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J. Biol. Chem., Vol. 283, Issue 16, 10671-10678, April 18, 2008

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Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae^*

René Jørgensen¹², Alexandra E. Purdy¹³, Robert J. Fieldhouse, Matthew S. Kimber, Douglas H. Bartlett, Supported by National Institutes of Health Grant AI46600-02 and University of California Marine Council Grant CEQI0047⁴, and A. Rod Merrill, Supported by CIHR and Canadian Cystic Fibrosis Foundation⁵

From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada and the Marine Biology Research Division, Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-0202

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC₅₀ = 4.6 ± 0.4 ng/ml) and crustaceans (Artemia nauplii LD₅₀ = 10 ± 2 µg/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-Å resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.

Received for publication, December 7, 2007 , and in revised form, February 4, 2008.

The atomic coordinates and structure factors (codes 2Q5T and 2Q6M) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Table S1.

¹ Both authors contributed equally to this work.

² Supported by Canadian Institutes for Health Research (CIHR) and Carlsberg Foundation fellowships.

³ Recipient of a pre-doctoral fellowship from Howard Hughes Medical Institute.

⁴ To whom correspondence may be addressed: Marine Biology Research Division, Center for Marine Biotechnology and Biomedicine, 8750 Biological Grade Scripps Institution of Oceanography, University of California-San Diego, La Jolla, CA 92093-0202. Tel.: 858-534-5233; Fax: 858-534-7313; E-mail: dbartlett{at}ucsd.edu. ⁵ To whom correspondence may be addressed: Dept. of Molecular and Cellular Biology, University of Guelph, Science Complex, 50 Gordon St., Guelph, Ontario N1G 2W1, Canada. Tel.: 519-824-4120 (ext. 53805); Fax: 519-837-1802; E-mail: rmerrill{at}uoguelph.ca.

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