Release of endotoxic lipopolysaccharide by sensitive strains of Escherichia coli submitted to the bactericidal action of human serum.

Abstract

Free endotoxin was assayed in filtered samples of E. coli suspensions submitted to the bactericidal and bacteriolytic action of 10% human serum. The Limulus amoebocyte lysate test, a passive hemolysis inhibition assay based on O antigenic specificity and the determination of 3-OH-myristic acid by mass spectrometry were used as assay methods differing from one another with regard to the part of the endotoxin macromolecule involved in the reaction. The biological activity of endotoxin was assessed in a mouse lethality test. The bactericidal and bacteriolytic action of human serum on sensitive strains of E. coli released quantities of endotoxic lipopolysaccharide (LPS) amounting to 3,000-12,000 ng/ml, for an inoculum of 1--3 x 10(8) colony-forming units. The material thus appearing in the medium was shown to react with the Limulus amoebocyte lysate, to be lethal for actinomycin D-sensitized mice and to bear O antigen, as well as 3-OH-myristic acid, a marker of lipid A. Samples of serum depleted of lysozyme by adsorption onto bentonite, and displaying a strictly bactericidal effect, released approximately 80% of the quantity of LPS appearing in the medium in a control experiment performed with untreated serum. The LPS release is therefore mainly linked to the bactericidal effect of antibody and complement. The amount of LPS released depended on the concentration of divalent cations in the medium, being reduced by an increase in the concentration of calcium and magnesium beyond the values optimal for complement activity. This effect was already observed for an increase in the concentration of divalent cations too low to alter the bactericidal or bacteriolytic effects. The significance of the release of endotoxin by complement dependent bactericidal reactions occurring in vivo is discussed.