A flagellar sheath protein of Vibrio cholerae CA401 (Inaba) was characterized. Purity of the preparation was indicated by a single band on polyacrylamide gel electrophoresis gels and on Ouchterlony plates prepared with antibody against crude sheath material. The sheath protein was composed of three polypeptides with minimal molecular weights of 61,500, 60,000, and 56,500. The presence of sheath protein on the flagellum as well as on the outer membrane of the cell was demonstrated by ferritin labeling experiments with antiserum. Sheath protein antibody reacted similarly in labeling experiments and agglutination tests with a classical Ogawa strain and two nonagglutinating V. cholerae isolates, indicating that the sheath protein may represent the common Vibrio H antigen. Antibody specific for lipopolysaccharide labeled the cell but not the sheathed flagellum, which demonstrated that the sheath is not a simple extension of the outer membrane of the cell.

We have previously demonstrated, by using a detergent-solubilized system, the existence of specific diphtheria toxin-binding glycoproteins on the surface of toxin-sensitive cells. We have now tested the effect of tunicamycin treatment on the sensitivity of cells in culture to diphtheria toxin and have investigated the toxin sensitivity of mutant cells with known defects in glycosylation of asparagine-linked glycoproteins. Treatment of CHO-Kl cells with tunicamycin, which blocks the synthesis of both high-mannose-type and complex-type oligosaccharide chains of asparagine-linked glycoproteins, resulted in a 50to 100-fold decrease in sensitivity to diphtheria toxin. In contrast, CHO-Kl mutants, defective in the synthesis of either high-mannose-type or complex-type oligosaccharides, showed no difference in toxin sensitivity compared with that of their parental cell lines. When we used an acid shock system, which is believed to result in receptor-dependent direct toxin penetration at the cell surface, the toxin sensitivity of tunicamycin-treated cells was not restored to that of untreated cells, suggesting that tunicamycin treatment results in a decrease in functional toxin receptors. Direct binding studies with 125I-labeled toxin demonstrated that this decrease in functional receptors is due to a decrease in the affinity of the receptors rather than to a change in the number of receptors. Taken together, these data are consistent with the interpretation that the diphtheria toxin receptor is a glycoprotein and suggest that the toxin binds neither to carbohydrate residues unique to the high-mannose-type oligosaccharides nor to those unique to the complex-type oligosaccharides. Furthermore, these data are consistent with the hypothesis that diphtheria toxin binds to the peptide backbone of the glycoprotein receptor.Diphtheria toxin (DT) is synthesized as a single polypeptide chain (molecular weight, 58,342) with two disulfide bridges and no free sulfhydryl groups. Limited proteolysis yields an amino-terminal fragment, the A fragment (molecular weight, 21,167), and a carboxy-terminal fragment, the B fragment (molecular weight, 37,195), which remain associated by an interchain disulfide bond (1, 2, 26). The A fragment inhibits protein synthesis through the NADdependent ADP ribosylation of elongation factor 2 present in the cytosol of eucaryotic cells (2, 26). The B fragment is responsible for interaction of the toxin with specific receptors on toxin-sensitive cells (23,47). It is widely held that the DT receptor possesses a protein component, since treatment of toxin-sensitive cells with such proteases as trypsin, pronase (21, 24), or bromelain (K. Hranitzky, D. A. Hart, and L. Eidels, Fed. Proc. 43:1956, 1984; manuscript in preparation) results in a significant decrease in sensitivity to the toxin. In contrast, treatment of toxin-sensitive cells with neuraminidase has been reported to result in a slight increase in sensitivity to DT (21,35), suggesting that carbohydrate structures that become exposed on removal of sialic...

Vibrio cholerae bacteria of the serological variety O1 were consistently isolated from water samples by passing the water with added Tween 20 through columns packed with polystyrene beads coated with antibodies against the O1 antigenic determinants. The beads from the columns were washed, transferred to alkaline peptone broth, and incubated. The O1 serovars were isolated and identified by established procedures.