Glycobiology Analysis Manual, 2nd Edition

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria. Lipopolysaccharide is localized in the outer layer of the membrane and is, in noncapsulated strains, exposed on the cell surface.


Intact bacterial lipopolysaccharides are macromolecules of molecular mass 10‑20 kDa made up of three structural components (see Figure 1):

  • A hydrophobic lipid section, lipid A, which is responsible for the toxic properties of the molecule,
  • A hydrophilic core polysaccharide chain, and
  • A repeating hydrophilic O-antigenic oligosaccharide side chain that is specific to the bacterial serotype.1
General structure for bacterial lipopolysaccharides

Figure 1. General structure for bacterial lipopolysaccharides. See the text for detailed explanation regarding each section. Abbreviations: KDO: 3‑deoxy-α-D-mannooctulosonic acid; Hep: Heptulose (ketoheptose); NGa: Galactosamine; NGc: Glucosamine.

The lipid A core is made up of a β-glucosamine-(1→6)-glucosamine-1-phosphate base with fatty acid esters attached to both carbohydrates. The acyl chain length and number of acyl groups may vary between bacterial species but are relatively conserved within a species. The inner polysaccharide core typically contains between 1 and 4 molecules of the KDO (3‑deoxy-α-D-manno-octulosonic acid) attached to the disaccharide core. KDO is specifically associated with lipopolysaccharide, and biologically active lipid A was thought to require at least one KDO residue for bacterial survival.2 However, an Escherichia coli K-12 suppressor strain that is KDO deficient demonstrates that the KDO requirement is not absolute for viability.3

The KDO-containing inner core also is modified with heptulose (ketoheptose) monosaccharides, the most common of which is L-glycero-α-D-manno-heptopyranose. The inner core glycan residues are typically phosphorylated or modified with phosphate-containing groups, e.g., pyrophosphate or 2‑aminoethylphosphate. The phosphate groups of lipopolysaccharides increase the overall negative charge of the cell membrane and help to stabilize the structure.

The outer core of the lipopolysaccharide contains more common hexoses, including glucose, galactose, and N-acetylglucosamine and is structurally more diverse than the inner core.

The O-antigen is a repeating oligosaccharide unit typically comprised of two to six sugars. The O-antigen is the primary structural constituent of lipopolysaccharide that differentiates bacteria. The distinctive O-antigen structures have been used to identify and assign serogroups to Escherichia coli, Salmonella enterica, and Vibrio cholerae.4 Lipopolysaccharides from rough mutant strains of E. coli lack the O-antigen portion of the structure.

The core section and the lipid A section of a lipopolysaccharide may have some variability in structure, while the O-antigen has a high degree of structural variability as well as variability in the number of repeating units. These differences result in a significant amount of heterogeneity in LPS preparations. Since LPS is heterogeneous and tends to form aggregates of varying sizes, there is a reported "molecular mass" range for these aggregates of 1‑4 million Dalton or greater. When the LPS is treated with sodium dodecyl sulfate (SDS) and heat, the molecular mass is ~50‑100 kDa.5

Functions and Applications

Within Gram-negative bacteria, the membrane lipopolysaccharides protect the bacterium against the action of bile salts and lipophilic antibiotics.6

Lipopolysaccharides are heat stable endotoxins and have long been recognized as a key factor in septic shock (septicemia) in humans1,7 and, more generally, in inducing a strong immune response in normal mammalian cells. The lipid A moiety has been identified as critical to the endotoxin activity of lipopolysaccharide. This was demonstrated, Galanos, et al., by finding identical bioactive results, including endotoxic activity, between synthetic and natural-sourced E. coli lipid A preparations.8 The active receptor for lipopolysaccharide has been identified as the CD14/TLR4/MD2 receptor complex, which promotes the secretion of proinflammatory cytokines including tumor necrosis factor-α and interleukin-1.9 While the lipid A component is primarily responsible for immune response activation, the polysaccharide component of Salmonella enterica LPS is also necessary for NF-κB activation.10

Lipopolysaccharide preparations have been used in research for the elucidation of LPS structure,11 metabolism,12 immunology,13 physiology,14 toxicity,15 and biosynthesis.16 They have also been used to induce synthesis and secretion of growth promoting factors such as interleukins.17,18 Because of its connection to septicemia, lipopolysaccharide has been studied to identify possible targets for antibodies and inhibitors to LPS biosynthesis.19,20

Extraction and Purification

Lipopolysaccharides can be prepared by extraction from TCA,21 phenol,22,23 or phenol-chloroform-petroleum ether (for rough strains).24 TCA extracted lipopolysaccharides are structurally similar to the phenol extracted ones, with similar electrophoretic patterns and endotoxicity. The primary differences are in the amounts of nucleic acid and protein contaminants remaining after extraction. The TCA extracts contain ~2% RNA and ~10% denatured proteins, while phenol extracts contain up to 60% RNA and <1% protein. Subsequent purification by gel filtration chromatography removes much of protein present in the phenolextracted LPS, but results in a preparation that contains 10‑20% nucleic acids. Further purification using ion exchange chromatography yields an lipopolysaccharide product which contains <1% protein and <1% RNA.




