SDS-PAGE analysis of protein and lipopolysaccharide of extracellular vesicules and Sarkosyl-insoluble membranes from Bacteroides gingivalis

Abstract

We have compared outer membranes (OM) of Bacteroides gingivalis ATCC 33277 isolated by the following 3 techniques: 1) high speed centrifugation after mechanical cell shearing; 2) sonication of the bacteria, followed by solubilization of the cytoplasmic membrane with N-Laurylsarconsinate (Sarkosyl), after which the Sarkosyl-insoluble membranes were recovered by centrifugation; 3) ammonium sulfate precipitation of extracellular vesicules from culture supernatant, followed by centrifugation and dialysis. Electron microscopy showed that the 3 preparations consisted of closed vesicules. Analysis by SDS-PAGE revealed that all 3 contained up to 28 polypeptides, most of which were common to each extract. The extracellular vesicules and Sarkosyl-insoluble preparation yielded similar protein patterns, although quantitative differences were observed. The sheared-cell preparation contained 8 additional proteins. The level of contamination of OM material by peptidoglycan and cytosol components was 1.8% in the sheared-cell preparation, and was null or lower than 0.8% in the other preparations. All 3 preparations showed the presence of LPS with a multiple banding pattern typical of smooth LPS. The sheared-cell preparation had a slightly lower LPS content than the other 2 preparations. Since extracellular vesicules are naturally released during bacterial growth, and are relatively simple to obtain, such native entities seem an appropriate source of OM components for use in studying the immunobiology of B. gingivalis surface antigens.