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 |
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Authors: | Rick, PD Hubbard, GL Kitaoka, M Nagaki, H Kinoshita, T Dowd, S Simplaceanu, V Ho, C |
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Affiliation: | Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA. |
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Abstract: | The polysaccharide chains of enterobacterial common antigen (ECA) consistof 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, andGlcNAc is N -acetyl-d-glucosamine. The major form of ECA (ECAPG) consistsof polysaccharide chains that are believed to be covalently linked todiacylglycerol through phosphodiester linkage; the phospholipid moietyfunctions to anchor molecules in the outer membrane. The ECA trisacchariderepeat unit is assembled as a polyisoprenyl-linked intermediate which hasbeen tentatively identified as Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongationpresumably occurs by a block-polymerization mechanism. However, theidentity of the polyisoprenoid carrier-lipid has not been established.Accordingly, the current studies were conducted in an effort tostructurally characterize the polyisoprenyl lipid-carrier involved in ECAsynthesis. Isolation and characterization of the lipid carrier wasfacilitated by the accumulation of a ManNAcA-GlcNAc-pyrophosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonellatyphimurium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAcresidues for ECA synthesis. Analyses of lipid II preparations by fast atombombardment tandem mass spectroscopy (FAB-MS/MS) resulted in theidentification of the lipid-carrier as the 55-carbon polyisoprenyl alcohol,undecaprenol. These analyses also resulted in the identification of a novelglycolipid which copurified with lipid II. FAB-MS/MS analyses of thisglycolipid revealed its structure to be 1,2-diacyl- sn-glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP- disaccharide). Anexamination of purified ECAPGby phosphorus-31 nuclear magnetic resonancespectroscopy confirmed that the polysaccharide chains are linked todiacylglycerol through phosphodiester linkage. Thus, DGP-disaccharide doesnot appear to be an intermediate in ECAPGsynthesis. Nevertheless, althoughthe available evidence clearly indicate that lipid II is a precursor ofDGP-disaccharide, the function of this novel glycolipid is not yet known,and it may be an intermediate in the biosynthesis of a molecule other thanECAPG. |
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