Lipid A and Bacterial Lipopolysaccharides

Lipopolysaccharides and their lipid A components are vital constituents of the outermost membrane leaflet of most Gram-negative bacteria (and of some marine cyanobacteria) that provide an efficient permeability barrier and are essential for their protection from environmental stresses and for symbiosis with other organisms.

In host animals, lipopolysaccharides are responsible for virulence towards host organisms and resistance to drugs, and they are modulators of innate immune responses that range from localized inflammation to sepsis. The importance of these molecules for infection was first demonstrated in humans towards the end of the 19th century when it was shown that heat-killed and lysed cholera bacteria were themselves toxic. After characterization of this 'endotoxin' in the 1930s as consisting of lipid and sugar moieties (first designated 'antigenic glycolipid'), the term 'lipopolysaccharide' was introduced in the 1940s.

1.  Introduction to the Cell Wall of Gram-negative Bacteria and its Lipids

The cell wall or 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 outer membrane is an asymmetric bilayer, and the outer leaflet of this in most species is a semi-rigid, highly ordered structure that consists predominantly of lipopolysaccharides (~75%) of which lipid A, a complex phosphoglycolipid, is a key component that serves as a hydrophobic anchor for the macromolecules. The term lipid A was first coined by Westphal and Luderitz, who in 1952 developed the solvent extraction method still in use today; 'lipid B' was the name given to the conventional phospholipids.

Schematic representation of the cell wall of Gram-negative cell bacteria
Schematic representation of the cell wall of Gram-negative cell bacteria. © Jeff Dahl, CC BY-SA 4.0 via Wikimedia Commons

The aqueous compartment between the inner and outer membranes is termed the periplasm and contains peptidoglycan molecules in a thin layer arranged parallel to the cell surface; this is an elastic heteropolymer of glycan strands interconnected by short peptide chains that protects bacterial cells from lysis by internal osmotic pressure and from external stress conditions. This layer is linked covalently to a proteolipid (such as Braun's lipoprotein) in the inner leaflet of the outer membrane, which also contains conventional glycerophospholipids, i.e., phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. In the outer membranes, the exchange process between the bacterial cell and its environment is controlled through porins with size-exclusion properties. Both leaflets of the inner membrane contain mainly the conventional glycerophospholipids.

Cyanobacteria produce lipopolysaccharides with related structures, while other biologically relevant lipopolysaccharides include lipochito-oligosaccharides (Nod factors), which are produced by nitrogen-fixing rhizobia. Lipopolysaccharides are indispensable macromolecules for the growth and survival of Gram-negative bacteria, providing an effective permeability barrier at the environmental interface to cationic antimicrobial peptides and antibiotics used in clinical practice, while enhancing intracellular survival and contributing to the evasion of the immune defences by mimicry of host molecules. As they can act as toxins and stimulate strongly the innate immune system in eukaryotic host species, such lipopolysaccharides and their lipid A components are of great pharmacological interest.

2.  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, but improved extraction methods and the discovery that the lipid component could be cleaved from the rest of the molecule by mild acidic hydrolysis led to the unravelling of the detailed structures. Modern mass spectrometric methods matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization together with NMR spectroscopy have been invaluable aids.

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. While the polysaccharide component interacts with the external environment, including the defences of the animal or plant host species, lipid A provides the anchor that secures the molecule in the bacterial outer membrane. It is always attached to two units of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), which is considered to be a marker for lipopolysaccharides, and the complete entity is best described as Kdo2-lipid A. This was long thought to be the minimal lipopolysaccharide entity essential to sustain the growth of most Gram-negative bacteria, 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 that are still viable.

Structural formula of the basic lipopolysaccharide from E. coli

Lipid A is a unique and distinctive phosphoglycolipid, the general structure of which is highly conserved among species, although variations in the fine structure can arise from the type of hexosamine present, the degree of phosphorylation, and in the nature, chain length, number and position of the acyl groups. All lipid A molecules contain two glucosamine (2,3‑diamino-2,3-dideoxy-D-glucose) residues, which are present as a β(1→6)-linked dimer and contain α‑glycosidic and non-glycosidic phosphoryl groups in the 1 and 4’ positions with four to seven saturated acyl chains, i.e., (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.

