Extracellular structure of polysialic acid explored by on cell solution NMR

Area of Science:

• Biochemistry
• Microbiology
• Structural Biology

Background:

• The capsular polysaccharide alpha(2-->8) polysialic acid (PSA) from Neisseria meningitidis serogroup B and Escherichia coli K1 is poorly immunogenic in adult humans.
• Despite poor immunogenicity, specific serum antibodies are generated during infection, indicating a discrepancy in antigen presentation.

Purpose of the Study:

• To investigate the structural characteristics of cell-bound PSA in vivo to understand its poor immunogenicity.
• To characterize antigen structures directly on bacterial cells.

Main Methods:

• Development and application of on-cell multidimensional solution Nuclear Magnetic Resonance (NMR) spectroscopy.
• Utilized 13C, 15N-labeled PSA and heteronuclear NMR techniques (HSQC, HNCA, HNCO) for in vivo analysis on E. coli bacteria.

Main Results:

• Structural analysis confirmed that free and cell-bound PSA exhibit similar structures.
• NMR data indicated that the poor immunogenicity is not attributable to major structural differences between purified PSA and cell-associated PSA.
• Observed 2- to 3-fold larger 13C linewidths for PSA on cells compared to free PSA, suggesting dynamic differences.

Conclusions:

• The structural similarity between free and cell-bound PSA suggests that factors beyond gross structural differences contribute to PSA's low immunogenicity.
• The observed differences in linewidths may offer insights into the in vivo behavior and presentation of PSA.
• The study demonstrates the utility of on-cell NMR for in vivo kinetic studies of bacterial polysaccharides.