Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic “A-B” paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The “B” components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated “B” components form homoheptameric rings that subsequently dock with an “A” component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, “A” molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria

The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. Here we report that binding of C2II to the surface of target cells requires cleavage of C2II by trypsin. Trypsin cleavage causes oligomerization of the activated C2II (C2IIa) to give SDS-stable heptameric structures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme component C2I is blocked by bafilomycin but not by brefeldin A or nocodazole, indicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells involves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cleaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface and assembly with C2I, (iv) receptor-mediated endocytosis of both C2 components into endosomes, and finally (v) translocation and release of C2I into the cytosol after acidification of the endosomal compartment.

The ephrins, ligands of Eph receptor tyrosine kinases, have been shown to act as repulsive guidance molecules and to induce collapse of neuronal growth cones. For the first time, we show that the ephrin-A5 collapse is mediated by activation of the small GTPase Rho and its downstream effector Rho kinase. In ephrin-A5–treated retinal ganglion cell cultures, Rho was activated and Rac was downregulated. Pretreatment of ganglion cell axons with C3-transferase, a specific inhibitor of the Rho GTPase, or with Y-27632, a specific inhibitor of the Rho kinase, strongly reduced the collapse rate of retinal growth cones. These results suggest that activation of Rho and its downstream effector Rho kinase are important elements of the ephrin-A5 signal transduction pathway.

Rb؉ efflux when cells were exposed to low pH. Toxin B also induced pH-dependent channel formation in artificial lipid bilayer membranes. Clostridium sordellii lethal toxin, another member of the family of large clostridial cytotoxins, also induced increased 86 Rb ؉ release at low pH. The results suggest that large clostridial cytotoxins including C. difficile toxin B and C. sordellii lethal toxin undergo structural changes at low pH of endosomes that are accompanied by membrane insertion and channel formation.

Our previous experiments indicated that GTPases, other than RhoA, are important for the maintenance of endothelial barrier integrity in both intact microvessels of rats and mice and cultured mouse myocardial endothelial (MyEnd) cell monolayers. In the present study, we inhibited the endothelial GTPase Rac by Clostridium sordellii lethal toxin (LT) and investigated the relation between the degree of inhibition of Rac by glucosylation and increased endothelial barrier permeability. In rat venular microvessels, LT (200 ng/ml) increased hydraulic conductivity from a control value of 2.5 +/- 0.6 to 100.8 +/- 18.7 x 10-7 cm x s(-1) x cm H2O(-1) after 80 min. In cultured MyEnd cells exposed to LT (200 ng/ml), up to 60% of cellular Rac was glucosylated after 90 min, resulting in depolymerization of F-actin and interruptions of junctional distribution of vascular endothelial cadherin (VE-cadherin) and beta-catenin as well as the formation of intercellular gaps. To understand the mechanism by which inhibition of Rac caused disassembly of adherens junctions, we used laser tweezers to quantify VE-cadherin-mediated adhesion. LT and cytochalasin D, an actin depolymerizing agent, both reduced adhesion of VE-cadherin-coated microbeads to the endothelial cell surface, whereas the inhibitor of Rho kinase Y-27632 did not. Stabilization of actin filaments by jasplakinolide completely blocked the effect of cytochalasin D but not of LT on bead adhesion. We conclude that Rac regulates endothelial barrier properties in vivo and in vitro by 1) modulation of actin filament polymerization and 2) acting directly on the tether between VE-cadherin and the cytoskeleton.

Clostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I. The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I. This toxin complex is taken up by receptor-mediated endocytosis. In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol. We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin. ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors. Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I. At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I. Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa. The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro. The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors. In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells. The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II.

