Cellular Uptake of Clostridium botulinum C2 Toxin: Membrane Translocation of a Fusion Toxin Requires Unfolding of Its Dihydrofolate Reductase Domain † Gerd Haug, Christian Wilde, Jost Leemhuis, Dieter K. Meyer, Klaus Aktories, and Holger Barth* Institut fu ¨r Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-UniVersita ¨t Freiburg, Albertstrasse 25 (Otto-Krayer-Haus), D-79104 Freiburg, Germany ReceiVed August 11, 2003; ReVised Manuscript ReceiVed October 14, 2003 ABSTRACT: The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP- ribosylating toxins. C2 toxin is composed of two separated nonlinked proteins. The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells. The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol. After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol. For this step, the cellular heat shock protein Hsp90 is essential. We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I. Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component. The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa. Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain. Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin. In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited. The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes. Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells. The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited. Bacterial protein toxins, which modify their substrates in the cytosol of target cells, must develop strategies to transport their enzymatic domain across cellular membranes (1). Therefore, AB-type toxins show a bipartite organization with different functional domains, an enzyme domain (A) and a binding/translocation domain (B) (2). The B-domain mediates cell binding and the translocation of the A-domain into the cytosol. However, the mechanism by which the B-domain mediates translocation of the A-domain of bacterial toxins is still not completely understood. In the family of binary ADP-ribosylating toxins, the A- and B-domains are two separated and nonlinked toxin components (for review see ref 3). Both components, the enzyme component and the binding component, have to assemble on the surface of the target cell to exhibit cytotoxic effects (4). The family of binary actin-ADP-ribosylating toxins consists of the Clostridium botulinum C2 toxin (5), Clostridium perfringens iota toxin (6, 7), Clostridium spiro- forme toxin (7-9), Clostridium difficile ADP-ribosyltrans- ferase (10, 11), and the VIP toxin (vegetative insecticidal protein) from Bacillus cereus (12). These toxins ADP- ribosylate G-actin at arginine-177 (13), leading to disas- sembly of actin filaments, breakdown of the actin cyto- skeleton, and rounding up of cultured monolayer cells (3, 14, 15). In this study, we want to investigate the mechanism by which C2 toxin translocates into the cytosol of its target cells. C2 toxin is composed of the enzyme component C2I 1 (49 kDa) and the binding/translocation component C2II (80 kDa) (5), which has to be activated by trypsin cleavage (16). An ∼20 kDa peptide is cleaved from the N-terminus, and the resulting active C2IIa (∼60 kDa) forms ring-shaped hep- tamers in solution (17). The C2IIa heptamers bind to complex and hybrid carbohydrate structures on the surface of target cells (18). After assembly of C2I to C2IIa heptamers, the toxin complex is taken up via receptor-mediated endocytosis and reaches endosomal compartments. Upon acidification of endosomes, C2IIa forms pores in endosomal membranes, and C2I is delivered into the cytosol (17). However, it is still not known whether C2I translocates directly through the C2IIa pore [inner diameter 1-2 nm (17)]. If so, an (at least partial) unfolding of the C2I protein during translocation † This work was supported by the Deutsche Forschungsgemeinschaft (SFB 388/C8 and SFB 505/B6). * Corresponding author. Tel: +49-0761-2035308. Fax: +49-0761- 2035311. E-mail: Holger.Barth@pharmakol.uni-freiburg.de. 1 Abbreviations: Baf, bafilomycin A1; C., Clostridium; C2I, enzyme component of C. botulinum C2 toxin; C2II, binding component of C. botulinum C2 toxin; DHFR, dihydrofolate reductase; HBSS, Hank’s balanced salt solution; Hsp, heat shock protein; MTX, methotrexate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- phoresis. 15284 Biochemistry 2003, 42, 15284-15291 10.1021/bi0354278 CCC: $25.00 © 2003 American Chemical Society Published on Web 11/26/2003