Annu Rev Biochem 2002 71 635 700 DOI 10 1146 annurev biochem 71 110601 135414 Copyright 2002 by Annual Reviews All rights reserved First published as a Review in Advance on April 30 2002 LIPOPOLYSACCHARIDE ENDOTOXINS Christian R H Raetz1 and Chris Whitfield2 1 Department of Biochemistry Duke University Medical Center Durham North Carolina 27710 e mail raetz biochem duke edu 2 Department of Microbiology University of Guelph Guelph Ontario N1G 2W1 Canada e mail cwhitfie uoguelph ca Key Words lipid A biosynthesis Gram negative bacteria outer membranes TLR4 LpxC MsbA f Abstract Bacterial lipopolysaccharides LPS typically consist of a hydrophobic domain known as lipid A or endotoxin a nonrepeating core oligosaccharide and a distal polysaccharide or O antigen Recent genomic data have facilitated study of LPS assembly in diverse Gram negative bacteria many of which are human or plant pathogens and have established the importance of lateral gene transfer in generating structural diversity of O antigens Many enzymes of lipid A biosynthesis like LpxC have been validated as targets for development of new antibiotics Key genes for lipid A biosynthesis have unexpectedly also been found in higher plants indicating that eukaryotic lipid A like molecules may exist Most significant has been the identification of the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells TLR4 belongs to a family of innate immunity receptors that possess a large extracellular domain of leucine rich repeats a single trans membrane segment and a smaller cytoplasmic signaling region that engages the adaptor protein MyD88 The expanding knowledge of TLR4 specificity and its downstream signaling pathways should provide new opportunities for blocking inflammation associated with infection CONTENTS ENDOTOXINS AS ACTIVATORS OF INNATE IMMUNITY LIPID A BIOSYNTHESIS IN ESCHERICHIA COLI AND SALMONELLA TYPHIMURIUM The Constitutive Lipid A Endotoxin Pathway Regulated Pathways for the Covalent Modification of Lipid A Origin of L Ara4N Modified Lipid A in Polymyxin Resistant Mutants ROLE OF THE ABC TRANSPORTER MsbA IN LIPID A AND PHOSPHOLIPID EXPORT GENOMIC INSIGHTS INTO LIPID A BIOSYNTHESIS AND DIVERSITY The Constitutive Lipid A Pathway As A Target For New Antibiotics The Constitutive Pathway in Bacteria with Unusual Lipid A Structures Presence of Lipid A Biosynthesis Genes in Plants 0066 4154 02 0707 0635 14 00 636 641 641 643 647 648 651 651 653 654 635 636 RAETZ y WHITFIELD STRUCTURE AND BIOSYNTHESIS OF CORE OLIGOSACCHARIDES Structure of Core Oligosaccharides Role of Core in Outer Membrane Stability The Deep Rough Phenotype Genetics and Biosynthesis of Core Oligosaccharide Assembly of the Inner Core Assembly of the Outer Core Ligation of O Polysaccharide to Lipid A Core Acceptor Contributions of Core Biosynthesis to LPS Heterogeneity Phase Variation and the Biosynthesis of Lipooligosaccharide STRUCTURE AND BIOSYNTHESIS OF O POLYSACCHARIDES Structure of O Polysaccharides Biosynthesis of O Polysaccharides Initiation Reactions The Wzy Dependent Pathway The ABC Transporter Dependent Pathway The Synthase Dependent Pathway Seroconversion Reactions EXPORT OF LPS TO THE CELL SURFACE FUTURE DIRECTIONS FOR LPS RESEARCH 655 655 661 662 665 668 669 670 671 672 672 674 675 676 681 684 685 688 689 ENDOTOXINS AS ACTIVATORS OF INNATE IMMUNITY Lipid A endotoxin the hydrophobic anchor of lipopolysaccharide LPS is a glucosamine based phospholipid that makes up the outer monolayer of the outer membranes of most Gram negative bacteria 1 5 There are 106 lipid A residues and 107 glycerophospholipids in a single cell of Escherichia coli 6 The minimal LPS required for the growth of E coli consists of the lipid A and Kdo 3 deoxy D manno oct 2 ulosonic acid domains Figures 1 2 1 7 8 In wild type strains additional core and O antigen sugars may be present Figure 1 5 7 9 11 Although generally not required for growth in the laboratory these help bacteria resist antibiotics the complement system and other environmental stresses Many Gram negative bacteria including pathogens synthesize lipid A species resembling the one found in E coli Figure 2 1 3 4 Early ambiguities concerning the structure of lipid A have generally been resolved see 1 3 4 Given their conserved architecture most types of lipid A molecules are detected at picomolar levels by an ancient receptor of the innate immune system present on macrophages and endothelial animal cells 12 13 The receptor recently identified as TLR4 toll like receptor 4 14 15 is a membrane spanning protein that is distantly related to the IL1 receptor 12 13 In macrophages lipid A activation of TLR4 triggers the biosynthesis of diverse mediators of inflammation such as TNF and IL1 16 17 and activates the production of costimulatory molecules required for the adaptive Figure 1 Model of the inner and outer membranes of E coli K 12 Only the Kdo and lipid A regions of LPS are required for the growth of E coli and most other Gram negative bacteria 2 Exceptions to this general rule include certain spirochetes in which all lipid A biosynthesis genes are absent 141 Thermotoga maritima 137 and Neisseria meningitidis Type B in which lipid A deficient lpxA knockouts can be constructed 133 provided the polysialic acid capsule is present 134a LIPOPOLYSACCHARIDE ENDOTOXINS 637 638 RAETZ y WHITFIELD LIPOPOLYSACCHARIDE ENDOTOXINS 639 immune response 13 In mononuclear and endothelial cells lipid A also stimulates tissue factor production 18 19 These events are desirable for clearing local infections and they act in synergy When overproduced systemically in the setting of severe sepsis however the various mediators and clotting factors can damage small blood vessels and precipitate Gram negative septic shock accompanied by disseminated intravascular coagulation and multiple organ failure 20 23 Synthetic E coli lipid A by itself causes a similar spectrum of effects when injected into animals 3 24 supporting the proposed role of lipid A in Gram negative sepsis The characteristic structural features of E coli lipid A Figure 2 especially its two phosphate groups and its two acyloxyacyl moieties are needed to trigger the endotoxin response in human cells 3 25 27 Many of the initial events in the interaction of lipid A with animal cells have been elucidated in the past ten years 14 15 28 29 A special lipid transfer protein in plasma delivers lipid A from bacteria or bacterial membrane fragments to CD14 on the surfaces