PURIFICATION, PORE-FORMING ABILITY, AND ANTIGENIC RELATEDNESS OF THE MAJOR OUTER-MEMBRANE PROTEIN OF SHIGELLA-DYSENTERIAE TYPE-1

The major outer membrane protein (MOMP), the most abundant outer membr ane protein, was purified to homogeneity from Shigella dysenteriae typ e 1. The purification method involved selective extraction of MOMP wit h sodium dodecyl sulfate in the presence of 0.4 M sodium chloride foll owed by size exclusion chromatography with Sephacryl S-200 HR. MOMP wa s found to form hydrophilic diffusion pores by incorporation into arti ficial liposome vesicles composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that MOMP of S. dysenteriae type 1 exhibi ted significant porin activity. However, the liposomes containing heat -denatured MOMP were barely active. The molecular weight of MOMP found by size exclusion chromatography was 130,000, and in sodium dodecyl s ulfate-l0% polyacrylamide gel it moved as an oligomer of 78,000 molecu lar weight. Upon boiling, fully dissociated monomers of 38,000 molecul ar weight were seen for S. dysenteriae type 1. However, among the four Shigella spp., the monomeric MOMP generated upon boiling ranged from 38,000 to 35,000 in molecular weight. Antibody raised in BALB/c mice i mmunized with MOMP of S. dysenteriae type 1 reacted strongly with puri fied MOMP of S. dysenteriae type 1 in an enzyme-linked immunosorbent a ssay (ELISA). The antibody reacted with whole-cell preparations of S. dysenteriae type 1 in an ELISA, suggesting that MOMP possessed surface components. Moreover, MOMP could be visualized on the bacterial surfa ce by immunoelectron microscopy with anti-MOMP antibody. S. dysenteria e type 1 MOMP-specific immunoglobulin eluted from MOMP bound to a nitr ocellulose membrane was found to cross-react with MOMP preparations of S. flexneri, S, boydii, and S. sonnei, indicating that MOMPs were ant igenically related among Shigella species. The strong immunogenicity, surface exposure, and antigenic relatedness make MOMP of Shigella spec ies an immunologically significant macromolecule for study.