Endotoxin liberation studied by biological and chemical methods

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Abstract
Release of endotoxin (or lipopolysaccharides, LPS) from four meningococcal strains was studied with a chemical and a biological technique. Two strains were endotoxin-liberating (E+; 270E+ and 840E+) and two had no or low endotoxin release E-; 270E- and 840E-). LPS was quantitated by gas chromatography (GC) of LPS-specific hydroxy fatty acids, in parallel with assay of endotoxin by Limulus Amebocyte Lysate (LAL), in cell suspensions of equal O.D. and in filtered samples. The GC and LAL methods showed a reasonably good agreement in the determination of LPS in filtrates, which had distinctly higher levels (approx. 10-100 times) for the E+ strains than the E- strains, in accordance with earlier LAL studies. This difference was not due to overproduction of LPS in the E+ strains, since all four strains had the same level of LPS (by GC) in cell suspensions of equal O.D. Here the agreement between the GC and LAL methods was substantially less, with lower values by LAL for the two E- strains. The chemical composition of purified LPS was determined by methanolysis and GC for the four strains and for two additional strains 247 and 714 with a high degree of genetic similarity with strains 270E- and 840E-, respectively. Amounts of unphosphorylated L-glycero-D-manno heptose and 2-keto-3-deoxyoctonic acid were the same in all 6 LPS. Otherwise distinct differences were found between LPS of the 6 strains. LPS of the two E+ strains formed one group with about 2.4 mol of galactose (gal), 1.4 mol of glucose (glc) and 2.8 mol of glucosamine (glcN) in the carbohydrate chain. Another group, LPS of all the E- strains except 270E-, had 1.1 mol of gal, 2.8 mol of glc and 1.3 mol of glcN in the LPS chain. LPS 270E- also had 1.3 mol of glcN but derived strongly form all other LPS by a complete lack of gal and glc. On the basis of genetic evidence strain 270E- is regarded as a "rough" LPS mutant of strain 247. The atypical chemistry of LPS 270E- may explain an observed hydrophobicity of this LPS, and it may be related to the previously described sulfonamide sensitivity. Whether the chemical differences observed for LPS of the E+ and E- strains is a mere coincidence remains to be elucidated by detailed studies of more strains of known tendency of endotoxin liberation.