In the lipid A illustrated from E. coli (106 lipid A residues in each cell), there are six fatty acids in total. The four hydroxy fatty acids are C14 in chain length, and the hydroxyl 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 exceptions to this type of fatty acid pattern, and bacterial species show variability in the chain-length and number of fatty acid residues (2 to 9), or with amino-sugars replacing phosphate residues or with methyl substituents. 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 to 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 penta-acyl 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. Some structural heterogeneity can arise from specific environmental pressures, and apparently minor modifications can alter the overall bioactivity of a lipopolysaccharide appreciably.

Basic Lipid A structure of Synechococcus CC911Bradyrhizobium species (slow-growing nodulating rhizobia) have at least one molecule of a hopanoid, carboxyl-bacteriohopanediol or its 2-methyl derivative, linked covalently to the (ω‑1)‑hydroxy group of a very-long-chain fatty acid (often C30), which spans the entire membrane to reinforce the stability and rigidity of the outer membrane especially and thereby facilitates the dual life cycle of the organisms, i.e., outside and inside the plant; they also have a 25‑hydroxy-C26 fatty acid that is not linked to the hopanoid.

In Rhizobiaceae species, the basic structure is a little more variable, and the phosphate 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 lipopolysaccharides of Francisella species have many 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; some remodelling of this can occur in response to acid stress. In some species, the disaccharide unit can have a different composition.

Lipopolysaccharides from marine cyanobacteria such as the genus Synechococcus (illustrated) 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 heptose, Kdo and phosphate residues and instead have a single galacturonic acid attached to glucosamine. Other species of marine cyanobacteria can have differing carbohydrate compositions. Whether these represent primitive structures or an adaptation to the marine environment is a matter of speculation.

In all of these species, 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.

Structure - bacterial lipopolysaccharidePolysaccharide components: The hetero-polysaccharide chains of the intact lipopolysaccharides extend outwards for about 10 nm from the surface of the outer membrane to form an external barrier, which is stabilized by lipopolysaccharide-associated divalent cations, e.g., Mg2+ and Ca2+, linking adjacent molecules through salt bridges that neutralize the repulsive forces and enhance packing. The result is an oriented and tightly cross-linked leaflet that protects bacteria from a variety of hydrophilic host-defence molecules and xenobiotics including some antimicrobial peptides and permits growth and survival of bacteria in harsh environments including those within eukaryotic hosts.

In each bacterial species, these are biosynthetically, structurally and functionally distinct, and they consist of two parts - a core unit with inner and outer parts 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 the O-specific chain determines the appearance, ‘smooth’ or ‘rough’, of a bacterial colony, and 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.

In a few pathogens, including null-strains of Bordetella pertussis, Neisseria meningitidis and Haemophilus influenzae, the lipopolysaccharides lack an O-chain and are sometimes termed 'lipooligosaccharides' and tend to have more complex and highly decorated core structures that are responsible for bacterial specificity. Forms of these lacking lipopolysaccharides entirely can survive in laboratory culture.

In all species, the core polysaccharide is attached directly to lipid A and is structurally more uniform than the O-chain, usually comprising 10 sugars The inner part of the core region tends to be more conserved within a bacterial species and is composed of the characteristic components heptose, mainly in the L-glycero-D-manno configuration, and several 3‑deoxy-D-manno-octulosonic (or 2-keto-3-deoxyoctonic) acid (Kdo) units; the latter is located at the reducing end of the oligosaccharide chain and is essential for its biological activity. The outer core covers the inner core and often contains additional heptoses, which are usually substituted by charged phosphate groups, including by phosphoethanolamine or pyrophosphoethanolamine, resulting in an accumulation of charge in this inner region. In some Enterobacteriacea, the core polysaccharide contains the enterobacterial common antigen (ECA), which is built of trisaccharide repeating units.

