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Infection and Immunity, April 2006, p. 2207-2214, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2207-2214.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Pore-Forming and Enzymatic Activities of Bordetella pertussis Adenylate Cyclase Toxin Synergize in Promoting Lysis of Monocytes

Marek Basler, Jiri Masin, Radim Osicka, and Peter Sebo*

Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology, CZ-142 20, Prague 4, Czech Republic

Received 1 September 2005/ Returned for modification 8 October 2005/ Accepted 26 January 2006

Bordetella adenylate cyclase (AC) toxin-hemolysin (CyaA) targets myeloid phagocytes expressing the {alpha}Mß2 integrin (CD11b/CD18) and delivers into their cytosol an AC enzyme that converts ATP into cyclic AMP (cAMP). In parallel, CyaA acts as a hemolysin, forming small membrane pores. Using specific mutations, we dissected the contributions of the two activities to cytolytic potency of CyaA on J774A.1 murine monocytes. The capacity of AC to penetrate cells and deplete cytosolic ATP was essential for promoting lysis and the enzymatically inactive but fully hemolytic CyaA-AC– toxoid exhibited a 15-fold-lower cytolytic capacity on J774A.1 cells than intact CyaA. Moreover, a two- or fourfold drop of specific hemolytic activity of the CyaA-E570Q and CyaA-E581P mutants was overpowered by an intact capacity to dissipate cytosolic ATP into cAMP, allowing the less hemolytic proteins to promote lysis of J774A.1 cells as efficiently as intact CyaA. However, an increased hemolytic activity, due to lysine substitutions of glutamates 509, 516, and 581 in the pore-forming domain, conferred on AC– toxoids a correspondingly enhanced cytolytic potency. Moreover, a threefold increase in hemolytic activity could override a fourfold drop in capacity to convert cellular ATP to cAMP, conferring on the CyaA-E581K construct an overall twofold increased cytolytic potency. Hence, although appearing auxiliary in cytolytic action of the toxin on nucleated cells, the pore-forming activity can synergize with ATP-depleting activity of the cell-invasive AC enzyme and complement its action toward maximal cytotoxicity.

* Corresponding author. Mailing address: Institute of Microbiology CAS, Videnska 1083, CZ-142 20 Prague 4, Czech Republic. Phone: (420) 241 062 762. Fax: (420) 241 062 152. E-mail: sebo{at}biomed.cas.cz.

Editor: J. T. Barbieri

Infection and Immunity, April 2006, p. 2207-2214, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2207-2214.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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