Antibodies against Affinity-Purified, Surface-Exposed Outer Membrane Proteins ofEdwardsiella ictaluriBlock Invasion into Fathead Minnow Epithelial Cells

Abstract

Surface-exposed outer membrane proteins (OMPs) of Edwardsiella ictaluri were isolated by selective solubilization of inner membrane proteins from total membrane preparations. Purification of biotin-labeled, insoluble, surface-exposed proteins using streptavidin columns was performed, and single-dimension sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed four major OMPs, with apparent molecular weights of 22, 31, 59, and 72 kilodaltons (kDa). Purified surface-exposed proteins corresponded to proteins isolated from total outer membrane preparations resolved by SDS–PAGE, showing that surface-exposed proteins are within the outer membrane fraction and can be successfully isolated using affinity purification. Polyclonal antiserum against these surface-exposed OMPs was produced in New Zealand white rabbits, and protein recognition was determined using in-gel Western analysis. Rabbit antisera recognized three of the four protein bands (22, 31, and 59 kDa). The produced antisera blocked invasion of cells from fathead minnow Pimephales promelas by virulent E. ictaluri, showing that at least one of these proteins is involved in initial bacterial–host cell interactions.