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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, USA. E-mail: pbarmstrong{at}ucdavis.edu
ABSTRACT
Lipopolysaccharide (LPS) (a.k.a., endotoxin) is an essential component of the outer leaflet of the outer membrane of Gram-negative bacteria and is a potent activator of the innate immune system of animals. Lipid A, the glycolipid core of LPS, is the agent responsible for disease and death from Gram-negative sepsis, an important cause of human mortality and morbidity. Although it is generally accepted that lipid A is restricted to the prokaryotes, recent efforts to purify molecules from green algae with structural features unique to lipid A have met with success. Furthermore, the vascular plant Arabidopsis thaliana has been found to contain genes that encode all of the enzymes of the biosynthetic pathway for lipid A. It is not known whether vascular plants synthesize lipid A or where lipid A might be located in the tissues. For the present study, we used affinity reagents for lipid A to probe green alga and tissues of the garden pea for a light microscopic localization of lipid A in these eukaryote cells. We find staining for lipid A in free-living and endosymbiotic green algae and in the chloroplasts of vascular plants, indicating that this molecule is not restricted to prokaryotes, but is found also in select eukaryotes.—Armstrong, M. T., Theg, S. M., Braun, N., Wainwright, N., Pardy, R. L., Armstrong, P. B. Histochemical evidence for lipid A (endotoxin) in eukaryote chloroplasts.
Key Words: lipopolysaccaride • E. coli • LALF • prokaryote
LIPOPOLYSACCHARID (LPS) CONSTITUTES THE principal lipid of the outer leaflet of the outer membrane of Gram-negative bacteria and the cyanobacteria (1)
. Lipid A, the phospholipid core of LPS, is a complex amphipathic glucosamine-based phospholipid with unusual ß-hydroxy fatty acids that is derivatized with a disaccharide of the novel sugar, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) (2)
. Lipid A is the causative agent of Gram-negative sepsis, a potentially lethal condition claiming more than 100,000 lives annually in the U.S., and with no known cure (3)
, making it a target for intensive investigation. Lipid A activates the innate immune system of a variety of animals, including arthropods (4;5) and mammals (6)
, and plays a role in nitrogen-fixing nodules of legumes (7)
. It is generally thought that lipid A is unique to Gram-negative cells and that this moiety is diagnostic for these bacteria (8)
. Thus, the discovery of a lipid A-like molecule in unicelluar algae was unanticipated. The algal molecule contains Kdo and ß-hydroxy fatty acids and thus is chemically similar to bacterial lipid A (9
10
11)
. Furthermore, 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 (2)
, although it is not known whether vascular plants synthesize lipid A or where lipid A might be located in the tissues. The object of the present study is to investigate the presence and to determine the location of lipid A-like molecules in green algae and vascular plants.
MATERIALS AND METHODS
Organisms
Escherichia coli (strain DH5
) were harvested from log phase cultures grown in Luria broth. Chlorella sp (strain NC64A, American Type Culture Collection (ATCC) 50528) was cultured under axenic conditions (11)
. Paramecium busaria and Chlorohydra viridis were obtained from Ward’s (Cat 87W1305 87W2120, Rochester, NY, USA). Pea chloroplasts were isolated as in ref. 12
.
Affinity labeling
Whole cells and tissues were fixed in freshly prepared 4% paraformaldehyde in PBS (10 min, room T), then quenched with 0.1 M glycine and blocked with 5 mg/ml bovine serum albumin (BSA). Cells and paraffin-sectioned tissues were exposed to recombinant Limulus antilipopolysaccharide factor (LALF) in Tris-buffered saline (TBS), followed by treatment with rabbit anti-LALF and Cy2-goat anti-rabbit IgG (Jackson Laboratories, Baltimore, MD, USA), or to polymyxin B labeled with the fluorophore biodipy (Molecular Probes, Eugene, OR, USA; cat P-13235, lot 4481–1). Biodipy-labeled polymyxin B was preincubated with bacterial LPS (Sigma cat. L-3129) for use as a negative control for biodipy-polymyxin B staining. Paraffin sections of pea seedlings and hydra were prepared according to standard methods. Because of the potential presence of contaminating LPS, solutions were prepared in pyrogen-free water (Sigma W3500), and all relevant glassware and microscope slides (Fisher Superfrost/Plus cat 12–550-15) were decontaminated by baking 4 h at 180°C. A Zeiss Axioplan light microscope and a Leica DMRBE confocal microscope were used for microscopy.
