Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis

The polysaccharide chains of enterobacterial common antigen (ECA) consist of linear trisaccharide repeat units with the structure -->3)-alpha-D-Fuc4NAc-(1-->4)-beta-D-ManNAcA-(1-->4)-alpha-D-GlcNAc-(1-->, where Fuc4NAc is 4-acetamido-4,6-di-deoxy-D-galactose, ManNAcA is N-acetyl-D- mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. The major form of ECA (ECA(PG)) consists of polysaccharide chains that are believed to be covalently linked to diacylglycerol through phosphodiester linkage; the phospholipid moiety functions to anchor molecules in the outer membrane. The ECA trisaccharide repeat unit has been tentatively identified as Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongation presumably occurs by a blocking-polymerization mechanism. However, the identity of the polyisoprenoid carrier-lipid has not been established. Accordingly, the current studies were conducted in an effort to structurally characterize the polyisoprenyl lipid-carrier involved in ECA synthesis. Isolation and characterization of the lipid carrier was facilitated by the accumulation of a ManNA-cA-GlcNAc-pyrophosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonella typhimurium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA synthesis. Analyses of lipid II preparations by fast atom bombardment tandem mass spectroscopy (FAB-MS/MS) resulted in the identification of a novel glycolipid which copurified with lipid II. FAB-MS/MS analyses of this glycolipid revealed its structure to be 1,2-diacyl-sn-glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP-disaccharide). An examination of purified ECA(PG) by phosphorus-31 nuclear magnetic resonance spectroscopy confirmed that the polysaccharide chains are linked to diacylglycerol through phosphodiester linkage. Thus, DGP-disaccharide does not appear to be an intermediate in ECA(PG) synthesis. Nevertheless, although the available evidence clearly indicate that lipid II is a precursor of DGP-disaccharide, the function of this novel glycolipid is not yet known, and it may be an intermediate in the biosynthesis of a molecule other than ECA(PG).