The aim of this study was to compare two methods for quantification of changes in intracellular potassium concentration (decrease from ∼140 to ∼20mM) due to the action of a pore-forming toxin, the adenylate cyclase toxin (CyaA) from the pathogenic bacterium Bordetella pertussis. CyaA was incubated with stably transfected K1 Chinese hamster ovary cells expressing the toxin receptor CD11b/CD18 and the decrease in potassium concentration in the cells was followed by inductively coupled plasma mass spectrometry (ICP-MS). It is shown that this method is superior in terms of sensitivity, accuracy, and temporal resolution over the method employing the potassium-binding benzofuran isophthalate-acetoxymethyl ester fluorescent indicator. The ICP-MS procedure was found to be a reliable and straightforward analytical approach enabling kinetic studies of CyaA action at physiologically relevant toxin concentrations (<1000ng/ml) in biological microsamples.

Institute of Analytical Chemistry Academy of Sciences of the Czech Republic 60200 Brno Czech Republic

Laboratory of Molecular Biology of Bacterial Pathogens Cell and Molecular Microbiology Division Institute of Microbiology Academy of Sciences of the Czech Republic 142 20 Prague 4 Czech Republic

Laboratory of Molecular Biology of Bacterial Pathogens Cell and Molecular Microbiology Division Institute of Microbiology Academy of Sciences of the Czech Republic 142 20 Prague 4 Czech Republic; Department of Genetics and Microbiology Faculty of Science Charles University Prague Prague 2 Czech Republic

References provided by Crossref.org

The adenylate cyclase toxin RTX domain follows a series templated folding mechanism with implications for toxin activity

Kingella kingae RtxA Cytotoxin in the Context of Other RTX Toxins

Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins

Almost half of the RTX domain is dispensable for complement receptor 3 binding and cell-invasive activity of the Bordetella adenylate cyclase toxin

Residues 529 to 549 participate in membrane penetration and pore-forming activity of the Bordetella adenylate cyclase toxin

Bordetella Pertussis Adenylate Cyclase Toxin Does Not Possess a Phospholipase A Activity; Serine 606 and Aspartate 1079 Residues Are Not Involved in Target Cell Delivery of the Adenylyl Cyclase Enzyme Domain

Bordetella pertussis Adenylate Cyclase Toxin Disrupts Functional Integrity of Bronchial Epithelial Layers

Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes

The conserved tyrosine residue 940 plays a key structural role in membrane interaction of Bordetella adenylate cyclase toxin

Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells

Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme