Endotoxic Glycolipid from a Heptoseless Mutant of Salmonella minnesota

Abstract

The endotoxin of a heptoseless mutant of Salmonella minnesota R595 was extracted with phenol-water. Most of this material was found distributed in the insoluble fraction of the extract. The results showed that the R595 endotoxin behaved as a lipid rather than as a lipopolysaccharide (LPS). The preparation, although it does not contain O-specific polysaccharides, does contain 2-keto-3-deoxyoctonic acid (KDO), hexosamine, and several other unidentified compounds. Therefore, the term “glycolipid” is used in this paper instead of lipopolysaccharide. The crude glycolipid fraction, which was soluble in a mixture of chloroform-methanol (8:2), was purified by a procedure including fractionation with organic solvents and by different-column chromatographic methods. Although a chromatographic fraction of the glycolipid showed homogeneity in most systems investigated, the presence of contaminants could not be excluded. Chemical analysis of the glycolipids showed the absence of hexoses and heptoses. Constituents which were found were hexosamine, KDO, fatty acids, and phosphorus, which showed a relatively simple chemical composition. Partial acidic hydrolysis of the glycolipid yielded hexosamine-phosphates, as described in “Lipid A” fractions of smooth LPS preparations. Thin-layer chromatography of the partially hydrolyzed glycolipid showed a pattern similar to “Lipid A” fractions of other strains. The biological activity of the glycolipid was at the same level as that of other gram-negative endotoxins. Pyrogenicity, Shwartzman reactivity, and chick embryo ld 50 values were as high or higher than those of purified Serratia marcescens endotoxin preparations, but mouse ld 50 measurements gave significantly lower results.