The lipid A moiety of lipopolysaccharide forms the outer monolayer of the outer membrane of most gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes. Following attachment of the core oligosaccharide, nascent core-lipid A is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Diverse covalent modifications of the lipid A moiety may occur during its transit from the outer surface of the inner membrane to the outer membrane. Lipid A modification enzymes are reporters for lipopolysaccharide trafficking within the bacterial envelope. Modification systems are variable and often regulated by environmental conditions. Although not required for growth, the modification enzymes modulate virulence of some gram-negative pathogens. Heterologous expression of lipid A modification enzymes may enable the development of new vaccines.

The LIPID MAPS Consortium (www.lipidmaps. org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo) 2 -Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo 2 -Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and 1 H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo 2 -Lipid A is comparable to LPS by these criteria. Its activity is reduced by . The LIPID MAPS consortium is developing quantitative methods for evaluating the composition, biosynthesis, and function of all macrophage lipids (1). These amphipathic substances not only are structural components of biological membranes but also play important roles in the pathophysiology of inflammation, atherosclerosis, and growth control. Additional lipid functions should emerge from the comprehensive analysis of macrophage lipids. Electrospray ionization/mass spectrometry (ESI/MS) (2, 3), coupled with prefractionation methods like reversephase liquid chromatography (LC), is being applied systematically to set the stage for the seamless integration of lipid metabolism into the broader fields of genomics, proteomics, and systems biology. To facilitate this endeavor, LIPID MAPS has introduced a new comprehensive classification system for biological lipids, amenable to computer-based data processing and substructure comparison (4). The eight LIPID MAPS categories are 1) fatty acyls, 2) glycerolipids, 3) glycerophospholipids, 4) sphingolipids, 5) sterol lipids, 6) prenol lipids, 7) saccharolipids,

Stoichiometric amounts of diacylglycerol were generated during the EptB-catalyzed transfer of pEtN to Kdo 2 -lipid A. EptB is an inner membrane protein of 574 amino acid residues with five predicted trans-membrane segments within its N-terminal region. An in-frame replacement of eptB with a kanamycin resistance cassette rendered E. coli WBB06 (but not wild-type W3110) hypersensitive to CaCl 2 at 5 mM or higher. Ca 2؉ hypersensitivity was suppressed by excess Mg 2؉ in the medium or by restoring the LPS core of WBB06. The latter was achieved by reintroducing the waaC and waaF genes, which encode LPS heptosyl transferases I and II, respectively. Our data demonstrate that pEtN modification of the outer Kdo protected cells containing heptose-deficient LPS from damage by high concentrations of Ca 2؉ . Based on its sequence similarity to EptA(PmrC), we propose that the active site of EptB faces the periplasmic surface of the inner membrane.The envelope of Gram-negative bacteria consists of an inner membrane (1, 2), a peptidoglycan cell wall (3), and an outer membrane (4). The latter is an asymmetric bilayer with glycerophospholipids on its inner surface and lipopolysaccharide (LPS) 1 on the outside surface (5, 6). LPS consists of three covalently linked portions (6, 7) as follows: 1) the lipid A moiety, a glucosamine-based saccharolipid 2 that serves as the hydrophobic membrane anchor of LPS; 2) the core region, a nonrepeating oligosaccharide modified with phosphate-containing substituents; and 3) the O-antigen, a distal repeating oligosaccharide, which is absent in most strains of Escherichia coli K12 (6). The lipid A moiety and the 3-deoxy-D-manno-octulosonic acid (Kdo) residues of the core are essential for growth of E. coli and most other Gram-negative bacteria (6). Strains lacking all LPS sugars distal to Kdo are termed heptose-deficient or "deep rough" (6, 8). These mutants are viable under laboratory conditions (9), but are hypersensitive to serum and antibiotics (4), and often show reduced virulence in animal models (7).Under certain conditions, strains of E. coli and Salmonella synthesize LPS molecules modified with a phosphoethanolamine (pEtN) group at position 7 of the outer Kdo residue (9) (Fig. 1). Brabetz et al. (9) have reported that E. coli WBB06, which harbors a deletion spanning the heptosyl transferase genes waaC(rfaC) and waaF(rfaF), contains heptose-deficient LPS, modified with pEtN at position 7 of the outer Kdo sugar. Kanipes et al. (10) later demonstrated that pEtN addition to LPS in WBB06 is a consequence of the presence of Ca 2ϩ in the growth medium used by Brabetz et al. (9) and is unrelated to the deletion of the heptosyl transferase genes. A pEtN transferase activity is present in membranes of WBB06 grown in the presence of 5-50 mM Ca 2ϩ (10). The enzyme is stimulated by exogenous phosphatidylethanolamine (PE) and is selective for the outer Kdo residue of Kdo 2 -lipid A and related substrates (10). Addition of EDTA to the in vitro pEtN transferase assay was found to be inhibitory.We have...

