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J. Biol. Chem., Vol. 282, Issue 17, 12419-12429, April 27, 2007

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Segments Crucial for Membrane Translocation and Pore-forming Activity of Bordetella Adenylate Cyclase Toxin^*

Marek Basler, Oliver Knapp¹, Jiri Masin, Radovan Fiser^¶, Elke Maier, Roland Benz, Peter Sebo, and Radim Osicka²

From the Institute of Microbiology of the Academy of Sciences of the Czech Republic, Videnska 1083, CZ-142 20 Prague 4, Czech Republic, the ^¶Department of Genetics and Microbiology, Faculty of Science, Charles University, CZ-128 44, Prague 2, Czech Republic, and Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany

Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic -helix_502–522 containing a pair of negatively charged Glu⁵⁰⁹ and Glu⁵¹⁶ residues. Another predicted transmembrane -helix_565–591 comprises a Glu⁵⁷⁰ and Glu⁵⁸¹ pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu⁵¹⁶ increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu⁵¹⁶ by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu⁵⁷⁰ and Glu⁵⁸¹ by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu⁵⁷⁰ in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA.

Received for publication, December 7, 2006 , and in revised form, March 2, 2007.

^* This work was supported by Ministry of Education, Youth, and Sports Grant 1M0506 (to J. M. and M. B.), Academy of Sciences of the Czech Republic Grant IAA5020406 and the European Union 6th FP contract LSHB-CT-2003–503582 THERAVAC (to P. S.), Deutsche Forschungsgemeinschaft SFB 487 Project A5 (to R. B.), and Academy of Sciences of the Czech Republic Institutional Research Concept 50200510. 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 Table 1.

¹ Present address: Interactions Bactéries Cellules, Institut Pasteur, 28 rue du Dr. Roux, F-75724 Paris cedex 15, France.

² To whom correspondence should be addressed: Institute of Microbiology CAS, Videnska 1083, CZ-142 20 Prague 4, Czech Republic. Tel.: 420-241-062-770; Fax: 420-241-062-152; E-mail: osicka{at}biomed.cas.cz.

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