THE HOST CELL CHAPERONE HSP90 IS ESSENTIAL FOR CELLULAR UPTAKE OF CLOSTRIDIAL BINARY ACTIN-ADP-RIBOSYLATING TOXINS

Holger Barth, Gerd Haug and Klaus Aktories

Institut für Experimentelle und Klinische Pharmakologie und Toxikologie (Otto-Krayer-Haus), Albertstraße 25, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg

e-mail: Holger.Barth@pharmakol.uni-freiburg.de

The family of binary actin-ADP-ribosylating toxins consists of Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, Clostridium difficile ADP-ribosyltransferase (CDT) and Clostridium spiroforme toxin. These toxins are composed of two individual non-linked proteins, a binding/translocation component and an enzyme component. The enzyme component ADP-ribosylates G-actin in the cytosol of host cells. This leads to depolymerization of actin filaments and to rounding up of cultured monolayer cells.

We used the Clostridium botulinum C2 toxin, to study the cellular uptake mechanism of this toxin family. The trypsin-activated binding component C2IIa (60 kDa) forms ring-shaped heptamers (~420 kDa) which bind to cells and mediate the uptake of the enzyme component C2I (~49 kDa). In acidic endosomes C2IIa inserts into membranes, forms pores (inner diameter ~2 nm) and C2I translocates into the cytosol. For translocation, the C2I protein becomes unfolded.

We used the specific inhibitors Radicicol and Geldanamycin to investigate the role of the host cell heat shock protein Hsp90 in the translocation process of binary actin-ADP-ribosylating toxins. By pretreating cells with Hsp90 inhibitors we could transiently block the intoxication of cells by C2 toxin, Iota toxin and CDT. Hsp90 inhibitors also blocked direct translocation of C2 toxin across the cell membrane.

The data indicate that the cellular chaperone Hsp90 is required for the cellular uptake of C2 toxin, iota toxin and CDT.