More about E. coli, lipid A and endotoxin signaling
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 (Fig. 1) (1-4) . There are 106 lipid A residues and 107 glycerophospholipids in an Escherichia coli cell (1,5) . The minimal LPS required for the growth of E. coli consists of lipid A and two Kdo units (Fig. 2) (1,4) .In wild type strains, additional core and O-antigen sugars are present (Fig. 1) (6-10) . These are not required for growth, but enhance survival during environmental stresses, and help bacteria evade the immune system. In Haemophilus influenzae, the heptose residues of the core are needed for virulence in infant rats.
Fig.1. Schematic molecular model of the inner and outer membranes of E. coli K-12. Colored ovals and rectangles represent sugar residues, as indicated, whereas circles represent polar headgroups of various lipids. (Red, ethanolamine-phosphate; purple, ethanolamine pyrophosphate; yellow, glycerol-phosphate; blue ovals, glucosamine units; gray ovals, N-acetylmuramic acid units). Abbreviations: Kdo, 3-deoxy-D-manno-octulosonic acid; LPS, lipopolysaccharide (1, 2).
Fig. 2. Structure of Kdo2-lipid A in E. coli K-12 compared to phosphatidylethanolamine, the major membrane phospholipid. Lipid A is much more complex in structure than phosphatidylethanolamine. Lipid A is glucosamine-based, and contains no glycerol. The acyl chains are shorter, fully saturated and hydroxylated if attached to glucosamine. Both Kdo and lipid A are required for growth, possibly because they are required for outer membrane protein folding. An x-ray structure of LPS bound to the outer membrane protein FhuA has recently been solved (80). You need to select RasMol (under Viewer:) and All Atom Model (under Complexity: ) and then follow “more information” instructions to download the Chime Plug-In. Try to locate the LPS and its lipid A moiety.
Many Gram-negative bacteria, including most pathogens, synthesize lipid A species resembling the one found in E. coli (1,4,13) (Fig. 2). Lipid A molecules are detected at picomolar levels by the innate immune system of animals (2,3,14,16) . Lipid A triggers the biosynthesis of diverse mediators of inflammation. TNF-a and IL1-b are especially important in animals (17,18) , because they may act in synergy when overproduced during a systemic infection to precipitate the syndrome of septic shock (17,19) . Synthetic E. coli lipid A causes similar symptoms when injected into animals (3,20) , supporting the pathophysiological role of lipid A. The characteristic structural features of E. coli lipid A (Fig. 2), such as its two phosphate groups, its acyloxyacyl moieties and its glucosamine disaccharide are needed to trigger the sepsis-like (endotoxin) response (2,3,21,22).
The initial events in the interaction of lipid A with animal cells have recently been elucidated (23-26). A special lipid transfer protein in serum delivers lipid A from bacteria (or bacterial membrane fragments) to CD14 on the surfaces of animal cells (Fig. 3) (15,16,27).The subsequent recognition of lipid A by Toll receptor proteins (24,28-30), primarily TLR-4, is the first step in signal transduction (Fig. 3) (23-26). However, binding of lipid A to the TLR-4 has not yet been demonstrated, and may involve other components, such as CD14 (31,32) and MD2 (Fig. 3) (33).
Fig. 3. Detection of lipid A endotoxin by animal cells and function of the signaling receptor TLR-4 in innate immunity. The above paradigm is based mainly upon studies of human, mouse and hamster cells (23-26). The TLR-4 receptor may be oligomerized upon binding of lipid A. As indicated by the upper arrow, cell activation can occur without CD14, but requires several orders of magnitude more lipid A. Overproduction of TNF-a and TLI-b during a severe infection can damage small blood vessals, causing fluid leakage and shock. Synthetic lipid A analogues and certain precursors are potent endotoxin antagonists via TLR-4 (22,26,66,67).
In insects an immediate response to LPS by the innate immune system is the production of cationic antibacterial peptides (34-36). Toll receptor proteins were first discovered in the context of Drosophila development (35,37,38). They were subsequently found to play additional roles in innate immunity and to have homologues in animal systems (23,28).