The Salmonella and related bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental stimuli. Some lipid A modifications are required for virulence and resistance to cationic antimicrobial peptides. We now demonstrate that membranes of Salmonella typhimurium contain a novel hydrolase that removes the 3′-acyloxyacyl residue of lipid A in the presence of 5 mM Ca 2+ . We have identified the gene encoding the S. typhimurium lipid A 3′-O-deacylase, designated lpxR, by screening an ordered S. typhimurium genomic DNA library, harbored in Escherichia coli K-12, for expression of Ca 2+ -dependent 3′-O-deacylase activity in membranes. LpxR is synthesized with an N-terminal type I signal peptide and is localized to the outer membrane. Mass spectrometry was used to confirm the position of lipid A deacylation in vitro and the release of the intact 3′-acyloxyacyl group. Heterologous expression of lpxR in the E. coli K-12 W3110, which lacks lpxR, resulted in production of significant amounts of 3′-O-deacylated lipid A in growing cultures. Orthologues of LpxR are present in the genomes of E. coli 0157:H7, Yersinia enterocolitica, Helicobacter pylori, and Vibrio cholerae. The function of LpxR is unknown, but it could play a role in pathogenesis because it might modulate the cytokine response of an infected animal.Salmonella typhimurium and related organisms are enteric Gram-negative bacteria. S. typhimurium cause gastroenteritis in human hosts but in mice can produce a fatal, typhoid-like sepsis (1,2). These bacteria invade the epithelial cells and M cells of Peyer patches and then pass into the lymphatic system by colonizing phagocytic cells. Subsequently, the bacteria survive and multiply within modified vacuoles of macrophages that can ultimately produce macrophage apoptosis (3-6).* This research was funded by National Institutes of Health Grants AI-064184 (to M. S. T.), GM-51310 (to C. R. H. R.), and GM-64402 (to R. J. C.). Lipopolysaccharide (LPS) 3 is the principal component of the outer leaflet of the outer membrane of Gram-negative bacteria. Recognition of LPS by the mammalian innate immune system results in the production of cell adhesion proteins in endothelial cells and of proinflammatory molecules such as tumor necrosis factor-α and interleukin-1β in monocytes (7,8). Lipid A, the hydrophobic anchor of LPS, produces most of these responses (9,10) after its detection by Toll-like receptor 4 (TLR-4) (11-13). Lipid A of S. typhimurium and Escherichia coli is a β1′-6-linked disaccharide of glucosamine, phosphorylated at the 1 and 4′ positions and acylated at the 2, 3, 2′, and 3′ positions with R-3-hydroxymyristate (Fig. 1A) We now report a novel, Ca 2+ -dependent 3′-O-deacylase present in the membranes of S. typhimurium (Fig. 2). The structural gene (lpxR) encoding the 3′-O-deacylase was identified and expressed in E. coli K-12. LpxR, like PagP and PagL, is localized to the outer membrane. Expression of LpxR in the E. coli K-12 strain W3110 results in a signific...

A group of bacterial flavoproteins related to thioredoxin reductase contain an additional < 200-amino-acid domain including a redox-active disulfide center at their N-termini. These flavoproteins, designated NADH:peroxiredoxin oxidoreductases, catalyze the pyridine-nucleotide-dependent reduction of cysteine-based peroxidases (e.g. Salmonella typhimurium AhpC, a member of the peroxiredoxin family) which in turn reduce H 2 O 2 or organic hydroperoxides. These enzymes catalyze rapid electron transfer (k cat . 165 s 21 ) through one tightly bound FAD and two redox-active disulfide centers, with the N-terminal-most disulfide center acting as a redox mediator between the thioredoxin-reductase-like part of these proteins and the peroxiredoxin substrates. A chimeric protein with the first 207 amino acids of S. typhimurium AhpF attached to the N-terminus of Escherichia coli thioredoxin reductase exhibits very high NADPH:peroxiredoxin oxidoreductase and thioredoxin reductase activities. Catalytic turnover by NADH:peroxiredoxin oxidoreductases may involve major domain rotations, analogous to those proposed for bacterial thioredoxin reductase, and cycling of these enzymes between two electron-reduced (EH 2 ) and four electron-reduced (EH 4 ) redox states.

