Protein Fraction with Immunogenic Potential and Low Toxicity Isolated from the Cell Wall of Neisseria meningitidis Group B

Abstract

Several fractions were extracted from the cell envelope (CE) of Neisseria meningitidis group B and characterized with regard to their morphology, antigenicity, protein composition, and toxicity. Whole bacterial cells were suspended in a medium of low ionic strength and disrupted in a French pressure cell. The crude CE thus obtained were separated into cell membrane (CM) enriched and cell wall (CW) enriched fractions on sucrose density gradients. In addition, CM and CW fractions were separated from CE on the basis of differential solubility in the nonionic detergent, Triton X-100. The Triton-insoluble fraction, containing primarily CW components, was further treated with a mixture of Triton and ethylenediaminetetraacetic acid, which was shown to remove additional protein and most of the lipopolysaccharide. Electron microscope examination of the various fractions revealed typical unit membrane structures in the case of CM, or large, open segments in the case of CW. The Triton-insoluble and especially the Triton-ethylenediaminetetraacetic acid-insoluble fractions consisted of small vesicular structures. All fractions, except the Triton-soluble fraction, when assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were shown to contain one major protein component accounting for more than 50% of the total. Sera from rabbits immunized with the various fractions formed precipitin lines in immunodiffusion tests against the homologous and some of the heterologous fractions. High-titer bactericidal antibodies were also demonstrated in these sera when tested against the homologous strains. Toxicity studies in rats sensitized with lead acetate indicate that the level of contamination of Triton-insoluble/Tritonethylenediaminetetraacetic acid-insoluble fractions with lipopolysaccharide was significantly smaller than that of the other fractions.

Electron Microscopical Study of Neisseria Meningitidis Releasing Various Amounts of Free Endotoxin

“…These cell evaginations are propably not artefacts during preparation since these structures can also be found with negative staining which is a more gentle procedure (7). Trilaminar structures and blebs are found in the culture supernatants and in the cell envelope fractions (8). Hill & Weiss (8) demonstrated large, open, segmented trilaminar structures from the cell wall enriched fractions containing most endotoxin.…”

Heat-modifiable outer membrane proteins of Neisseria meningitidis and their organization within the membrane

“…A simnilar heat-modifiable protein has also been described in N. gonorrhoeae (10,33). Only a small portion of protein b can be solubilized from the outer membrane by nonionic detergents (6,11), and antibody against this protein is highly bactericidal (6), indicating that on September 21, 2020 by guest http://jb.asm.org/ Downloaded from…”

Immunochemical analysis of inner and outer membranes of Escherichia coli by crossed immunoelectrophoresis

“…In contrast, cell components analyzed by immunochemical techniques often retain sufficient biological activity to permit functional characterization (2,37,52). However, gram-negative cell envelopes have rarely been analyzed in this way (18,49), largely because of the poor resolution of these complex systems achieved by classical immunochemical methods (e.g., immunodiffusion and immunoelectrophoresis).…”

“…Concanavalin A was incorporated into the affinity gels at a concentration of 3 mg/ml, and wheat germ agglutinin was incorporated at a concentration of either 100 ,ug/ml or 3 mg/ml. Electrophoresis in the second direction was performed at 2 V/cm for18 to 24 h. Identification of immunoprecipitates by enzyme-staining (zymogram) techniques. The methods described by Owen and Salton (36) were used to detect immunoprecipitates possessing the following enzyme activities: malate dehydrogenase (EC 1.1.1.37), succinate dehydrogenase (EC 1.3.99.1), NADH dehydrogenase (EC 1.6.99.3), and ATPase (EC 3.6.1.3).…”