E T Rietschel, T Kirikae, F U Schade, U Mamat, G Schmidt, H Loppnow, A J Ulmer, U Zähringer, U Seydel, F Di Padova
FASEB Journal 1994-02-01
Endotoxins of Gram-negative microbes fulfill as components of the outer membrane a vital function for bacterial viability and, if set free, induce in mammalians potent pathophysiological effects. Chemically, they are lipopolysaccharides (LPS) consisting of an O-specific chain, a core oligosaccharide, and a lipid component, terme...Read More
I M Helander, B Lindner, H Brade, K Altmann, A A Lindberg, E T Rietschel, U Zähringer
European Journal of Biochemistry 1988-11-15
The chemical structure of the lipopolysaccharide of a deep-rough mutant (strain I-69 Rd-/b+) of Haemophilus influenzae was investigated. The hydrophilic backbone of lipid A was shown to consist of a beta-(1',6)-linked D-glucosamine disaccharide with phosphate groups at C-1 of the reducing D-glucosamine and at C-4' of the non-red...Read More
Timothy C Meredith, Parag Aggarwal, Uwe Mamat, Buko Lindner, Ronald W Woodard
ACS Chemical Biology 2006-02-17
Gram-negative bacteria possess an asymmetric lipid bilayer surrounding the cell wall, the outer membrane (OM). The OM inner leaflet is primarily composed of various glycerophospholipids, whereas the outer leaflet predominantly contains the unique amphiphilic macromolecule, lipopolysaccharide (LPS or endotoxin). The majority of a...Read More
Gabrielle Samuel, Peter Reeves
Carbohydrate Research 2003-11-14
The O-antigen is an important component of the outer membrane of Gram-negative bacteria. It is a repeat unit polysaccharide and consists of a number of repeats of an oligosaccharide, the O-unit, which generally has between two and six sugar residues. O-Antigens are extremely variable, the variation lying in the nature, order and...Read More
B Jann, K Reske, K Jann
European Journal of Biochemistry 1975-12-01
Lipopolysaccharide preparations from R(rough) Escherichia coli O8-,SR(semirough) Salmonella typhimurium and S (smooth) strains E. coli O8 and Citrobacter 396 were disintegrated with sodium dodecylsulfate and subjected to polyacrylamide gel electrophoresis in the presence of 1% sodium dodecylsulfate. The results obtained were com...Read More
6. Analysis of lipopolysaccharides of Gram-negative bacteria. Mayer, H. et al. Methods Microbiol., 18, 157-207 (1985).
J Schletter, H Heine, A J Ulmer, E T Rietschel
Archives of Microbiology 1995-12-01
Endotoxin (lipopolysaccharide, LPS), a constituent of the outer membrane of the cell wall of gram-negative bacteria, exerts a wide variety of biological effects in humans. This review focuses on the molecular mechanisms underlying these activities and discusses structure-function relationships of the endotoxin molecule, its inte...Read More
C Galanos, O Lüderitz, E T Rietschel, O Westphal, H Brade, L Brade, M Freudenberg, U Schade, M Imoto, H Yoshimura
European Journal of Biochemistry 1985-04-01
The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell ...Read More
Eva M Pålsson-McDermott, Luke A J O'Neill
Immunology 2004-10-01
An understanding of lipopolysaccharide (LPS) signal transduction is a key goal in the effort to provide a molecular basis for the lethal effect of LPS during septic shock and point the way to novel therapies. Rapid progress in this field during the last 6 years has resulted in the discovery of not only the receptor for LPS - Tol...Read More
Masashi Muroi, Ken-Ichi Tanamoto
Infection and Immunity 2002-11-01
The lipid A portion has been identified as the active center responsible for lipopolysaccharide (LPS)-induced macrophage activation. However, we found that Salmonella (Salmonella enterica serovars Abortusequi, Minnesota, and Typhimurium) lipid A is inactive in human macrophages, despite its LPS being highly active. Thus we inves...Read More
S M Strain, S W Fesik, I M Armitage
Journal of Biological Chemistry 1983-03-10
Lipopolysaccharide (LPS) isolated from Escherichia coli D31m4, a heptoseless mutant, was studied by 13C and 31P NMR spectroscopy. Modified isolation and purification procedures are described which permitted high resolution NMR spectra to be obtained from samples of intact LPS. 31P NMR was used to monitor the purity and native he...Read More
