A genetic approach is described for exploring the mechanism by which diphtheria toxin undergoes pH-dependent membrane insertion and transfer of its enzymic A fragment into the cytoplasm of mammalian cells. The cloned toxin expressed inEscherichia coliis secreted to the periplasmic space, where it is processed normally and folds into a native structure. When bacteria synthesizing the toxin are exposed to pH 5, they die rapidly. The toxin undergoes a conformational change that is believed to allow it to be inserted into the bacterial inner membrane and form channels, which proves lethal for the cell. The membrane insertion event mimics the process by which the toxin inserts into the endosomal membrane of mammalian cells, leading to release of the enzymic A fragment into the cytoplasm. The observation of pH-dependent bacterial lethality provides the basis for a positive genetic selection method for mutant forms of the toxin that are altered in ability to undergo membrane insertion or pore formation.
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