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Department of Molecular and Cellular Biology, University of California, Davis, California
1 Department of Molecular and Cellular Biology, University of California, Davis, CA 95616
2 School of Biological Sciences, University of Nebraska, Lincoln, NE 68583
3 Marine Biological Laboratory, Woods Hole, MA 02543
The lipopolysaccharides (LPS) are ubiquitous components of the outer leaflet of the outer membrane of all gram-negative bacteria and are the principal toxic products of these organisms (1). The membrane anchor of LPS is lipid A, a central phosphodisaccharide unit that is attached to multiple ß-hydroxy fatty acid chains. LPS also contains the novel sugar, 3-deoxy-D-manno-octulosonic acid (KDO). Although generally believed to be restricted to prokaryotes, specifically the gram-negative eubacteria and the cyanobacteria (2), an LPS-like molecule has recently been reported from a eukaryote, the green alga Chlorella sp., strain NC64A. The algal molecule includes KDO, lipid A, and ß-hydroxy fatty acids and is thus chemically similar to bacterial LPS (3). The subject of this study is the localization of the LPS-like molecule in the algal cell.
One of the defenses raised against LPS by the horseshoe crab, Limulus, is a small, 101-amino acid, cationic protein—Limulus anti-LPS factor (LALF) (4)—which is released from the secretory granules of the blood cells during their exocytosis response to LPS challenge (5). LALF binds and neutralizes bacterial LPS (6). In the present study, we use the specific LPS-binding activity of LALF to localize the LPS-like molecule in eukaryotic and prokaryote cells.
The reactivity of LALF is apparently specific for LPS, with an amphipathic loop of LALF serving as the LPS-binding motif (7). A standard assay for bacterial LPS is the Limulus amebocyte lysate (LAL) test. This test (Charles River Endosafe, used according to accompanying instructions) reported 7.3 U/ml for 50 µg/ml of algal LPS. This activity was diminished to 0.08 U/ml in the presence of 50 µg/ml of LALF.
For immunohistochemical investigation, Chlorella cells, strain NC64A, were grown under bacteria-free conditions, then were freeze-dried and fixed in freshly-prepared 4% paraformaldehyde dissolved in phosphate-buffered saline for 10 min, then blocked successively with 0.1 M glycine in Tris-buffered saline (TBS) and with 5 mg/ml bovine serum albumin (BSA) in the same buffer. The cells were then exposed to 0.1 mg/ml LALF in TBS, then were treated with an anti-LALF antiserum (rabbit) in TBS + BSA, followed by exposure to a fluorescein-labeled goat anti-rabbit IgG second antibody in the same buffer. Finally, the cells were examined with a Zeiss Axiophot 2 fluorescence microscope.
The LALF labeling procedure stained the outer surface of aldehyde-fixed Escherichia coli cells, which served as our positive control for the method (Fig. 1A, 1B). Cells of the alga Chlorella showed a similar staining pattern, with LALF staining the outer surface of the cell, the cell wall (Fig. 1C, 1D). The cells failed to stain when LALF was omitted from the reaction scheme. This observation indicates that the LPS-like molecule of Chlorella is positioned at the appropriate location for a true lipopolysaccharide and adds substance to the still controversial claim that LPS is not confined to the prokaryotes, but is also present in certain eukaryotes.
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Literature Cited
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