Clostridium perfringens epsilon toxin binds to erythrocyte MAL receptors and triggers phosphatidylserine exposure

Abstract Epsilon toxin (ETX) is a 33‐kDa pore‐forming toxin produced by type B and D strains of Clostridium perfringens. We previously found that ETX caused haemolysis of human red blood cells, but not of erythrocytes from other species. The cellular and molecular mechanisms of ETX‐mediated haemolysis are not well understood. Here, we investigated the effects of ETX on erythrocyte volume and the role of the putative myelin and lymphocyte (MAL) receptors in ETX‐mediated haemolysis. We observed that ETX initially decreased erythrocyte size, followed by a gradual increase in volume until lysis. Moreover, ETX triggered phosphatidylserine (PS) exposure and enhanced ceramide abundance in erythrocytes. Cell shrinkage, PS exposure and enhanced ceramide abundance were preceded by increases in intracellular Ca2+ concentration. Interestingly, lentivirus‐mediated RNA interference studies in the human erythroleukaemia cell line (HEL) cells confirmed that MAL contributes to ETX‐induced cytotoxicity. Additionally, ETX was shown to bind to MAL in vitro. The results of this study recommend that ETX‐mediated haemolysis is associated with MAL receptor activation in human erythrocytes. These data imply that interventions affecting local MAL‐mediated autocrine and paracrine signalling may prevent ETX‐mediated erythrocyte damage.

in cell volume, followed by mitochondrial disappearance, cell membrane blistering and rupture, ATP release, nuclear size reduction, and increased propidium iodide (PI) uptake. 4,8,9 The formation of pores in the affected cells leads to a rapid outflow of K + in the cells, the inflow of Cland Na + , followed by an increase in intracellular ([Ca 2+ ] i ). 10 Previously, we found that ETX is highly specific to human red blood cells, but does not cause haemolysis of erythrocytes in other species (murine, rabbit, sheep, goat, cattle, equine, dog, monkey). 11 This finding prompted us to further study the mechanisms of ETX-induced haemolysis.
Some bacterial toxins cause erythrocyte haemolysis through cell shrinkage, membrane blebbing and exposure of phosphatidylserine (PS) at the cell surface. 12 These include Escherichia coli α-haemolysin (HlyA), 13 Pseudomonas aeruginosa pyocyanin 14 and listeriolysin. 12 The MAL receptor was found to be required for ETX cytotoxicity in oligodendrocytes, 15 human T lymphocytes 16 and polarized epithelial cells. 17,18 The relative simplicity of erythrocytes makes these cells a suitable model for addressing the basic mechanisms of ETX-induced cell damage. Here, we investigated the role of MAL receptors in ETX-mediated toxicity and lysis of human erythrocytes. Our results showed that ETX initially causes a significant decrease in erythrocyte size, followed by an increase in cell volume leading to lysis. Moreover, ETX insertion caused an increase in [Ca 2+ ] i , enhanced ceramide abundance and promoted PS exposure in the outer leaflets of erythrocyte membranes. We also found that ETX-mediated death of HEL cells requires MAL and that ETX was shown to bind to MAL in vitro. Together, these data suggest that MAL receptors play an important role in ETXmediated haemolysis.

| Preparation of erythrocytes
Human blood was collected from healthy volunteers by venipuncture into evacuated blood collection tubes containing ethylenediaminetetraacetic acid-2K. Erythrocytes were washed three times with 0.01 M phosphate-buffered saline (PBS) (1000 × g, 4°C, 5 min). The serum layer was removed, and the pellet was the red blood cells.
The both plasmids were transformed into E coli BL21 (DE3) cells. To purify the His/GST-tagged ETX proteins, the bacterial pellets were resuspended in lysis buffer and cells were lysed by sonication on ice. The lysates were centrifuged at 3300 g for 15 minutes at 4°C.
The clarified supernatants were purified using a Ni 2+ /GST affinity chromatography column (GE Healthcare, Pittsburgh, PA, USA) as previously described. The purified proteins were analysed by 15% SDS-PAGE. We selected purified toxins with a purity greater than 98% for subsequent experiments.

| Measurements of haemolytic activity
The separated erythrocytes were diluted to a 5% solution with different controls are used in each figure. In general, maximal haemolysis in each figure was defined as 1(complete haemolysis caused by 10% Triton-100).

| Cell culture and treatment
The HEL cells were purchased from the China Infrastructure of

| MTS assay
HEL cells (3-4 × 10 4 cells/well) were seeded in 96-well plates and incubated at 37°C for 24 hours in a 5% CO 2 . Freshly purified toxin proteins were used in this assay. MTS assays were performed as described by the manufacturer.

