HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 14, 10272-10282, April 6, 2007

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 282/14/10272    most recent M610254200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pust, S. Articles by Barth, H. Search for Related Content PubMed PubMed Citation Articles by Pust, S. Articles by Barth, H. Social Bookmarking                      What's this?

A Cell-permeable Fusion Toxin as a Tool to Study the Consequences of Actin-ADP-ribosylation Caused by the Salmonella enterica Virulence Factor SpvB in Intact Cells^*

Sascha Pust¹, Henrike Hochmann¹, Eva Kaiser, Guido von Figura, Karin Heine, Klaus Aktories, and Holger Barth²

From the Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, D-89081 Ulm, Germany and Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany

The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N- and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells.

Received for publication, November 2, 2006 , and in revised form, January 24, 2007.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SPP 1150/BA 2087. 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 Movies 1 and 2.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 49-731-50065503; Fax: 49-731-50065502; E-mail: holger.barth{at}uni-ulm.de.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				K. Heine, S. Pust, S. Enzenmuller, and H. Barth ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death Infect. Immun., October 1, 2008; 76(10): 4600 - 4608. [Abstract] [Full Text] [PDF]