compared with each other. The order parameters for these carbons are lower, and it is possible to obtain some estimates of the local motion correlation times.

Multiple-field carbon-13 NMR relaxation measurements have been performed on the two vicinally disubstituted trisaccharides -D- (2). Trisaccharide 1 has trans disubstitution of the terminal -glucosyl residues, while trisaccharide 2 has cis disubstitution. Carbon-13 T 1 and T 2 relaxation and NOE measurements were performed outside the extreme-narrowing regime at three temperatures 283, 303, and 323 K and four magnetic field strengths 6.3, 9.4, 11.7, and 14.1 T. The obtained relaxation rates and NOE factors, averaged for each sugar residue, were fitted to overall and internal correlation times and order parameters using the Lipari-Szabo "model-free" formalism. The 2,3-disubstituted O-methyl glycoside showed fairly high-order parameters, S 2 ≈ 0.9, for both trisaccharides at all three temperatures, while the terminal glucosyl residues of 1 and 2 showed lower and slightly temperature-dependent order parameters, S 2 ≈ 0.8. In 1, the terminal residues were found to have similar motional properties and an internal correlation time of ∼60 ps could be obtained from a simultaneous three-parameter fit at 303 K. Similar results were obtained for trisaccharide 2. This study shows that the branch-point residue displays more restricted mobility than the terminal residues in both trisaccharides and that internal motions can be detected for small oligosaccharides using NMR relaxation measurements and the Lipari-Szabo model-free formalism.