Immunochemical identification and biological characterization of cytotoxic necrotizing factor from Escherichia coli

The aim of this study was to identify Escherichia coli cytotoxic necrotizing factor (CNF) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and to investigate the possible dissociation of CNF from hemolysin (Hly), which is often produced by CNF-producing strains. CNF was purified from cell lysates of a CNF-producing strain by using ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and preparative nondenaturing PAGE. All eluates from successive longitudinal slices of a preparative polyacrylamide gel were tested for cytoxicity and analyzed by SDS-PAGE; CNF activity was quantitatively correlated with a protein of 115 kilodaltons (kDa). This procedure increased both cytotoxicity and lethal activity (about 300-fold). We then compared SDS-PAGE protein patterns of enriched lysates from eight field and mutant E. coli strains producing both CNF and Hly, Hly alone, or neither; the 115-kDa band was present solely in CNF-producing strains, irrespective of Hly production. A neutralizing antiserum was produced against unpurified CNF from strain BM2-1 and then extensively adsorbed with cells and extracts of a CNF-defective mutant from BM2-1. The adsorbed antiserum possessed antitoxin activity and neutralized both lethal and necrotic effects of cell lysates from all the CNF-producing strains tested. In an immunoblot of enriched extract from BM2-1, the adsorbed antiserum recognized, besides the 115-kDa protein, another protein of 59 kDa, which was present in the CNF-defective mutant from BM2-1 and was not associated with cytotoxicity. We can conclude from these findings that CNF is a protein of 115 kDa associated with both cytotoxicity and in vivo toxicity, distinct from Hly, and present in all presumed CNF-producing strains tested.