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J. Biol. Chem., Vol. 283, Issue 1, 445-452, January 4, 2008

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Characterization of the Enzymatic Activity of the Actin Cross-linking Domain from the Vibrio cholerae MARTX_Vc Toxin^*

Dmitri S. Kudryashov¹, Christina L. Cordero², Emil Reisler, and Karla J. Fullner Satchell³

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

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins, which alter host cells through a number of mechanisms resulting in diarrheal disease. Among the secreted toxins is the multifunctional, autoprocessing RTX toxin (MARTX_Vc), which disrupts actin cytoskeleton by covalently cross-linking actin monomers into oligomers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we demonstrate unambiguously that ACD utilizes G- and not F-actin as a substrate for the cross-linking reaction and hydrolyzes one molecule of ATP per cross-linking event. Furthermore, major actin-binding proteins that regulate actin cytoskeleton in vivo do not block the cross-linking reaction in vitro. Cofilin inhibits the cross-linking of G- and F-actin, at a high mole ratio to actin but accelerates F-actin cross-linking at low mole ratios. DNase I completely blocks the cross-linking of actin, likely due to steric hindrance with one of the cross-linking sites on actin. In the context of the holotoxin, the inhibition of Rho by the Rho-inactivating domain of MARTX_Vc (Sheahan, K. L., and Satchell, K. J. F. (2007) Cell. Microbiol. 9, 1324–1335) would accelerate F-actin depolymerization and provide G-actin, alone or in complex with actin-binding proteins, for cross-linking by ACD, ultimately leading to the observed rapid cell rounding.

Received for publication, May 11, 2007 , and in revised form, September 26, 2007.

^* This work was supported in part by an Investigator in the Pathogenesis of Infectious Disease award from the Burroughs Wellcome Fund and United States Public Health Service Grant AI051490 (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 American Heart Association Western States Affiliates Fellowship 0425119Y.

² Supported by National Research Service Award Predoctoral Fellowship F31-AI52490. Present address: The Joint Commission, Oakbrook Terrace, IL 60181.

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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