The Binary Clostridium botulinum C2 Toxin as a Protein Delivery System

Abstract

Clostridium botulinum C2 toxin belongs to the family of binary actin-ADP-ribosylating toxins. Further members of this toxin family are iota toxin from Clostridium perfringens (1), the Clostridium spiroforme toxin (2), the Clostridium difficile ADPribosyltransferase (3), and the recently crystallized vegetative insecticidal protein (VIP) 1 from Bacillus cereus (4). These toxins are composed of two separated proteins, a binding/translocation component and an enzyme component, which must assemble on the surface of target cells to exhibit cytotoxicity. This mechanism of toxin up-take is also shared by anthrax toxin, whose binding and translocation component protective antigen (PA) is related to the binding components of the actin-ADPribosylating toxins (5). C2 toxin consists of the ADP-ribosylating enzyme component C2I (M r ϳ 50,000) and the binding component C2II (M r ϳ 80,000). For cellular uptake of C2 toxin, trypsin-activated C2IIa forms a heptamer (6) that interacts with the C2I enzyme component and binds to the cellular receptor, a hybrid and/or complex carbohydrate structure (7). Both C2IIa and C2I are internalized via receptor-mediated endocytosis. In an acidic endosomal compartment, the C2IIa oligomers insert in the membrane and form pores (6). Most likely, C2I is translocated through this pore across the endosomal membrane into the cytosol to ADP-ribosylate G-actin at Arg-177 (8). As a consequence, actin polymerization is blocked, and actin filaments disassemble. Site-directed mutagenesis of C2I (9) and of the related ADP-ribosyltransferase iota from C. perfringens (10) indicate that the catalytic site of these enzymes is located in their C-terminal part. This is in line with the recently reported crystal structure and the suggested catalytic mechanism of the ADP-ribosyltransferase VIP2 (4).The N-terminal domain of C2I (amino acid residues 1-225) is enzymatically inactive but interacts with the C2IIa oligomer and mediates cellular uptake of C2I (11). This allows C2I to act as an adaptor for C2IIa-mediated delivery of fusion proteins into cells. In the C2I 1-225 -C3 fusion toxin, C2I 1-225 was used to deliver C3 transferase from Clostridium limosum (M r ϳ 23,000) (11), which inactivates Rho GTPase by ADP-ribosylation at . C3 transferase by its own does not enter cells efficiently because it lacks any specific binding and translocation unit (13). We characterized the C2I/C2IIa contact site in more detail by the use of various truncations of C2I and their fusions with C3 transferase. Here, we report that amino acid residues 1-87 of C2I are sufficient for C2IIa-mediated delivery of C2I fusion toxins into cells. EXPERIMENTAL PROCEDURESMaterials-Cell culture medium was from Biochrom (Berlin, Germany), fetal calf serum was from PAN Systems (Aidenbach, Germany), and cell culture materials were from Falcon (Heidelberg, Germany). The C2II binding component from C. botulinum C2 toxin was purified as recombinant GST fusion protein in Escherichia coli and activated with trypsin as described (6). Antiserum against C2I ...

G13-dependent Activation of MAPK by Thyrotropin

“…Since a variant of this toxin, TcdB 1470, which exhibits no inhibitory activity toward Rho proteins (48), was also effective, we conclude that Rho is not involved in this pathway. This conclusion is supported by the fact that a fusion toxin with clostridial C3 activity, which acts as a more specific Rho inhibitor (49,50), did not prevent TSH-induced p44/42 MAPK activation in both cell lines. Furthermore, treatment with H89, which also acts as a Rho-kinase inhibitor besides its inhibitory effect on PKA (61), did not prevent stimulation of MAPK in FTC-133 WT TSHR cells.…”

“…To further exclude an involvement of Rho in TSH-promoted MAPK activation, we used C2IN-C3, a toxin with C3-transferase activity, thus acting as a specific Rho inhibitor. Since C2IN-C3 does not enter the cell, it was co-administered with C2IIa, the transport unit of C2-toxin, to ensure intracellular delivery (49,50). Treatment of FTC-133 WT TSHR or PHT cells with C2IN-C3 plus C2IIa did not inhibit stimulation of MAPK by TSH (Fig.…”

The Host Cell Chaperone Hsp90 Is Essential for Translocation of the Binary Clostridium botulinum C2 Toxin into the Cytosol

“…The components of C2 toxin (C2I, C2IS16C, C2II) and the components of iota toxin (Ia and Ib) were purified as recombinant glutathione S-transferase proteins as described previously (21,22). C2II was activated with trypsin as described (10).…”

“…Binding of C2 Toxin by Flow Cytometry-C2IS16C was labeled with Alexa-488, and binding of C2 toxin to CHO cells was determined as described earlier in detail (21). Cell-associated fluorescence was detected by using a FACSCalibur fluorescence-activated cytometer (BD Biosciences).…”

Clostridium botulinum C2 Toxin

“…Labeled proteins were stored at 4°C (protected from light) and used within 2 weeks. Biological activity of labeled proteins was analyzed as described previously (23). Iodination of C2I was performed using IODO-BEADS according to the manufacturer's (Pierce) protocol using 100 Ci of Na 125 I per 100 g of C2I.…”