Endotoxin-like properties of an extract from a symbiotic,eukaryotic Chlorella-like green alga

Abstract

Hot phenol-water extraction of axenic cultures of the eukaryotic symbiotic green alga, Chlorella, yielded a substance having many of the characteristics of bacterial endotoxin or lipopolysaccharide (LPS). This material caused gelation of extracts of Limulus amoebocyte lysate at concentrations similar to those manifest by LPS from Gram-negative enteric bacteria. Activity was reduced substantially by incubation with polymyxin B sulfate and Limulus endotoxin-neutralizing protein (ENP), both of which have been shown to neutralize the biological activity of LPS from Gram-negative enteric bacteria. Partially purified biologically active material was found to contain 3-deoxy-D-manno-octulosonic acid (KDO), a sugar characteristically found in LPS. Acid hydrolysis of the Chlorella extract yielded a precipitate with characteristics of lipid A. Further hydrolysis followed by methylation yielded products with retention times on gas chromatography indistinguishable from 3-hydroxylauric and 3-hydroxymyristic acids. Results of transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining are consistent with those obtained with purified enteric bacterial LPS. Exhaustive precautions against potential experimental artifacts due to cross contamination by endotoxin from Gram-negative bacteria, either in algal cultures or associated with glassware allowed the conclusion that the green alga, Chlorella (strain NC64A) synthesizes a LPS-like molecule.

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References

Karakashian MW Symbiosis in Paramecium bursaria. Symp Soc Exp Biol 1975; 29: 145-173.

Pardy RL , Muscatine L. Recognition of symbiotic algae by Hydra viridis. A quantitative study of the uptake of living algae by aposymbiotic H. viridis. Biol Bull 1973; 145: 565-579.

Pardy RL , Royce CL Insights into symbiotic acquisition of organelles from studies of the green hydra symbiosis. In: Lewin RA. (Ed) Origins of Plastids: Symbiogenesis, Prochlorophytes, and the Origins of Chloroplasts. Bodega Bay Symposium Series, Chapman and Hall, 1992; 45-52.

Vanetten JL , Lane LC , Meints RH Viruses and virus-like particles of eukaryotic algae. Microbiol Rev 1991; 55: 586-620.

Karakashian S. Growth of Paramecium bursaria as influenced by the presence of algal symbionts. Physiol Zool 1963; 36: 52-67.

Nichols HW , Bold HC Trichosarcina polymorpha Gen. et Sp. Nov. J Phycol 1965; 1: 34-38.

Z Goldman RC , Lieve L. Electrophoretic separation of lipopolysaccharide monomers differing in polysaccharide length. Methods Enzymol 1987; 138: 267-275.

Karkhanis YD , Zeltner JY , Jackson JJ et al. A new and improved microassay to determine 2-keto-3-deoxyoctanoate in lipopolysaccharide of Gram-negative bacteria. Anal Biochem 1978; 85: 595-601.

Qureshi N. , Takayama K. , Mascagni P. et al. Complete structural determination of lipopolysaccharide obtained from deep rough mutant of Escherichia coli. j Biol Chem 1988; 263: 11971-11976.

10.

Qureshi N. , Takayama K. , Ribi E. Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium. J Biol Chem 1982; 257: 11808-11815.

11.

Wollenweber H-W. , Rietschel ET Analysis of lipopolysaccharide (lipid A) fatty acids. J Microbiol Methods 1990; 11: 195-211.

12.

Cooperstock MS Inactivation of endotoxin by polymyxin B. Antimicrob Agents Chemother 1974; 6: 422-425.

13.

Wainwright NR , Miller RJ , Paus E. et al. Endotoxin binding and neutralizing activity of a protein from Limulus polyphemus. In: Nowotny A , Spitzer JJ , Zeigler EJ. (Eds) Cellular and Molecular Aspects of Endotoxin Reactions. Amsterdam: Elsevier, 1990; 315-325.

14.

Warren HS , Glennon ML , Wainwright N. et al. Binding and neutralization of endotoxin by Limulus anti-lipopolysaccharide factor. Infect Immun 1992; 60: 2065-2513.

15.

Maniatis T. , Fritsch EF , Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982; 188-209.

