LIPID A and BACTERIAL LIPOPOLYSACCHARIDES
STRUCTURE, OCCURRENCE and BIOLOGY
1. Structure and Occurrence
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, consisting of glycerophospholipids, and a highly distinctive outer membrane. The latter is an asymmetric bilayer, the outer leaflet of which in most species consists predominantly of lipopolysaccharides of which Lipid A is a key component. They are of great scientific interest in that they are toxins and stimulate strongly the innate immune system in eukaryotic host species. Proteins take up much of the remaining surface of the outer leaflet of the outer membrane, while the inner leaflet is composed of conventional glycerophospholipids, mainly phosphatidylethanolamine, phosphatidylglycerol and cardiolipin.
Early attempts to determine the structures of such lipopolysaccharides 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, improved extraction methods and the discovery that the lipid component could be cleaved from the rest of the molecule by mild acidic hydrolysis lead to the unravelling of the detailed structures. Modern mass spectrometric methods, especially with matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization, have been invaluable aids. Thus, lipopolysaccharides derived from different groups of Gram-negative bacteria have a common basic structure structure comprising two parts - a covalently bound lipid component, termed lipid A, and a hydrophilic hetero-polysaccharide. 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.
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 in the 1 and 4’ positions, and (R)-3-hydroxy fatty acids at positions O-2, O-3, O-2' and O-3' 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 of the most studied organism Escherichia 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 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). In Rhodobacter sphaeroides, the amide-linked fatty acids of the disaccharide backbone are 3-oxo-tetradecanoate, while some species contain 2-hydroxy acids. Agrobacterium and Rhizobiaceae species, which are plant pathogens and symbionts, respectively, tend to have pentacyl units with four C12 to C20 3-hydroxy acids and one very-long-chain (ω-1)-hydroxy acid such as 27-hydroxyoctacosanoic acid (sometimes with 3-hydroxy-butyric acid linked in turn), attached to one of the 3-hydroxyl groups. Lipopolysaccharides from marine cyanobacteria of the genus Synechococcus differ significantly from those of all other species in that the lipid moieties consist of tri- and tetraacylated structures with hydroxy- (odd-chain) and nonhydroxy-fatty acids connected to the diglucosamine backbone.
There are also exceptions to the basic structure of the lipid A component. In Rhizobiaceae species, the core structure is a little more variable, and the phosphates residues may be absent or substituted with glucuronic acid. Together with the distinctive fatty acyl pattern, this may be a strategy of the organism to weaken or evade the response of the plant and so enable symbiosis. The lipopolysaccharide of Francisella species have a number of unusual features, not least in that the lipid A exists partly in the free form, i.e. not linked to Kdo, core sugars and O-specific chain. In comparison to the lipid A from E. coli, the phosphate group at the 1-position of the β-(1–6)-linked diglucosamine unit is replaced by α-linked galactosamine and there is no phosphate at the 4'-position, while the fatty acid components are C18 and C16 in chain-length. Lipopolysaccharides of the genus Synechococcus, in addition to lipid anomalies, differ importantly in that they have 4-linked glucose as their main saccharide component instead of heptose and Kdo, while the lipid A lacks phosphate and contains a single galacturonic acid. Whether these represent primitive structures or an adaptation to the marine environment is a matter of speculation.
The hetero-polysaccharide chains of the intact lipopolysaccharides extend outwards for a distance of about 10 nm from the surface of the outer membrane, which has an important function in nutrient uptake and also provides the organisms with remarkable permeability barriers that confer resistance to many different detergents and antibiotics. The large number of fully saturated fatty acyl groups in each molecule of lipid is believed to create a gel-like lipid interior of low fluidity that inhibits the penetration of hydrophobic solutes into the membrane. Also, it is believed that this barrier is stabilized by lipopolysaccharide-associated cations (e.g. Mg2+) linking adjacent molecules through salt bridges that neutralize the repulsive forces. The result is an oriented and tightly cross-linked leaflet that protects bacteria from a variety of hydrophobic host-defence molecules including some antimicrobial peptides. It permits growth and survival of bacteria in harsh environments including those within eukaryotic hosts.
In each bacterial species, the hetero-saccharide 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, the presence or absence of which determines the appearance – ‘smooth’ or ‘rough’ – of a bacterial colony. Thus, the ‘rough’ type lipopolysaccharides lack the O-specific chain, while a ‘semi-rough’ or short-chain type contains only one O-chain repeating unit attached to the core oligosaccharide–lipid A.
