α-Hemolysin (HlyA) from Escherichia coli lyses mammalian erythrocytes by creating nonselective cation pores in the membrane. Pore insertion triggers ATP release and subsequent P2X receptor and pannexin channel activation. Blockage of either P2X receptors or pannexin channels reduces HlyA-induced hemolysis. We found that erythrocytes from Python regius and Python molurus are remarkably resistant to HlyA-induced hemolysis compared to human and Trachemys scripta erythrocytes. HlyA concentrations that induced maximal hemolysis of human erythrocytes did not affect python erythrocytes, but increasing the HlyA concentration 40-fold did induce hemolysis. Python erythrocytes were more resistant to osmotic stress than human erythrocytes, but osmotic stress tolerance per se did not confer HlyA resistance. Erythrocytes from T. scripta, which showed higher osmotic resistance than python erythrocytes, were as susceptible to HlyA as human erythrocytes. Therefore, we tested whether python erythrocytes lack the purinergic signalling known to amplify HlyA-induced hemolysis in human erythrocytes. P. regius erythrocytes increased intracellular Ca2+ concentration and reduced cell volume when exposed to 3 mM ATP, indicating the presence of a P2X7-like receptor. In addition, scavenging extracellular ATP or blocking P2 receptors or pannexin channels reduced the HlyA-induced hemolysis. We tested whether the low HlyA sensitivity resulted from low affinity of HlyA to the python erythrocyte membrane. We found comparable incorporation of HlyA into human and python erythrocyte membranes. Taken together, the remarkable HlyA resistance of python erythrocytes was not explained by increased osmotic resistance, lack of purinergic hemolysis amplification, or differences in HlyA affinity.
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We would like to thank Anne B. Strandsby, Edith Møller, Christian Westberg, and Rasmus Buchanan for their technical assistance. We also thank Jeppe Praetorius for assistance with production of fluorescent HlyA and binding studies of fluorescent HlyA to erythrocyte ghosts.
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Supplementary material 3: ATP-induced shrinkage of P. regius erythrocytes stimulated with 3 mM ATP. Pictures were captured every 30 seconds for 90 minutes after the addition of 3 mM ATP. The frame rate of the movie is 10 pictures s−1, i.e. 1 second in the movie corresponds to 5 minutes. The ATP-induced shrinkage persists for the duration of the experiment, no cells were ever observed to lyse when stimulated with ATP (AVI 5288 kb)
Supplementary material 4: HlyA-induced shrinkage and lysis of P. regius erythrocytes. Pictures were captured every 30 seconds for 90 minutes after the addition of HlyA at EC50. The frame rate of the movie is 10 pictures s−1, i.e. 1 second in themovie corresponds to 5 minutes. Lysis can be observed in a few cells on the lower right quadrant of the movie (AVI 4951 kb)
Supplementary material 5: PPADS blocks HlyA-induced shrinkage and lysis of P. regius erythrocytes. Pictures were captured every 30 seconds for 90 minutes after the addition of HlyA at EC50 and PPADS (500 μM). The frame rate of the movieis 10 pictures s−1, i.e. 1 second in the movie corresponds to 5 minutes. Neither shrinkage nor hemolysis was observed in the presence of PPADS (AVI 5284 kb)
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Larsen, C.K., Skals, M., Wang, T. et al. Python Erythrocytes Are Resistant to α-Hemolysin from Escherichia coli . J Membrane Biol 244, 131–140 (2011). https://doi.org/10.1007/s00232-011-9406-2
- Escherichia coli
- Purinergic receptor
- Python molurus
- Python regius