Purification and Physico-Chemical Analysis of Fractions from the Culture Supernatant of Escherichia coli O78K80: Free Endotoxin and a Non-Toxic Fraction

Abstract

SUMMARYThe extracellular production of ' free endotoxin ' from Escherichia coli serotype 078K80 is discussed. Isolation of purified toxic and non-toxic fractions from crude extracellular material and the physico-chemical properties of these fractions are described. Purified free endotoxin possessed similar properties to endotoxins extracted from the bacteria by conventional procedures ; a purified non-toxic extracellular fraction corresponded to the so-called ' native hapten '. When dissolved in buffers containing sodium dodecylsulphate, the non-toxic fraction was not disaggregated, but the endotoxin produced sub-units of a smaller particle size than the non-toxic material. From these experiments it was concluded that the non-toxic material did not constitute simple sub-units of endotoxin. I N T R O D U C T I O NThe term ' endotoxin ' describes certain lipopolysaccharides of high molecular weight found in Gram-negative bacterial cell walls. Although Shear and his associates (e.g. Shear & Turner, 1943) have isolated tumour-necrotizing substances (endotoxins) from supernatant fluids of cultures of Gram-negative bacteria, most other workers have preferred to extract endotoxins from the bacteria themselves. An exception has been Work and her collaborators (Bishop & Work, 1965, Taylor, Knox & Work, 1966' who found endotoxic lipopolysaccharides in culture supernatant fluids of lysinerequiring mutants of Escherichia coli grown under lysine-limited conditions.The release of large quantities of free endotoxin into the culture supernatant fluids of Gram-negative bacteria, under favourable growth conditions, has recently been described (Crutchley, Marsh & Cameron, 1967a). The present paper will deal with the fractionation of supernatant fluids of cultures of Escherichia coli serotype 078K80 and the subsequent physical and chemical characterization of the fractions ; the accompanying paper (Crutchley et al. 1967 b) will discuss the biological properties of these fractions.Origin of toxicity. charide, was essential for toxicity; but a more recent investigation of endotoxins from rough Salmonella strains led them to suggest more strongly that the lipid moiety ('lipid A') was 'the factor decisive for at least some endotoxic effects' (Liideritz & Westphal, 1966). They also believe that, with respect to endotoxic activity, combined polysaccharide may function only as a solubilizing agent. However, the American school of Landy, Ribi and their associates (Ribi, Haskins, Landy & Milner, 1961a;Fukushi et al. 1964) have generally found very little lipid in their highly toxic fractions from several of the Enterobacteriaceae ; all their lipid extracts, including lipid A, possessed less than 1 % of the biological activity of the parent endotoxins. The balance of their published evidence suggests that a carbohydrate moiety is implicated in the toxicity, although certain other factors may be involved (Ribi, Haskins, Landy & Milner, 1961b).There have also been conflicting reports about the minimal molecular size of the complex required fo...

Biological Studies on Free Endotoxin and a Non-toxic Material from Culture Supernatant fluids of Escherichia coli 078 K 80

“…In addition to free endotoxin, non-toxic material of lower molecular weight was present in culture fluids of Escherichia coli serotype 078 K 80 (Marsh & Crutchley, 1967). This substance was similar to native hapten described by Anacker et al (1964) or as it is now called, native protoplasmic polysaccharide (Rudbach et al 1967).…”

“…The body temperatures remained low for as long as 12 hr, whereas the diarrhoea lasted for only 2 hr. These findings suggested that culture fluids from aerated cultures contained endotoxin which we propose to call 'free endotoxin', to distinguish it from material extracted from whole bacteria by chemical methods (Crutchley et al 1967).…”

“…Treatment of the supernatant fluids of such cultures with ammonium sulphate yielded a precipitate which, after purification on diethylaminoethyl cellulose and Sephadex G-IOO was found to contain two main fractions, one indistinguishable from endotoxin and the other a lower molecular weight non-toxic material which lacked most of the biological properties of endotoxin. This latter fraction we have tentatively identified (Marsh & Crutchley, 1967) as native hapten, or as it is now called, native protoplasmic polysaccharide (Rudbach et al I 967). The present paper describes the biological properties and serological relationships of these two fractions : free endotoxin and the non-toxic material from culture supernatants of Escherichia coli 078 K 80.…”

“…This was described previously (Marsh & Crutchley, 1967). A flow diagram for the isolation of free endotoxin and non-toxic material is shown in Fig.…”

“…Medium. The casein sucrose yeast-extract medium used (D. C. Edwards, unpublished) was described previously (Marsh & Crutchley, 1967); it will be referred to as casein sucrose.…”