The LipidWeb blank

Lipid A and Bacterial Lipopolysaccharides

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, containing glycerophospholipids and integral membrane proteins, 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 (the term was first coined by Westphal and Luderitz, while 'lipid B' was the name given to the conventional phospholipids). The compartment between these membranes is termed the periplasm and contains a layer of peptidoglycan molecules. Proteolipids (lipoproteins) take up much of the remaining surface of the outer leaflet of the outer membrane, while the inner leaflet is contains mainly conventional glycerophospholipids, phosphatidylethanolamine, phosphatidylglycerol and cardiolipin predominantly, with some proteolipids. Cyanobacteria produce lipopolysaccharides with related structures. Other biologically relevant lipopolysaccharides include lipo-chitooligosaccharides (Nod factors) produced by nitrogen-fixing rhizobia.

Lipopolysaccharides are indispensable macromolecules for the growth and survival of the organisms, providing an effective barrier that prevents harmful toxic compounds and antibiotics, including those of clinical value, from entering the cells. The function of the lipid A component is to anchor lipopolysaccharides to the outer membrane. They are of particular scientific interest in that they are toxins and stimulate strongly the innate immune system in eukaryotic host species.

1.  Structure and Occurrence of Lipid A and Lipopolysaccharides

Early attempts to determine the structures of lipid A and 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 are now known have a common basic 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 in the bacterial outer membrane, while the polysaccharide component interacts with the external environment, including the defences of the animal or plant host species. It is always attached to two units of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), which is considered as a marker of lipopolysaccharides, and the complete entity is best described as Kdo2-lipid A.

Structural formula of the basic lipopolysaccharide from E. coli

Lipid A is a unique and distinctive phosphoglycolipid, the structure of which is highly conserved among species. All contain glucosamine (2,3-diamino-2,3-dideoxy-D-glucose) residues, 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 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 acid of the disaccharide backbone is 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. Bradyrhizobium (slow-growing rhizobia) has at least one molecule of carboxyl-bacteriohopanediol or its 2-methyl derivative linked covalently to the (ω-1)-hydroxy group of a very-long-chain fatty acid, which reinforces the stability and rigidity of the outer membrane.

Basic Lipid A structure of Synechococcus CC911Lipopolysaccharides from marine cyanobacteria such as 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. They lack phosphate residues and instead have a single galacturonic acid attached to glucosamine. Whether these represent primitive structures or an adaptation to the marine environment is a matter of speculation.

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.

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, cationic antimicrobial peptides 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. 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, and it is now recognized that some species retain the pyrophosphate substituent. Other structural modifications sometimes observed include phosphorylation and phosphoethanolamine addition.

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.

Again, lipopolysaccharides from marine cyanobacteria such as the genus Synechococcus differ appreciably in the core region in that they lack heptose and Kdo and instead have a chain of 4-linked glucose units, with one rhamnose in some strains, but no phosphate residues. Some of these structures include long fatty acyl chains with varying degrees of unsaturation. Similarly, the lipid A components in bacteria from extreme environments can differ appreciably in structure from the norm, and they together with their biosynthetic enzymes are being studied for their potential therapeutic value.

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. On the other hand, when separated from the lipid A component, the O-antigens do not display endotoxic activity.

2.  Biosynthesis of Kdo2-Lipid A

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 (sometimes termed the 'Raetz' pathway), the first six of which are required for bacterial growth (and are targets for the development of new antibiotics). It is only possible to give a brief outline of the pathway here, and more detailed information is available in the reading lists below. Biosynthesis begins with the transfer of one molecule of 3-hydroxy-14:0 (in E. coli) from linkage to acyl carrier protein to position O-3 of UDP-N-acetylglucosamine by means of the enzyme LpxA. This is deacetylated by LpxC (the committed step in the pathway), followed by the transfer of a second 3-hydroxy-14:0 to the free amino group by the enzyme LpxD. LpxA and LpxD act in effect as 'hydrocarbon rulers' and determine the length of hydroxyacyl chains incorporated, ensuring that all are the same. UDP-diacyl-GlcN is then cleaved by the pyrophosphatase LpxH to form the phosphorylated intermediate termed lipid X (a key molecule in the discovery of the pathway), which is in turn condensed with a further molecule of UDP-diacyl-GlcN by LpxB (the disaccharide synthase) to produce a β(1→6)-linked disaccharide, which carries 3-hydroxy-residues at positions 2, 3, 2' and 3' together with an α-linked phosphate at O-1.

Biosynthesis of Lipid A

The next reactions in the sequence are catalysed sequentially by the integral membrane proteins LpxK, KdtA, LpxL and LpxM. Kinase LpxK phosphorylates the 4'‑position of the disaccharide-1-P to form lipid IVA, KdtA then transfers two Kdo residues from CMP-Kdo to the non-reducing GlcN of lipid IVA before the remaining acyl groups are added (LpxL and LpxM) to produce the basic Kdo2-lipid A molecule.

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, for example further acylation, and phosphate or carbohydrate addition, that may not be essential for growth but strengthen the permeability barrier and influence the virulence of some pathogens. Finally, several transport proteins, designated LptA to LptG, are required to transfer the completed lipopolysaccharide across the periplasm and then to the outer surface of the cell; these are also seen as potential targets for antibiotics.

The O-antigen is also synthesised by membrane-associated enzyme complexes in the cytoplasm and requires C55-undecaprenyl phosphate as an acceptor for assembly of the oligosaccharide chain via the action of many different glycosyltransferases, but this is a topic for carbohydrate experts.