R S Munford, J M Andersen, J M Dietschy
Journal of Clinical Investigation 1981-12-01
When gram-negative bacterial lipopolysaccharides (LPS) are injected intravenously into the rabbit or rat, they bind to plasma lipoproteins, particularly high density lipoproteins (HDL). The present studies were performed to examine the mechanisms by which LPS-HDL complexes are removed from the circulation and taken up by various...Read More
D C Morrison, J A Rudbach
Contemporary topics in molecular immunology 1981-01-01
14. International Review of Biochemistry. Galanos, C., et al. Biochemistry of Lipids III, Vol. 14, 2309, T. E. Goodwin, ed., University Park Press, Baltimore, MD USA (1977).
H J Kurtz, J Quast
American Journal of Veterinary Research 1982-02-01
Escherichia coli endotoxin was continuously infused IV into 31 pigs. The response was related to the infusion rate. Of 13 pigs given endotoxin at the rate of 15.4 and 23.1 micrograms/kg of body weight/hour, 10 had bilateral renal cortical necrosis similar to a generalized Shwartzman lesion. All 13 pigs given endotoxin at the rat...Read More
P D Rick, D A Young
Journal of Bacteriology 1982-05-01
A new mutant of Salmonella typhimurium was isolated which possesses a temperature-sensitive defect in the synthesis of 3-deoxy-D-manno-octulosonic acid. The defect in 3-deoxy-D-manno-octulosonic acid synthesis is due to a temperature-sensitive 3-deoxy-D-manno-octulosonate-8-phosphate synthetase, and the mutant accumulates an inc...Read More
R R Skelly, P Munkenbeck, D C Morrison
Infection and Immunity 1979-02-01
The murine immune response to a haptenated lipopolysaccharide (LPS) lacking repeating oligosaccharide determinants was studied. The LPS was extracted from a rough strain of bacteria (Salmonella minnesota R595) and chemically haptenated with either trinitrophenol or fluorescein isothiocyanate. These preparations of hapten-R595 LP...Read More
Charles H Lang, Christine Silvis, Nobuko Deshpande, Gerald Nystrom, Robert A Frost
Shock 2003-06-01
The presence of increased levels of proinflammatory cytokines in the blood is associated with decreased muscle protein synthesis and the erosion of lean body mass in many catabolic conditions. However, little is known regarding the role of endogenous cytokine synthesis in muscle per se. The purpose of the present study was to ch...Read More
J A Yethon, C Whitfield
Current Drug Targets: Infectious Disorders 2001-08-01
Lipopolysaccharide (LPS) constitutes the lipid portion of the outer leaflet of Gram-negative bacteria, and is essential for growth. LPS is also known to be responsible for the variety of biological effects associated with Gram-negative sepsis. In recent years, tremendous progress has been made in determining the exact chemical s...Read More
Sven Muller-Loennies, Lore Brade, C Roger MacKenzie, Franco E Di Padova, Helmut Brade
Journal of Biological Chemistry 2003-07-11
Septic shock due to infections with Gram-negative bacteria is a severe disease with a high mortality rate. We report the identification of the antigenic determinants of an epitope that is present in enterobacterial lipopolysaccharide (LPS) and recognized by a cross-reactive monoclonal antibody (mAb WN1 222-5) regarded as a poten...Read More
21. Bacterial lipido-protinopolysaccharides ('O' somatic antigens) extraction with trichloroacetic acid. Staub, A. M. Methods in Carbohydrate Chem., Vol. 5, 92-93, John Wiley & Sons, Inc., Hoboken, NJ USA (1965).
22. Bacterial lipopolysaccharides extraction with phenolwater and further applications of the procedure. Westphal, O. and Jann, K. Methods in Carbohydrate Chem., Vol. 5, 92‑93, John Wiley & Sons, Inc., Hoboken, NJ USA (1965).
23. Isolation of lipopolysaccharides from bacteria. Leive, L. and Morrison, D. C. Methods Enzymol., 28, 254-262 (1972).
C Galanos, O Lüderitz, O Westphal
European Journal of Biochemistry 1969-06-01
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