Again, lipopolysaccharides from marine cyanobacteria such as the genus Synechococcus differ appreciably in that they lack heptose and Kdo in the core region and instead have a chain of 4-linked glucose units, with one rhamnose in some strains but no phosphate residues, while some 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.

As a result of diversity in the nature of the monosaccharides, their alternative configurations and the innumerable types of glycosidic linkage, the O‑specific chain in most bacterial species is unique and characteristic. In pathogenic bacteria, it is the O-chains that are in contact with the tissues of the host organism during infection and provide protection against the lytic action of the host defences as well as from the effects of antibiotics. As they are antigenic, they form the basis for serotype classification of bacterial genera and so are also termed 'O-antigens', although they do not display endotoxic activity when separated from the lipid A component. 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. 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. In Pseudomonas aeruginosa and some phytopathogens, 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.

Lipid A is the membrane anchor for capsular polysaccharides in Bacteroides fragilis in the human gut microbiome. In this organism, the polysaccharide component is zwitterionic and contains more than 100 repeating units of a tetrasaccharide consisting of D‑galactopyranose, 2,4-dideoxy-4-amino-D-FucNAc, DN‑acetylgalactosamine and D‑galactofuranose with 4,6‑pyruvate. It is considered to be the model symbiotic immunomodulatory molecule that confers benefits to the host in respect of autoimmune, inflammatory and infectious diseases.

3.  Biosynthesis of Kdo2-Lipid A and Lipopolysaccharides.

Lipid A and each of the two polysaccharide regions are synthesised independently in the cytoplasmic compartment of Gram-negative bacteria, and the essential details of the process are now known for the lipid A component at least. 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). There are three protein-bound acyl donor substrates together with UDP-N-acetylglucosamines (UDP-GlcNAc), ATP and CMP-3-deoxy-D-manno-octulosonic acid (CMP-Kdo). While some variation exists between species, mainly in relation to acylation, the enzymes of this pathway are highly conserved in Gram-negative bacteria. It is only possible to give a brief outline of the various steps here, but 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 its linkage to acyl carrier protein to position O‑3 of UDP‑N‑acetylglucosamine by means of the enzyme LpxA. This is deacetylated by LpxC, which is the committed and rate-limiting step in the pathway and interacts with the peptidoglycan synthesis enzyme MurA to coordinate the two processes. Deacetylation is followed by the transfer of a second 3-hydroxy-14:0 to the free amino group by the enzyme LpxD with LpxA and LpxD acting in effect as 'hydrocarbon rulers' to determine the length of hydroxyacyl chains incorporated and to 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 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, a critical biosynthetic step and regulatory node, then KdtA transfers two Kdo residues from CMP-Kdo to the non-reducing GlcN of lipid IVA before the remaining acyl groups are added (by LpxL and LpxM) to produce the basic Kdo2-lipid A molecule. While acyl transferases in some organisms have strict substrate requirements, others are more tolerant of both the fatty acid and its acyl acceptor with the result of a heterogeneous lipid A composition of the outer membrane.

After the completion of the Raetz pathway to Kdo2-lipid A, the core oligosaccharide is attached at the cytoplasmic side of the inner membrane, when the nascent core-lipid A is flipped to the periplasmic surface of the inner membrane by a specific transporter (MsbA). Both the inner and the outer core regions of the polysaccharide are derived from sequential addition of sugar moieties, and in this process phosphorylation occurs. Biosynthesis of the outer core ends with the addition of a L-glycero-D-manno-heptopyranose to the penultimate D-glucopyranose, and this final heptose unit acts as the acceptor of the O‑antigen after the core-lipid A precursor is flipped to the periplasmic face of the inner membrane. The O-antigen is synthesised by membrane-associated enzyme complexes in the cytoplasm and requires C55-undecaprenyl phosphate as an acceptor for assembly of the oligosaccharide chain via four different routes and the action of many different glycosyltransferases, but this is a topic for carbohydrate experts.

