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J. Biol. Chem., Vol. 282, Issue 5, 2808-2820, February 2, 2007

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Third Activity of Bordetella Adenylate Cyclase (AC) Toxin-Hemolysin

MEMBRANE TRANSLOCATION OF AC DOMAIN POLYPEPTIDE PROMOTES CALCIUM INFLUX INTO CD11b⁺ MONOCYTES INDEPENDENTLY OF THE CATALYTIC AND HEMOLYTIC ACTIVITIES^*

Radovan Fier, Jií Maín, Marek Basler, Jan Krek^¶, Veronika puláková, Ivo Konopásek, and Peter ebo¹

From the Department of Genetics and Microbiology, Faculty of Science, Charles University, CZ-128 44, Prague 2 and the Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology, and the ^¶Department of Cellular Neurophysiology, Institute of Physiology, Academy of Sciences of the Czech Republic, CZ-142 20, Prague 4, Czech Republic

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the _M2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca²⁺]_i) in target cells. The CyaA-mediated [Ca²⁺]_i increase in CD11b⁺ J774A.1 monocytes was inhibited by extracellular La³⁺ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca²⁺ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC^– toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca²⁺]_i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca²⁺]_i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca²⁺]_i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca²⁺ influx elicited by the respective AC^– toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.

Received for publication, October 24, 2006 , and in revised form, November 30, 2006.

^* This work was supported by Grant 146/2005/B-BIO/PrF from Charles University (to R. F.), Grant 1M0506 from the Ministry of Education, Youth, and Sports (to J. M. and M. B.), European Union 6th FP Contract LSHB-CT-2003-503582 THERAVAC (to P. S.), and Grant IAA5020406 (to J. K.) and the Institutional Research Concept 50200510 from the Academy of Sciences of the Czech Republic. 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 Figs. S1–S6.

¹ To whom correspondence should be addressed. Tel.: 420-241-062-762; Fax: 420-241-062-152; E-mail: sebo{at}biomed.cas.cz.

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