Grant-funded Project Nr. 146/2005/B-BIO/PřF
Final Report

Project title:Mechanism of changes in intracellular calcium concentration in cells after interaction with adenylate-cyclase toxin of bacterium Bordetella pertussis
Research leader:Mgr. Radovan Fišer, 2003
Co-researcher: RNDr. Ivo Konopásek, CSc.; Mgr. Jiří Mašín; Veronika Špuláková; RNDr. Jan Krůšek, CSc.; Ing. Marek Basler
Period of project:2005-2006
Overall grant:290 000 CZK

Project Results

The Bordetella adenylate cyclase (CyaA, ACT or AC-Hly) is a multifunctional toxin that paralyzes bactericidal activities of phagocytes expressing the integrin CD11b/CD18 (CR3 or Mac-1). Besides permeabilizing cells by formation of cation-selective channels, CyaA delivers into cells an adenylate cyclase enzyme (AC) that converts cytosolic ATP to cAMP. We found that CyaA is able to elicit entry of extracellular calcium ions into CD11b+ myeloid cells by a mechanism independent of the catalytic and hemolytic capacities of the toxin. The elevation of cytosolic [Ca2+]i concentration was not triggered by the mere interaction of CyaA with the integrin receptor and was not affected by inhibitors of conventional calcium channels, such as nifedipine, SK&F-96365, flunarizine, 2-APB, or thapsigargin. The increase of [Ca2+]i was, however, compromised in the presence of La3+ ions and following modifications of toxin structure that interfered with membrane translocation of the AC domain polypeptide, such as the Glu509 to Pro509 substitutions. Structural alterations within the AC domain due to insertion of various heterologous oligopeptides and disruption of the ATP and calmodulin binding sites by dipeptide insertion strongly affected the kinetics and extent of [Ca2+]i influx promoted by the respective CyaA-AC- toxoids. It is concluded that membrane translocation and structure of the AC domain determine the capacity of the toxin to elevate the cellular [Ca2+]i concentration.

These results were published in the article:
Fiser, R., Masin, J., Basler, M., Krusek, J., Spulakova, V., Konopasek, I., and Sebo, P. (2007)
Journal of Biological Chemistry 282(5), 2808-2820

Posters:
- BASLER, M.; MAŠÍN, J.; FIŠER, R.; OSIČKA, R.; KRŮŠEK, J.; KONOPÁSEK, I.; BENZ, R.; ŠEBO P. Synergy between ATP-depleting, hemolytic and intracellular calcium-increasing capacities accounts for cytotoxic activity of Bordetella adenylate cyclase toxin. 12th European Conference on Bacterial Protein Toxins (ETOX), June 25 - 29, 2005, Canterbury, England
- FIŠER, R.; MAŠÍN, J.; BASLER, M.; KRŮŠEK, J.; ŠPULÁKOVÁ, V.; KONOPÁSEK, I., ŠEBO, P., Membrane translocation of Bordetella adenylate cyclase toxin promotes calcium entry into CD11b+ J774A.1 macrophage cells. 12th European Conference on Bacterial Protein Toxins (ETOX) , June 25 - 29, 2005, Canterbury, England
- FIŠER, R.; MAŠÍN, J.; BASLER, M.; KRŮŠEK, J.; ŠPULÁKOVÁ, V.; KONOPÁSEK, I., ŠEBO, P., Bordetella adenylate cyclase toxin provokes calcium influx into CD11b+ J774A.1 macrophages during adenylate cyclase domain translocation across the plasma membrane. 2nd FEMS Congress of  European Microbiologists, Spain, Madrid, July 4 – 8, 2006
- FIŠER, R.; MAŠÍN, J.; BASLER, M.; KRŮŠEK, J.; ŠPULÁKOVÁ, V.; KONOPÁSEK, I.;, ŠEBO, P., Membrane translocation of Bordetella adenylate cyclase toxin promotes calcium entry into CD11b+ J774A.1 macrophage cells. 40th Annual Scientific Meeting of the European Society for Clinical Investigation, Praha, 15 – 18 March 2006