| [Ca 2+ ]i imaging of human erythrocytes
Erythrocytes (~10 6 cells/mL) were first incubated with Fluo-4 AM (5 μM) for 25 minutes at 37°C in the dark, washed once with Ca 2+containing buffer and then centrifuged at 1000 g for 10 minutes at room temperature. After addition of ETX (0.2 μM), the increase in fluorescence at 488 nm over time was measured.

| Determine ceramide abundance
Ceramide concentrations in human erythrocytes were also assessed using the FACSaria. Erythrocytes (~10 6 cells/mL) were preincubated for 1 hours with ETX (0.2 μM) at 37°C, washed once in Ca 2+ -containing saline and centrifuged at 1000 × g for 10 minutes at room temperature.
The cells were incubated for 2 hours with anti-ceramide polyclonal antibody at 37°C, then washed once with Ca 2+ -containing saline. After incubation with FITC-conjugated secondary antibodies and washing once in Ca 2+ -containing saline, fluorescence at 488 nm was measured.

| Estimate reactive oxygen species (ROS)
To estimate the production of reactive oxygen species (ROS), eryth-

| Determine MAL receptor and anti-CD235a antibody expression
Erythrocytes (~106 cells/mL) were first incubated with anti-MAL antibodies (1:100) for 1 hours at 37°C, washed once with PBS buffer and then centrifuged at 1000 g for 10 minutes at room temperature.
The erythrocytes were incubated with PE-conjugated anti-human CD235a antibody and FITC-conjugated secondary antibody at 37°C for 30 minutes, washed once with PBS buffer and then detected by flow cytometry. After flow cytometry, human erythrocytes were attached to glass slides and covered with cover slips. The slides were imaged under a laser confocal scanning microscope (SP8, LEICA). quickly placed on glass slides. The slides were placed in a laser confocal scanning microscope for continuous imaging at 594 nm.

| MAL protein detection by Western blotting
**MAL protein expression in human erythrocytes, rat erythrocytes, mouse erythrocytes, HEL cells and HEL-∆MAL cells was assessed by Western blotting. The HEL and HEL-∆MAL cells were seeded in a 10 cm diameter culture plate overnight; then, 1 × 10 6 human, rat and mouse erythrocytes were taken and washed twice with PBS.
The cells were collected by centrifugation, and 500 μL of protein lysate supplemented with a protease inhibitor cocktail (1:100) was added. The lysed cells were placed in 1.5-mL tubes and fully lysed on ice for 30 minutes. The pellet was discarded by centrifugation, and the supernatant of the Pierce™ BCA Protein Assay Kit was quantified. Total protein (40 μg) was electrophoresed on a 15% SDS-PAGE gel, electrotransferred onto a nitrocellulose membrane and identified by Western blot. The membranes were probed with rabbit anti-MAL polyclonal antibody followed by secondary polyclonal HRP-conjugated goat anti-rabbit antibody. The blots were imaged using an AE-1000 cool CCD image analyser.

| Binding of mScarlet-ETX and MAL to HEL cells or human erythrocytes
HEL cells were cultured, and erythrocytes were prepared as de- the stained cells were dropped onto a glass slide to mount, and the slides were imaged using a confocal microscope.

| Propidium iodide (PI) staining
Cells were harvested and incubated with 0.2 μmol/L ETX at 37°C for 1 hours. The cells were washed once with PBS and resuspended in 200 μL of annexin V binding buffer containing 4 μL of 0.5 mg/mL PI. The stained cells were washed and resuspended with PBS and placed onto a glass slide for confocal microscopy.

| Statistical analysis
Data analysis and statistics data are presented as mean ± SD. Each experiment was repeated at least three times. When multiple comparisons were made between groups, significant differences were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni test. A P-value < 0.05 was considered to be a statistically significant.

| ETX induces human erythrocyte shrinkage, swelling and lysis
Since ETX and E coli HlyA belong to β-pore-forming toxin family, 4,19 we first assessed whether ETX induced erythrocyte shrinkage, swelling and lysis in a manner similar to HlyA. 13,20 Here, we stained the cell membranes of human erythrocytes with PKH26, incubated the cells with ETX and imaged the cells using confocal microscopy. Within the first 1 minute, ETX induced the formation of spinous cells in erythrocytes, which may reflect shrinkage of the cells ( Figure 1A,B). Similar results were obtained by light microscopy ( Figure 1C), as compared to the negative control ( Figure 1D). The cells swell after contraction, their morphology changes from spinous cells to spherocytes, and eventually rupture (Figure 1 A,B,D). Figure 1D shows images of human erythrocytes at 0, 1, 5, 10 and 60 minutes after ETX stimulation (0.2 μmol/L), with a gradual decrease in the number of red blood cells after 1 minute, confirming that ETX can cause erythrocyte lysis. Figure 1E,F shows the average data of contracted and lysed erythrocytes over time from three experiments. After incubation with ETX for 1 minute, the percentage of shrinking erythrocyte to total cells peaked. Over time, ETX causes a gradual increase in human erythrocyte lysis. It can be concluded that these changes in cellular appearance were a consequence of changes in cell volume.

