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Lipopolysaccharides

Synonym: LPS

Product Information

Lipopolysaccharides (LPS) are characteristic components of the cell wall of Gram negative bacteria; they are not found in Gram positive bacteria. They are localized in the outer layer of the membrane and are, in noncapsulated strains, exposed on the cell surface. They contribute to the integrity of the outer membrane, and protect the cell against the action of bile salts and lipophilic antibiotics.1

Lipopolysaccharides are made up of a hydrophobic lipid (lipid A, which is responsible for the toxic properties of the molecule), a hydrophilic core polysaccharide chain, and a hydrophilic O-antigenic polysaccharide side chain. In most cases, O-specific chains are built of repeating units of oligosaccharides which exhibit a strain-specific structural diversity. The sugar constituents, their sequence, and their mode of linkage determine the serological O specificity of respective strains. They are the main determinants of the classifications of the serotypes of Salmonella species. The diversity of O chains in Enterobacteriaceae may have developed during evolution to allow enteric bacteria to escape the host’s immune system by developing new specificities on their cell surface (specific to the bacterial serotype).1

Since lipopolysaccharides confer antigenic properties on the cell, they have been termed O antigens. As the main antigen, lipopolysaccharides are involved in various host-parasite interactions. They seem to protect Gram negative bacteria from phagocytosis and lysis.1 Bacteria with common serotypes have surface antigens (group O, group H, or LPS) which generate the same antibody response. Examples of serotypes are O55:B5 and O26:B6 for the E. coli bacterium. The designations are immunological classifications, which specify which antibody recognized which strains. Different strains may have some common antigenic determinants.

If a wild strain of bacterium is irradiated with UV light or exposed to mutagenic compounds, it will mutate. The few mutations that are not lethal result in viable mutants (rough strains) which are generally not found in nature, and which possess some unique characteristics. The genes that encode lipopolysaccharide formation may also be altered in the mutants, and LPS with shorter polysaccharide chains may be formed. Ra, Rb, Rc, Rd, Re, etc. (where a, b, c, etc... designate 1st, 2nd, 3rd, etc... degree, respectively) designate the polysaccharide length of a given LPS. Ra and Re designate the mutants with the longest and shortest chain lengths, respectively.2 The most extreme mutants are the Re mutants which produce an LPS which is made up of Lipid A and 3-deoxy-D-manno-octulosonic acid (2-Keto-3-deoxyoctonate, KDO) as the sole constituent of the core.2  Lipid A and lipopolysaccharides from rough strains are tested for KDO content.3

Purified endotoxin is generally referred to as lipopolysaccharide or LPS, to distinguish it from the more natural complexed cell membrane associated form. The core portion of the polysaccharide chain is common to LPS from wild and mutant bacterial strains.

Removal by hydrolysis of the polysaccharide chain from LPS produces lipid A, either as the naturally occurring, cytotoxic diphosphoryl form4 or the less toxic, monophosphoryl form.5,6 The longer the polysaccharide chain is, the longer and more difficult the hydrolysis. LPS with a long polysaccharide chain has a relatively low lipid A content, which must be purified from a large amount of hydrolysis byproducts (oligosaccharides and saccharide monomers). Thus, the yield of lipid A is low and recovery is poor. LPS with a short polysaccharide chain (LPS from mutant bacteria) is therefore used to produce lipid A products. Removal of the fatty acid portions of lipid A results in a detoxified LPS7 with an endotoxin level about 10,000 times lower than that of the parent LPS.

The molecular structure of LPS has been studied.8,9 Since LPS is heterogeneous and tends to form aggregates of varying sizes, the molecular weight is not very meaningful. However, there is a reported range of 1-4 million or greater. When the LPS is treated with SDS and heat, the molecular weight is in the range of 50 to 100 kDa. In their purest form, in the presence of strong surface active agents, and in the absence of divalent cations, bacterial endotoxins consist of 10-20 kDa macromolecules. In the absence of surface active agents and in the presence of divalent cation sequestering agents such as EDTA, LPS is believed to arrange itself into a micellar structure with a molecular weight of approximately 1,000 kDa. This is the smallest form of bacterial LPS that is likely to exist in aqueous liquids. In the presence of divalent cations such as Ca2+ and Mg2+, a bilayer structure appears to exist that passes through a 0.2 μm membrane, but does not pass through a 0.025 μm membrane. LPS vesicles up to 0.1 μm in diameter may also be formed in water in the presence of divalent cations. The self aggregation of LPS is generally a function of the lipid A component of the molecule, which also confers the ability to bind to hydrophobic surfaces.