Lipid A biosynthesis
Over the past ten years, the enzymology and molecular genetics of lipid A biosynthesis in E. coli have been elucidated in our laboratory (1,2,39,40). The first step is the acylation of the sugar nucleotide UDP-GlcNAc (Fig. 4) (41,42). The E. coli UDP-GlcNAc acyltransferase (LpxA) is selective for beta-hydroxymyristate, consistent with the composition of E. coli_ lipid A (41,42). UDP-GlcNAc acyltransferase has an absolute requirement for acyl carrier protein (ACP) thioesters as donor substrates (41-43). The equilibrium constant (~0.01) for the acylation of UDP-GlcNAc is unfavorable (43). The biological implication is that the deacetylation of the product, UDP-3-O-(acyl)-GlcNAc (Fig. 4), by LpxC is the committed reaction of the pathway . LpxC is the product of a conserved, single-copy gene in diverse Gram-negative bacteria (44-46), and displays no sequence similarity to other deacetylases or amidases. The early steps of the pathway are excellent targets for the design of new antibiotics.
Fig. 4. Structure and biosynthesis of Kdo-lipid A in E. coliK-12. Only the Kdo and lipid A portions of LPS are required for cell growth. The red symbols indicate the relevant structural genes. A single enzyme catalyses each reaction (1, 2). In almost all sequenced bacteria, the genes encoding the enzymes of lipid A biosynthesis are present in single copy. LpxA and LpxC are the most highly conserved. The structure in the inset is a potent LpxC inhibitor with antibiotic activity comparable to ampicillin (79).
Following deacetylation, a second β – hydroxymyristate moiety is incorporated by LpxD to generate UDP-2,3-diacylglucosamine (Fig. 4) (50). Again, only ACP thioesters are substrates (50). The sequences of LpxA and LpxD are distantly related (BLASTP LpxA) because of multiple, contiguous hexad repeats in both proteins (50,51). The x-ray structure of E. coli LpxA (α homotrimer) shows that the hexads specify a novel protein fold, a left-handed helix of short parallel b-sheets (52) (Fig. 5).Three hexads (18 amino acid residues) form one coil of the β – helix (52). The active site of E. coli LpxA, located between the subunits (Fig. 5), functions as an accurate hydrocarbon ruler that incorporates 14-carbon acyl chains two orders of magnitude faster than 12- or 16-carbon chains (40,53).
Fig. 5. Ribbon diagrams of LpxA. The left ribbon diagram is a monomer of LpxA (262 amino acids). The left-handed b-helix is present the bottom three fourths of the protein. The right ribbon diagram shows the native homotrimer, viewed from the bottom. The active sites are located between the subunits just above the loop-outs. Note the precise stacking of the coils of the b-helix on top of each other. Rotate the monomer and carefully inspect the b-helix in LPXA. See Fig. 1 for Chime Plug-In downloading instructions.
Disaccharide formation and 4’-Kinase
UDP-2,3-diacylglucosamine (Fig. 4) is cleaved at its pyrophosphate bond by LpxH to form 2,3-diacylglucosamine-1-phosphate (lipid X) (54). A b, 1’-6 linked disaccharide is then generated by the condensation of another molecule of UDP-2,3-diacylglucosamine with lipid X (Fig. 4) (55,56). The disaccharide synthase is encoded by lpxB, which is co-transcribed with lpxA in E. coli (57,58).
A specific kinase next phosphorylates the 4’ position of the disaccharide to form lipid IVA (Fig. 4) (59). The kinase gene (lpxK) (60,61) is downstream in an operon with msbA in E. coli. MsbA (Fig. 6) is an essential, inner membrane ABC transporter (62) that may function in the initial stages of lipid A (63,64) and glycerophospholipid (64) export, about which very little is known. The kinase product, lipid IVA (Fig. 4), possesses some of the properties of endotoxins (65). In mouse cells it is a potent endotoxin-like agonist, but in human cells it is an endotoxin antagonist (22). The pharmacology of lipid IVA is determined by whether the target cells express mouse or human TLR-4 (Fig. 3) (66,67).
