N-Methylation in polylegionaminic acid is associated with the phase-variable epitope of Legionella pneumophila serogroup 1 lipopolysaccharide. Identification of 5-(N,N-dimethylacetimidoyl)amino and 5-acetimidoyl(N-methyl)amino-7-acetamido-3,5,7,9-tetradeoxynon-2-ulosonic acid in the O-chain polysaccharide.
Previously, a phase-variable epitope was detected in the virulent wild-type strain RC1 of Legionella pneumophila serogroup 1 subgroup OLDA using a lipopolysaccharide-specific monoclonal antibody, mAb 2625 [Lüneberg, E., Zähringer, U., Knirel, Y. A., Steinmann, D., Hartmann, M., Steinmetz, I., Rohde, M., Kohl, J. & Frosch, M. (1998) J.Exp. Med. 188, 49-60]. In the present study, an isogenic mutant strain, termed 5215, was constructed by deletion of genes involved in the biosynthesis of the mAb 2625 epitope. Mutant 5215 was as virulent as the parental wild-type RC1 but did not bind mAb 2625. The two strains showed no difference in the core oligosaccharide and lipid A but in the O-chain polysaccharide structure, which is a homopolymer of 5-acetimidoylamino-7-acetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic acid (a derivative of legionaminic acid). NMR spectroscopic studies revealed a hitherto unknown modification of bacterial polysaccharides in the wild-type strain, namely N-methylation of the 5-acetimidoylamino group on a single legionaminic acid residue that is located, most likely, proximal to the core oligosaccharide. Two major N-methylated substituents, the (N,N-dimethylacetimidoyl)amino and acetimidoyl(N-methyl) amino groups, could be allocated to the long- and middle-chain O-polysaccharide species, respectively. N-Methylation of legionaminic acid that was absent from the isogenic mutant 5215 and from the spontaneous phase variant 811, correlated with the presence of the mAb 2625 epitope.
Kooistra
Oliver
O
Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany.
Lüneberg
Edeltraud
E
Knirel
Yuriy A
YA
Frosch
Matthias
M
Zähringer
Ulrich
U
D015153:Blotting, Western; D002849:Chromatography, Gas; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry*; D016952:Legionella pneumophila / Q000737:chemistry*; D008745:Methylation; D019906:Nuclear Magnetic Resonance, Biomolecular; D019081:O Antigens / Q000737:chemistry*; D011135:Polysaccharides, Bacterial / Q000737:chemistry*
D000939:Epitopes; D019081:O Antigens; D011135:Polysaccharides, Bacterial