Toxin-Based Therapeutic Approaches

Shapira, Assaf; Benhar, Itai

Anthrax toxin (AnTx) is a major virulence factor secreted by the gram-positive, spore-forming bacterium Bacillus anthrachis. The toxin, which damages cells and impairs host defenses, belongs to a family of toxins called "binary toxins" which are characterized by consisting of minimally two discrete nontoxic proteins that must be combined to elicit toxicity. AnTx consist of three non-toxic plasmid-encoded multidomain proteins: protective antigen (PA/PrAg; 83 kDa), lethal factor (LF; 90 kDa) and edema factor (EF; 89 kDa) (for review, see [440] [441] [442] ). Intoxication begins with the binding of PA to either of its two known cellular receptors ATR/TEM8 [443] and CMG2 [444] . PA then undergoes an activating cleavage by a member of the furin family of cellular proteases [418, 420] , into an N terminal PA63 (63 kDa) and C terminal PA20 (20 kDa) fragments. Following cleavage, receptor-bound PA63 self associate to form a ring-shaped homoheptamer [445] , called the prepore, which may then form complexes with up to three molecules of LF and/or EF (each bound LF/EF molecule "occupies" two neighboring PA subunits). The resulting complexes, which are known as lethal toxin (LeTx) and edema toxin (EdTx), respectively, are then internalized via clathrin-dependent receptor-mediated endocytosis [446, 447] and delivered to early endosomes where the complex is sorted to the vesicular regions and preferentially incorporated into intraluminal vesicles [448] . Subsequently, the prepore undergoes an acidic pH-dependent conformational change to form a cationselective, ion-conducting channel [449] [450] [451] . This channel/pore is thought to participate in the unfolding of LF and EF, and functions in their translocation into the lumen of the intraluminal vesicles or into the cytoplasm, probably with the aid of cytosolic components [452] [453] [454] [455] [456] . Following transportation to late endosomes, back fusion of intraluminal vesicles with the limiting membrane delivers the toxic factors, which were "trapped" inside the vesicles lumen, to the cytoplasm [448] (for review about the cellular routing of the anthrax toxin, see [442, 457] ). Once in the cytoplasm, LF functions as a zinc metalloproteinase that specifically cleaves the N-termini of MKK/MEK proteins (kinases of mitogen-activated protein kinases), blocking their signaling activity [458] [459] [460] [461] [462] [463] [464] [465] [466] . EF is a Ca 2+ /calmodulin activated adenylate cyclase (AC) that acts by elevating the intracellular level of cyclic AMP (cAMP), upsetting water homeostasis, destroying the delicate balance of intracellular signaling and impairing neutrophil functions [440, [467] [468] [469] [470] [471] [472] (Figure 6 ). In the left panel, the colors of the different proteins correspond to those in the scheme; 2. PA binds to cellular receptor (ATR/TEM8; CMG2); 3. Cell-surface furin protease cleaves PA into an N terminal PA63 (63 kDa) and C terminal PA20 (20 kDa) fragments; 4. Receptor bound PA63 self associates into a homoheptamer ("prepore") that can bind up to 3 molecules of LF and/or EF; 5. The complex internalized via clathrin-dependent receptor mediated endocytosis; 6. In the early endosomes (EE), the complex is sorted to the vesicular region and preferentially incorporated into intraluminal vesicles. The acidic environment of the endosome induces a conformational change in the prepore that turns into a channel/pore and functions in the translocation of LF and/or EF to the l