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.
De Rycke
J
J
Station de Pathologie de la Reproduction, Institut National de la Recherche Agronomique, Nouzilly, France.
Phan-Thanh
L
L
Bernard
S
S
D000818:Animals; D001427:Bacterial Toxins / Q000032:analysis* / Q000302:isolation & purification / Q000633:toxicity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003601:Cytotoxicity Tests, Immunologic; D003603:Cytotoxins / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins; D006367:HeLa Cells; D006460:Hemolysin Proteins / Q000032:analysis / Q000096:biosynthesis; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D009500:Neutralization Tests
D001427:Bacterial Toxins; D003603:Cytotoxins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C060296:cytotoxic necrotizing factor type 1