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Histochemical evidence for lipopolysaccharide (endotoxin) in eukaryotes.

Lipopolysaccharide is a component of the outer membrane of gram-negative bacteria and is the principal mediator of gram-negative bacterial toxicity to animals. Toxicity is mediated by lipid A, the membrane-forming element of lipopolysaccharide. Although usually thought to be restricted to prokaryotes, recent evidence suggests the presence of lipopolysaccharide in certain eukaryotes. Glycolipids with all of the unique components of bacterial lipopolysaccharide, including the lipid A moiety, have been isolated from axenically-grown green algae; and the vascular plant Arabidopsis thaliana has been found to contain genes that encode all enzymes of the lipid A biosynthetic pathway. We used two lipid A binding agents to localize LPS in cells: LALF (Limulus antilipopolysaccharide factor), a 12 kDa protein from the secretory granules of the blood cells of the horseshoe crab; and polymyxin B, an antibiotic of fungal origin. LALF binding was detected with an antibody, and biodipy conjugated to polymyxin B was used to localize that agent. Our positive control, the gram-negative bacterium Escherichia coli, showed staining of the surface with both agents. We also found intense staining of the free-living alga Chlorella (strain NC64A--a green alga) and the related species Prototheca (strain 289--an achlorophylic, white alga that is an obligate heterotroph and is also an opportunistic pathogen of livestock and humans), and the commensal algae of Paramecium bursaria and Chlorohydra viridissima. Chloroplasts isolated from pea seedlings and chloroplasts of paraffin-sectioned pea seedling leaves stained with both staining agents. To minimize the possibility that the LPS found on algae and chloroplasts derives from bacteria-produced LPS that subsequently binds to the surfaces of these cells and organelles, we processed axenically grown algae and all specimens destined for paraffin embedding and sectioning with fixatives and other reagents prepared in LPS-free water (Sigma). Staining with LALF and biodipy-polymyxin B was intense. These results suggest that endogenously synthesized LPS is present at the surfaces of free-living and commensal green algae and is localized to the chloroplasts of vascular plants.

This research was supported by NSF Grant 0344360 to PBA.

Peter B. Armstrong (1,2), Margaret T. Armstrong (1,2), Steven M. Theg (1), Nikolai Braun (1), Norman Wainwright (2), and R. L. Pardy (3)

(1) University of California, Davis, California

(2) Marine Biological Laboratory, Woods Hole, Massachusetts

(3) University of Nebraska, Lincoln, Nebraska

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Author:Armstrong, Peter B.; Armstrong, Margaret T.; Theg, Steven M.; Braun, Nikolai; Wainwright, Norman; Pa
Publication:The Biological Bulletin
Geographic Code:1USA
Date:Oct 1, 2004
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