| ETX increases [Ca 2+ ] i in human erythrocytes
ETX forming a heptameric toxin pore is a β-barrel pore characterized by an arrangement of 14 amphoteric β-strands. 4 The significant initial ETX-induced shrinkage of erythrocytes implied that ion efflux early during this process exceeded ion influx. To test whether the contraction caused by ETX has a similar mechanism, we verified if ETX triggered a change in [Ca 2+ ] i . Figure 2A Figure 2D). Subsequently, we added calcium ion-binding chelating agent (BAPTA-AM) to the calcium-free buffer without inhibiting ETX-induced haemolysis similar to Figure 2D (data not shown).
These data indicate that ETX causes influx of calcium ions, but the influx of calcium ions is not important for ETX-induced haemolysis.
The volume reduction caused by ETX showed that the formation of pores induced the flow out of net ions rather than flow in.
As the concentration of K + was the most important intracellular cation, there may be an outflow of K + during the shrinkage. 21 We first tested that if the shrinkage was caused by Ca 2+ -activated K + efflux, but clotrimazole and TRAM-34 (Ca 2+ -activated K + channel (K Ca 3.1

| ETX triggers PS exposure in the outer leaflet of the erythrocyte membrane
Increased [Ca 2+ ] i levels trigger an early senescent response in erythrocytes, including exposure of PS. 22 Therefore, we explored whether ETX could cause PS exposure and FITC-conjugated annexin V was used for this assay, which has high affinity for PS. We incubated red blood cells with 0.2 μM ETX and observed ~14% of cells with significant annexin V staining ( Figure 3A,B). These data demonstrated that ETX triggered PS exposure in the outer leaflet of the erythrocyte membrane.

| ETX enhanced erythrocyte ceramide abundance, but did not trigger production of reactive oxygen species
We further explored other mechanisms involved in ETX-induced erythrocyte death. A previous study showed that PS exposure was related to ceramide accumulation. 12,23 Additionally, ETX oligomer formation is induced by activation of neutral sphingomyelinase and production of ceramide. 24 Thus, we speculated that the toxic effects of ETX might be related to sphingomyelinase activity. We therefore examined whether ETX treatment influenced sphingomyelinase activation, which mediates ceramide formation in erythrocytes. Exposure to 0.2 μM ETX increased ceramide formation in erythrocytes ( Figure 3C and D). However, inhibitors of neutral sphingomyelinase (GW4869) and the neuraminidase inhibitor (N-oleoylethanolamine) did not inhibit ETX-induced haemolysis ( Figure 3G and H). We further investigated the effect of GW4869 on ETX-induced ceramide production. Figure 3C showed GW4869 inhibited ETX-induced ceramide formation. These data indicate that ETX-induced haemolysis enhanced

| ETX cytotoxicity in HEL cells requires MAL receptors
We have previously found that ETX is highly specific for human erythrocytes, but it has no toxic effects on erythrocytes from other species. 9 We incubated mouse, rat and human erythrocytes with mScarlet-ETX and found that mScarlet-ETX bound to human erythrocytes ( Figure 4A) but not to mouse ( Figure 4B) and rat erythrocytes ( Figure 4C). These findings suggest that ETX-binding receptors are present on human erythrocytes but absent in mouse and rat erythrocytes.
MAL has recently been found to be involved in the cytotoxicity of ETX. 25 We stained human, mouse and rat erythrocytes and found that MAL was expressed in human erythrocytes ( Figure 4A), but not in mouse ( Figure 4B) and rat erythrocytes ( Figure 4C). We also confirmed the expression of MAL receptors only in human red blood cells by Western blotting ( Figure 4E). Confocal microscopy revealed that expression of MAL proteins was mostly localized to the plasma membrane. Most mScarlet-ETX binds around the cell membrane ( Figure 4A and D). We confirmed that ETX caused HEL cell death and that these cells expressed MAL protein ( Figure 4E). Most of the mScarlet-ETX was located around HEL cells and co-localized with MAL proteins ( Figure 4D).
To demonstrate that MAL expression is involved in the cytotoxic effects of ETX, MAL was depleted in HEL cells via lentivirus-mediated RNA interference. Several MAL-deleted clones (HEL-∆MAL) were obtained, and the toxic effects of ETX on HEL-∆MAL were analysed.

