A panel of ten site-directed mutants of Clostridium perfringens &-toxin was generated. All of the mutated proteins expressed in Escherichia coli were recognized in immunoblots by a neutralizing mAb raised against wild-type native otoxin. The cytotoxicity of the site-directed mutated toxins was assayed in vitro against MDCK cells. One mutation resulting in loss of activity in the assay was identified. This non-toxic protein was derived by substituting a proline for the histidine at residue 106 of the toxin. Immunization of mice with the non-toxic mutated c-toxin resulted in the induction of a specific antibody response and immunized mice were protected against 1000 LD,, doses of wildtype recombinant c-toxin.

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The K88 fimbrial adhesin enables certain strains of enterotoxigenic Escherichia coli to adhere to the porcine small intestine. In this study, the ability of the K88 adhesin to bind to glycosphingolipids was monitored by modified enzyme-linked immunosorbent assay and thin-layer chromatography overlay binding analysis. The binding of the K88 adhesin to glycosphingolipid-coated microtiter plates was saturable, with 50% maximal binding occurring with gangliotriaosylceramide, gangliotetraosylceramide, and lactosylceramide at 67 ± 21, 117 + 21, and 73 + 22 pM, respectively. Thin-layer chromatography overlay binding analysis demonstrated that serotype 08:K87:K88ab:H19 E. coli bound to hydroxylated galactosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, and lactosylceramide but not to globotriaosylceramide, nonhydroxylated galactosylceramide, glucosides, glucosylceramide, or a mixture of ceramides. The K88 adhesin did not bind by either assay to globoside, the Forssman glycolipid, GM1, GM2, GM3, GDla, GDlb, GD2, GD3, GQlb, or GTlb. The binding pattern observed with the K88 adhesin suggests that o1-linked galactosyl residues are the minimum determinant required for binding, provided they are presented correctly. It is suggested that

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The Yersinia pestis pH 6 antigen was expressed by, and purified from, Escherichia coli containing cloned psa genes. By an enzyme-linked immunosorbence-based assay, purified pH 6 antigen bound to gangliotetraosylceramide (GM1A), gangliotriaosylceramide (GM2A), and lactosylceramide (LC) (designations follow the nomenclature of L. Svennerholm [J. Neurochem. 10:613–623, 1963]). Binding to GM1A, GM2A, and LC was saturable, with 50% maximal binding occurring at 498 ± 4, 390, and 196 ± 3 nM, respectively. Thin-layer chromatography (TLC) overlay binding confirmed that purified pH 6 antigen bound to GM1A, GM2A, and LC and also revealed binding to hydroxylated galactosylceramide. Intact E. coli cells which expressed the pH 6 antigen had a specificity similar to that of purified pH 6 in the TLC overlay assay except that nonhydroxylated galactosylceramide was also bound. The binding patterns observed indicate that the presence of β1-linked galactosyl residues in glycosphingolipids is the minimum determinant required for binding of the pH 6 antigen.