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* Department of Molecular and Cellular Biology, University of California, Davis, California, USA;
Department of Plant Biology, University of California, Davis, California, USA;
Marine Biological Laboratory, Woods Hole, Massachusetts, USA; and
School of Biological Sciences, University of Nebraska, Lincoln, Nebraska, USA
1Correspondence: Department of Molecular and Cellular Biology, University of California, One Shields Ave., Davis, CA 95616-8535, USA. E-mail: pbarmstrong{at}ucdavis.edu
SPECIFIC AIM
The lipopolysaccharides (LPS) are a set of glycolipids that constitute major constituents of the outer leaflet of the outer membrane of the Gram-negative bacteria and are thought be restricted to this class of prokaryotes (Fig. 1
). We investigated the presence and localization of lipid A, the lipid core of LPS, in eukaryotic cells using specific lipid A binding reagents for histochemical staining and light microscopy.
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PRINCIPAL FINDINGS
1. The lipid A binding agents, polymyxin B and LALF, can be used to localize lipid A in cells
Biodipy-conjugated polymyxin B, an antibiotic of fungal origin, and the lipid A binding protein, LALF (Limulus antilipopolysaccharide factor), a 12 kDa protein from the secretory granules of the blood cells of the horseshoe crab, can be used to localize lipid A in prokaryotic cells. Biodipy-conjugated polymyxin B can be visualized directly; indirect immunohistochemistry using an anti-LALF antibody (Ab) was used to visualize the localization LALF. Both probes bound to the surface of the positive control, the Gram-negative bacterium, Escherichia coli (Fig 2
A–C).
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2. Both lipid A binding agents stain green algae
The monocellular algae, Chlorella (strain NC64A) (Fig. 2D
) and Prototheca (strain 289), bound polymyxin B and LALF. Both agents also stained cells of endosymbiotic green algae in the ciliate protozoan, Paramecium bursaria (Fig. 2E
), and the coelenterate, Chlorohydra viridissima (Fig. 2F
). Care was taken to ensure that exogenous bacterium-derived LPS was not present in these systems. Algae were cultured under axenic conditions, then fixed and processed with reagents prepared with LPS-free water. Algal cells and Paramecium bursaria were processed as intact cells. Presumably intracellular elements such as intracellular algae would be protected from contact with exogenous LPS in the external environment. Paraffin sections of Chlorohydra were attached to LPS-free glass slides in LPS-free water. This significantly reduces the chance that staining is of LPS from bacterial sources that might have bound to the surfaces of the algal cells and suggests that lipid A staining is of endogenously produced glycolipid.
3. Both probes stained the chloroplasts of the garden pea
The vascular plant, Arabidopsis thalii, possesses the entire complement of genes to encode the enzymes of the lipid A biosynthetic pathway, suggesting that this glycolipid may be present in vascular plants. Isolated chloroplasts of the pea seedling (Fig 2G
) and the chloroplasts in paraffin sections of pea seedling leaf tissue (Fig. 2I
) stained with both agents, indicating that this proposition is valid and that lipid A is found in the chloroplast. The staining is not a general membrane staining because organelles other than the endosymbiotic algae of Paramecium and Chlorohydra and chloroplasts of the garden pea did not stain with either lipid A probe. The autofluorescence of chlorophyll did not contribute to the staining signal of the chloroplasts (Fig. 2H
)
CONCLUSIONS AND SIGNIFICANCE
LPS constitutes the principal lipid of the outer leaflet of the outer membrane of Gram-negative bacteria and the cyanobacteria; lipid A, its lipid core, is the causative agent of Gram-negative sepsis, a potentially lethal condition claiming >100,000 lives annually in the U.S. Lipid A is a potent activator of the innate immune systems of metazoans. It is thought that lipid A is unique to Gram-negative bacteria and that this moiety is diagnostic for this class of prokaryotes. Thus, the discovery of a lipid A-like molecule in unicellular algae, members of the eukaryotes, was unanticipated. Biochemical characterization of the algal molecule has documented the presence of several chemical moieties found in bacterial lipid A. The vascular plant Arabidopsis thaliana (Angiospermae, Dicotyledonae) has been found to contain genes that encode all of the enzymes of the lipid A biosynthetic pathway, although it has not been shown that vascular plants synthesize lipid A or where lipid A might be located in the tissues. This study indicates that lipid A is indeed present and in chloroplasts. The observation that the lipid A binding probes, polymyxin B and LALF, bind green algae and the chloroplasts of vascular plants supports the hypothesis that lipid A is not restricted to the Gram-negative bacteria, but is found, as well, in select eukaryotes.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5484fje
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  M.-A. Newman, J. M. Dow, A. Molinaro, and M. Parrilli Invited review: Priming, induction and modulation of plant defence responses by bacterial lipopolysaccharides Innate Immunity, April 1, 2007; 13(2): 69 - 84. [Abstract] [PDF]
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