The outer leaflet of the outer membranes of Gram-negative bacteria is covered with a remarkable glycolipid known as lipopolysaccharide (LPS). In Escherichia coli, the lipid A anchor of LPS is a hexa-acylated disaccharide of glucosamine, bearing phosphate groups at the 1 and 4' positions. The minimal LPS required for growth of E. coli contains lipid A and Kdo sugars. The biosynthesis of lipid A is well characterized. Inhibition of any one of the enzymes catalyzing the first seven steps of the pathway in E. coil causes cell death. Lipid A is therefore an interesting target for designing new antibacterial agents. Emerging genomic sequences of diverse bacteria indicate that these enzymes are present in virtually all Gram-negative organisms. An unanticipated genomic surprise, however, is that orthologs of key enzymes for lipid A biosynthesis are also present in higher plants. Lipid A (endotoxin) is the active component of LPS that stimulates immune cells. During severe Gram-negative infections, the lipid A moiety of LPS can cause excessive activation of macrophages and endothelial cells. The resulting systemic over-production of certain inflammatory mediators and clotting factors damages small blood vessels. A full response to endotoxin leads to Gram-negative septic shock with multiple organ failure and death. An exciting potential therapeutic approach to this problem has emerged with the discovery that certain lipid A-like molecules, including some precursors, are endotoxin antagonists. The primary signaling receptor for lipid A is now known to be the TLR4 protein, which is distantly related to the IL-1 receptor. In earlier work, the P. I. discovered the nine constitutive enzymes for lipid A assembly in E. coli, and the genes encoding them. In the proposed work, the specific aims are: I) elucidation of the biosynthesis of lipid A variants containing four amide-linked fatty acids; II) re-engineering of the lipid A pathway in living E. coli cells; III) characterization of new PmrA and PhoP regulated enzymes that modify lipid A; IV) studies of cold shock and high Ca++ induced lipid A modifications in E coli: and V) analysis of lipid flip-flop and export in E. coli.
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