Pathways for the reduction of protein disulfide bonds are found in all organisms and are required for the reductive recycling of certain enzymes including the essential protein ribonucleotide reductase. An Escherichia coli strain that lacks both thioredoxin reductase and glutathione reductase grows extremely poorly. Here, we show that a mutation occurring at high frequencies in the gene ahpC, encoding a peroxiredoxin, restores normal growth to this strain. This mutation is the result of a reversible expansion of a triplet nucleotide repeat sequence, leading to the addition of one amino acid that converts the AhpC protein from a peroxidase to a disulfide reductase. The ready mutational interconversion between the two activities could provide an evolutionary advantage to E. coli.

The deacetylation of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (UDP-3-O-acylGlcNAc) by LpxC is the committed reaction of lipid A biosynthesis. CHIR-090, a novel N-aroyl-Lthreonine hydroxamic acid, is a potent, slow, tight-binding inhibitor of the LpxC deacetylase from the hyperthermophile Aquifex aeolicus, and it has excellent antibiotic activity against P. aeruginosa and E. coli, as judged by disk diffusion assays. We now report that CHIR-090 is also a two-step slow, tight-binding inhibitor of Escherichia coli LpxC with K i = 4.0 nM, K i * = 0.5 nM, k 5 = 1.9 min -1 and k 6 = 0.18 min -1 . CHIR-090 at low nM levels inhibits LpxC orthologues from diverse Gram-negative pathogens, including Pseudomonas aeruginosa, Neisseria meningitidis, and Helicobacter pylori. In contrast, CHIR-090 is a relatively weak competitive and conventional inhibitor (lacking slow, tight-binding kinetics) of LpxC from Rhizobium leguminosarum (K i = 340 nM), a Gram-negative plant endosymbiont that is resistant to this compound. The K M (4.8 μM) and the k cat (1.7 s -1 ) of R. leguminosarum LpxC with UDP-3-O-(R-3-hydroxymyristoyl)-Nacetylglucosamine as the substrate are similar to values reported for E. coli LpxC. R. leguminosarum LpxC therefore provides a useful control for validating LpxC as the primary target of CHIR-090 in vivo. An E. coli construct in which the chromosomal lpxC gene is replaced by R. leguminosarum lpxC is resistant to CHIR-090 up to 100 μg/mL, or 400 times above the minimal inhibitory concentration for wild-type E. coli. Given its relatively broad spectrum and potency against diverse Gram-negative pathogens, CHIR-090 is an excellent lead for the further development of new antibiotics targeting the lipid A pathway.The emergence of multi-drug resistant bacteria in hospital and community clinics has created an urgent need for new antibiotics (1,2). About half of the multi-drug resistant bacteria are Gram-negative pathogens (2), including strains of Escherichia coli, Pseudomonas aeruginosa (1), and Acinetobacter baumannii (3). Inhibitors that exploit traditional antibiotic targets, such as peptidoglycan, DNA replication or protein biosynthesis (4), are becoming less effective (2). These obstacles could be overcome by developing inhibitors of novel targets required for bacterial growth (5,6).The biosynthesis of the lipid A component of lipopolysaccharide (LPS), a unique, outermembrane lipid that shields Gram-negative bacteria from environmental stresses (7,8), is a promising target for new antibiotic development (9-12). The lipid A moiety of LPS is a hexaacylated disaccharide of glucosamine (7,8) (Figure 1). Although inhibition of any one of the first six enzymes of lipid A biosynthesis is lethal to E. coli (8), the most promising target identified to date is LpxC (9-12), a unique deacetylase that is selective for UDP-3-O-(R-3-*Author to whom correspondence should be addressed: C. R. H. Raetz at (919) Fax (919) LpxC is a zinc-dependent amidase with a catalytic mechanism related to that of carboxypeptida...

The development of safe live, attenuated Salmonella vaccines may be facilitated by detoxification of its lipopolysaccharide. Recent characterization of the lipid A 1-phosphatase, LpxE, from Francisella tularensis allowed us to construct recombinant, plasmid-free strains of Salmonella that produce predominantly 1-dephosphorylated lipid A, similar to the adjuvant approved for human use. Complete lipid A 1-dephosphorylation was also confirmed under low pH, low Mg2+ culture conditions, which induce lipid A modifications. lpxE expression in Salmonella reduced its virulence in mice by five orders of magnitude. Moreover, mice inoculated with these detoxified strains were protected against wild-type challenge. Candidate Salmonella vaccine strains synthesizing pneumococcal surface protein A (PspA) were also confirmed to possess nearly complete lipid A 1-dephosphorylation. After inoculation by the LpxE/PspA strains, mice produced robust levels of anti-PspA antibodies and showed significantly improved survival against challenge with wild-type Streptococcus pneumoniae WU2 as compared to vector-only immunized mice, validating Salmonella synthesizing 1-dephosphorylated lipid A as an antigen delivery system.