Gram-negative bacteria express multiple tailoring enzymes that work sequentially to further modify lipid A structures during transport of the intact lipopolysaccharide molecule from the outer surface of the cytoplasmic membrane to the inner surface of the outer membrane. The enzymes responsible for these modifications are usually expressed in response to environmental signals and enable bacteria to adapt to host defences and immune surveillance. In the periplasmic leaflet, enzymes can modify the phosphate moieties in the 1- and 4′-positions of the disaccharide backbone by hydrolysis or by the addition of cationic sugars or phosphoethanolamine, to prevent binding to host defence molecules so influencing the virulence of some pathogens and their resistance to antibiotics.

Transport of the mature lipopolysaccharide from the periplasmic side of the inner membrane to its final destination, the outer leaflet of the outer membrane, is carried out by the Lpt (Lipopolysaccharide transport) machinery, which consists of seven proteins (LptA to LptG) that link together in a complex or protein bridge spanning the entire cell wall, i.e., cytoplasm to inner membrane to periplasm to outer membrane. This apparatus can be considered as three main parts: an inner membrane ABC transporter, which uses hydrolysis of ATP in the cytoplasm to provide the energy to extract lipopolysaccharide from the inner membrane; a periplasmic bridge, which shields the hydrophobic component of the lipopolysaccharide from the aqueous periplasm; an outer membrane translocon, which catalyses the final insertion of the complex molecule into the outer leaflet of the outer membrane. Cardiolipin is reported to aid this process.

The enzymes of the biosynthetic pathway and the transport proteins, which can differ according to species, are seen as potential targets for the development of novel antibiotics against Gram-negative pathogens, either to kill them or to increase the permeability of the outer membrane to other antibiotics. While some are undergoing clinical trials, none have yet been approved for clinical use.

Glycerophospholipids: The conventional phospholipids, phosphatidylethanolamine (~80%), phosphatidylglycerol (~15%) and cardiolipin (~5%), are synthesised at the inner leaflet of the inner membrane and must be flipped across to the outer leaflet before some are transported across the periplasm to the inner leaflet of the outer membrane. The transport mechanisms are poorly understood, but it is evident that transport can occur in both directions, possibly via contact sites between the inner and outer membranes or by protein-mediated systems. (R)-3-hydroxymyristate is a metabolic precursor for palmitic acid biosynthesis for incorporation into phospholipids, as well as for synthesis of lipid A.

4.  Lipid A as an Endotoxin in Bacterial Infections

As the bacterial lipid A is one of the most conserved structures within all Gram-negative bacterial species, it is a pathogen associated molecule that is recognized by innate immune systems across all kingdoms of life, and under optimum conditions, this can result in the clearance of a bacterial infection in a timely manner. However, it can cause localized inflammation and if uncontrolled to disseminated sepsis. When bacteria multiply and then die and break up, a heat-stable lipopolysaccharide is liberated that functions as a powerful bacterial toxin, which has been termed an endotoxin to differentiate it from the heat-labile exotoxins released by live bacteria.

The lipid A component is known to be the endotoxic and pyrogenic centre of the entire lipopolysaccharide molecule, and it is responsible for most 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, such as toll-like receptor 4 (TLR4) on immune cells (e.g., monocytes, macrophages, neutrophils and dendritic cells), and it stimulates a robust inflammatory response by activation of caspase-4 and caspase-5 in humans and thence secretion of pro-inflammatory cytokines. As one example and rather simplistically, lipopolysaccharide binds to a large hydrophobic pocket in TLR4 via its lipid chains, while its phosphate group with its two negative charges can interact directly with the myeloid differentiation factor 2 (MD-2) leading to formation of a heterodimer complex that recognizes a common 'pattern' (Microbe-Associated Molecular Pattern or MAMP) in structurally diverse lipopolysaccharide molecules and then activation of a signalling cascade. Minor changes in the structure of lipid A can affect this binding and thence the effects on signal transduction. Bruce A. Beutler was awarded a share of the 2011 Nobel Prize in Physiology or Medicine for his discoveries in this area.