16.

Sturm S. , Fortnagel P. , Timmis KN Immunoblotting procedure for the analysis of electrophoretically-fractionated bacterial lipopolysaccharide. Arch Microbiol 1984; 40: 198-201.

17.

Mayberry-Carson KJ , Roth IL , Smith PF Ultrastructure of lipopolysaccharide isolated from Thermoplasma acidophilum. J Bacteriol 1975; 121: 700-703.

18.

Shands Jr JW , Graham JA , Nath K. The morphologic structure of isolated bacterial lipopolysaccharide. J Mol Biol 1967; 25: 15-21.

19.

Ingraham JL , Maaloe O. , Neidhardt FC Growth of the Bacterial Cell. Sunderland, MA: Sinauer and Associates, Inc., 1983; 1-48.

20.

Nagi N. , Ohno N. , Adachi Y. et al. Application of Limulus test (G pathway) for the detection of different conformers of (1-3)-β-D-glucans. Bio Pharm Bull 1993; 16: 822-828.

21.

Appelmelk BJ , Su D. , Verweij-van Vught Amjj et al. Polymyxin B-horseradish peroxidase conjugates as tools in endotoxin research. Anal Biochem 1992; 207: 311-316.

22.

Bader J. , Teuber M. Action of polymyxin B on bacterial membranes. 1. Binding to the O-antigenic lipopolysaccharide of Salmonella typhimurium. Z Naturforsch 1973; 28C: 422-430.

23.

Morrison DC , Jacobs DM Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides. Immunochemistry 1976; 13: 813-818.

24.

Hoess A. , Watson S. , Siber GR et al. Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5 Å resolution. EMBO J 1993; 12: 3351-3356.

25.

Bannatyne RM , Hamett NM , Lee K-Y. et al. Inhibition of the biologic effects of endotoxin on neutrophils by polymyxin B sulfate . J Infect Dis 1977; 136: 469-474.

26.

Cooperstock M. , Riegle L. Polymyxin B inactivation of lipopolysaccharide in vaccines of Gram-negative bacteria. Infect Immun 1981; 33:315-318.

27.

Morrison DC , Jacobs DM Inhibition of lipopolysaccharide-initiated activation of serum complement by polymyxin B. Infect Immun 1976; 13:298-301.

28.

Morita T. , Ohtsuko S. , Nakamura T. et al. Isolation and biological activities of Limulus anticoagulant (anti-LPS factor) which interacts with lipopolysaccharide (LPS). J Biochem 1985; 97: 1611-1620.

29.

Neter E. , Gorzynski EA , Westphal O. et al. The effects of antibiotics on enterobacterial lipopolysaccharides (endotoxins), hemagglutination and hemolysis. J Immunol 1958; 80: 66-72.

30.

Lynn WA , Golenbock DT Lipopolysaccharide antagonists. Immunol Today 1992; 13: 271-276.

31.

Morrison DC , Rudbach JA

Endotoxin-cell-membrane interactions leading to transmembrane signaling. Contemp Top

Mol Immunol 1981; 8: 187-218.

32.

Fujiwara T. , Ogawa T. , Sobue S. et al. Chemical, immunobiological and antigenic characterizations of lipopolysaccharides from Bacteroides gingivalis strains. J Gen Microbiol 1990; 136: 319-326.

33.

Russell Rrb , Johnson KG SDS-polyacrylamide gel electrophoresis of lipopolysaccharides. Can J Microbiol 1975 ; 21:2013-2018.

34.

Olins AL , Warner RC Physicochemical studies on a lipopolysaccharide from the cell wall of Azotobacter vinelandii. J Biol Chem 1967 ; 242: 4994-5001.

35.

Jones JW , McFadden HW , Chandler FW et al. Green algal infection in a human. Am J Clin Pathol 1983; 80: 102-107.

36.

Meints RH , Burbank DE , van Etten JL et al. Properties of the Chlorella receptor for the virus PBCV 1. Virology 1988; 164: 15-21.

37.

Meints RH , Pardy RL Quantitative demonstration of cell surface involvement in a plant-animal symbiosis: lectin inhibition of reassociation. J Cell Sci 1980; 43: 239-251.