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Infection and Immunity, October 2001, p. 6004-6011, Vol. 69, No. 10
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.10.6004-6011.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Characterization of the Enzymatic Component of the ADP-Ribosyltransferase Toxin CDTa from Clostridium difficile

Irene Gülke, Gunther Pfeifer, Jan Liese, Michaela Fritz, Fred Hofmann,dagger Klaus Aktories, and Holger Barth*

Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany

Received 12 April 2001/Returned for modification 22 May 2001/Accepted 19 June 2001

Certain strains of Clostridium difficile produce the ADP-ribosyltransferase CDT, which is a binary actin ADP-ribosylating toxin. The toxin consists of the binding component CDTb, which mediates receptor binding and cellular uptake, and the enzyme component CDTa. Here we studied the enzyme component (CDTa) of the toxin using the binding component of Clostridium perfringens iota toxin (Ib), which is interchangeable with CDTb as a transport component. Ib was used because CDTb was not expressed as a recombinant protein in Escherichia coli. Similar to iota toxin, CDTa ADP-ribosylates nonmuscle and skeletal muscle actin. The N-terminal part of CDTa (CDTa1-240) competes with full-length CDTa for binding to the iota toxin binding component. The C-terminal part (CDTa244-263) harbors the enzyme activity but was much less active than the full-length CDTa. Changes of Glu428 and Glu430 to glutamine, Ser388 to alanine, and Arg345 to lysine blocked ADP-ribosyltransferase activity. Comparison of CDTa with C. perfringens iota toxin and Clostridium botulinum C2 toxin revealed full enzyme activity of the fragment Ia208-413 but loss of activity of several N-terminally deleted C2I proteins including C2I103-431, C2I190-431, and C2I30-431. The data indicate that CDTa belongs to the iota toxin subfamily of binary actin ADP-ribosylating toxins with respect to interaction with the binding component and substrate specificity. It shares typical conserved amino acid residues with iota toxin and C2 toxin that are suggested to be involved in NAD-binding and/or catalytic activity. The enzyme components of CDT, iota toxin, and C2 toxin differ with respect to the minimal structural requirement for full enzyme activity.

* Corresponding author. Mailing address: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany. Phone: 49-0761-2035301. Fax: 49-0761-2035311. E-mail: barthh{at}uni-freiburg.de.

dagger Present address: Institut für Toxikologie der Medizinischen Hochschule Hannover, D-30623 Hannover, Germany.

Infection and Immunity, October 2001, p. 6004-6011, Vol. 69, No. 10
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.10.6004-6011.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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