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Adenylate Cyclase Toxin Subverts Phagocyte Function by RhoA Inhibition and Unproductive Ruffling¹

Jana Kamanova^*, Olga Kofronova^*, Jiri Masin^*, Harald Genth, Jana Vojtova^*, Irena Linhartova^*, Oldrich Benada^*, Ingo Just and Peter Sebo^2,*

^* Cellular and Molecular Microbiology Division, Institute of Microbiology of the Academy of Sciences of the Czech Republic, Prague 4, Czech Republic and Institute of Toxicology, Hannover Medical School, Hannover, Germany

Adenylate cyclase toxin (CyaA or ACT) is a key virulence factor of pathogenic Bordetellae. It penetrates phagocytes expressing the _Mβ₂ integrin (CD11b/CD18, Mac-1 or CR3) and paralyzes their bactericidal capacities by uncontrolled conversion of ATP into a key signaling molecule, cAMP. Using pull-down activity assays and transfections with mutant Rho family GTPases, we show that cAMP signaling of CyaA causes transient and selective inactivation of RhoA in mouse macrophages in the absence of detectable activation of Rac1, Rac2, or RhoG. This CyaA/cAMP-induced drop of RhoA activity yielded dephosphorylation of the actin filament severing protein cofilin and massive actin cytoskeleton rearrangements, which were paralleled by rapidly manifested macrophage ruffling and a rapid and unexpected loss of macropinocytic fluid phase uptake. As shown in this study for the first time, CyaA/cAMP signaling further caused a rapid and near-complete block of complement-mediated phagocytosis. Induction of unproductive membrane ruffling, hence, represents a novel sophisticated mechanism of down-modulation of bactericidal activities of macrophages and a new paradigm for action of bacterial toxins that hijack host cell signaling by manipulating cellular cAMP levels.

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.

¹ This work was supported by the European Union 6th FP Contract LSHB-CT-2003-503582 THERAVAC (to J.V.), Deutsche Forschungs-gemein-schaft Grant SPP1150 GE1247/1-3 (to I.J. and H.G.), the Institutional Research Concept MSM0021620858 (to J.M.), and Grants 1M0506 (to J.K.) and 2B06161 (to I.L.) from the Ministry of Education, Youth, and Sports and concept AV0Z50200510 and the Grant No. GA310/08/0447 (to P.S.) of the National Science Foundation of the Czech Republic.

² Address correspondence and reprint requests to Dr. Peter Sebo, Institute of Microbiology AS CR, v.v.i., Videnska 1083, Prague 4, Czech Republic. E-mail address: sebo{at}biomed.cas.cz

³ Abbreviations used in this paper: DC, dendritic cell; AC, adenylate cyclase enzyme domain; PKA, protein kinase A; GEF, guanine nucleotide exchange factor; CR3, complement receptor 3; db-cAMP, N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate; LY, lucifer yellow; BMM, bone marrow macrophage-like cells; CyaA, B. pertussis adenylate cyclase; CyaA-AC^–, enzymatically inactive adenylate cyclase toxoid; ROK, Rho kinase.