The core polysaccharide is structurally more uniform than the O-chain. 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). The Kdo residue is located at the reducing end of the oligosaccharide chain and is essential for its biological activity. These saccharide units are usually substituted by charged phosphate groups, resulting in an accumulation of charge in this inner region. The molecule from E. coli, which is illustrated in Figure 1 above, is 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 IVA'), have recently been produced. Indeed, lipid IVA may be the minimum structure required for the viability of the organism. During the biosynthesis of lipid A, intermediates are formed with a pyrophosphate residue in position 1 and a monophosphate in position 4’ of the disaccharide unit. It is now recognized that some species retain the pyrophosphate substituent.
The O-specific chains are distinctive and characteristic. Smooth-type Gram-negative bacteria synthesise lipopolysaccharides that differ in the length, branching and fine structure of this part of the molecule. The polysaccharide chains consist of repetitive subunits that extend out from the bacteria, and they can include from one to 25 chemically identical repeating oligosaccharide units, which in turn contain from 2 to 7 monosaccharide residues. As a result of diversity in the nature of the monosaccharides, their alternative configurations and the innumerable types of glycosidic linkage, the O-chain in most bacterial species is unique. While each repeating unit may only contain a limited number of monosaccharide residues, there are more than a hundred types that can be selected in addition to many kinds of non-carbohydrate substituents. For example, in some phytophathogens, most of the O-specific chains have a backbone of rhamnose residues, which may be of the D or L configuration and in α or β anomeric forms, often in the same structure. The references cited below afford more detailed information.
In pathogenic bacteria, it is the O-chains that come in contact with the host organism during infection, and as they are antigenic, they form the basis for serotype classification of bacterial genera, and so are also termed 'O-antigens'. They protect the bacterium against the lytic action of the host defences as well as from the effects of antibiotics. When separated from the lipid A component, the O-antigens do not display endotoxic activity.
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, the first six of which are required for bacterial growth (and are targets for the development of new antibiotics). Those requiring further information should consult the reading list below, but it is of interest that the lipid undecaprenyl phosphate is the intermediate involved in the transfer of carbohydrate units. 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, which must also be transported from the cytoplasmic side of the membrane, is attached. Various modifications of the lipid A can then occur that may not be essential for growth, but strengthen the permeability barrier and influence the virulence of some pathogens.
3. Lipid A as an Endotoxin
When bacteria multiply and then die and break up, a heat-stable lipopolysaccharide is liberated, which functions as a powerful bacterial toxin that has been termed an endotoxin to differentiate it from the heat-labile exotoxins released by live bacteria. This was first demonstrated towards the end of the 19th century when it was shown that heat-killed cholera bacteria were themselves toxic. The lipid A component, in particular, is known to be responsible for many of the toxic effects of infections with Gram-negative bacteria. Because of its conserved structure in diverse pathogens of this kind, it is recognized as a pathogen-associated molecule by a specific receptor, toll-like receptor 4 (TLR4), on immune cells (monocytes, macrophages, neutrophils and dendritic cells especially) and stimulates them to secrete pro-inflammatory cytokines. At high concentrations, these induce high fever, increased heart rate, and in the worst cases can lead to septic shock and death by lung or kidney failure. The response to lipopolysaccharide exposure is highly complex, and the rates of transcription of hundreds of genes are affected.
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. Most living organisms produce antimicrobial peptides that possess dual functions in that they kill bacteria and neutralize the endotoxic effect of lipopolysaccharides, and this is also the subject of much research.
The observed biological 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 determinant role in the toxicity and biological activity of the molecule, and the secondary (estolide) fatty acid constituents appear especially important in this context. Indeed, quite subtle changes in fatty acid composition can lead to profound changes in toxicity and in the immune response. Penta-acyl lipid A forms in some bacterial species show highly variably potency depending on the precise composition, but the tetra-acyl lipid IVA, i.e. without the estolide-bound fatty acids, lacks endotoxic activity although the virulence can remain high. Thus, the pathogen Yersinia pestis normally synthesises lipid A with six fatty acid chains in the fleas that act as carriers, but in a human host it produces lipid A with only four fatty acyl chains. This escapes attack by the immune system, as it does not activate the TLR4 receptor.
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. The phosphate groups are also important to toxicity, as they are involved in binding to receptor molecules. Removal of phosphate groups or addition of amine groups to the hydrophilic region enables the bacterial lipopolysaccharides to evade the host immune system.
To counter the direct 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.
Similarly, lipopolysaccharides are important molecules in the interactions between bacteria and plants, both in relation to symbiosis and to pathogenesis. They protect bacteria from plant-derived antimicrobial substances, and conversely they trigger defence responses following challenge by pathogens.
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, shielding them from a variety of aggressive conditions. It is not produced simply to aggravate humans.
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James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland.
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