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J. Biol. Chem., Vol. 281, Issue 43, 32366-32374, October 27, 2006

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The Actin Cross-linking Domain of the Vibrio cholerae RTX Toxin Directly Catalyzes the Covalent Cross-linking of Actin^*

Christina L. Cordero¹, Dmitry S. Kudryashov², Emil Reisler, and Karla J. Fullner Satchell³

From the Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611 and the Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins to alter host cells and to elicit diarrheal disease. Among the secreted toxins is the multifunctional RTX toxin, which causes cell rounding and actin depolymerization by covalently cross-linking actin monomers into dimers, trimers, and higher multimers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we further investigated the role of the ACD in the actin cross-linking reaction. We show that the RTX toxin cross-links actin independently of tissue transglutaminase, thus eliminating an indirect model of ACD activity. We demonstrate that a fusion protein of the ACD and the N-terminal portion of lethal factor from Bacillus anthracis (LF_NACD) has cross-linking activity in vivo and in crude cell extracts. Furthermore, we determined that LF_NACD directly catalyzes the formation of covalent linkages between actin molecules in vitro and that Mg²⁺ and ATP are essential cofactors for the cross-linking reaction. In addition, G-actin is proposed as a cytoskeletal substrate of the RTX toxin in vivo. Future studies of the in vitro cross-linking reaction will facilitate characterization of the enzymatic properties of the ACD and contribute to our knowledge of the novel mechanism of covalent actin cross-linking.

Received for publication, June 1, 2006 , and in revised form, August 23, 2006.

^* This work was supported in part by a Biomedical Research Support Program award from the Howard Hughes Medical Institute and United States Public Health Service Grant AI051490 from NIAID, National Institutes of Health (to K. J. F. S.), and by United States Public Health Service Grant GM-077190 and National Science Foundation Grant MCB 0316269 (to E. R.). 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.

¹ Supported by National Research Service Award Predoctoral Fellowship F31-AI52490.

² Supported by Fellowship 0425119Y from the American Heart Association, Western States Affiliates.

³ To whom correspondence should be addressed: Dept. of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Tarry 3-713, Chicago, IL 60611. Tel.: 312-503-2162; Fax: 312-503-1339; E-mail: k-satchell{at}northwestern.edu.

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