A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.

• MeSH
• Actinobacillus pleuropneumoniae metabolism   MeSH
• Adenylate Cyclase Toxin antagonists & inhibitors   chemistry   metabolism   MeSH
• Bacterial Proteins antagonists & inhibitors   chemistry   metabolism   MeSH
• Bordetella pertussis metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Erythrocytes metabolism   MeSH
• Hemolysis * MeSH
• Hemolysin Proteins antagonists & inhibitors   chemistry   metabolism   MeSH
• Hexokinase MeSH
• Cells, Cultured MeSH
• Lipid Bilayers metabolism   MeSH
• Macrophages MeSH
• Mice MeSH
• Osmotic Pressure MeSH
• Cell Membrane Permeability MeSH
• Pyridoxal Phosphate analogs & derivatives   MeSH
• Rosaniline Dyes MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenylate Cyclase Toxin MeSH
• ApxI toxin, Bacteria MeSH Browser
• Bacterial Proteins MeSH
• coomassie Brilliant Blue MeSH Browser
• Hemolysin Proteins MeSH
• Hexokinase MeSH
• Lipid Bilayers MeSH
• pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid MeSH Browser
• Pyridoxal Phosphate MeSH
• Rosaniline Dyes MeSH