Extracellular structure of polysialic acid explored by on cell solution NMR.
The capsular polysaccharide of the pathogens Neisseria meningitidis serogroup B and of Escherichia coli K1, alpha(2 --> 8) polysialic acid (PSA), is unusual, because when injected into adult humans, it generates little or no antibody. In contrast, people infected with these pathogens generate specific serum antibodies. A structural study on cells is used to address this anomaly by characterizing antigen structures in vivo. We introduce on cell multidimensional solution NMR spectroscopy for direct observation of PSA on E. coli bacteria. Using 13C,15N-labeled PSA, we applied a combination of heteronuclear NMR methods, such as heteronuclear single quantum coherence, HNCA, and HNCO, in vivo. Analysis reveals that free and cell-bound PSA are structurally similar, indicating that the poor immunogenicity of PSA is not due to major structural differences between cells and purified PSA. The 13C linewidths of PSA on cells are 2 to 3 times larger than the corresponding ones in free PSA. The possible implications of the differences between free and on cell PSA are discussed. In addition, we demonstrate the suitability of the method for in vivo kinetic studies.
Azurmendi
Hugo F
HF
Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 20852-1448, USA.
Vionnet
Justine
J
Wrightson
Lauren
L
Trinh
Loc B
LB
Shiloach
Joseph
J
Freedberg
Darón I
DI
D048168:Escherichia coli K12 / Q000737:chemistry*; D005110:Extracellular Space / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D012794:Sialic Acids / Q000737:chemistry*; D012996:Solutions
D012794:Sialic Acids; D012996:Solutions; C021319:polysialic acid