N‐Methylation in polylegionaminic acid is associated with the phase‐variable epitope of Legionella pneumophila serogroup 1 lipopolysaccharide

Abstract

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.

A structural comparison of lipopolysaccharide biosynthesis loci of Legionella pneumophila serogroup 1 strains

“…This supported the assumption that the mAb 3/1-specific epitope generated by the O -acetyltransferase Lag-1 masks the N -methyl group and hinders binding of mAb 10/6 [48]. This is in agreement with earlier observations which reported a correlation between ORF 8 and N -methylated legionaminic acid residues for the mAb 3/1 - strain RC1 [52]. However, the fact that mutants of both strains, 130b and Görlitz 6543, lost the reactivity with mAb 20/1, indicated that ORF 8 and/or ORF 11 are also involved in the generation or modification of another epitope which is not blocked by the O -acetyl group.…”

“…It is reported that the putative methyltransferase encoded by ORF 8 is responsible for N -methylation of the 5-acetimydyol amino group of the legionaminic acid [48,52]. This is supported by studies on the legionaminic acid pathway of Campylobacter .…”

“…However, recent transcriptomic data obtained from strain Paris revealed a four-gene operon which lacks ORF 8 [42]. For all strains regardless of the distance in the phylogenetic tree BLASTP predicted a methyltransferase function for ORF 8 [48,52] and a siliac acid synthetase function (neuB family) for ORF 9 [21]. …”

Virulence Properties of the Legionella Pneumophila Cell Envelope

“… Chemical structure of L. pneumophila LPS (modified from Kooistra et al, 2002b) . Structure indicates its various regions: O-specific chain, core region consisting of the outer core and inner core and lipid A.…”

Targeted Metabolomics Analysis of Campylobacter coli VC167 Reveals Legionaminic Acid Derivatives as Novel Flagellar Glycans

“…NMR experiments performed in 95% H 2 O and at lower pH (pH 3.7) revealed exchangeable NH resonances and confirmed the location of the N-methylated acetamidino group at C5 (data not shown). By comparing carbon chemical shifts to those reported for the N-methylated acetamidino structures in Legionella pneumophila (36), the 5NAm…”