Mikrobiol. Z. 2019; 81(2):3-13. Russian.
doi: https://doi.org/10.15407/microbiolj81.02.003

Influence of Isolation Methods on Composition and Biological Properties
of Escherichia coli Lipopolysaccharides

Brovarskaya O.S., Varbanets L.D.

Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine

The purpose of this work was to study the influence of isolation methods on composition and biological activity of Escherichia coli lipopolysaccharides (LPS). Methods. Microbiological, biochemical and serological methods were used. LPS were obtained from the cells by the classic Westphal method (LPSw), Boivin (LPSb), and an alkaline lipopolysaccharide (LPSa). Results. LPSw, LPSb and LPSa were characterized by a different yield of 0.33, 0.25 and 0.05%, respectively, from the mass of microbial cells. All LPS preparations contained the main components of LPS: carbohydrates, KDO and fatty acids of lipid A. It was also shown that the method of Boivin and Westphal makes it possible to isolate higher molecular weight, and alkaline extraction method - lower molecular weight fractions of LPS. The main components of the hydrophobic part of the LPS in lipid A were 3-OH-C14:0, C12:0, C14:0, C16:0 acids, and the polysaccharide part of the LPS was represented by glucose, galactose and heptose, as well as by one unidentified monosaccharide. LPSw exhibited the largest serological and toxic activity, and LPSb - less toxic, but more pyrogenic. LPSa characterized the smallest pyrogenicity and was slightly toxic. Conclusions. The method of extraction of LPS influences its composition and biological properties.

Keywords: Escherichia coli 58, lipopolysaccharide, monosaccharide and fatty acid composition, biological activity.

Full text (PDF, in Russian)

  1. Rosenfeld Y, Shai Y. Lipopolysaccharide (Endotoxin)-host defense antibacterial peptidesinteractions: Role in bacterial resistance and prevention of sepsis. Biochimica et Biophysica Acta. 2006; 1758:1513–1522. https://doi.org/10.1016/j.bbamem.2006.05.017
  2. Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002; 71(1):635–700. https://doi.org/10.1146/annurev.biochem.71.110601.135414
  3. Van Amersfoort, ES, Van Berkel TJ, Kuiper J. Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock. Clin Microbiol Rev. 2003; 16:379–414. https://doi.org/10.1128/CMR.16.3.379-414.2003
  4. Westphal O. Jann K. Bacterial lipopolysaccharides – extraction with phenol. Methods Carbohydr Chem. 1965; 5:83–91.
  5. Boivin A, Mesrobeanu J, Mesrobeanu L. Technique pour la preparation des polysaccharides microbiens specifiques. C.r. Soc biol. 1933; 113(21):490–492.
  6. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determinationn of sugars and related substranses. Anal Chem. 1956; 28(3):350–356. https://doi.org/10.1021/ac60111a017
  7. Lowry OH, Rosenbrough NJ, Farr LA., Randal RJ. Protein measurement with the Folin reagent. J Biol Chem. 1951; 193(5):265–275.
  8. Spirin AS. [Spectrophotometric determination of total nucleic acids]. Biochemistry. 1958; 23(5):656–662. Russian.
  9. Varbanets LD, Zdorovenko GM, Knirel YuA. [Methods of endotoxin investigations]. Kyiv: Naukova Dumka, 2006; 237. Russian.
  10. Laemmli UK. Cleavage of proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227:680–685. https://doi.org/10.1038/227680a0
  11. Tsai CM, Frash CE. A sensitive silver stain for detecting lipopolysaccharides in polyacrilamide gels. Anal Biochem. 1982; 119:115–119. https://doi.org/10.1016/0003-2697(82)90673-X
  12. Galanos C, Lüderitz O, Rietschel ET. Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities. Eur J Biochem. 1985; 148:1–5. https://doi.org/10.1111/j.1432-1033.1985.tb08798.x
  13. Muthannan AR. Determination of 50% endpoint titer using a simple formula. World J Virol 2016; 12; 5(2):85–86.
  14. Ouchterlony O. Diffusion in gel methods for immunological analysis. Prog Allergy. 1962; 6:3–15. https://doi.org/10.1159/000391328
  15. Rezania S, Amirmozaffari N, Tabarraei B, Jeddi-Tehrani M, Zarei O, Alizadeh R, Masjedian F, and Zarnani A. Extraction, Purification and Characterization of Lipopolysaccharide from Escherichia coli and Salmonella typhi. Avicenna J Med Biotechnol. 2011; 3(1):3–9.
  16. Alexander C, Rietschel ET, Bacterial lipopolysaccharides and innate immunity. J. Endotoxin Res. 2001; 7:167–202. https://doi.org/10.1179/096805101101532675
  17. Proctor RA. Handbook of endotoxin. Vol. 3. Cellular biology of endotoxin. Ed. Berry LJ. Amsterdam: Elsevier, 1985; 454.
  18. Galanos C, Freudenberg MA, Reuter W. Galactosamineinduced sensitization to the lethal effects of endotoxin. Proc Natl Acad Sci. USA. 1979; 76:5939–5943. https://doi.org/10.1073/pnas.76.11.5939
  19. Zdorovenko EL, Varbanets LD, Bin Liu, Valueva OA, Wang Q, Shashkov AS, Garkavaya EG, Brovarskaya OS, Wang Lei, Knirel YuA. Structure and gene cluster of the O-antigen of Escherichia coli L-19, a candidate for a new O-serogroup. Microbiology (Elsevier). 2014; 160:2102–2107. https://doi.org/10.1099/mic.0.080804-0
  20. Varbanets LD, Zdorovenko EA, Garkavaya EG, Brovarskaya OS. Lipopolysaccharide of Escherichia coli M-17. Microbiology (Moscow). 2012; 81(3):324–331. https://doi.org/10.1134/S0026261712020154