title: 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.
abstract: 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-dideoxy-d-galactose, ManNAcA is N -acetyl-d- mannosaminuronic acid, and GlcNAc is N -acetyl-d-glucosamine. The major form of ECA (ECAPG) 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 is assembled as a polyisoprenyl-linked intermediate which has been tentatively identified as Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongation presumably occurs by a block-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 ManNAcA-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 the lipid-carrier as the 55-carbon polyisoprenyl alcohol, undecaprenol. These analyses also 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 ECAPGby 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 ECAPGsynthesis. 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 ECAPG.
authors.affiliation: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
authors.forename: P D
authors.initials: PD
authors.lastname: Rick
authors.forename: G L
authors.initials: GL
authors.lastname: Hubbard
authors.forename: M
authors.initials: M
authors.lastname: Kitaoka
authors.forename: H
authors.initials: H
authors.lastname: Nagaki
authors.forename: T
authors.initials: T
authors.lastname: Kinoshita
authors.forename: S
authors.initials: S
authors.lastname: Dowd
authors.forename: V
authors.initials: V
authors.lastname: Simplaceanu
authors.forename: C
authors.initials: C
authors.lastname: Ho
mesh_terms: D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000737:chemistry*; D002240:Carbohydrate Sequence; D004755:Enterobacteriaceae / Q000276:immunology; D005994:Glycerophosphates / Q000032:analysis / Q000737:chemistry*; D006017:Glycolipids / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010759:Phosphorus Isotopes; D012486:Salmonella typhimurium / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D016339:Spectrometry, Mass, Fast Atom Bombardment
chemical_list: D000942:Antigens, Bacterial; D005994:Glycerophosphates; D006017:Glycolipids; D010759:Phosphorus Isotopes; C017286:enterobacterial common antigen