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A novel biological synthesis and purification of lipid A precursors
Author(s) -
Metzger Louis Eugene,
Raetz Christian R. H.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.876.1
Subject(s) - lipid a , lipopolysaccharide , bacterial outer membrane , lipid ii , bacteria , biochemistry , innate immune system , chemistry , gram negative bacteria , toll like receptor , biology , microbiology and biotechnology , receptor , peptidoglycan , enzyme , escherichia coli , gene , immunology , genetics
Gram‐negative bacteria possess an asymmetric outer membrane in which the outer leaflet contains both phospholipids and lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram‐negative bacteria from antibiotics and other environmental stressors. The lipid A anchor of LPS is a glucosamine‐based saccharolipid that is further modified with core and O‐antigen sugars. In addition to serving a structural role as the hydrophobic anchor of LPS, lipid A is recognized by the innate immune system in animal cells and macrophages. In macrophages, lipid A has been found to stimulate TLR4 (toll like receptor 4), which in turn activates the inflammatory cascade. Given lipid A's role in both pathogen viability and virulence, the genetic engineering of Gram‐negative bacteria to produce non‐canonical lipid A may play an important role in the development of attenuated vaccine strains. Here, we engineer E. coli to produce a non‐canonical lipid A precursor, 2,3‐diamino‐2,3‐dideoxy‐D‐glucopyranose lipid X. We then purify this compound from bacterial homogenate, verify its structure, and evaluate the specificity of various orthologues of LpxB, the bacterial lipid A disaccharide synthase, toward this non‐canonical substrate. This study presents the first biological synthesis and purification of this lipid A precursor. Funding: NIH 5R01GM0531310‐15 to CR and NSF GRF2005029353 to LM.