Toxin-Based Therapeutic Approaches

Shapira, Assaf; Benhar, Itai

Diphtheria toxin (DT), secreted by pathogenic strains of the bacterium Corynebacterium diphtheria, is the prototype for the family of ADP-ribosylating toxins. It belongs to a group of toxins called AB toxins that consist of two fragments (A and B). The B fragment is responsible for cell entry (binding to a cell surface receptor and subsequent translocation into the cell cytoplasm) while the internalized A fragment intoxicates the cell by virtue of its enzymatic activity [119, 120] . The toxin is secreted as a single protein of 535 amino acids and is composed of three functional domains: the N terminal domain (residues represents the catalytic (C) A fragment/domain (DTA/DT-A). The C terminal portion of the toxin (amino acids represents the B fragment and is divided into two functional domains: the translocation domain (T) (amino acids and the receptor binding domain (R) (amino acids [121] . The native diphtheria toxin binds, via its R domain, to heparin binding epidermal growth factor precursor on the cell membrane, where it is cleaved by cell-surface furin or furin-like protease [122, 123] . The di-chain protein that is still linked by a single disulfide bond between cysteine 186 and cysteine 201 is internalized into clathrin coated pits and reaches the lumen of a developing endosome (where furin-mediated cleavage of toxin molecules that escaped cleavage by cell-surface proteases, may occur [122] ). Upon endosome acidification, the T domain undergoes a conformational change that leads to exposure of hydrophobic areas that are inserted into the membrane, forming a channel through which the catalytic domain translocates and escapes from the endosome, probably with the aid of cytosolic factors [124] [125] [126] [127] [128] [129] . In the cell cytoplasm, the catalytic domain exerts its toxic activity by transferring adenosine di-phosphate-ribose (ADP-ribose) moiety from nicotinamide dinucleotide (NAD) to a modified histidine residue (diphthamide) at position 715 in the eukaryotic translation elongation factor (eEF2). This action results in the inactivation of the latter, inhibition of protein synthesis, and programmed cell death [13, [130] [131] [132] [133] (Figure 3 ). It was also reported that delivery of a single molecule of the catalytic domain into the cytosol is sufficient to kill a cell, demonstrating the extreme potency of this bacterial toxin [134].