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 (PI(4,5)P2) in the plasma membrane. This leads to the formation of molecular complexes in the cell that initiate the TLR4 signalling. As part of the mechanism, lipid A induces S‑palmitoylation and thence activation of phosphatidylinositol 4-kinase, which generates phosphatidylinositol 4-monophosphate, the precursor of PI(4,5)P2. The strength of these effects is dependent on the precise structure of the lipid A, but the result is recruitment of immune cells to the site of infection to attack the foreign pathogen, a response that is beneficial for clearing minor bacterial infections. On other occasions, lipid A can trigger systemic inflammation to cause tissue damage, and in the worst cases, this can lead to septic shock and death of the host by lung or kidney failure. While the response to lipopolysaccharide exposure is highly complex, and the rates of transcription of hundreds of genes are affected, one relatively constant factor appears to the involvement of PI(4,5)P2 in the process.

One of the exceptions to this general mechanism is the Francisella novicida endotoxin, which is not recognized by the TLR4 receptor and is thus able to evade the host innate immune system. Instead, this stimulates the cyclooxygenase-2-dependent inflammatory pathway and is responsible for the lethality of such infections through overproduction of proinflammatory effectors including prostaglandin E2. Endotoxins of bacteria from very different ecosystems to which humans have never been exposed, such as those from deep oceans, may not be detected by pattern recognition receptors in humans.

Scottish thistleThe observed biological effects are partly due to the primary structure of the lipid A moiety, as this 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 to be important in this context with quite subtle differences in fatty acid composition leading 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, while penta-acyl lipid A forms in some bacterial species show highly variably potency depending on the precise composition. The pathogen Yersinia pestis normally synthesises lipid A with six fatty acid chains in the fleas that act as carriers at 21 to 27°C, but in a human host at 37°C it produces lipid A with only four fatty acyl chains because of a single-nucleotide polymorphism that results in a premature stop in translation of a specific lipid A acyltransferase. This lipid A form escapes attack by the immune system as it does not activate the TLR4 receptor and is crucial for the infectivity and pathogenesis of the organism. Lipid A from invasive strains of Neisseria meningitides is hexa-acylated, whereas lipid A of carrier strains is penta-acylated. In general, increasing acylation reduces the permeability of the bacterial membranes to antibacterial agents. Remarkably, the presence of an R-2-hydroxymyristic acid moiety in the lipid A of Enterobacter cloacae has been linked to death of neonates from septic shock.

Full recognition of lipopolysaccharides by pattern-recognition receptors requires the complete complement of six acyl chains that is normally present in the lipid portion. To counter the direct toxic effects, there is an endogenous lipase or acyloxyacyl hydrolase in the liver and spleen of the host that selectively removes the secondary fatty acyl chains from bacterial lipopolysaccharides and prevents their recognition by the mammalian signalling receptors. This is conserved evolutionarily among animal species and reduces substantially the risk of prolonged inflammatory reactions during infections by Gram-negative bacteria. By transforming lipopolysaccharides from stimulants to inhibitors, the enzyme reduces tissue injury, prevents prolonged immunosuppression after infection and limits the entry of stimulatory forms into the bloodstream. It prevents lipopolysaccharide-induced cholesterol accumulation in macrophages and thence inhibits foam cell accumulation in carotid arterial lesions. The phospholipid transfer protein in plasma transfers amphiphilic lipids between circulating lipoproteins and between lipoproteins, cells and tissues, and it may assist in the neutralization and clearance of bacterial lipopolysaccharides or endotoxins.

In contrast, as potent stimulators of the innate immune system, endotoxins can sometimes bring about a significant enhancement of resistance to infection that is beneficial to the host and indeed may be necessary for the proper maturation and development of host immunity. Chemically modified endotoxins have therapeutic potential, and there are some reports that certain lipopolysaccharide molecules, including the total lipopolysaccharides from the human gut microbiome, show antagonistic activity against toxic lipopolysaccharides. By competing with them for the binding to the TLR4/MD-2 complex, they prevent the transmission of the downstream signal responsible for eliciting inflammatory responses. B. vulgatus from the human gut produces a highly unusual lipid A consisting of a heterogeneous mixture of tetra- and penta-acylated species phosphorylated only at the reducing glucosamine unit, and this results in a weak immune response that may aid the bacterium to persist within the intestinal environment. Thus, the structural diversity of lipid A has been harnessed to create a vaccine adjuvant ('MPL®') that enhances a beneficial adaptive immune response safely against a co-inoculated antigen. From all standpoints, these interactions continue to be the subject of intensive study in humans.