LPS can be prepared by TCA,10 phenol,11 or phenolchloroform- petroleum ether (for rough strains)12 extraction. The TCA extracted lipopolysaccharides are structurally similar to the phenol extracted ones. Their electrophoretic pattern and endotoxicity are similar. The main differences are in the amounts of nucleic acid and protein contaminations. The TCA extract contains approximately 2% RNA and approximately 10% denatured proteins. The phenol extract contains up to 60% RNA and less than 1% protein. Purification by gel filtration chromatography removes much of protein present in the phenol-extracted LPS, but leaves a product that still contains 10-20% nucleic acids. Further purification using ion exchange chromatography, will yield an LPS product which contains <1% protein and <1% RNA. Sigma offers LPS with various levels of protein and/or RNA.

Sigma's lipopolysaccharides contain endotoxin levels of not less than 500,000 EU (endotoxin units)/mg unless otherwise noted. One nanogram of endotoxin is equivalent to 5 EU (Limulus lysate assay) and 10 EU (chromogenic assay).

LPS preparations are used extensively for research in the elucidation of LPS structure,13 metabolism,14 immunology,15 physiology,16 toxicity,17 and biosynthesis.18  They have also been used to induce synthesis and secretion of growth promoting factors such as interleukins.19

FITC (fluorescein isothiocyanate), TRITC (tetramethyrhodamine isothiocyanate), and TNP (trinitrophenyl) conjugates have been prepared by reacting LPS with either FITC, TRITC or 2,4,6-trinitrobenzenesulfonic acid, respectively.20 They are used in research on the T-independent B cell immune response to bacterial LPS.20

Precautions and Disclaimer

For Laboratory Use Only. Not for drug, household or other uses.

Storage/Stability

Solutions at 1 mg/ml in buffer or culture medium are stable for approximately one month at 2-8 °C. Frozen aliquots can be stored up to 2 years. Repeated freeze/thaw cycles are not recommended. Solutions should be stored in silanized containers, since LPS can bind to plastics and certain types of glass (especially at concentrations of <0.1 mg/ml). If the LPS concentration is >1 mg/ml, adsorption to the sides of the vial is negligible. If glass containers are used, solutions should be vortexed for at least 30 minutes to redissolve the adsorbed product.

Preparation Instructions

The product is soluble in water (5 mg/ml) or cell culture medium (1 mg/ml) yielding a hazy, faint yellow solution. A more concentrated, though still hazy, solution (20 mg/ml) has been achieved in aqueous saline after vortexing and warming to 70-80 °C.21 Lipopolysaccharides are molecules that form micelles in every solvent. Hazy solutions are observed in water and phosphate buffered saline. Organic solvents do not give clearer solutions. Methanol yields a turbid suspension with floaters, while water yields a homogeneously hazy solution.

For cell culture use, LPS should be reconstituted by adding 1 ml of sterile balanced salt solution or cell culture medium to a vial (1 mg) and swirling gently until the powder dissolves. Solutions can be further diluted to the desired working concentration with additional sterile balanced salt solutions or cell culture media.

LPS Table

Source
Organism            
Extraction Method                       
Gel Filtration
Gel Filtration γ-irr.                      
Ion-exchange
Detoxified Gel Filtration FITC Label
Gel Filtration TNP Label
O26:B6 E. coli
Phenol - L8274
TCA - L3755                                 
L2762
L2654        
O55:B5 E. coli
Phenol - L2880
TCA - L4005
L2637
L6529 L4524 L9023 F8666  
O111:B4 E. coli
Phenol - L2630
TCA - L4130
L3012
L4391 L3024 L3023 F3665 T3382
O127:B8 E. coli
Phenol - L3129
TCA - L3880
L3137
L4516 L5024      
O128:B12 E. coli
Phenol - L2755
L2887
        T6769
E. coli EH-100
(Ra mutant)
Ph/Chl/Pet - L9641
           