3.  Lipid A as an Endotoxin in Bacterial Infections

As this bacterial lipopolysaccharide is one of the most conserved structures within all Gram negative bacterial species, it is an important pathogen associated molecule that is recognized by mammalian innate immune systems, and this can result in the clearance of a bacterial infection in a timely manner. Thus, when bacteria multiply and then die and break up, a heat-stable lipopolysaccharide is liberated, and this 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 many different receptors, e.g. toll-like receptor 4 (TLR4), myeloid differentiation protein 2 (MD-2), G-protein-coupled receptors, integrins, receptor-like kinases and caspases, on immune cells (monocytes, macrophages, neutrophils and dendritic cells especially) and stimulates a robust inflammatory response through secretion of pro-inflammatory cytokines. As one example and rather simplistically, a lipopolysaccharide binds to a large hydrophobic pocket in TLR4 via its lipid chains, while its phosphate groups can interact directly with the receptor leading to formation of a heterodimer complex that is active in immune signalling. TLR4 is unique in that it exists as a transmembrane protein that enables the transmission of information on lipopolysaccharide detection to the cytosol, where the dimerized TLR4 domains are detected by a protein TIRAP, which binds to phosphatidylinositol 4,5-bisphosphate in the plasma membrane. This leads to the formation of molecular complexes in the cell that initiate the TLR4 signalling. While the response to lipopolysaccharide exposure is highly complex, and the rates of transcription of hundreds of genes are affected, one constant factor appears to the involvement of phosphatidylinositol 4,5-bisphosphate in the process.

As a potent stimulator of the innate immune system, this leads to a significant enhancement of the resistance to infection and is beneficial to the host; modified lipid As have therapeutic potential. On the other hand, this response must be balanced as at high concentrations of endotoxin, it induces high fever, increased heart rate, and in the worst cases can lead to septic shock and death by lung or kidney failure. From both standpoints, these interactions have been the subject of intensive study, especially in humans, and the mechanisms are fairly well understood.

Scottish thistleThe 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. As a generality, lipid A forms with shorter chain-fatty acids tend to be less toxic than those with longer-chain substituents. 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 at 21-27°C, but in a human host at 37°C 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. Similarly, lipid A from invasive strains of Neisseria meningitides is hexa-acylated, whereas lipid A of carrier strains is penta-acylated. Together with the addition of positively charged substituents to the lipid A phosphate groups, increasing acylation reduces the permeability of the bacterial membranes to antibacterial agents.

While the O-antigen component may be less important to toxicity, it aids the solubility and transport of the lipopolysaccharide molecule and enhances the bacteria’s ability to establish infection; its antigenicity is of course of great importance and it promotes adhesion to epithelial cells. The phosphate groups and other polar moieties are also important to toxicity, as they are involved in binding to receptor molecules, and they can inhibit the action of antimicrobial agents. For example, the substituents at the 4'-phosphate of glucosamine II are responsible for the bacterial resistance to polycationic antibiotics such as polymyxins. If the hydroxyl group at the 4'-phosphate of glucosamine II is not substituted, polymyxin will attach to it and the bacteria will be susceptible to the antibiotic. On the other hand, if the hydroxyl group carries a substituent, polymyxin cannot join and the bacteria will be resistant. Most living organisms produce antimicrobial peptides of this kind that possess dual functions in that they kill bacteria and neutralize the endotoxic effect of lipopolysaccharides, and this is the subject of much research.

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. Although no plant receptors have yet been identified, it is apparent that both lipid A and the core oligosaccharide from pathogenic bacteria are potent inducers of immune responses, including a rapid increase in the concentration of cytosolic calcium, the induction of antibacterial oxygen species and the expression of antimicrobial peptides, presumably by different signalling pathways although the molecular mechanism is not yet known.

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. On the other hand, because of the increasing emergence of multidrug-resistant bacteria, there is a critical need for the development of novel antibiotics, and the detailed knowledge that has been gained of the biosynthetic pathway for Kdo2-lipid A is seen as providing a target for pharmaceutical intervention. Agonists of the TLR4 receptor are also being sought, including synthetic lipid A analogues and the less toxic forms from marine bacteria.

4.  Other Bacterial Lipopolysaccharides

Lipo-chitooligosaccharides are a class of signalling molecules produced by nitrogen-fixing rhizobia that play important roles in plant-microbe interactions. They are nodulation factors (Nod factors), which are essential for establishment of the nitrogen-fixing root nodule symbiosis with legume plants. In general, they consist of a carbohydrate (chitin) backbone consisting of three to five N-acetylglucosamine (GlcNAc) units, sulfated on O6 of the reducing residue and with a non-reducing terminal glucosamine unit linked by an amide bond to a fatty acid. In different bacterial species, the number of GlcNAc units, the degree of acetylation, the presence or absence of a sulfate group and the number of double bonds in the unsaturated fatty acid can vary. In that secreted by the bacterium Sinorhizobium meliloti (illustrated), which interacts with plants of the genus Medicago, the fatty acid component is mainly 2E,9Z-hexadecadienoate, but small amounts of palmitate or palmitoleate may be found also.

Nodulation factor from Sinorhizobium meliloti

Similar compounds have now been isolated from one species of mycorrhizal fungi (Rhizophagus irregularis). These are less diverse structurally, but like Nod factors they stimulate root development and induce calcium spiking and transcriptional change in leguminous plants. There is evidence that two complementary receptor systems operate in the host plants, one triggering a signalling cascade that leads to cell division while the second controls the intracellular entry of rhizobia. As many different microbial and fungal species may be involved in the interactions, these receptors must be able to accommodate a wide range of Nod factor structures.

Recommended Reading

Lipid listings Credits/disclaimer Updated: October 18th, 2017 Author: William W. Christie LipidWeb icon