Role of Mg2+ and pH in the modification of Salmonella lipid A after endocytosis by macrophage tumour cells

Mol Microbiol. 2005 Jan;55(2):425-40. doi: 10.1111/j.1365-2958.2004.04409.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cell Line
  • Endocytosis*
  • Gene Expression Regulation, Bacterial*
  • Hydrogen-Ion Concentration
  • Lipid A / metabolism*
  • Macrophages / immunology
  • Macrophages / microbiology*
  • Magnesium / metabolism*
  • Mice
  • Phosphorus Radioisotopes / metabolism
  • Salmonella / genetics
  • Salmonella / growth & development*
  • Salmonella / metabolism

Substances

  • Bacterial Proteins
  • Lipid A
  • Phosphorus Radioisotopes
  • Magnesium