The morphological heterogeneity of lipopolysaccharides (LPSs) among salmonella mutants with different LPS chemotypes was analyzed in silver-stained polyacrylamide gels. The biochemical differences in the LPS chemotypes were reflected in the unique profiles of the purified LPSs. The LPS profiles in the whole-cell lysates were also unique for each chemotype. (Whole-cell lysates were assessed by a method which preferentially silver stains LPS and by a proteinase K digest of whole-cell lysates. The silver-stained LPS profiles of proteinase K-digested lysates were similar to the homologous purified LPS and could be used to preliminarily characterize the LPS chemotype before purification.) In summary, biochemical variation in LPS composition can be detected in silver-stained polyacrylamide gels.

Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.

In an effort to decrease deaths from gram-negative bacteremia and endotoxin shock, we treated bacteremic patients with human antiserum to endotoxin (lipopolysaccharide) core. Antiserum was prepared by vaccinating healthy men with heat-killed Escherichia coli J5; this mutant lacks lipopolysaccharide oligosaccharide side chains, so that the core, which is nearly identical to that of most other gram-negative bacteria, is exposed for antibody formation. In a randomized controlled trial, patients were given either J5 antiserum or preimmune control serum intravenously, near the onset of illness. The number of deaths in the bacteremic patients was 42 of 109 (39 per cent) in controls and 23 of 103 (22 per cent) in recipients of J5 antiserum (P = 0.011). In those with profound shock, mortality was 30 of 39 (77 per cent) in controls and 18 of 41 (44 per cent) in recipients of J5 antiserum (P = 0.003). We conclude that human antiserum to the lipopolysaccharide core can substantially reduce deaths from gram-negative bacteremia.

Treatment of Gram-Negative Bacteremia and Shock with Human Antiserum to a Mutant Escherichia coli

Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria

A highly sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels.

Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Enterobacteriaceae cells growing in liquid media shed fragments of their outer membranes. These fragments, which may constitute a biologically important form of gram-negative bacterial endotoxin, have been reported to contain proteins, phospholipids, and lipopolysaccharides (LPS). In this study we compared the sizes of LPS molecules in shed membrane fragments and outer membranes from cells growing in broth cultures. Using conditional mutants of Salmonella typhimurium which incorporate specific sugars into LPS, we analyzed radiolabeled LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique revealed that S. typhimurium LPS are more heterogeneous than previously known; molecules possessing from 0 to more than 30 O-chain repeat units were identified in outer membranes, supernatant fragments, and purified LPS. The size distributions of LPS molecules in outer membranes and supernatant fragments were similar; supernatant fragments appeared to be slightly enriched in molecules with long O-polysaccharide chains. Our results indicate the LPS molecules of many sizes are synthesized, translocated to outer membranes, and released into culture supernatants. Since the hydrophilic O-polysaccharides extend from bacterial surfaces into the aqueous environment, our findings suggest that the cell surface topography of this bacterium may be very irregular. We also speculate that heterogeneity in the degree of polymerization of O-antigenic side chains may influence the interactions of the toxic moiety of LPS (lipid A) with host constituents.

P D Rick

Papers

Size heterogeneity of Salmonella typhimurium lipopolysaccharides in outer membranes and culture supernatant membrane fragments.