LIPID A and BACTERIAL LIPOPOLYSACCHARIDES

STRUCTURE, OCCURRENCE and BIOLOGY

The envelope of Gram-negative bacteria, including those of human pathogens such as Escherichia coli and Salmonella enterica, is composed of two distinct lipid membranes: an inner membrane and outer membrane. The outer membrane is an asymmetric bilayer, the outer leaflet of which consists predominantly of lipopolysaccharides with proteins taking up much of the remaining surface. The polysaccharide chains extend outwards from the surface of the bacterium for a distance of about 10 nm, and this may permit growth and survival of bacteria in harsh environments including those within eukaryotic hosts. The inner leaflet is composed simply of conventional glycerophospholipids, mainly phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. The outer membrane has an important function in nutrient uptake but also provides the organisms with remarkable permeability barriers that confer resistance to many different detergents and antibiotics.

Early attempts to determine the structures of these compounds were greatly hindered by their amphipathic nature and their strong tendency to form aggregates by hydrophobic bonding or via cross-linking through ionic species. However, the discovery that the lipid component could be cleaved from the rest of the molecule by mild acidic hydrolysis soon lead to the unraveling of the detailed structures. Modern mass spectrometric methods, especially with matrix-assisted laser desorption/ionisation (MALDI), have been an invaluable aid. Thus, lipopolysaccharides derived from different groups of Gram-negative bacteria have a common basic structure, consisting of a covalently bound lipid component, termed lipid A, and a hydrophilic heteropolysaccharide. Lipid A provides the anchor that secures the molecule within the membrane, while the polysaccharide component interacts with the external environment, including the defences of the animal or plant host species.

Structural formula of the lipopolysaccharide from E. coli.

Lipid A is a unique and distinctive phosphoglycolipid, the structure of which is highly conserved among species. All contain D-gluco-configured pyranosidic hexosamine residues (or 2,3-diamino-2,3-dideoxy-D-glucose), which are present as β(1-6)-linked dimers. The disaccharide contains α-glycosidic and non-glycosidic phosphoryl groups, and (R)-3-hydroxy fatty acids in ester and amide linkages, of which two are usually further acylated at their 3-hydroxyl group. However, variations in the fine structure can arise from the type of hexosamine present, the degree of phosphorylation, the presence of phosphate substituents, and importantly in the nature, chain length, number, and position of the acyl groups. In the lipid A of E. coli illustrated, the hydroxy fatty acids are C₁₄ in chain length, and the hydroxy groups of the two (R)-3-hydroxy fatty acids of the distal GlcN-residue (GlcN II), and not those of the GlcN-residue at the reducing side (GlcN I), are acylated by non-hydroxy fatty acids (12:0 and 14:0). Some molecular species contain an additional fatty acid attached to the amide-linked 3-hydroxy acid and the phosphate group may be substituted with ethanolamine-phosphate (of GlcN I).

There are a few important exceptions to this type of fatty acid pattern. For example, in Rhodobacter sphaeroides, the amide-linked fatty acids of the disaccharide backbone are 3-oxo-tetradecanoic acid, while some species contain 2-hydroxy acids and others have very-long-chain (ω-1)-hydroxy acids such as 27-hydroxyoctacosanoic acid. In the lipid A of Helicobacter pylori in comparison to that of E. coli, there are four rather than six fatty acids with a longer average chain-length (16-18).

Scottish thistle In each bacterial species, the heterosaccharide unit is in two parts - an inner core, and an outer O-specific chain consisting of a complex polymer of oligosaccharides, which determines the serological or antigenic specificity of the lipopolysaccharide and is often termed an O-antigen. Different bacteria synthesise lipopolysaccharide molecules that differ in the length and fine structure of the O-specific chains. The core polysaccharide is structurally more uniform than the O-chain, the diversity being found primarily in an outer core region. The inner part of the core region is composed of the characteristic components heptose, mainly in the L-glycero-D-manno configuration, and 3-deoxy-D-manno-octulosonic (or 2-keto-3-deoxyoctonic) acid (Kdo). These are usually substituted by charged phosphate groups, resulting in an accumulation of charge in this inner region. The molecule from E. coli that is illustrated is thus 3-deoxy-D-manno-octulosonic acid (Kdo)₂-lipid A.

For some time it was thought that the minimum structure for cell viability in E. coli had the di-Kdo moiety, but viable mutants lacking Kdo and with the basic tetra-acyl form of lipid A, i.e. lacking the two secondary acyl groups (and termed 'lipid IV_A'), have recently been produced. Indeed, lipid IV_A may be the minimum structure required for the viability of the organism.

Lipid A is synthesised in the cytoplasmic compartment of Gram-negative bacteria, and the essential details of the process are now known. In brief in E. coli, lipid A is synthesised on the cytoplasmic surface of the inner membrane by a conserved pathway of nine distinct enzymes. Once the core oligosaccharide is in place, the nascent core-lipid A is flipped to the outer surface of the inner membrane by a specific transporter when the O-antigen polymer is attached. Various modifications of the lipid A can then occur that may not be essential for growth but influence the virulence of some pathogens.

Scottish thistle When bacteria multiply and when they die and break up, lipopolysaccharide is liberated and then functions as a powerful bacterial toxin that has been termed endotoxin. The lipid A component , in particular, is known to be responsible for many of the toxic effects of infections with Gram-negative bacteria. At high concentrations, it induces high fever, increased heart rate, and in the worst cases can lead to septic shock and death by lung or kidney failure. However, lipid A is also an active immuno-modulator, able to induce non-specific resistance to both bacterial and viral infections at low concentrations. From both standpoints, it has been the object of intensive study, not only in humans but also in plants, and the mechanisms are fairly well understood as is the biosynthetic pathway, a target for drug therapy (see the literature cited below). The observed effects are partly due to the primary structure of the lipid A moiety, but also to the fact that it adopts a specific conformation that enhances the activity by enabling binding to specific host molecules. It is evident that the number, positions, and chain-lengths of the fatty acid constituents have a role in the toxicity and biological activity of the molecule. Thus, the tetra-acyl lipid IV_A lacks endotoxic activity. While the polysaccharide component may be less important to toxicity, it aids the solubility and transport of the lipopolysaccharide molecule, and it does have some biological properties in its own right including its antigenicity.

To counter these toxic effects, there is an endogenous lipase or acyloxyacyl hydrolase in the liver and spleen that selectively removes the secondary fatty acyl chains from bacterial lipopolysaccharides and prevents their recognition by the mammalian signalling receptors. This reduces substantially the risk of prolonged inflammatory reactions during infections by Gram-negative bacteria.

It should be recognized that the existence of lipid A-containing lipopolysaccharide in the most ancient and primitive Gram-negative bacteria demonstrates that it is absolutely required for their survival and is not produced simply to aggravate humans.

The Lipid Library

LIPID A and BACTERIAL LIPOPOLYSACCHARIDES

STRUCTURE, OCCURRENCE and BIOLOGY

Recommended Reading