E. coli F-583
(Rd mutant)
Ph/Chl/Pet - L6893
           
E. coli J5
(Rc mutant)
Ph/Chl/Pet - L5014
           
E. coli K-235
Phenol - L2143
L2018           
Klebsiella pneumoniae   
Phenol - L4268
           
Pseudomonas aeroginosa 10
Phenol - L9143
TCA - L7018
L8643
         
Salmonella
abortus equi
Phenol - L5886
TCA - L6636
L1887
         
Salmonella enteritidis
Phenol - L6011
L2012
L7770 L4774      
Salmonella minnesota
Phenol - L6261
TCA - L7011
L2137 L4641        
Salmonella minnesota strain Re595
(Re mutant)
Ph/Chl/Pet - L9764
           
Salmonella typhimurium
Phenol - L6511
TCA - L7261
L2262 L6143        
Salmonella typhimurium strain SL1181
(Re mutant)
Ph/Chl/Pet -  L9516         
           
Salmonella typhimurium strain TV119
(Ra mutant)
Ph/Chl/Pet - L6016
           
Salmonella typhosa
Phenol - L6386
TCA - L7136
L2387 L7895        
Serratia marcescens
Phenol - L6136
           

* = discontinued product number
Ph/Chl/Pet = phenol:chloroform:petroleum ether

 

