␣-Hemolysin from Escherichia coli uses endogenous amplification through P2X receptor activation to induce hemolysis Marianne Skalsa, Niklas R. Jorgensenb, Jens Leipzigera, and Helle A. Praetoriusa,1 aDepartment of Physiology and Biophysics, Water and Salt Research Center, Aarhus University, Ole Worms Alle 1160, 8000 Aarhus C, Denmark; and bDepartment for Clinical Biochemistry, Roskilde Hospital, Koegevej 3-7, 4000 Roskilde, Denmark Edited by Sucharit Bhakdi, University of Mainz, Mainz, Germany, and accepted by the Editorial Board January 6, 2009 (received for review July 22, 2008) Escherichia coli is the dominant facultative bacterium in the normal and are referred to as P2X1–7. All P2X receptors are permeable to intestinal flora. E. coli is, however, also responsible for the majority small monovalent cations and some have significant calcium per- of serious extraintestinal infections. There are distinct serotypical meability (11). Here we show that human, murine, and equine differences between facultative and invasive E. coli strains. Inva- erythrocytes use a combination of P2X1 and P2X7 receptor acti- sive strains frequently produce virulence factors such as ␣-hemolysin vation for full HlyA-induced hemolysis to occur. This is particularly (HlyA), which causes hemolysis by forming pores in the erythrocyte interesting, as prolonged stimulation of P2X7 receptors are known membrane. The present study reveals that this pore formation to increase the plasma membrane permeability to an extent that triggers purinergic receptor activation to mediate the full hemo- eventually leads to lysis of certain cells (12). In macrophages it has lytic action. Non-selective ATP-receptor (P2) antagonists (PPADS, been shown that pannexin1, a recently discovered pore-forming suramin) and ATP scavengers (apyrase, hexokinase) concentration protein, is required for this increment in permeability (12, 13). Our dependently inhibited HlyA-induced lysis of equine, murine, and data also support a role for pannexin channels in addition to P2X human erythrocytes. The pattern of responsiveness to more selec- channels in HlyA-induced hemolysis. tive P2-antagonists implies that both P2X1 and P2X7 receptors are involved in HlyA-induced hemolysis in all three species. In addition, Results PHYSIOLOGY our results also propose a role for the pore protein pannexin1 in HlyA-Induced Hemolysis Requires Activation of Purinergic Receptors. HlyA-induced hemolysis, as non-selective inhibitors of this channel ␣ significantly reduced hemolysis in the three species. In conclusion, Supernatant from the -hemolysin (HlyA)–producing E. coli–strain activation of P2X receptors and possibly also pannexins augment ARD6 lyses equine, human, and murine erythrocytes (Fig. 1). hemolysis induced by the bacterial toxin, HlyA. These findings Figure 1 shows the HlyA-induced hemolysis as a function of time. potentially have clinical perspectives as P2 antagonists may ame- Time-lapse experiments with murine and human erythrocytes liorate symptoms during sepsis with hemolytic bacteria. attached to coverslips revealed that HlyA-induced hemolysis is a sequential process. Within the first 20 minutes, HlyA induced alpha-hemolysin ͉ E. coli ͉ erythrocytes ͉ hemolysis ͉ P2X crenation of the red blood cells as a result of cell shrinkage, followed by a gradual volume increase and finally lysis of the cells (Figs. 1A he dominant facultative intestinal bacterium Escherichia coli (E. and 1B, Movie S1). This sequential shrinkage and swelling also Tcoli) frequently induces serious extraintestinal infections as applies at the single-cell level. Thus, it is not different populations neonatal meningitis, peritonitis, Gram-negative bacteriemia, and of red blood cells that either shrink or swell but, rather, that a single urinary infections including pyelonephritis (1, 2). There are, how- erythrocyte first shrinks and then swells as a consequence of HlyA ever, distinct serotypical differences between the facultative E. coli application. The erythrocyte suspension (1.25%) was incubated Ϫ and the ones that invade the tissue and cause infection. The invasive with dilute E. coli supernatant (50 ␮l ⅐ ml 1). Erythrocytes from the E. coli strains frequently produce virulence factors such as the three tested species showed marked difference in the responsive- exotoxin ␣-hemolysin (HlyA) (1, 3). The frequency by which ness to HlyA (Fig. 1C) with the lowest sensitivity to HlyA in human hemolytic E. coli strains can be isolated from patient samples erythrocytes. In all of the following experiments, the amount of increases with the severity of disease (1). added E. coli supernatant was adjusted to produce Ϸ50% hemolysis HlyA is a 107 kDa (4) protein that induces hemolysis by creating after 60 minutes’ incubation. Ϸ2-nm-wide pores in the erythrocyte membrane. These pores are We generally use filtered E. coli (ARD6) supernatant to induce thought to increase