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J. Biol. Chem., Vol. 280, Issue 44, 36657-36663, November 4, 2005

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Binding of Escherichia coli Hemolysin and Activation of the Target Cells Is Not Receptor-dependent^*

Angela Valeva1, Ivan Walev, Helene Kemmer, Silvia Weis, Isabel Siegel, Fatima Boukhallouk, Trudy M. Wassenaar, Triantafyllos Chavakis¶2, and Sucharit Bhakdi

From the Institute of Medical Microbiology and Hygiene, University of Mainz, D-55101 Mainz, Germany, Molecular Microbiology and Genomics Consultants 55576, Zotzenheim, Germany, and ^¶Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892

Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (HlyA) to be radioactively labeled with tritiated N-ethylmaleimide without affecting biological activity. It thus became possible to study the binding characteristics of HlyA as well as of toxin mutants in which one or both acylation sites were deleted. All toxins bound to erythrocytes and granulocytes in a nonsaturable manner. Only wild-type toxin and the lytic monoacylated mutant stimulated production of superoxide anions in granulocytes. An oxidative burst coincided with elevation of intracellular Ca²⁺, which was likely because of passive influx of Ca²⁺ through the toxin pores. Competition experiments showed that binding to the cells was receptor-independent, and preloading of cells with a nonlytic HlyA mutant did not abrogate the respiratory burst provoked by a subsequent application of wild-type HlyA. In contrast to a previous report, expression or activation of the ₂ integrin lymphocyte function-associated antigen-1 did not affect binding of HlyA. We conclude that HlyA binds nonspecifically to target cells and a receptor is involved neither in causing hemolysis nor in triggering cellular reactions.

Received for publication, July 15, 2005 , and in revised form, August 30, 2005.

^* This work was supported by Deutsche Forschungsgemeinschaft 490, Project C1/D3. 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.

² Supported by the Intramural Research Program, NCI, National Institutes of Health.

¹ To whom correspondence should be addressed: Inst. of Medical Microbiology and Hygiene, University of Mainz, Hochhaus am Augustusplatz, D-55101 Mainz, Germany. Tel.: 49-6131-3936363; Fax: 49-6131-3932359; E-mail: avaleva{at}uni-mainz.de.

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