Scottish thistleWhile the O-antigen component may be a lesser factor in toxicity, it aids the solubility and transport of the lipopolysaccharide molecule, promotes adhesion to epithelial cells and enhances the ability of the bacteria to establish infection. The phosphate groups and other polar moieties of the core polysaccharide (and of lipid A) are relevant 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 such as GlcpN or 2-amino-ethyl groups, polymyxin cannot bind and the bacteria will be resistant. Inhibition of the lipid A phosphoethanolamine transferase is therefore believed to have potential as a therapeutic approach. Many organisms produce antimicrobial peptides akin to polymyxins that possess dual functions in that they kill bacteria and neutralize the endotoxic effect of lipopolysaccharides, and this is the subject of much research.

There is concern that neurodegeneration and Parkinson's disease especially could be affected by lipopolysaccharides derived from Gram-negative bacteria in the intestines if they are transported to the central nervous system via the vagus nerve when bound to proteins such as alpha-synuclein in vascular disorders, including the 'leaky gut syndrome'. They can cross the blood brain barrier during aging to accumulate in brain, where they can activate TLR4 to trigger the release of various pro-inflammatory cytokines that promote neuroinflammation with harmful effects towards cognitive impairment and other neurodegenerative diseases, including Alzheimer's disease, amyotrophic lateral sclerosis and multiple sclerosis.

In the interactions between bacteria and plants, as in animals, lipopolysaccharides are important molecules 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 the mechanisms are not fully understood, 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. The receptor kinase Lipooligosaccharide-Specific Reduced Elicitation (LORE) has been shown to mediate plant immune responses to medium-chain 3-hydroxy fatty acids of bacterial lipopolysaccharides in a chain length- and hydroxylation-specific manner with free (R)‑3‑hydroxydecanoic acid as the strongest immune elicitor.

It should be recognized that the existence of lipid A-containing lipopolysaccharides in the most ancient and primitive Gram-negative bacteria demonstrates that they are absolutely required for their survival, shielding them from a variety of aggressive conditions that include host immune sensors. They are 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. Antagonists of the TLR4 receptor are also being sought that include synthetic lipid A analogues and the less toxic forms from marine bacteria, extremophiles and the intestinal microbiome.

Analysis: While analysis of lipid A per se is a daunting technical challenge, requiring advanced mass spectrometric and other spectroscopic techniques allied to liquid chromatography, it is relatively a much easier task to detect and quantify the 3‑hydroxy-fatty acid constituents, mainly 3‑hydroxytetradecanoic acid, in plasma and other tissues as a means of evaluating endotoxin levels in patients infected with Gram-negative bacteria.

5.  Other Bacterial Lipopolysaccharides

Lipo-chitooligosaccharides are a class of signalling molecules produced by nitrogen-fixing rhizobia that function 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 O-6 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 (e.g., alfalfa), the fatty acid component is mainly 2E,9Z‑hexadecadienoate, but small amounts of palmitate or palmitoleate may be found also. Intriguingly, the lipopolysaccharide and lipid A component from this species suppress both the early and late responses to pathogen attack in alfalfa.

Nodulation factor from Sinorhizobium meliloti

Similar Nod-like 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.

Mycobacteria synthesize intracellular 6-O-methylglucose–containing lipopolysaccharides, and that from M. tuberculosis has acetyl, isobutyryl, succinyl and octanoyl groups attached to glucose in the terminal region of the molecule, and it induces protective T cell formation in host animals. There are many other classes of bacterial lipopolysaccharides, but for reasons of practical convenience, these are discussed elsewhere on this website in relation to glycolipid surfactants, phosphoglycolipids (lipoteichoic acids) and glycophospholipids (capsular lipopolysaccharides).

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