Influence of the length of the lipooligosaccharide alpha chain on its sialylation in Neisseria meningitidis

Abstract

The sialylation of lipooligosaccharide (LOS) in Neisseria meningitidis plays a role in the resistance of the organism to killing by normal human serum. The length of the alpha chain extending out from the heptose I [Hep (I)] moiety of LOS influenced sialylation of N. meningitidis LOS in vitro and in vivo. The alpha chain required a terminal Gal and a trisaccharide or longer oligosaccharide to serve as an acceptor for sialylation. The disaccharide lactose (Galbeta1-4Glc) in the alpha chain of immunotype L8 LOS could not function as an acceptor for the sialyltransferase, probably due to steric hindrance imposed by the neighboring Hep (II) with phosphorylethanolamine and another group attached.

FIG. 1.

Schematic presentation of N. meningitidis LOS. The α chains of L2, L3, L4, L5, L7, and L9 LOSs contain LNnT structures (14). The alternative α chain of L1 LOS is a trisaccharide, Galα1-Galβ1-Glc. The L6 LOS lacks the terminal Gal in the LNnT structure, and the L8 LOS is a lactosyl disaccharide (20). The terminal Gals of LNnT and the L1 trisaccharide are often partially sialylated. Ac, acetyl residue.

FIG. 2.

Sialylation of purified LOSs from N. meningitidis strains grown in Catlin medium. (A) Silver-stained LOSs after SDS-PAGE; (B) LFA blot for detecting sialylated LOSs; (C) MAL blot for detecting α2,3-linked sialic acid. Lanes 1, M986 LOS markers with a pair of doublets (the upper 4.1-kDa component is sialylated and the lower 4-kDa component is nonsialylated); lanes 2 and 2A, nonsialylated 126E (L1) LOS without and with addition of CMP-NeuNAc, respectively; lanes 3 and 3A, M986NCV (L3,7); lanes 4 and 4A, A1 (L8); lanes 5 and 5A, 7880 (L10); lanes 6, the control with 126E STase and CMP-NeuNAc only. Sample loads were 100 ng of LOS for silver staining (23) and double that amount for lectin blots. The arrows indicate the location of the sialylated 4.1-kDa LOS; the asterisk indicates the location of the nonsialylated 3.6-kDa L8 LOS.

FIG. 3.

Sialylation of LOS in N. meningitidis grown in TSB with and without CMP-NeuNAc. (A) Silver-stained LOSs on SDS-PAGE; (B) LFA blot for detecting sialylated LOSs; (C) MAL blot for detecting α2,3-linked sialic acid. Lanes 1, M986 LOS markers as in Fig. 2; lanes 2 and 2A, 126E (C:L1) strain grown in the absence and presence of 200 μg of CMP-NeuNAc/ml, respectively; lanes 3 and 3A, strain M986-NCV (L3,7); lanes 4 and 4A, strain A1 (A:L8); lanes 5 and 5A, strain 7880 (A:L10); lanes 6 and 6A, strain M978 (B:L8). The arrows and asterisks are as described in the legend of Fig. 2.