RESULTS
We used two lipid A binding agents to identify and localize lipid A in cells: LALF (Limulus antilipopolysaccharide factor), a 12 kDa protein from the secretory granules of the blood cells of the horseshoe crab (13)
, and polymyxin B, an antibiotic of fungal origin (14)
. The two probes for lipid A both bind strongly to that molecule (15
16
17
18)
. Polymyxin B also binds certain anionic phospholipids such as phosphatidic acid (19)
and phosphatidyl glycerol (20)
. LALF appears to be more selective for lipid A (21;22). LALF binding was detected by immunohistochemical staining, and the presence of the fluorescent dye conjugate biodipy-polymyxin B identified the binding targets of polymyxin B. We also tested an antilipid A monoclonal antibody (mAb) and human LPS binding protein (LBP), but neither reagent stained our positive control, the Gram-negative bacterium E. coli, making them unsuitable as staining probes. Perhaps both molecules were too large to gain access to the lipid A moiety buried in the outer leaflet of the outer membrane of the bacterium.
The positive control, the Gram-negative bacterium Escherichia coli, showed affinity labeling of the surface with both LALF and polymyxin B (Fig. 1
A). Staining failed to occur when LALF was omitted, but first and second antibodies were present (Fig. 1B, C
). We also found intense staining of the axenically grown free-living green algae, Chlorella (strain NC64A)(Fig. 1D
) and the related species Prototheca (strain 289, an achlorophyli, white algae that is an obligate heterotroph and an opportunistic pathogen of livestock and humans; data not shown). The intracellular symbiotic green algae of the ciliate protozoan, Paramecium bursaria (Fig. 1E
), and of the green coelenterate, Chlorohydra viridissima (Fig. 1F
), also stained. Chloroplasts isolated from pea seedlings (Fig. 1G
) and chloroplasts of paraffin-sectioned pea seedling leaves (Fig. 1I
) stained with both lipid A binding agents. Isolated membranes from pea seedling chloroplasts (a gift from Prof. Dr. Soll) stained with LALF and biodipy-polymyxin B (data not shown). Chlorophyl, an abundant molecule of green algae and chloroplasts, is strongly fluorescent at longer wavelengths than those used for the fluorescein and biodipy reporter dyes used in these assays (fluorescence emission for chlorophyl, 640–685 nm) and did not contribute to the fluorescent signal for confocal (not shown) or epifluorescence (Fig. 1H
) images.
|
Bacterial LPS is a frequent contaminant of fluids and surfaces. Contamination of biological samples with bacterial LPS has been the cause of a number of false attributions of novel biological activities to particular agents where the responses were actually due, not to the agents themselves, but to contaminating LPS (23
24
25)
. To minimize the possibility that contaminating LPS of bacterial origin was bound to the surfaces of the cells and organelles, we processed axenically grown algae and all specimens with fixatives and other reagents prepared in LPS-free water (Sigma). Presumably the natural intracellular location of the symbiotic algae of Paramecium (Fig. 1E
) and Chlorohydra (Fig. 1F
) and of chloroplasts (Fig. 1I
) would protect them from exposure to bacterial LPS from the environment prior to paraffin sectioning. Our results suggest that the lipid A detected on these cells and organelles is endogenously synthesized.
DISCUSSION
It is generally believed that LPS and its membrane anchor, lipid A, are found uniquely in select prokaryotes. Recently this view has been challenged by reports that glycolipids with the chemical signature moieties of lipid A can be purified from green algae and by the observation that genes encoding the proteins of the enzymatic pathway for biosynthesis of lipid A are present in vascular plants. Genetic studies do not show that lipid A is actually synthesized by vascular plants, nor do they indicate its localization in the cell. Our histochemical observations indicate that lipid A is synthesized by vascular plants and is localized to the chloroplasts. Chloroplasts are thought to be evolved endosymbiotic relatives of present-day Gram-negative cyanobacteria (26)
. It is possible that the genes encoding the biosynthetic pathway responsible for lipid A biosynthesis were introduced into the primitive collectives that represented the ancestral eukaryote cells of vascular plants via these endosymbiotic cyanobacteria. The adaptive significance of lipid A in the chloroplast is uncertain but it can be presumed to be important because lipid A is still present even after hundreds of millions of years of evolutionary integration of the chloroplast into the economy of the plant cell.
ACKNOWLEDGMENTS
This research was supported by NSF Grant 0344360 (P.B.A.).
Received for publication February 10, 2006. Accepted for publication May 15, 2006.
REFERENCES
This article has been cited by other articles:
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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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