Role of Mg 2+ and pH in the modification of Salmonella lipid A after endocytosis by macrophage tumour cells

Summary 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 Mg 2+ 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 32 P i , 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 Mg 2+ concentrations of endosomes, we found that lipid A of S. typhimurium grown in an acidic, low‐Mg 2+ 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 Mg 2+ alone. Escherichia coli K‐12 W3110 modified its lipid A molecules in response to growth under acidic but not low‐Mg 2+ conditions. Growth in a high‐Mg 2+ , 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 Mg 2+ , 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.