the permeability and thereby produce cell hemolysis unless otherwise stated. This approach was chosen to swelling, which finally ruptures the erythrocyte. Thus, increasing ensure that our results would also apply in vivo where HlyA is the osmolality of the extracellular solution with cell-impermeate released from E. coli together with various other components. sugars inhibits the HlyA-induced hemolysis completely (5). If When choosing this approach, we did, however, have to verify that HlyA-induced hemolysis is merely a consequence of inserting the hemolysis induced by HlyA-producing E. coli could in fact be non-selective pores into the plasma membrane of red blood cells, it ascribed to HlyA. Therefore, we purified HlyA from our ARD6- is puzzling that the sensitivity to HlyA varies among species (6). This culture. After purification, a suspension of the purified HlyA was feature is not unique to HlyA, as the sensitivity to other pore- separated on a 5–15% sodium dodecyl sulfate (SDS) gel. A single formers such as ␣-toxin from Staphylococcus aureus also shows great interspecies variability (7). Regarding S. aureus, the interspe- cies variation was explained through differences in expression levels Author contributions: M.S., J.L., and H.A.P. designed research; M.S. performed research; of a specific receptor for ␣-toxin (8). This option has also been N.R.J. contributed new reagents/analytic tools; M.S. and H.A.P. analyzed data; and M.S. and suggested for HlyA-induced hemolysis (9) but is not yet generally H.A.P. wrote the paper. accepted (10). The authors declare no conflict of interest. In the present study, we investigate the possibility that HlyA This article is a PNAS Direct Submission. S.B. is a guest editor invited by the Editorial Board. requires P2-receptor activation to produce hemolysis. P2X recep- 1To whom correspondence should be addressed. E-mail: hp@fi.au.dk. tors are ligand-gated cation channels activated by extracellular This article contains supporting information online at www.pnas.org/cgi/content/full/ ATP. To date, seven subtypes of P2X receptors have been identified 0807044106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807044106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 1, 2021 A A B 1.5 Human 1.0 Mouse Horse 1.0 Mu Hum 0.5 1.0 * 0.5 0.5 Relative hemolysis Relative Relative hemolysis Relative 012 510 0 Control 10min 20 min 60 min 0 0 0 1 10 0 1 10 Apyrase (U ml-1) Hexokinase (U ml-1) 2.5 B Human erythrocytes C 60 50 2.0 1.0 2.5 50 C D Cont 40 PPADS 250µM 1.5 40 0.8 2.0 PPADS 500µM 30 PPADS 1000µM 30 OD540 1.0 0.6 20 1.5 20 0.5 10 Horse 0.4 OD 540 1.0 10 Mouse Number of cells (%) Number of cells 0 0 0 Human 0.2 01020304060 01020304060 0.5 0 10 20 30 40 50 60 hemolysis Relative Time (min) Time (min) Time (min) 0 0 0 100 1000 0.5 5 50 -1 Fig. 1. ␣-Hemolysin–induced hemolysis in equine, murine and human eryth- PPADS (µM) HlyA (ng ml ) rocytes. (A) Effect of ␣-hemolysin containing E. coli (ARD6, serotype Fig. 2. HlyA-induced hemolysis of erythrocytes is inhibited by ectoATPases OK:K13:H1) supernatant on human erythrocytes attached to a coverslip after and purinergic antagonist. E. coli supernatant (60 minutes) induces hemolysis 10, 20, and 60 minutes’ incubation at 37 °C (see also Movie S1). (B) Summarized of human (square), murine (filled circles), and equine (open circles) erythro- data. The total amount of crenated erythrocytes (open columns) and lysed cytes. (A) Concentration–response curves for the ATP scavenger apyrase. Inset erythrocytes (dotted columns) over time analyzed in image sequences col- shows a representative picture of supernatant from murine erythrocytes lected over 60 minutes, at 0.1 Hz (n ϭ 8 human). (C) The overall hemolysis is subjected to HlyA in the presence of 0, 1, 2, 5 or 10 U mlϪ1 apyrase. (B) Effect shown as an increase in optical density at 540 nm (OD540) reflecting the of hexokinase on the HlyA-induced lysis of human, murine, and equine hemoglobin concentration in the solution. The erythrocytes were incubated erythrocytes; inset shows the effect of hexokinase (10 U mlϪ1) on hemolysis with E. coli supernatant (50 ␮l⅐mlϪ1) from 0 to 60 minutes. n ϭ 5, 7, and 6 for induced by purified HlyA in murine and human erythrocytes). (C) Effect of the equine, murine, and human, respectively. non-selective P2 receptor antagonist PPADS on HlyA-induced lysis of erythro- cytes from all three species. (D) Concentration–response relationship of PPADS at various concentration of purified HlyA in human erythrocytes. Hemolysis 100-kDa band appeared after Coomassie R staining, and mass was measured as OD540. Values are mean Ϯ SEM; n ϭ 5–13. spectroscopy identified the band as HlyA (Fig. S1 A and B). As an additional control we used the supernatant from the E. coli strain D2103, a non-pathological laboratory strain of E. coli that does not tions (Fig. 2D) tested in human erythrocytes exposed to purified produce HlyA.