Silencing of MAL expression in HEL-∆MAL clones was confirmed by
Western blotting (Figure 4F). Cytotoxicity assays (MTS colorimetric assays) of HEL-∆MAL clones revealed ETX did not have a cytotoxic effect when MAL protein was not expressed ( Figure 5A). Furthermore, absence of ETX binding to HEL-∆MAL clones was demonstrated by confocal microscopy ( Figure 5B). These results clearly indicate that the absence of expression of MAL protein directly impairs the binding of ETX to cells and the cytotoxic effects of ETX.

| MAL protein is required for ETX-induced oligomeric complex and pore formation
The cytotoxic effect of ETX occurs through binding to specific receptors on target cells, followed by oligomerization and pore formation, The results confirmed that ETX induced pore formation in HEL cells but not in HEL-ΔMAL cells ( Figure 6A). Western blot analysis showed that there was no membrane complex formation after incubation of HEL-∆MAL cells with ETX ( Figure 6B). These results suggested that MAL protein is involved in ETX-induced pore and oligomeric complex formation.

| ETX directly interacts with MAL receptors in the cell membranes of human erythrocytes
The

| D ISCUSS I ON
ETX has been found to form toxin pores on target cell membranes since 2001. 8 Clinically, it has been reported that in patients infected with C perfringens, large-scale intravascular haemolysis occurs, sometimes with severe anaemia. 27,28 The mechanism of causing severe anaemia during C perfringens infection has not been elucidated, but it is considered that erythrocyte haemolysis caused by ETX may be necessary for the treatment of C perfringens infection. Recently, we found that ETX is highly specific for human erythrocytes but not sensitive to erythrocytes of other species (such as sheep and goats). 11 However, the mechanism of haemolysis caused by ETX remains unclear. In this study, we further studied and defined the mechanism by which ETX causes erythrocyte lysis.
In the present study, it was found that ETX caused a significant decrease in human erythrocyte volume, and then swelled and lysis. The initial volume reduction is caused by the inflow of Ca 2+ and the outflow of K + . In addition, ETX also triggers PS exposure on the cell membrane and increases the abundance of ceramide.
Mechanistically, intracellular Ca 2+ activity is a crucial participant in eryptosis signalling. 29 Our results indicate that the first event after adding ETX is an increase in [Ca 2+ ] i ( Figure 2 tion of sphingomyelinase and production of ceramide. 12 We found that ETX could trigger an increase in ceramide abundance in human erythrocytes using flow cytometry ( Figure 3C). Ceramide enhances the Ca 2+ sensitivity of cell membranes, which is similar to increasing cytosolic Ca 2+ activity and increases PS exposure. 30 15 human T lymphocytes 16 and polarized epithelial cells. 25 We confirmed the high expression of MAL receptor in human erythrocytes by co-staining with anti-CD235a antibody ( Figure Fig S1) and showed that the MAL protein was expressed only in the membrane of human erythrocytes by immunofluorescence and Western blot. mScarlet-ETX bound to human but not to mouse or rat erythrocytes, and co-localized with MAL on human erythrocyte cell membranes. Our previous data also indicated that ETX only caused human erythrocytes haemolysis, but had no effect on mouse and rat erythrocytes. Based on the association between haemolysis and MAL expression, we conclude that MAL receptors play an important role in erythrocyte haemolysis caused by ETX. We further investigated the effects of ETX in MAL-deficient HEL cells and found that HEL cells also expressed MAL receptors, which co-localized with mScarlet-ETX on the cell membrane, and that ETX had a cytotoxic effect on HEL cells but not on MAL-deficient HEL cells. In addition, ETX did not bind MAL-deficient HEL cells and could not form pores or oligomeric complexes on the membranes of these cells. Together, these dates support the hypothesis that MAL is the cellular receptor for ETX.
We further confirmed a direct interaction between ETX and MAL receptors on human erythrocytes and HEL cells in vitro using a pull-down assay ( Figure 7). As far as we know, this is the first time showing a direct bind of ETX to MAL proteins in vitro. All of these observations support the notion that the MAL protein is the likely receptor for ETX.
In conclusion, our results underscore that ETX can only induce haemolysis of human erythrocytes, but it cannot induce haemolysis of erythrocytes in animals. Thus, ETX is potentially toxic to humans and may cause disease in humans to be more severe than in animals. ETX causes haemolysis is a process of volume change in human erythrocytes with an increase in PS exposure. This mechanism may be critical for the clearance of ETX-inserted erythrocytes in blood circulation after C perfringens infection. In addition, given that MAL receptors play an important role in ETX binding, ETX-induced cytotoxicity, polymer formation and pore formation.
Together with our data confirming an in vitro interaction between ETX and MAL, we conclude that MAL is likely the receptor for ETX, this protein represents a potential target for treatment of ETX-induced diseases.

ACK N OWLED G EM ENTS
We are grateful to all the members for support on this project. We thank Liwen Bianji, Edanz Editing China (www.liwen bianji.cn/ac), for editing the English text of a draft of this manuscript.

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the author.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included in the article.