Activity of diphtheria toxin. II. Early events in the intoxication of HeLa cells.

The initial steps in the interaction of diphtheria toxin with HeLa cells were studied. It was demonstrated that lethal doses of toxin are rapidly adsorbed to the cell. The kinetics of uptake, as measured by lethality, indicated that a single toxin molecule is able to cause cell death. Studies on the effect of pH on intoxication showed that adsorption of toxin occurred over a wide pH range but was partially inhibited at high pH values. Experiments to determine the influence of the ionic environment on intoxication indicated that adsorption of toxin did not take place in the absence of salts and was partially inhibited in the presence of a polyanion. The evidence indicates that the initial binding of toxin to the cell is electrostatic in nature, involving positively charged surface groups. Attempts to demonstrate specific receptors for the attachment of toxin to cells were unsuccessful, suggesting that toxin adsorption may be a nonspecific process. The effect of energy inhibitors on intoxication was examined. Sodium fluoride, an inhibitor of glycolysis, almost completely prevented intoxication in HeLa cells, whereas inhibitors of respiration and oxidative phosphorylation had no effect. Sodium fluoride did not prevent adsorption of toxin but appeared to inhibit a later step in the intoxication process, perhaps the transport of toxin to subsurface or intracellular levels.