Fig. 6. Possible scheme for the export of newly made lipopolysaccharide. Kdo-lipid A is depicted in the same manner as in Fig. 1, in which blue ovals represent glucosamine residues and small white boxes are Kdo moieties. Lipid A-core and the precursor units of O-antigen are transported separately to the outer surface of the inner membrane, where they are then attached to each other after the O-antigen is polymerized. MsbA initiates lipid A-core transport, and RfbX may do the same for O-antigen precursor. Nothing is known about how nascent LPS gets across the periplasm and into the outer membrane (steps 3 and 4). Chemical structures of core and O antigen units (larger white boxes, as indicated) may vary a lot depending on the organism (1-4, 6-10), but Kdo-lipid A is highly conserved, and is sufficient in size and structure for transport to occur. The MsbA protein is currently under intense study in the laboratory.
Kdo transfer and late acylations
E. coli LPS contains two Kdo residues that are transferred to lipid IVA by a bifunctional enzyme (Fig. 4), encoded by kdtA (68). The related kdtA gene of H. influenzae specifies an enzyme that adds one Kdo moiety (69). It is not possible to determine whether a Kdo transferase is mono-, bi-, or even tri-functional (as in Chlamydia trachomatis)(70) from its sequence. The labile nucleotide CMP-Kdo(Fig. 4), synthesized by KdsA and KdsB (not shown in Fig. 4 ), serves as the Kdo donor.
The last steps of E. coli lipid A biosynthesis involve the addition of laurate and myristate residues to the distal glucosamine unit (Fig. 4), generating the so-called acyloxyacyl moieties (71). The “late” acyltransferases require the presence of the Kdo disaccharide in their substrate (71). Like LpxA and LpxD, they utilize acyl-ACPs as donors . The genes encoding the lauroyl and the myristoyl transferases (htrB and msbB respectively) (72,73) display some sequence similarity to each other (74,75). The msbB gene is not required for growth (75), but msbB mutants are greatly attenuated in their ability to activate human macrophages (76) and to cause septic shock in animals (77,78).
New targets for antibiotic design
As noted above, the lipid A pathway is an excellent target for the design of new antibiotics against Gram-negative bacteria, such as E. coli, Salmonella, and Pseudomonas, some strains of which have become resistant to existing antibiotics. Recently, potent LpxC inhibitors have been identified (79). An example of their activity in killing E. coli is shown in Fig. 7. The best one (see structure in Fig. 4 inset) is comparable to ampicillin, a classical b-lactam (79). Improved versions of lipid A biosynthesis inhibitors with better antibacterial activity are under investigation. A key goal is to determine an X-ray structure of Lpx C with a bound inhibitor molecule at the active site.
Fig. 7. Antibacterial activity of a potent LpxC inhibitor. In this experiment a lawn of E. coli is grown on a nutrient agar plate. Shortly after inoculation, discs containing 50 µg of test compound are put on top. The compounds diffuse out and kill the bacteria, creating a zone of clearing after 24 hours. The lipid A inhibitor L-161,240 (disc 1) and ampicillin (disc 3) show comparable halos. The enantiomer of L-161,240 (disc 2) is inactive, as is the buffer control (disc 4).
Rhizobium outer membranes and symbiosis with plants
Gram-negative bacteria, such as Rhizobium etli and Rhizobium leguminosarum, belong to a family of select microbes that fix nitrogen during symbiosis within the roots of leguminous plants (Fig. 8 and Fig. 9) (81,82). Lipopolysaccharides (LPS), which coat the outer membranes of the Rhizobiaceae, may play important role(s) in this process (83,84). Mutants of R. leguminosarum and R. etli that lack O-antigen cannot generate functional nodules (85-88), and LPS undergoes subtle structural modifications during symbiosis, possibly reflecting adaptations to the root microenvironment (89,90).
