Bacterial RTX Toxins Allow Acute ATP Release from Human Erythrocytes Directly through the Toxin Pore

• Published in: The Journal of biological chemistry 2014-07, Vol.289 (27), p.19098-19109
• Main Authors: Skals, Marianne, Bjaelde, Randi G., Reinholdt, Jesper, Poulsen, Knud, Vad, Brian S., Otzen, Daniel E., Leipziger, Jens, Praetorius, Helle A.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Aggregatibacter actinomycetemcomitans; Animals; ATP Release; Bacterial Proteins - chemistry; Bacterial Proteins - pharmacology; Bacterial Toxin; Connexins - deficiency; Connexins - genetics; Erythrocyte; Erythrocytes - cytology; Erythrocytes - drug effects; Erythrocytes - metabolism; Escherichia coli (E. coli); Escherichia coli Proteins - chemistry; Escherichia coli Proteins - pharmacology; Gene Knockout Techniques; Hemoglobins - metabolism; Hemolysin; Hemolysin Proteins - chemistry; Hemolysin Proteins - pharmacology; Hemolysis; Hemolysis - drug effects; Humans; Leukotoxin; Membrane Biology; Membranes, Artificial; Mice; Nerve Tissue Proteins - deficiency; Nerve Tissue Proteins - genetics; P2-receptors; Pannexin; Phosphatidylcholines - metabolism; Phospholipid Vesicle; Porosity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.571414

ATP is as an extracellular signaling molecule able to amplify the cell lysis inflicted by certain bacterial toxins including the two RTX toxins α-hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans. Inhibition of P2X receptors completely blocks the RTX toxin-induced hemolysis over a larger concentration range. It is, however, at present not known how the ATP that provides the amplification is released from the attacked cells. Here we show that both HlyA and LtxA trigger acute release of ATP from human erythrocytes that preceded and were not caused by cell lysis. This early ATP release did not occur via previously described ATP-release pathways in the erythrocyte. Both HlyA and LtxA were capable of triggering ATP release in the presence of the pannexin 1 blockers carbenoxolone and probenecid, and the HlyA-induced ATP release was found to be similar in erythrocytes from pannexin 1 wild type and knock-out mice. Moreover, the voltage-dependent anion channel antagonist TRO19622 had no effect on ATP release by either of the toxins. Finally, we showed that both HlyA and LtxA were able to release ATP from ATP-loaded lipid (1-palmitoyl-2-oleoyl-phosphatidylcholine) vesicles devoid of any erythrocyte channels or transporters. Again we were able to show that this happened in a non-lytic fashion, using calcein-containing vesicles as controls. These data show that both toxins incorporate into lipid vesicles and allow ATP to be released. We suggest that both toxins cause acute ATP release by letting ATP pass the toxin pores in both human erythrocytes and artificial membranes. Background: Hemolysis induced by the two RTX toxins HlyA and LtxA depends on ATP receptor activation. Results: HlyA and LtxA result in ATP release from human erythrocytes, which was unrelated to the main suggested ATP release pathway pannexin 1. Conclusion: ATP is released through a toxin pore. Significance: Adds new insights to the mechanism of these toxins.