Several intracellularly acting bacterial protein toxins, which are known to enter cells by endocytosis, are shown to produce channels. This finding also holds true for the C2-II binding component of C2-toxin of Clostridium botulinum. There is evidence that toxin translocation across the target cell membrane and channel formation by the binding component are related phenomena. Here, we demonstrate that C2-II is fully reconstituted when it is added to only one side of the lipid bilayer membrane. Chloroquine and some related compounds, known as potent drugs against malaria infection of humans, efficiently block the C2-II-mediated channel in a dose-dependent way. The half-saturation constant for binding of chloroquine and some of the related compounds to C2-II is in the micromolar to millimolar range. Their binding to the C2-II channel is asymmetric with respect to its addition to one side of the membrane, and the halfsaturation constant is smaller when both inhibitor and protein are added to the same side of the membrane, the cis-side, which corresponds to the external surface of target cells. In vivo experiments with Vero cells demonstrate that chloroquine and related compounds also efficiently block intoxication of the cells by C2-toxin in the same concentration range as they block in vitro the channels.he C2-toxin of Clostridium botulinum is a member of a family of bacterial ADPribosylating toxins that enter the eukaryotic target cells and develop their toxic activity inside the cells (1-3). The common feature of this family of toxins, to which C. T perfringens iota toxin and C. spiroforme toxin also belong, is an ADP-ribosyltransferase activity (4-8). C2-toxin ADP-Tribosylates in the target cell monomeric G-actin but not polymerized Factin at the position arginine-177 (9), leading to a complete depolymerization of the actin filaments and loss of the cell functions, linked to the actin cytoskeleton. Similar to all the other toxins of this family and other nonrelated toxins, this toxin consists of two separate components that are exported separately out of the Clostridium botulinum cells (2, 3, 10). One component possesses the enzymatic activity (C2-I) and the other is the binding protein (C2-II) that is essential for binding the enzyme component to the cell and internalizing C2-I into the cytosol. Recently, the cellular uptake of C2-toxin was described (11). For binding to its cellular carbohydrate receptor, C2-II must be activated by trypsin cleavage. This activation leads to formation of heptamers that form a ring-shaped pore structure. C2-I assembles, and both toxin components are internalized via receptor-mediated endocytosis. C2-toxin reaches the early endosomal compartment and, after decrease of pH, C2-I escapes into the cytosol. The activated binding component but not the nonactivated form of C2-I can induce import of C2-I in the cell (12, 13), Similarly, the removal of about 26 kDa from the secreted C2-II is critical for the formation of small ion-selective channels in artificial lipid bilayer membranes (...

The binary iota-toxin is produced by Clostridium perfringens type E strains and consists of two separate proteins, the binding component iota b (98 kDa) and an actin-ADP-ribosylating enzyme component iota a (47 kDa). Iota b binds to the cell surface receptor and mediates the translocation of iota a into the cytosol. Here we studied the cellular uptake of iota-toxin into Vero cells. Bafilomycin A1, but not brefeldin A or nocodazole, inhibited the cytotoxic effects of iota-toxin, indicating that toxin is translocated from an endosomal compartment into the cytoplasm. Acidification (pH < 5.0) of the extracellular medium enabled iota a to directly enter the cytosol in the presence of iota b. Activation by chymotrypsin induced oligomerization of iota b in solution. An average mass of 530 ؎ 28 kDa for oligomers was determined by analytical ultracentrifugation, indicating heptamer formation. The entry of iota-toxin into polarized CaCo-2 cells was studied by measuring the decrease in transepithelial resistance after toxin treatment. Iota-toxin led to a significant decrease in resistance when it was applied to the basolateral surface of the cells but not following application to the apical surface, indicating a polarized localization of the iota-toxin receptor.Bacterial toxins which act within the cytosol of eucaryotic cells have to be transported across lipid membranes to reach their targets. So far, two mechanisms are known by which the catalytic domain of toxins can be translocated into the cytosol. One group of toxins (e.g., diphtheria toxin, anthrax toxin, and Clostridium botulinum C2 toxin) is trafficked into endosomes after endocytosis, and acidification of this compartment leads to conformational changes in the translocation domain. This domain is then able to insert into the endosomal membrane, eventually resulting in delivery of the catalytic domain into the cytosol (2,19,34). A second mechanism employed by some toxins (e.g., cholera toxin) involves transport to the trans-Golgi network (TGN) after endocytosis. In a retrograde manner, these toxins are transported from the TGN to the endoplasmatic reticulum, where the catalytic domain is delivered to the cytosol (24, 32).Clostridium perfringens iota-toxin belongs to the family of binary toxins, in which the translocation and enzyme domains are located on two individual, nonlinked proteins (37, 38). Other members of the family are Clostridium botulinum C2 toxin (1, 9), Clostridium spiroforme toxin (27), Clostridium difficile ADP-ribosyltransferase (28), the vegetative insecticidal proteins from Bacillus cereus (14), and anthrax toxin from Bacillus anthracis (18). Iota a, the enzyme component of iotatoxin, modifies actin by ADP-ribosylation at arginine-177 (31, 39). The ADP-ribosylation leads to breakdown of the cytoskeleton by inhibiting actin polymerization (1). In contrast to C2 toxin of Clostridium botulinum, iota a ADP-ribosylates both muscle and nonmuscle actin whereas C2I, the enzyme component of C2 toxin, modifies only nonmuscle actin (20, 31). The binding c...