Physical properties of short- and long-O-antigen-containing fractions of lipopolysaccharide from Escherichia coli 0111:B4

Abstract

Aggregates of short-and long-chain 0-antigen-containing fractions of lipopolysaccharide were analyzed by electron spin resonance probing to reveal differences in their physical properties. The fluidities of the lipid regions of the two fractions were quite similar, although the long-chain lipopolysaccharide aggregates appeared to be more hydrated as reflected by the polarity determined with a lipid probe. In contrast, the head-group region of the long-chain fraction was dramatically more mobile than that of the short-chain sample. The binding of polycations (e.g., polymyxin B, spermine) to lipopolysaccharide aggregates was measured by the partitioning of a cationic spin probe. Less probe was displaced from the long-chain fraction and unseparated lipopolysaccharide than from the short-chain fraction by the addition of cations, suggesting that the long 0-antigen masks anionic sites on lipopolysaccharide. These results indicate that the aggregate shape and reactivity of lipopolysaccharide are affected by 0-antigen length. Thus, the biological activity of lipopolysaccharide may be modulated directly by the presence of 0-antigen and indirectly by the effects of 0-antigen on the lipopolysaccharide aggregate structure.Gram-negative bacteria possess an outer membrane which serves as a permeability barrier to toxic compounds. A key component in this barrier function is the lipopolysaccharide (LPS) which is a major part of the outer monolayer of the membrane. LPS forms a rigid, highly charged surface that prevents the diffusion of hydrophobic compounds across the bilayer (24). In addition, LPS can form blebs off the bacterium and is found in sera of patients suffering from septicemia. This LPS can cause adverse host responses, including fever, shock, and even death (22).In its interactions within a host or on the surface of the bacteria, LPS does not act in monomeric form but as large aggregates. The size and shape of these aggregates depend on temperature (3), ion content (4, 11, 17), and pH (R. T. Coughlin, A. A. Peterson, A. Haug, H. T. Pownall, and E. J. McGroarty, Biochim. Biophys. Acta, in press), as well as the composition of the LPS (17). Variations in biological activities between preparations may then be caused by variations in the physical properties of different aggregate structures. The LPS isolated from Rhodospirillum tenue is moderately toxic, whereas the lipid A prepared from it is 70 to 140 times more toxic than the intact LPS (21). Differences in toxicity may thus result from covering the lipid A by the core 0-antigen of R. tenue LPS or from differences in the physical state of the aggregates modulating whether the inner regions of LPS are exposed and able to bind to or react with target structures.LPS, as isolated from bacteria, is a heterogeneous collection of molecules which can vary as to substitution of the core and lipid A (20) and in the length of the 0-antigen polysaccharide chain (12,16,23,25,27). The presence of covalently bound phosphate and acidic sugars in the core and lipid A make LPS a hig...

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