Fig. 8. Stages in the biology of the nodulation process. Mutants of R. etli and R. leguminosarum lacking O-antigen are arrested in the late steps of nodule development.
Fig. 9. Membrane topography of the symbiosome. The intimate contact of the Rhizobium outer membrane with the plant peri-bacteroid membrane could involve remodeling of specific membrane proteins and lipids. Diagram adapted from Whitehead and Day, Physiologia Plantarum 100, 30-44, 1997.
Whether or not the lipid A portion of LPS also plays an important role in symbiosis is unknown. Well-characterized R. etli or R. leguminosarum mutants with altered lipid A structures have not been described. The studies of Carlson and coworkers have demonstrated that the chemistry of lipid A in R. etli and R. leguminosarum is remarkably different from that of most other lipid A molecules (91,92), suggesting the possibility of unique biological function(s). Interesting features of R. etli lipid A (Fig. 10) include the absence of phosphate moieties, the presence of a galacturonic acid residue at position 4’, substitution of the proximal glucosamine residue by a 2-aminogluconate unit, and the presence of an unusual C28 acyl chain (91,92).
Fig. 10. Highly unusual lipid A structure in Rhizobium etli. Peculiar features include the absence of phosphate groups, an aminogluconate unit and a C-28 acyl chain. E. coli lipid A is shown for comparison.
Enzymatic studies from our laboratory are consistent with the proposed structure of R. etli lipid A (93-97). R. etli initiates the biosynthesis of lipid A with UDP-N-acetylglucosamine and hydroxyacyl-ACP (Fig. 11), as does E. coli (93). After formation of the key intermediate Kdo2-lipid IVA (93), R. etli then employs unique enzymes, such as its 4’- and 1- phosphatases (94,95), to generate its own unusual lipid A precursors (Fig. 11). A special acyl carrier protein and a unique membrane enzyme for transferring 27-hydroxyoctacosanoic acid to Kdo2-lipid IVA have also recently been discovered (Fig. 11) (96). The C28 acyltransferase of R. etli resembles the HtrB acyltransferase of E. coli, which generates the acyloxyacyl residue at position 2’ (Fig. 4), in its strict dependence upon the presence of the Kdo disaccharide in the substrate (96). Further enzymatic characterizations of the R. etli system (98, 99) and construction of R. etli mutants with altered lipid A structures are currently in progress with the aim of examining the effects of such mutations on the outer membrane assembly and symbiosis.
Fig. 11. Partial scheme for the biosynthesis of the unusual lipid A of R. etli. The first seven enzymes are the same as in E. coli (Fig. 4). However, the later steps are unique to the Rhizobium system. The purpose of the project is to determine whether or not the distinct lipid A structural modifications seen in Rhizobium are required for symbiosis.
Functional genomics: lipid A in plants?
Recent versions of protein, DNA and EST databases indicate that higher plants and algae contain significant homologues of the Escherichia coli genes encoding enzymes of lipid A biosynthesis (Fig. 4)! In Arabidopsis thaliana, all putative lipid A biosynthesis genes are nuclear, and contain the key residues known to be essential for catalysis in the E. coli system. These amazing genomic observations imply that plants synthesize lipid A-like substance(s). Lipid A may have appeared in plants following symbiosis with cyanobacteria. It may have a structural role in certain plant membranes, as in bacterial outer membranes (Fig. 1). Alternatively, plant lipid A might function in signal transduction. Plants have numerous homologues of TLR-4 (Fig. 3), the lipid A signal-transducing receptor of animal cells. Arabidopsis thaliana also contains a gene encoding a homologue of serum lipopolysaccharide binding protein (LBP), which is required for lipid A transport and delivery (Fig. 3). Lipid A molecules may be minor components of plants that were overlooked in earlier biochemical studies. The laboratory is currently focussed on the cloning and functional characterization of the Arabidopsis lpx genes, and is searching for lipid A-like molecules in plants. We are also exploring the idea that the unusual structure of lipid A in R. etli (Fig. 10) may somehow be required during symbiosis to avoid inappropriate interactions with the plant lipid A system.
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