Mikrobiol. Z. 2020; 82(6):35-42.
doi: https://doi.org/10.15407/microbiolj82.06.035

Chemical Characterization and Biological Activity of Escherichia coli Lipopolysaccharides

O.S. Brovarska1, L.D. Varbanets1, S.V. Kalinichenko2

1Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine

2Mechnikov Institute of Microbiology and Immunology, NAMS of Ukraine
14/16 Pushkinskaya Str., Kharkiv, 61057, Ukraine

Lipopolysaccharides (LPS) are specific components of the cell envelope of gram-negative bacteria, located at the external surface of their outer membrane and performing a number of important physicochemical and biological functions. The widespread in nature are representatives of Enterobacteriaceae family. Among them there are saprotrophic, useful human symbionts, as well as causative agents of acute intestinal infections. The role of saprophytic intestinal microbiota is not limited only to its participation in the digestion process. The endotoxin released as a result of self-renewal of the cell pool of Escherichia coli partially enters the portal blood and performs antigenic stimulation of the macroorganism. In addition, a small amount of endotoxin can also be released by live gram-negative bacteria, which, given the large population of E. coli in the intestine, can create a sufficiently high concentration of endotoxin. Aim. The study of composition and biological activity of lipopolysaccharides of new E. coli strains, found in the human body. Methods. The objects of investigation were strains of Escherichia coli, isolated from healthy patients at the epidemiological center in Kharkiv. Lipopolysaccharides were extracted from dried cells by 45% phenol water solution at 65–68°С by Westphal and Jann method. The amount of carbohydrates was determined by phenol-sulfuric method. Carbohydrate content was determined in accordance to the calibration curve, which was built using glucose as a standard. The content of nucleic acids was determined by Spirin method, protein − by Lowry method. Serological activity of LPS was investigated by double immunodiffusion in agar using the method of Ouchterlony. Results. In all studied E. coli LPS (2884, 2890, 2892), glucose was dominant monosaccharide (40.5, 41.1, 67.3%, respectively). LPS also contained rhamnose (1.8, 22.9, 1.6%, respectively), ribose (3.5, 6.1, 3.6%, respectively) and galactose (4.1, 20.2, 18.3%, respectively). E. coli 2884 LPS also contained arabinose (1.0%) and mannose (44.8%), while E. coli strains 2890 and 2892 LPS contained heptose (9.7 and 7.8%, respectively). Lipid A composition was presented by fatty acids with a carbon chain length from C12 to C18. As the predominant components were 3-hydroxytetradecanoic (39.2–51.3%) as well as tetradecanoic (23.1–28.5%), dodecanoic (8.9–10.9%), hexadecanoic (4.3–7.2%) and octadecanoic (1.8–2.4%) acids. Unsaturated fatty acids: hexadecenoic (2.0–17.9%) and octadecenoic (3.4–4.2%) have been also identified. It was found that octadecanoic and octadecenoic acids were absent in the LPS of 2884 and 2892 strains, respectively. In SDS-PAAG electrophoresis, a bimodal distribution typical for S-forms of LPS was observed. The studied LPS were toxic and pyrogenic. Double immunodiffusion in agar by Ouchterlony revealed that the tested LPS exhibited an antigenic activity in the homologous system. In heterologous system E. coli 2892 LPS had cross reactivity with LPS of E. coli 2890 and М-17. Since the structure of the O-specific polysaccharide (OPS) of E. coli M-17 was established by us earlier, the results of serological reactions make it possible to suggest an analogy of the E. coli 2892 and 2890 OPS structures with that of E. coli М-17 and their belonging to the same serogroup. Conclusions. The study of the composition and biological activity of LPS of new strains of Escherichia coli 2884, 2890 and 2892, isolated from the body of almost healthy patients, expands our knowledge about the biological characteristics of the species.

Keywords: Escherichia coli, lipopolysaccharide, monosaccharide and fatty acid composition, toxicity, pyrogenicity, serological activity.

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  1. Alexander C, Rietschel ET. Bacterial lipopolysaccharides and innate immunity. J Endotoxin Res. 2001; 7(3):167−202. https://doi.org/10.1179/096805101101532675
  2. Westphal O. Jann K. Bacterial lipopolysaccharides – extraction with phenol. Methods Carbohydr Chem. 1965; 5:83−91.
  3. Varbanets LD, Zdorovenko GM, Knirel YuA. [Methods of endotoxin investigations]. Kyiv: Nauk Dumka; 2006. Russian.
  4. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substanses. Anal Chem. 1956; 28(3):350−356. https://doi.org/10.1021/ac60111a017
  5. Spirin AS. [Spectrophotometric determination of total nucleic acids]. Biochemistry. 1958; 23(5):656−662. Russian.
  6. Lowry OH, Rosenbrough NJ, Farr LA., Randal RJ. Protein measurement with the Folin reagent. J Biol Chem. 1951; 193(5):265−275.
  7. 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
  8. 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
  9. Albershein P, Nevis DJ, English PD, Karr A. A method for analysis of sugars in plant cell wall polysaccharides by gasliquid chromatography. Carbohyd Res. 1967; 5(3):340−345. https://doi.org/10.1016/S0008-6215(00)80510-8
  10. Brovarska OS, Varbanets LD. [Influence of isolation methods on composition and biological properties of Escherichia coli lipopolysaccharides]. Microbiol Z. 2019; 81(2):3–13. Russian. https://doi.org/10.15407/microbiolj81.02.003
  11. Ouchterlony O. Diffusion in gel methods for immunological analysis. Progr Allergy. 1962; 6:3−15. https://doi.org/10.1159/000313797
  12. Rezania S, Amirmozaffari N, Tabarraei B, Jeddi-Tehrani M, Zarei O, Alizadeh R, et al. Extraction, Purification and Characterization of Lipopolysaccharide from Escherichia coli and Salmonella typhi. Avicenna J Med Biotechnol. 2011; 3(1):3–9.
  13. Pollack M. Biological functions of lipopolysaccharide antibodies. In: Brade H, Opal S, Vogel S and Morrison D, editors. Endotoxin in health disease. New York-Basel: Marcel Dekker, Inc. 1999; p. 623–632. https://doi.org/10.1201/9781003064961-40
  14. Varbanets LD, Zdorovenko EL, Garkava EG, Brovarska OS. Escherichia coli M-17 lipopolysaccharide. Microbiology (Moscow). 2012; 81(3):324–331. https://doi.org/10.1134/S0026261712020154
  15. Zdorovenko EL, Varbanets LD, Bin Liu, Valueva OA, Wang Q, Shashkov AS, et al. 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
  16. Varbanets LD, Brovarskaya OS, Kalinichenko SV, Zdorovenko EL. Characteristics of the lipopolysaccharide Escherichia coli 126. Microbiology (Moscow). 2017; 86(1):56–63. https://doi.org/10.1134/S0026261717010167
  17. Stenutz R, Weintraub A, Widmalm G. The structures of Escherichia coli O-polysaccharide antigens. FEMS Microbiology Reviews. 2006; 30(3):382−403. https://doi.org/10.1111/j.1574-6976.2006.00016.x