Escherichia coli K-12 acquired the ability to produce a high titer of Shiga-like toxin after lysogenization by either of two different bacteriophages isolated from a highly toxinogenic Escherichia coli O157:H7 strain that causes hemorrhagic colitis. One of these phages and another Shiga-like toxin-converting phage from an Escherichia coli O26 isolate associated with infantile diarrhea were closely related in terms of morphology, virion polypeptides, DNA restriction fragments, lysogenic immunity, and heat stability, although a difference in host range was noted. These phages are currently the best-characterized representatives from a broader family of Shiga-like toxin-converting phages.

Microorganisms, in press); (iii) demonstration that the intracellular action of toxin causes an inhibition of protein synthesis (7, 8a, 97, 111; T.

Escherichia coli 0157:H7 strain 933 contains two distinct toxin-converting phages (933J and 933W). The biologic activities and antigenic relationship between the toxins produced by 933J and 933W lysogens of E. coli K-12, as well as the homology of the genes that encode the two toxins, were examined in this study. The 933J and 933W toxins, like Shiga toxin produced by Shigella dysenteriae type 1, were cytotoxic for the same cell lines, caused paralysis and death in mice, and caused fluid accumulation in rabbit ileal segments. The cytotoxic activity of 933J toxin for HeLa cells was neutralized by anti-Shiga toxin, whereas the activity of 933W toxin was not neutralized by this antiserum. In contrast, an antiserum prepared against E. coli K-12(933W) neutralized 933W toxin but not 933J toxin or Shiga toxin. For E. coli 933, most of the cell-associated cytotoxin was neutralized by anti-Shiga toxin, whereas most of the extracellular cytotoxin was neutralized by anti-933W toxin. However, a mixture of these antisera indicated the presence of both toxins in cell lysates and culture supernatants. Among 50 elevated cytotoxin-producing strains of E. coli, we identified 11 strains isolated from cases of diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome that produced cell-associated cytotoxins which were neutralized by the 933W antitoxin. Southern hybridization studies showed that the cloned toxin structural genes from phage 933J hybridized with DNA from phage 933W under conditions estimated to allow no more than 26% base-pair mismatch. These findings indicate that E. coli produces two genetically related but antigenically distinct cytotoxins with similar biologic activities which we propose to name Shiga-like toxins I and II. Strains of E. coli that produce elevated levels of Shiga-like toxin I or Shiga-like toxin II, or both, have been associated with the clinical syndromes of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome.

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Seventeen selected hybridoma cell lines that produced monoclonal antibodies against cholera enterotoxin (CT) were isolated and characterized. All of the monoclonal antibodies contained the kappa light chain; 14 were of the immunoglobulin Gl (IgGl) isotype and 3 were IgG2a. The 17 monoclonal antibodies were divided into a minimum of seven different specificity groups based on their abilities to bind to the following purified test antigens in solid-phase radioimmunoassays: CT, the A and B polypeptides of CT (CT-A and CT-B, respectively), and the heat-labile enterotoxins designated LTh and LTp from Escherichia coli. The binding of these antibodies to the following subunits and fragments of CT was also determined in Western blots: pentameric CT-B, monomeric CT-B, intact CT-A, and the Al fragment of CT-A. Each of the monoclonal antibodies was tested for neutralization of CT and for precipitation with CT in immunodiffusion tests. Antigenic determinants were identified on CT that were not present either on CT-A or CT-B. One class was unique for CT and another was shared with LTh and LTp. Antibodies directed against these holotoxin-specific determinants had no neutralizing activity. Most of the monoclonal antibodies that reacted strongly with CT-A or CT-B also reacted strongly with CT holotoxin; however, one class of antibody reacted strongly with CT-A but weakly with CT. Among the monoclonal antibodies against CT-A or CT-B, some were specific for CT and others crossreacted with LTh and LTp or with LTh only. The most potent neutralizing antibodies were against CT-B, and all of our monoclonal antibodies against CT-B had some neutralizing activity. In contrast, only some of the monoclonal antibodies against CT-A had neutralizing activity, and their specific activities were low. We found no direct correlations between the ability of monoclonal antibodies to neutralize CT and to cross-react with LTh or LTp. None of the epitopes recognized by our monoclonal anti-CT antibodies was present on CT-A and CT-B.Cholera enterotoxin (CT) is a heat-labile enterotoxin produced by Vibrio cholerae (9). It has an essential role in the pathogenesis of cholera and causes secretory diarrhea by activating adenylate cyclase in the mucosal epithelium of the small intestine (13). CT is closely related to the heat-labile enterotoxin (LT) of Escherichia coli (34). The mode of action of LT is similar to that of CT, and LT has been implicated in the pathogenesis of secretory diarrhea in humans and in animals (13).Both CT and LT have been purified to homogeneity (3,9,18,22), and the structural genes for both toxins have been cloned (30,35,37). Each toxin is composed of two different polypeptide subunits designated A and B. Each holotoxin contains one A polypeptide and five copies of the B polypeptide held together by noncovalent bonds (14,15). Comparisons of primary structures demonstrated that the A and B polypeptides from CT have extensive homology with those from LT (6,36). Hyperimmune antisera prepared against either of the purified toxins will ne...