LIPOPOLYSACCHARIDE ENDOTOXINSChristian R. H. Raetz1and Chris Whitfield21Department of Biochemistry, Duke University Medical Center, Durham, NorthCarolina 27710; e-mail: [email protected] of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada;e-mail: [email protected] Words lipid A biosynthesis, Gram-negative bacteria, outer membranes,TLR4, LpxC, MsbAf Abstract Bacterial lipopolysaccharides (LPS) typically consist of a hydropho-bic domain known as lipid A (or endotoxin), a nonrepeating “core” oligosaccharide,and a distal polysaccharide (or O-antigen). Recent genomic data have facilitatedstudy of LPS assembly in diverse Gram-negative bacteria, many of which are humanor plant pathogens, and have established the importance of lateral gene transfer ingenerating structural diversity of O-antigens. Many enzymes of lipid A biosynthesislike LpxC have been validated as targets for development of new antibiotics. Keygenes for lipid A biosynthesis have unexpectedly also been found in higher plants,indicating that eukaryotic lipid A-like molecules may exist. Most significant has beenthe identification of the plasma membrane protein TLR4 as the lipid A signalingreceptor of animal cells. TLR4 belongs to a family of innate immunity receptors thatpossess a large extracellular domain of leucine-rich repeats, a single trans-membranesegment, and a smaller cytoplasmic signaling region that engages the adaptor proteinMyD88. The expanding knowledge of TLR4 specificity and its downstream signalingpathways should provide new opportunities for blocking inflammation associatedwith infection.CONTENTSENDOTOXINS AS ACTIVATORS OF INNATE IMMUNITY ............636LIPID A BIOSYNTHESIS IN ESCHERICHIA COLI AND SALMONELLATYPHIMURIUM........................................641The Constitutive Lipid A (Endotoxin) Pathway.....................641Regulated Pathways for the Covalent Modification of Lipid A ...........643Origin of L-Ara4N-Modified Lipid A in Polymyxin Resistant Mutants ......647ROLE OF THE ABC TRANSPORTER MsbA IN LIPID A AND PHOSPHOLIPIDEXPORT ............................................648GENOMIC INSIGHTS INTO LIPID A BIOSYNTHESIS AND DIVERSITY . . . 651The Constitutive Lipid A Pathway As A Target For New Antibiotics .......651The Constitutive Pathway in Bacteria with Unusual Lipid A Structures ......653Presence of Lipid A Biosynthesis Genes in Plants ...................654Annu. Rev. Biochem. 2002. 71:635–700DOI: 10.1146/annurev.biochem.71.110601.135414Copyright © 2002 by Annual Reviews. All rights reservedFirst published as a Review in Advance on April 30, 20026350066-4154/02/0707-0635$14.00STRUCTURE AND BIOSYNTHESIS OF CORE OLIGOSACCHARIDES .....655Structure of Core Oligosaccharides ............................655Role of Core in Outer-Membrane Stability: The Deep-Rough Phenotype .....661Genetics and Biosynthesis of Core Oligosaccharide ..................662Assembly of the Inner Core ................................665Assembly of the Outer Core ................................668Ligation of O Polysaccharide to Lipid A-Core Acceptor ...............669Contributions of Core Biosynthesis to LPS Heterogeneity ..............670Phase Variation and the Biosynthesis of Lipooligosaccharide ............671STRUCTURE AND BIOSYNTHESIS OF O POLYSACCHARIDES.........672Structure of O Polysaccharides...............................672Biosynthesis of O Polysaccharides ............................674Initiation Reactions......................................675The Wzy-Dependent Pathway ...............................676The ABC-Transporter-Dependent Pathway........................681The Synthase-Dependent Pathway.............................684Seroconversion Reactions ..................................685EXPORT OF LPS TO THE CELL SURFACE ......................688FUTURE DIRECTIONS FOR LPS RESEARCH .....................689ENDOTOXINS AS ACTIVATORS OF INNATEIMMUNITYLipid A (endotoxin), the hydrophobic anchor of lipopolysaccharide (LPS), is aglucosamine-based phospholipid that makes up the outer monolayer of the outermembranes of most Gram-negative bacteria (1–5). There are ⬃106lipid Aresidues and ⬃107glycerophospholipids in a single cell of Escherichia coli (6).The minimal LPS required for the growth of E. coli consists of the lipid A andKdo (3-deoxy-D-manno-oct-2-ulosonic acid) domains (Figures 1, 2) (1, 7, 8). Inwild-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, thesehelp bacteria resist antibiotics, the complement system, and other environmentalstresses.Many Gram-negative bacteria, including pathogens, synthesize lipid A speciesresembling the one found in E. coli (Figure 2) (1, 3, 4). Early ambiguitiesconcerning the structure of lipid A have generally been resolved [see (1, 3, 4)].Given their conserved architecture, most types of lipid A molecules are detectedat picomolar levels by an ancient receptor of the innate immune system presenton macrophages and endothelial animal cells (12, 13). The receptor, recentlyidentified as TLR4 (toll-like receptor 4) (14, 15), is a membrane-spanning proteinthat is distantly related to the IL1 receptor (12, 13).In macrophages, lipid A activation of TLR4 triggers the biosynthesis ofdiverse mediators of inflammation, such as TNF-␣and IL1-␤(16, 17), andactivates the production of costimulatory molecules required for the adaptive636 RAETZ y WHITFIELDFigure 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 growthof E. coli and most other Gram-negative bacteria (2). Exceptions to this general rule include certain spirochetes in which all lipid Abiosynthesis genes are absent (141), Thermotoga maritima (137), and Neisseria meningitidis Type B in which lipid A-deficient lpxAknockouts can be constructed (133), provided the polysialic acid capsule is present (134a).637LIPOPOLYSACCHARIDE ENDOTOXINS638 RAETZ y WHITFIELDimmune response (13). In mononuclear and endothelial cells, lipid A alsostimulates tissue factor production (18, 19). These events are desirable forclearing local infections, and they act in synergy. When overproduced systemi-cally in the setting of severe sepsis, however, the various mediators and clottingfactors can damage small blood vessels and precipitate Gram-negative septicshock, accompanied by disseminated intravascular coagulation and multipleorgan failure