TY - JOUR
T1 - Role of Mg2+ and pH in the modification of Salmonella lipid A after endocytosis by macrophage tumour cells
AU - Gibbons, Henry S.
AU - Kalb, Suzanne R.
AU - Cotter, Robert J.
AU - Raetz, Christian R.H.
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2005/1
Y1 - 2005/1
N2 - 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 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 endosornes, 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-Mg 2+, mildly alkaline medium resulted in suppression of most lipid A modifications with the exception of the 2-hydroxymyristate in S. typhimurium. Although IpxO 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.
AB - 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 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 endosornes, 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-Mg 2+, mildly alkaline medium resulted in suppression of most lipid A modifications with the exception of the 2-hydroxymyristate in S. typhimurium. Although IpxO 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.
UR - http://www.scopus.com/inward/record.url?scp=13144306075&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=13144306075&partnerID=8YFLogxK
U2 - 10.1111/j.1365-2958.2004.04409.x
DO - 10.1111/j.1365-2958.2004.04409.x
M3 - Article
C2 - 15659161
AN - SCOPUS:13144306075
SN - 0950-382X
VL - 55
SP - 425
EP - 440
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 2
ER -