References

  1. Mayer, H. et al., Analysis of Lipopolysaccharides of Gram-Negative Bacteria. Methods in Microbiology 18, 157-207 (1985).
  2. Raetz, C. R. H., Biochemistry of Endotoxins. Annu. Rev. Biochem. 59, 129-170 (1990).
  3. Cynkin, M. A., Estimation of 3-Deoxy sugars by means of the Manonaldehyde-Thibarbituric Acid Reaction. Nature, 186, 155 (1960).
  4. Qureshi, N., et al., Position of ester groups in the lipid A backbone of lipopolysaccharides obtained from Salmonella typhimurium. J. Biol. Chem., 258(21), 12947-12951 (1983).
  5. Chang C. M., and Nowotny, A., Relation of Structure to Function in Bacterial O-antigens VII. Endotoxicity of “lipid A.” Immunochem., 12, 19 (1975).
  6. Qureshi N., and Takayama, N., Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium.J. BioI. Chem., 257, 11808-11815 (1982).
  7. Ding H. F., et al., Protective immunity induced in mice by detoxified Salmonella lipopolysaccharide. J. Med. Microbiol., 31(2), 95-102 (1990).
  8. Jann, B., et al., Heterogeneity of lipopolysaccharides. Analysis of polysaccharide chain lengths by sodium dodecylsulfatepolyacrylamide gel electrophoresis. Eur. J. Biochem., 60, 239-246 (1975).
  9. Leive, L., and Morrison, D. C., Isolation of Lipopolysaccharides from Bacteria. Methods in Enzymology, 28, 254-262 (1972).
  10. Staub, A. M., Bacterial Lipido-protinopolysaccharides (‘O’ Somatic Antigens) Extraction with Trichloroacetic Acid. Methods in Carbohydrate Chem., 5, 92-93 (1965).
  11. Westphal, O., and Jann, K., Bacterial Lipopolysaccharides Extraction with Phenol-Water and Further Applications of the Procedure. Methods in Carbohydrate Chem., 5, 83-91 (1965).
  12. Galanos, C., et al., A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem., 9(2), 245-249 (1969).
  13. Strain, S. M., et al., Characterization of lipopolysaccharide from a heptoseless mutant of Escherichia coli by carbon 13 nuclear magnetic resonance. J. BioI. Chem., 258(5), 2906-2910 (1983).
  14. Munford, R. S., et al., Sites of tissue binding and uptake in vivo of bacterial lipopolysaccharide-high density lipoprotein complexes: studies in the rat and squirrel monkey. J. Clin. Invest., 68(6), 1503-1513 (1981).
  15. Morrison, D. C., and Rudbach, J. A., Endotoxincell- membrane interactions leading to transmembrane signaling. Contemporary Topics in Molecular Immunology, 8, 187-218, P. Inman and J. Mandy, eds., Plenum Press, New York (1981).
  16. Galanos, C., et al., International Review of Biochemistry, Biochemistry of Lipids III, 14, 2309, T. E. Goodwin, ed., University Park Press, Baltimore (1977).
  17. Kurtz, H. J., et al., Effects of continuous intravenous infusion of Escherichia coli endotoxin into swine. Amer. J. Vet. Res., 43, 262-8 (1982).
  18. Rick, P. D., and Young, P. A., Isolation and characterization of a temperature-sensitive lethal mutant of Salmonella typhimurium that is conditionally defective in 3-deoxy-D-mannooctulosonate-8-phosphate synthesis. J. Bacteriol.,150, 447-55 (1982).
  19. Oppenheim, J. J., et al., Immunol. Today, 7, 45 (1986).
  20. Skelly, R., et al., Stimulation of T-independent antibody responses by hapten-lipopolysaccharides without repeating polymeric structure. Infect. Immun., 23(2), 287-293 (1979).
  21. Customer report
  22. Formsgaard, A., et al., Quantitation and biological activities of native tumour necrosis factor from PLS-stimulated human monocytes. APMIS, 98, 529-534(1990).
  23. Bleicher, P.A., et al., Mitogenic response of frog lymphocytes to crude and purified preparations of bacterial lipopolysaccharide (LPS). Dev. Comp. Immunol., 7, 483-407(1983).
  24. Sveen, K., and Skaug, N., Comparative mitogenicity and polyclonal B cell activation capacity of eight oral or nonoral bacterial lipopolysaccharides in cultures of spleen cells from athymic (nu/nu-BALB/c) and thymic (BALB/c) mice. Oral Micorbiol. Immunol., 7, 71-77 (1992).
  25. Hepper, K. P., et al., Plaque-forming cell response in BALB/c mice to two preparations of LPS extracted from Salmonella enteritidis. J. Immunol., 122, 1290-3 (1979).
  26. Gardiner, J. S., et al., In vitro formation of complement activation products by lipopolysaccharide chemotypes of Salmonella minnesota. Int. Arch. Allergy Appl. Immunol. 96(1), 51-54 (1991).
  27. Salter, M., et al., Widespread tissue distribution, species distribution and changes in activity of Ca2+-dependent and Ca2+-independent nitric oxide synthases. FEBS Lett. 291(1) , 145-149 (1991).
  28. 28.  Mond, J. J. and Brunswick, M., "Proliferative assays for B Cell Function". Current Protocols in Immunology, Coligan, et al., editors, (John Wiley & Sons, NY: 1991) Unit 3.10.
  29. 29. Geller, D. A., et al., Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc. Natl. Acad. Sci. USA 90(8), 3491-3495 (1993).
  30. 30. Wrenger, E. et al., Peritoneal Mononuclear Cell Differentiation and Cytokine Production in Intermittent and Continuous Automated Peritoneal Dialysis. Am. J. Kidney Dis., 31, 234-241 (1998).
  31. 31. Hawkins, D. L. et al., Human interleukin 10 suppresses production of inflammatory mediators by LPS-stimulated equine periotoneal macrophages. Vet. Immunol. Immunopathol., 66, 1-10 (1998). 
  32. 32. Xu H., et al., Leukocytosis and resistance to septic shock in intercellular adhesion molecule 1-deficient mice. J. Exp. Med., 180(1), 95-109 (1994)
  33. 33. Stuehr, D. J., and Marletta, M. A., Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc. Natl. Acad. Sci. USA, 82(22), 7738-7742 (1985).
  34. 34. Wang, J. et al., The effects of endotoxin on platelet-activating factor synthesis in cultured rat glomerular mesangial cells. Biochim. Biophys. Acta, 969(3), 217-224 (1988).
  35. 35. Cassatella, M. A., et al., Molecular basis of interferon-gamma and lipopolysaccharide enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components. J. Biol. Chem., 265(33), 20241-20246 (1990).