Uptake of diphtheria toxin and its fragment A moiety by mammalian cells in culture

Abstract

Evidence suggesting that diphtheria toxin reaches the cytoplasm of susceptible mammalian cells by two independent mechanisms is presented. A schematic model describing the two processes of toxin entry into the cell is developed. One process of toxin uptake considered to be physiologically significant is passage of the protein toxin through the plasma membrane. This most likely happens by binding of fragment B to receptors on the membrane and by subsequent toxinmembrane interaction so that ultimately fragment A, the enzymatically active moiety, is transported to the cell interior. This process, which ultimately leads to cessation of protein synthesis and cell death, involves a comparatively small number of toxin molecules. A second mechanism of toxin uptake is by classical pinocytosis. The majority of toxin taken into the cell is accomplished by this process. The fate of toxin taken into HEp-2 cells via pinocytosis is proteolysis by lysosomal enzymes. Thus, such vesicle-bound toxin is ordinarily not expressed biologically. Evidence suggesting that ammonium chloride provides total protection to diphtheria toxin-susceptible cells by preventing entry of toxin by the specific receptor-associated process is also provided; data showing that the ammonium salt immobilizes bound toxin on the plasma membrane of HEp-2 cells are presented. Finally, it is suggested that actively endocytic cells such as guinea pig macrophages interact with toxin in a significantly different manner than do nonphagocytic cells.

Bacterial toxins: cellular mechanisms of action.

“…Similar results were obtained by Bonventre et al (12) with HEp-2 and L-929 cells and by Saelinger et al (183,184) with HEp-2 cells and actively phagocytic mouse or guinea pig macrophages. Both studies supported the view that pinocytic uptake of toxin does not necessarily lead to cytotoxicity.…”

“…Whereas internalization and degradation of '25I-labeled diphtheria toxin appeared to proceed normally in the presence of ammonium chloride, a fraction of potentially active toxin molecules was maintained in a position accessible to neutralizing antitoxin for prolonged periods at 37°C (36,184). The initial interpretation of such data was that ammonium chloride protects cells by preventing the internalization of biologically relevant toxin molecules or that only a small fraction of toxin-specific receptors are productive receptors.…”

Pathogenesis of Shigella Diarrhea: XVII. A Mammalian Cell Membrane Glycolipid, Gb3, Is Required but Not Sufficient to Confer Sensitivity to Shiga Toxin

“…This raises the possibility that CHO cells may lack either the mechanism for translocation of Gb3-bound toxin from the cell surface to the cytoplasm or a second protein or glycoprotein receptor needed to interact with the uptake mechanism and/or direct the complex to the trans-Golgi region of the cell [31] and beyond to the endoplasmic reticulum [32], where Shiga toxin exerts its biochemical action. The minimal toxicity observed in Gb3-deficient cells incubated with very high concentrations of Shiga toxin is probably due to non-receptor-based toxin uptake, as reported for receptor-negative diphtheria toxinresistant mouse L cells [33]. It is also remotely possible that CHO cell ribosomes are not susceptible to the action of Shiga toxin.…”

Receptor-mediated entry of diphtheria toxin into monkey kidney (Vero) cells: electron microscopic evaluation

“…The intervening step(s) between DT binding and inactivation of elongation factor 2 is not completely understood. It is evident that DT may be internalized by several endocytic mechanisms (36). The wide range in sensitivity to DT exhibited by different animal species and cell lines may be determined by the endocytic pathway taken (29).…”