Endotoxin-Like Properties Of An Extract From A Symbiotic, Eukaryotic Chlorella-Like Green Alga

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 Gramnegative 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-mannooctulosonic 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 9-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.