Python Erythrocytes Are Resistant to a-Hemolysin from Escherichia coli Casper K. Larsen • Marianne Skals • Tobias Wang • Muhammad U. Cheema • Jens Leipziger • Helle A. Praetorius Received: 31 August 2011 / Accepted: 20 October 2011 / Published online: 8 November 2011 Ó Springer Science+Business Media, LLC 2011 Abstract a-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 con- centrations 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 Ca 2? concentration and reduced cell volume when exposed to 3 mM ATP, indicat- ing the presence of a P2X 7 -like receptor. In addition, scav- enging 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 puriner- gic hemolysis amplification, or differences in HlyA affinity. Keywords Erythrocyte  Escherichia coli  Purinergic receptor  Python molurus  Python regius  a-Hemolysin The gram-negative bacterium Escherichia coli plays a dual role in most mammals. E. coli is the most prevalent gram- negative bacterium in the human intestine (Cavalieri et al. 1984; Johnson and Stell 2000), where it contributes to normal intestinal function and protects against intestinal infection by pathogenic bacteria (Hudault et al. 2001) and is possibly involved in vitamin K production (Conly and Stein 1992). On the other hand, E. coli also gives rise to serious extraintestinal infections, such as urinary tract infections, neonatal meningitis, and peritonitis (Cavalieri et al. 1984; Johnson and Stell 2000). E. coli strains isolated from patients often produce virulence factors, which con- tribute to the severity of the infection and are rarely pro- duced by the commensal, noninvasive strains (Johnson and Stell 2000; Cavalieri et al. 1984). One such virulence factor is a-hemolysin (HlyA), a pore-forming toxin commonly produced by E. coli strains isolated from extraintestinal infections (Cavalieri et al. 1984; Johnson and Stell 2000). Electronic supplementary material The online version of this article (doi:10.1007/s00232-011-9406-2) contains supplementary material, which is available to authorized users. C. K. Larsen  M. Skals  J. Leipziger  H. A. Praetorius (&) Department of Biomedicine, Aarhus University, Ole Worms Alle 4, Build 1160, 8000 Aarhus C, Denmark e-mail: hp@fi.au.dk C. K. Larsen  T. Wang Zoophysiology, Department of Biological Sciences, Aarhus University, C.F. Moellers Alle ´ 3, Build 1311, 8000 Aarhus C, Denmark M. U. Cheema Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle ´ 3, Build 1223, 8000 Aarhus C, Denmark 123 J Membrane Biol (2011) 244:131–140 DOI 10.1007/s00232-011-9406-2