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J. Biol. Chem., Vol. 280, Issue 22, 21107-21114, June 3, 2005

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Functional Properties of the p33 and p55 Domains of the Helicobacter pylori Vacuolating Cytotoxin^*

Victor J. Torres, Susan E. Ivie, Mark S. McClain¶||, and Timothy L. Cover¶**

From the Departments of Microbiology and Immunology and ^¶Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2605 and ^**Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212

Helicobacter pylori secretes an 88-kDa vacuolating cytotoxin (VacA) that may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. VacA cytotoxic activity requires assembly of VacA monomers into oligomeric structures, formation of anion-selective membrane channels, and entry of VacA into host cells. In this study, we analyzed the functional properties of recombinant VacA fragments corresponding to two putative VacA domains (designated p33 and p55). Immunoprecipitation experiments indicated that these two domains can interact with each other to form protein complexes. In comparison to the individual VacA domains, a mixture of the p33 and p55 proteins exhibited markedly enhanced binding to the plasma membrane of mammalian cells. Furthermore, internalization of the VacA domains was detected when cells were incubated with the p33/p55 mixture but not when the p33 and p55 proteins were tested individually. Incubation of cells with the p33/p55 mixture resulted in cell vacuolation, whereas the individual domains lacked detectable cytotoxic activity. Interestingly, sequential addition of p55 followed by p33 resulted in VacA internalization and cell vacuolation, whereas sequential addition in the reverse order was ineffective. These results indicate that both the p33 and p55 domains contribute to the binding and internalization of VacA and that both domains are required for vacuolating cytotoxic activity. Reconstitution of toxin activity from two separate domains, as described here for VacA, has rarely been described for pore-forming bacterial toxins, which suggests that VacA is a pore-forming toxin with unique structural properties.

Received for publication, January 27, 2005 , and in revised form, April 6, 2005.

^* This work was supported by National Institutes of Health Grants AI39657 and DK53623 and by the Medical Research Department of the Department of Veterans Affairs (to T. L. C.). 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.

Supported by the GM070061-02 National Institutes of Health Ruth L. Kirschstein pre-doctoral fellowship.

^|| Supported in part by the Vanderbilt University Medical Center Intramural Discovery Grant Program.

To whom correspondence should be addressed: Div. of Infectious Diseases, A2200 MCN, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-322-2035; E-mail: timothy.L.cover{at}vanderbilt.edu.

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