title: Role of Mg2+ and pH in the modification of Salmonella lipid A after endocytosis by macrophage tumour cells.
abstract: Lipid A of Salmonella typhimurium is covalently modified with additional acyl and/or polar substituents in response to activation of the PhoP/PhoQ and/or PmrA/PmrB signalling systems, which are induced by growth at low Mg2+ concentrations and mild acid pH respectively. Although these conditions are thought to exist within macrophage phagolysosomes, no direct evidence for lipid A modification after endocytosis has been presented. To address this issue, we grew S. typhimurium inside RAW264.7 cells in the presence of 32Pi, and then isolated the labelled lipid A fraction, which was found to be extensively derivatized with phosphoethanolamine, aminoarabinose, 2-hydroxymyristate and/or palmitate moieties. S. typhimurium grown in tissue culture medium synthesized lipid A molecules lacking all these substituents with the exception of the 2-hydroxymyristate chain, which was still present. Using defined minimal media to simulate the intracellular pH and Mg2+ concentrations of endosomes, we found that lipid A of S. typhimurium grown in an acidic, low-Mg2+ medium closely resembled lipid A isolated from bacteria internalized by RAW264.7 cells. A subset of S. typhimurium lipid A modifications were induced by low Mg2+ alone. Escherichia coli K-12 W3110 modified its lipid A molecules in response to growth under acidic but not low-Mg2+ conditions. Growth in a high-Mg2+, mildly alkaline medium resulted in suppression of most lipid A modifications with the exception of the 2-hydroxymyristate in S. typhimurium. Although lpxO transcription was stimulated by growth on low Mg2+, the biosynthesis of lipid A species containing 2-hydroxymyristate was independent of PhoP/PhoQ and PmrA/PmrB in S. typhimurium. Our labelling methods should be applicable to studies of lipid A modifications induced by endocytosis of diverse bacteria.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Henry S
authors.initials: HS
authors.lastname: Gibbons
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Kalb
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Cotter
authors.forename: Christian R H
authors.initials: CR
authors.lastname: Raetz
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004705:Endocytosis; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D008050:Lipid A / Q000378:metabolism*; D008264:Macrophages / Q000276:immunology / Q000382:microbiology*; D008274:Magnesium / Q000378:metabolism*; D051379:Mice; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D012475:Salmonella / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008050:Lipid A; D010761:Phosphorus Radioisotopes; D008274:Magnesium